The SARS-CoV-2 virus has caused a global crisis, resulting in 0.5 billion infections and over 6 million deaths as of March 2022. Fortunately, infection and hospitalization rates were curbed due to the rollout of DNA and mRNA vaccines. However, the efficacy of these vaccines significantly drops a few months post immunization, from 88% down to 47% in the case of the Pfizer BNT162 vaccine. The emergence of variant strains, especially delta and omicron, have also significantly reduced vaccine efficacy. We propose peptide vaccines as a potential solution to address the inadequacies of the current vaccines. Peptide vaccines can be easily modified to target emerging strains, have greater stability, and do not require cold-chain storage. We screened five peptide fragments (B1–B5) derived from the SARS-CoV-2 spike protein to identify neutralizing B-cell peptide antigens. We then investigated adjuvant systems for efficient stimulation of immune responses against the most promising peptide antigens, including liposomal formulations of polyleucine (L10) and polymethylacrylate (PMA), as well as classical adjuvants (CFA and MF59). Immune efficacy of formulations was evaluated using competitive ELISA, pseudovirion neutralization, and live virus neutralization assays. Unfortunately, peptide conjugation to L10 and PMA dramatically altered the secondary structure, resulting in low antibody neutralization efficacy. Of the peptides tested, only B3 administered with CFA or MF59 was highly immunogenic. Thus, a peptide vaccine relying on B3 may provide an attractive alternative to currently marketed vaccines. The COVID-19 pandemic pandemic has been a major healthcare disaster. Since the first case was documented in December 2019 until now (February 2022), the virus has caused 415 million infections and 5.8 million global deaths. COVID-19 infection occurs through the inhalation of contaminated aerosol from an infected patient. SARS-CoV-2 (SARS-2) binds via its spike protein (SARS-2-S) to the angiotensin-converting enzyme-2 (ACE2) ,4,5.C-terminal residues, N-terminal residues of the RBM, or even outside of the RBM sequence 2+ m/z 979.6 , [M + 3H]3+ m/z 653.5 . tR = 17.97 min . Purity = 98%.B1 peptide (469STEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPY508) at the RBM of RBD. Yield: 45%. Molecular weight: 4425.8. ESI-MS: [M + 3H]3+ m/z 1476.8 , [M + 4H]4+ m/z 1108.0 . tR = 21.15 min . Purity ≥ 99%.B2 peptide (444GVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV483) at the RBM of RBD. Yield: 50%. Molecular weight: 4593.2. ESI-MS: [M + 3H]3+ m/z 1532.3 , [M + 4H]4+ m/z 1149.10 , [M + 5H]5+ m/z 919.5 . tR = 21.49 min . Purity ≥ 99%.B3 peptide (559FLPFQQFGRDIADT572) near the S1/S2 cleavage/priming site. Yield: 80%. Molecular weight: 1675.8. ESI-MS: [M + 1H]1+ m/z 1676.8 , [M + 2H]2+ m/z 839.4 , [M + 3H]3+ m/z 559.9 . tR = 19.35 min . Purity ≥ 99%.B4 peptide (366SVLYNSASFSTFKCYGVSPTKLNDLCFTNV395) at the N-terminus of RBD, but not in direct contact with ACE2. Yield: 55%. Molecular weight: 3348.6. ESI-MS: [M + 2H]2+ m/z 1675.2 , [M + 3H]3+ m/z 1117.1 , [M + 4H]4+ m/z 838.6 . tR = 17.2 min . Purity ≥ 99%.B5 peptide , [M + 2H]2+ m/z 695.8 . tR = 23.2 min . Purity ≥ 99%.PADRE peptide (AKFVAAWTLKAAA). Yield: 75%. Molecular weight: 1389.7. ESI-MS: [M + 1H]10-B1 peptide (L10-AIHADQLTPTWRVYSTG). Yield: 50%. Molecular weight: 3088.8. ESI-MS: [M + 2H]2+ m/z 1545.4 , [M + 3H]3+ m/z 1030.6 . tR = 17.97 min . Purity = 96%.L10-B2 (L10-STEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPY). Yield: 35%. Molecular weight: 5560.4. ESI-MS: [M + 3H]3+ m/z 1854.5 , [M + 4H]4+ m/z 1391.1 , [M + 5H]5+ m/z 1113.1 . tR = 30.2 min . Purity ≥ 99%.L10-B3 (L10-GVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV). Yield: 40%. Molecular weight: 5747.40. ESI-MS: [M + 4H]4+ m/z 1437.2 , [M + 5H]5+ m/z 1149.8 , [M + 6H]6+ m/z 958.9 . tR = 25.64 min . Purity ≥ 99%.L10-PADRE (L10-AKFVAAWTLKAAA). Yield: 45%. Molecular weight: 2521.27. ESI-MS: [M + 2H]2+ m/z 1260.6 , [M + 3H]3+ m/z 841.0 . tR = 37.10 min . Purity ≥ 99%.L1+ m/z 6.8 kDa. tR = 33.50 min . Purity = 97%.PMA-B3 (PMA-GVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV). Yield: 51%. Molecular weight: 6.9 ± 0.3 kDa. MALDI-ToF: [M + 1H]1+ m/z 3.77 kDa. tR = 33.2 min . Purity = 98%.PMA-PADRE (PMA-AKFVAAWTLKAAA). Yield: 43%. Molecular weight: 3.75 ± 0.3 kDa. MALDI-ToF: [M + 1H]Secondary Structure of Peptide AntigensAntigen secondary structure was evaluated by preparing a 1 mg/mL solution of each synthesized peptide in phosphate buffered saline (PBS). Aliquots of 0.2 mL were transferred into 1 mm pathlength quartz microcuvettes and measured via circular dichroism (CD) spectra using a Jasco-720 spectropolarimeter . Quantitative secondary structure content was determined by fitting the composite spectra to yield contribution of each pure individual component spectrum, i.e. α-helix, β-sheet, or random coil, obtained from reported polylysine spectra, adapted from the method introduced by Greenfield et al. ,47, as dLiposome Preparation and Encapsulation Efficiency−3 mmole), didodecyldimethylammonium bromide , and cholesterol  in 2 mL of chloroform in a 5 mL round-bottom flask. Polyleucine- or PMA-conjugated antigen peptide, single (0.6 mg) or double dose (1.2 mg), along with helper T-cell epitopes L10-PADRE or PMA-PADRE (0.6 mg), were dissolved in 2 mL of methanol and chloroform mixture (1:1). This was then added to the chloroform lipid solution in the round-bottom flask. The flask was attached to a rotary evaporator  and the mixture was evaporated under reduced pressure (95 mbar) for 2 h at 30 °C. The formed thin layer of film was left to dry overnight in a desiccator under vacuum to remove any residual solvents. The dry lipid film was then allowed to rehydrate and swell with 0.9 mL of MilliQ water. The resulting multivesicular liposomes were placed in an ice-cold water bath and sonicated three times each at 40% pulser and 40% power for 5 min using a 120 W sonicator probe . A volume of 0.1 mL of 10X PBS was added to the 0.9 mL preparation to yield 1 mL liposomal formulation in 1X PBS. The preparation was analyzed and characterized prior to immunization.Liposomes were prepared by dissolving and mixing dipalmitoylphosphatidylcholine , 250 µL of each formulation was transferred to 1 mL-thick walled polycarbonate ultracentrifuge tubes and diluted with 250 µL of PBS. The tubes were placed in an ultracentrifuge  equipped with a TLA120.2 rotor. Centrifugation was conducted at 100,000 RPM for 1 hr at 4 °C. To determine the concentration, 100 µL of the supernatant was put through HPLC using the same method employed in the characterization of peptides . In parallel, 60 µL of each fresh formulation was diluted with 60 µL of PBS and put through the same HPLC protocol for a reference concentration. EE % values were calculated using Equation (1).To evaluate encapsulation efficiency  at 173 °C and 25 °C. Refractive indices were set to 1.45 and 1.332 for dispersed phase and dispersion medium (PBS), respectively. The viscosity of the dispersion medium was 0.9103 cp at 25 °C. Sample temperature was allowed to equilibrate for 5 min before measurement; collected data were set as the average of 11 runs, with 10 s duration per sample. Measurements that exceeded 100,000 counts per second were expected to yield representative and reproducibly sized data. Measurements were conducted in triplicate. Scatter data was analyzed using the general-purpose analysis model within the instrument’s software.Subcutaneous Immunization of Mice319–S541, at a dose of 25 µg in 50 µL/mouse, while the negative control group was injected with pure PBS. Blood was collected from each mouse before immunization (naïve), then again 14 days after immunization, via cardiac puncture. The blood was centrifuged at 10,000× g for 10 min to extract the serum. Sera samples were stored at −80 °C until further analysis.For initial screening studies, 6- to 8-week-old female BALB/c mice were randomly divided into eight groups of five mice. The mice were immunized subcutaneously, once with 50 µL/mouse containing 25 μg of CFA-adjuvanted B1, B2, B3, B4, B5, or a mixture of B1–B5 peptides. The peptide antigens were administered with equivalent masses, i.e. 25 μg of a given peptide antigen was loaded into 50 μL in volume of CFA formulation and injected into each mouse. The positive control group was immunized with CFA-adjuvanted RBD , STo evaluate the vaccine candidates, 6- to 8-week-old female C57BL/6 mice were randomly divided into nine groups of five mice. Mice in the first six groups were immunized subcutaneously with 50 µL of vaccine formulation containing 25 μg of peptide antigen, as follows: LL-B1, LL-B2, LL-B3, LPMA-B3, B3-MF59, and L-B3-MF59. These peptide antigens were administered with equivalent masses, i.e. 25 μg of a given peptide antigen was loaded into a 50 μL volume of liposomal formulation and injected into each mouse. Another group was immunized with LL-B3 at a higher antigen dose of 50 µg in 50 µL/mouse. CFA-adjuvanted RBD (25 µg in 50 µL/mouse) was used to immunize the positive control group, and PBS was injected into mice in the negative control group. Mice were boosted 14 and 28 days after primary immunization, except for those in the CFA-adjuvanted RBD group, which were immunized only once. Naïve blood samples were collected before immunization and immune blood samples were collected on days 12, 26, and 42 after primary immunization via tail bleed. Sera were obtained and stored using the same approach as above.Both studies were conducted according to regulations set by the National Health and Medical Research Council (NHMRC) of Australia . All animal procedures and protocols were approved by The University of Queensland Animal Ethics Committee (AEC), AEC Approval Number: SCMB/AIBN/069/17.Evaluation of Serological Immunogenicity10 value of 2. The plates were emptied and washed three times with PBST buffer. Secondary antimouse IgG-Fc-HRP antibodies were diluted by adding 33 µL to 100 mL of 0.5% skim milk; 100 µL of this solution was then added to each well of the plates. The plates were emptied and washed three times with PBST buffer. O-phenylenediamine dihydrochloride substrate (OPD) was prepared and 100 µL was added to each well; this was left to react for 20 min at room temperature. The reaction was stopped through the addition of 50 µL of 1N H2SO4 solution to each well. The absorbance was measured at 450 nm wavelength using a SpectraMax microplate reader. Positive readings were tracked down the serial dilution of each serum sample until the response was not significant, i.e., the sum between the average and three times the standard deviation (3 × SD) of the naïve sera absorbance readings.Antipeptide sera samples were tested for immunogenicity and antibody avidity via enzyme-linked immunosorbent assay (ELISA) by coating either the same peptide or RBD, respectively. First, 96-well plates were coated with carbonate coating buffer containing 0.5 µg of peptide antigen or RBD. Plates were emptied, then blocked by coating the wells with 5% skim milk. The plates were emptied and washed with washing PBST buffer (PBS with 0.5% Tween 20). Serum samples were added to the plates and serially diluted in 0.5% skim milk starting with 100-fold dilutions, i.e., LogNeutralization Efficacy AssaysThe neutralization efficacy of mouse serum was evaluated through various methods: ACE-2-RBD binding inhibition, pseudovirion neutralization, and live virus neutralization assays . For fac2SO4 solution to each well. Absorbance was measured at 450 nm wavelength using a microplate reader . To determine the content of bound or missing ACE2 in each well, calibration curves (n = 4) were fit to logistic function (R2 ≈ 0.98), Equation (2). The best fit equation was used with absorbance (y) values of each well to determine bound ACE2 content (x) by interpolation. The inhibition percentage was determined from the missing amount of ACE2, Equation (3). Where y is the absorbance reading, 1A and 2A are minimum and maximum absorbance readings, respectively; x is ACE2 concentration in ng/100 µL/well; x0 is the x-axis center point; p is the power exponent; and the ACE2total value is 250 ng/100 µL/well.Briefly, each well of a 96-well sample plate was coated with 100 µL of carbonate coating buffer containing 250 ng/mL of RBD. A separate calibration curve plate was also coated. Plates were blocked using 2% bovine serum albumin (BSA). Serum samples were added to group-designated sample plates, including the PBS group, and diluted in 0.5% BSA at a starting concentration of 5% dilution, followed by 2-fold serial dilutions four times down the plate. A volume of 100 µL of Fc-tagged human angiotensin converting enzyme (ACE2-Fc) solution (2.5 µg/mL) was added to each well of all sample plates. For the calibration curve plate, ACE2-Fc was serially diluted from 250 ng/well to 25 ng/well. Goat antihuman IgG-Fc-HRP secondary antibodies were added to all plates. OPD substrate was prepared and 100 µL was added to each well; the plates were left to react for 20 min at room temperature. The reaction was stopped by adding 50 µL of 1N H4) in a 96-well plate in a total volume of 150 µL, and the cells were incubated for 72 h at 37°C and 5% CO2. After incubation, medium volume was aspirated and replaced with 100 µL of BioGloTM  and left for 5 min to lyse the cells and allow luciferase enzyme reaction. Finally, a volume of 100 µL of cell lysate was transferred to a black-walled plate for luminescence measurement using a microplate reader . Samples were tested in duplicate, and neutralization values were determined as the percentage RLU of positive (immune serum-free) virus control. Neutralization efficacy is presented as the reciprocal serum dilution that achieved 50% neutralization compared to controls (luciferase fluorescence intensity).Pseudovirion neutralization assay was conducted as described previously ,51. Brie4) cells were seeded in 96-well plates with DMEM medium and incubated overnight at 37 °C and 5% CO2. After incubation, the medium was discarded, mouse serum was serially diluted 5-fold across the plate, and 260 FFU of viral inoculum was added to each well and left to incubate for 1 h at 37 °C. In a similar process, as a control, a concentration of 10 µg/mL of S309 neutralizing antibodies was also serially diluted 5-fold, then incubated with the same inoculum of SARS-CoV-2 virus and the seeded Vero E6 cells. The overlay medium was added to the cells, and the plates were re-incubated for 14 h at 37 °C and 5% CO2. The cells were then fixed with 80% acetone. The plates were dried and blocked using blocking buffer [milk diluent sera  and 0.1% Tween in PBS] for 1 h at room temperature. The plates were probed with antispike antibody (CR3022), then IR dye®-conjugated secondary antibodies , both diluted in blocking buffer by addition to each of the seeded cell wells. The plates were read using an Odyssey Infrared Imaging System infrared high-resolution scanner LI-COR CLX . Spots denoting the number of infected Vero E6 cells were counted using ImageJ Fiji software version 1.53e .Live virus neutralization assays (PRNTs) were conducted as described previously ,52. BrieStatistical Analysisp < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).Parametric one-way ANOVA with Dunnett’s multiple comparison test was used to evaluate all assays. Significance was set at 329–S421) encloses several critical binding residues essential for binding to human ACE2 receptors [444–S508 is in intimate contact with the ACE2 receptor [469–S508) and B3 (S444–S483). In addition, peptide B5 (S366–S395), which encloses the SARS-CoV-conserved epitope footprint of moderately neutralizing antibody CR3022 [623–S639) and B4 (S559–S572); these are located at or near the peptide priming site that separates the S1 and S2 subunits of the spike protein.The RBD sequence  or PMA, anchored to the liposomes, characterized, and used to subcutaneously immunize C57BL/6 mice. The conjugates were compared to CFA-adjuvanted RBD as a positive control. The clinically approved adjuvant MF59® was also employed to formulate promising peptide antigens. Mouse serum was collected to assess immunogenicity and avidity via antipeptide and anti-RBD ELISA assays, and to assess neutralization efficacy using competitive ELISA as a quick screening tool. This was followed by more in-depth evaluation of the promising groups’ neutralization efficacy using pseudovirion neutralization assays and live virus (plaque reduction) neutralization assays.The most promising peptide antigens, B1–B3, were then conjugated to polyleucine ; however, larger aggregates (microparticles) were also detected . The polp < 0.001 and p < 0.0001, respectively) following single subcutaneous immunization (p > 0.05). B3–B5-immunized mice produced antibodies that recognized B3-B5 peptide epitopes  sequence that is present in alpha, beta, and gamma lineages reduced the efficacy of the ChAdOx-1 nCoV-19  DNA vaccine from 76% against the original strain to 22% against the N501Y strains, with particular variation against the beta variant (B.1.351) [10/liposome system may boost its ability to stimulate antibody production. Moreover, convalescent patient serum was found to contain antibodies against the B1 peptide, which implies that it may also contain a partial or complete neutralizing epitope within its short sequence, e.g., S475–S499 [B1 and B2 were included in the follow up adjuvant screening studies to act as  negative controls. This was considered pertinent, as the mutation NB.1.351) . However475–S499 . Consequ10- or PMA-conjugated peptide antigens proved to be powerful adjuvants for peptide vaccines [All mice were immunized three times, except for those in the CFA-adjuvanted RBD group, which were immunized only once. The liposome formulations with Lvaccines ,39,40; tvaccines . C57BL/610 = 3.75 and 3.3, respectively) following single dose immunization (p > 0.05). After the second immunization, anti-B3 IgG titers were high: log10 IgG titers of 5–6 for CFA-adjuvanted RBD, B3-MF59, LL-B3 (50 µg), and LL-B3 (25 µg), similar to those induced by CFA-adjuvanted RBD mice (p <0.05). Notably, neither the B2 nor B1 vaccines generated antipeptide IgG titers in the serum after two immunizations. CFA-adjuvanted RBD mouse serum generated significant anti-B2 IgG titers  in the serum (B3-MF59 and LL-B3 (50 µg) induced the highest anti-B3 IgG titers (lognization . Furtherhe serum .p > 0.05), except for LB3-MF59 (10 = 3) similar to anti-B2 IgG titers of the CFA-adjuvanted RBD group (p > 0.05) (p < 0.0001).All B3-immunized mice generated high anti-B3 IgG titers following the third immunization, comparable to CFA-adjuvanted RBD mice (LB3-MF59 A. Mice i > 0.05) C. Furthe > 0.05) B. Howeve10 and MF59 (with soluble antigens) induced IgG titers as high as CFA-adjuvanted RBD after the first immunization. The antibodies produced had high avidity, recognizing RBD coated on ELISA plates. However, the addition of L10-conjugated peptide antigen to MF59 reduced immunogenicity significantly, likely due to the peptides self-assembling separately from the emulsion oil globules . CFA-adjuvanted B3 sera was also able to neutralize ACE2/RBD binding in the second immunization experiment; however, the neutralization capacity was lower than in the first experiment. This may reflect the fact that different breeds of mice were used between the experiments. B3-MF59-immunized sera were the most neutralizing of those tested in the second study and were comparable to the CFA-adjuvanted RBD group; however, variability between mice within both groups was very high. The neutralization/inhibition efficacy of B3-MF59, LPMA-B3, and LL-B3 sera dropped swiftly with dilution; 50% inhibition was achieved at serum dilutions of ≤1/40.Immunized mouse sera were evaluated for its neutralization efficacy using competitive ELISA  and pseup > 0.05). However, sera from C57BL/6 mice in the adjuvant screening study did not significantly neutralize the virus compared to PBS group (p > 0.05) and were far less neutralizing compared to the first study (involving BALB/c mice).The pseudovirion neutralization test assesses the neutralization efficacy of immune sera without the need to use pathogenic live virus, eliminating the rigorous containment requirements to conduct the assay, while still maintaining high relevance to real infection processes in vivo . Thus, iAlum-adjuvanted RBD was recently reported to be weakly neutralizing in C57BL/6 mice , even th10/liposomes, PMA/liposomes, and MF59′s suitability as adjuvants and delivery systems for peptide antigens may depend on several factors, including how an enclosed neutralizing epitope is presented to or recognized by B-cell receptors. Modification of the N-terminal fragment of peptide antigen (B3) with polyleucine or PMA, as well as anchoring of conjugates to liposomes, may reduce the accessibility of the N-terminus fragment of the antigen to the environment and recognition by B-cell receptors. Recently, mice immunized with CFA-adjuvanted peptides derived from spike protein were explored for potential neutralizing epitopes [439–S454 and S455–S469, with nAb titers ranging from 30 to 60 in pseudovirion neutralization assays [439–S454, S455–S469, or S435–S479) overlap/coincide with the N-terminus of B3 (S444–S483) [N-terminus-anchored B3 epitopes. Nonetheless, CFA-adjuvanted B3 triggered the production of highly neutralizing antibodies, equal to or greater than those from immunization with RBD/CFA (p > 0.05). Thus, alternative orientation/anchoring strategies of B3 epitopes, e.g., via conjugating the L10 moiety to the C-terminus of B3, may improve the efficacy of the conjugates. However, synthesis of such compounds could be very challenging.Lepitopes ,65. Seve44–S483) ,65. ThisIn summary, we identified a promising peptide-based SARS-CoV-2 vaccine antigen (B3). However, to produce effective B3-based vaccine alternatives, powerful adjuvant systems are required as CFA is not safe for humans, and the alternative adjuvants tested in this study were not adequately effective. Furthermore, the introduction of a helix or strand promoting adjuvant moieties altered the secondary structure of the peptides, and potentially rendered them inadequately neutralizing. It would be interesting to evaluate whether similar effects would still occur if peptide antigens were conjugated to random coil-supporting poly(hydrophobic amino acids), for example: polyproline or polyaspartic acids . AlternaWe identified a new SARS-2-S B-cell epitope. B3 (S444–S483) proved to be the most immunogenic peptide antigen tested. CFA-adjuvanted B3-immunized mice produced sera with significant neutralization efficacy against SARS-CoV-2. Adjuvating systems relying on liposomal formulations of polyleucine or polymethyl acrylate increased the immunogenicity of B3; however, they did not help to achieve similar neutralization efficacy. Emulsion-based adjuvants, such as CFA and MF59, improved the avidity of the antibodies generated and their neutralization efficacy against SARS-CoV-2. Further effort to optimize peptide antigen delivery is required.


Pancreatic ductal adenocarcinoma (PDAC) represents the fourth cause of cancer-associated death in the West. This type of cancer has a very poor prognosis notably due to the development of chemoresistance when treatments including gemcitabine and Abraxane (Nab-paclitaxel) were prescribed. The identification of new treatment circumventing this chemoresistance represents a key challenge. Previous studies demonstrated that the activation of orexin receptor type 1 (OX1R), which was ectopically expressed in PDAC, by its natural ligand named orexin-A (OxA), led to anti-tumoral effect resulting in the activation of mitochondrial pro-apoptotic mechanism. Here, we demonstrated that OxA inhibited the pancreatic cancer cell (AsPC-1) growth and inhibited the tumor volume in preclinical models as effectively as gemcitabine and Nab-paclitaxel. Moreover, the combination therapy including OxA plus gemcitabine or OxA plus Nab-paclitaxel was additive on the inhibition of cancer cell growth and tumor development. More importantly, the treatment by OxA of chemoresistant tumors to gemcitabine or Nab-paclitaxel obtained by successive xenografts in mice revealed that OxA was able to induce a strong inhibition of tumor development, whereas no OxA resistance was identified in tumors. The OX1R/OxA system might be an innovative and powerful alternative treatment of chemoresistant PDAC. Pancreatic cancers widely represented >90%) by pancreatic ductal adenocarcinoma (PDAC) are the most lethal human digestive cancer with a very poor 5-year-survival rate, which is <10% . Moreove0% by panIn this context, identification of new targets with antitumoral action on drug-resistant cancer cells represents an essential challenge. Since several years, our group demonstrated that orexin receptor type 1 (OX1R) but not orexin receptor type 2 (OX2R) was expressed in digestive cancer cells including pancreatic, colon, and liver cancers  where it2/95% air in Roswell Park Memorial Institute (RPMI 1640) medium supplemented with 10% (v/v) fetal calf serum, 100 µg/ml streptomycin, 100 U/ml penicillin, and 1% (v/v) ZellShield  to prevent mycoplasma contamination. AsPC-1 cells were incubated in the presence or in the absence of 0.1 µM orexin-A (OxA) , 0.01 µM gemcitabine, 0.1 µM Nab-paclitaxel, 0.01 µM gemcitabine + 0.1 µM OxA, or 0.1 µM Nab-paclitaxel + 0.1 µM OxA. These doses of compounds corresponded to the dose that induced half of the effect on the cell growth inhibition. After 48 h of treatment, total cells were harvested and counted to determine the cell viability, as previously reported (https://synergyfinder.fimm.fi/). In sequential treatment, AsPC-1 cells were incubated in the presence of 0.1 µM orexin-A or 0.01 µM gemcitabine or 0.1 µM Nab-paclitaxel for 48 h. After pretreatment, compounds were substituted for another 48 h by 0.01 µM gemcitabine or 0.1 µM Nab-paclitaxel for cells pretreated by OxA and 0.1 µM OxA for cells pretreated by 0.01 µM gemcitabine or 0.1 µM Nab-paclitaxel, respectively. Control corresponded to the absence of treatment for 96 h.PDAC cell line, AsPC-1 , was cultured at 37°C in 5% COreported . To quanImmunohistochemistry was performed on formalin-fixed paraffin-embedded 3-µm slices of spheroids or tumor tissue from xenografts with an automated immunohistochemical stainer according to the manufacturer’s guidelines . Slides were immunolabeled with antibodies against OX1R  or activated caspase-3 .6 AsPC-1 cells or 2.106 cancer cells isolated from pancreatic tumor patient were subcutaneously injected into the two flanks of five nude mice (to limit mouse number according to 3Rs rules) per arm corresponding to the development of 10 tumors per arm, as previously reported  consisted of subcutaneously inoculating AsPC-1 cells into nude mice followed by three intraperitoneal injections/week of PBS, 120 µg gemcitabine, 230 µg Nab-paclitaxel, or 100 µg OxA during 8 weeks before reaching the endpoints. Then, mice were euthanized, and tumors were resected. Tumors were sliced using scalpels and then incubated in dissociation medium containing culture medium added with collagenase IV (400 U/ml). The second run (run02) consisted of subcutaneously injecting dissociated cells corresponding to PBS-, gemcitabine-, Nab-paclitaxel-, or OxA-treated cells from run01 into a new batch of nude mice. Mice from run02 were again treated by three intraperitoneal 100-µl injections/week of PBS, 120 µg gemcitabine, 230 µg Nab-paclitaxel, or 100 µg OxA until endpoints defined in the protocol were reached. Tumor volumes were estimated as described above. At the end of the experiment, mice were euthanized and necropsied. Tumors were resected and weighted. Resected tumors were either formalin fixed or cell dissociated for RNA-seq and spheroid analyses.Spheroid formation was obtained from AsPC-1 cells resistant to gemcitabine or Nab-paclitaxel or OxA isolated from xenografted tumors obtained in run02 experiments (see above). Briefly, xenografted tumors corresponding to control, gemcitabine treated, and Nab-paclitaxel treated were collected and dissociated, and tumoral cells were grown in 96-well plate  containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4.5 g/L of glucose, 10% (v/v) fetal calf serum, 100 µg/ml streptomycin, and 100 U/ml of 3,000 cells/well under agitation at 200 rpm, and were treated or not with 1 µM OxA, 0.01 µM gemcitabine, 0.1 µM Nab-paclitaxel, 1 µM OxA + 0.01µM gemcitabine, or 1 µM OxA + 0.1 µM Nab-paclitaxel up to 10–15 days. The volume of spheroids was determined using the plugin SpheroidSizer  under MaR or ASPC-1/pacliR cells isolated from xenografted tumors acquired in run02 experiment (see above). After dissociation, tumor cells were cultivated in RPMI medium supplemented with 10% (v/v) fetal calf serum, 100 µg/ml streptomycin, 100 U/ml penicillin, and 1% (v/v) ZellShield in the presence of PBS (control), or 1 µM OxA for 24 h. Total RNA was extracted using Trizol reagent  and then treated with DNAse I RNAse free  to eliminate genomic DNA. The integrity of the total RNA was verified by agarose gel electrophoresis and quantified using NanoDrop One . RNA-seq was performed on genomic platform , and sequencing data were managed with the modular and scalable workflow engine, Eoulsan 2.5, which used DESeq 2 for normalization and differential analysis . DESeq 2 files were analyzed with iDEP.94 (https://www.genome.jp/kegg/). Heatmaps were generated with Morpheus (https://software.broadinstitute.org/morpheus).Transcriptomic analysis was performed on AsPC-1/gem iDEP.94 , and theR, or AsPC-1 Nab-pacliR cells were washed twice with phosphate-buffered saline and treated with 1 µM OxA in medium or with medium only at 37°C for 24 h. Cells were collected and lysed with RIPA buffer . The mixture was gently agitated for 30 min at 4°C and centrifuged at 15,000×g for 15 min at 4°C, followed by collection of the supernatant. Solubilized proteins (100 µg) in Laemmli buffer (incubated for 5 min at 98°C) were loaded onto a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and immunoblotted with antibodies described below. Anti-SHP-2/SHPTP-2  antibody was from Millipore ; anti-FoxM1 (D12D5) , anti-cleaved PARP (Asp214) , anti-PARP (46D11) , anti-elF2a (Ser51) , anti-ATF-3 (D2Y5W) , anti-ATF-4 (D4D8) , or anti b-actin (8H10D10)  antibodies were from Cell Signaling Technology ; anti-DNA polymerase theta antibody (1C11)  was from Novus Biologicals ; and anti-NrF2 (ab137550)  and anti-Bcl-2 (E17)  antibodies were from Abcam . Immunoreactive proteins were secondary labeled with ECL™ anti-rabbit IgG antibody  (NA934)  or with anti-mouse IgG-peroxidase antibody (A9044)  , visualized by ECL immunodetection  and quantified by chemoluminescence analysis.Semiconfluent AsPC-1, AsPC-1 gempost-hoc Bonferroni’s tests when groups of mean values were more than 2. In different figure legends, samples sizes were indicated, and statistical significance was defined as p<0.05.Results were expressed as mean ± standard error of the mean (SEM). All data were analyzed with GraphPad Prism v8.4.3 software . The Student’s t-test  was used for comparison of two mean values. A normality test (Agostino–Pearson test) was applied to validate the use of an ANOVA with As previously reported, OxA was able to induce a cell apoptosis through the recruitment of the tyrosine phosphatase SHP2 in pancreatic cancer cell line AsPC-1 . The proCurrently, Nab-paclitaxel represents a molecule widely used in the treatment arsenal of metastatic PDAC, associated or not to gemcitabine . To comp6 AsPC-1 cells at day 1 after implantation. As shown in One hundred micrograms of OxA (4 mg/kg) or 120 µg gemcitabine (5 mg/kg) or 100 µg OxA plus 120 µg gemcitabine was intraperitoneally injected three times/week in nude mice subcutaneously xenografted with 2.106 AsPC-1 cells in nude mice, intraperitoneal injection three times/week of 100 µg OxA (4 mg/kg) or 230 µg (9.2 mg/kg) Nab-paclitaxel and 100 µg OxA plus 230 µg Nab-paclitaxel on day 1 after implantation showed that OxA and Nab-paclitaxel were able to significantly reduce the tumor volume of 60% and 55%, respectively, as compared to control mice  for 40 days , were suHistological analyses show that OX1R was expressed in gemcitabine and Nab-paclitaxel tumors ; then, they were treated with PBS, OxA, gemcitabine, or Nab-paclitaxel  (To determine whether OxA induced tumor growth inhibition bGeSTAT) . As showbGeSTAT) . MoreovebGeSTAT) , 19, wasbGeSTAT) , we detein vivo, also had an additive effect with the two drugs. It was well known that repetitive activation of GPCRs by agonist ligands led to a decrease in GPCR response associated to desensitization and cell internalization processes . The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Apafis No. 17199-201810221522166v4.ACn, TV, and PN designed the experiments. ACn, TV, VG, and PN performed experiments. ACd, ACn, ACh, VR, TV, and DM have performed and analyzed histologic studies. ACn has written the manuscript. All authors have read, edited and approved the final version of manuscript.Our work was supported by the “Institut National de la Santé et de la Recherche Medicale” (INSERM), the “Université de Paris,” The “Institut National du Cancer (INCA)” , and the “Ligue Contre le Cancer” (grant numbers R16020HH and GB/MA/CD/EP-12062).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Fractions were examined for their molecular structure, effect on ruminal enzyme activities, and principal leaf protein (Rubisco) solubilization in vitro. All fractions except FRL-70 contained flavonoids including (+) catechin, (−) epicatechin, (+) gallocatechin, (−) epigallocatechin, and their -4-phloroglucinol adducts. The fractions FRL-50 and FRL-DW significantly (p < 0.05) inhibited the activity of ruminal glutamic oxaloacetic transaminase and glutamic pyruvic transaminase. All fractions inhibited glutamate dehydrogenase activity (p < 0.05) with increasing concentration, while protease activity decreased 15–18% with increasing concentrations. Fractions FRL-50 and FRL-DW completely inhibited the activity of cellulase enzymes. Solubilization of Rubisco was higher in F. religiosa (22.36 ± 1.24%) and F. racemosa (17.26 ± 0.61%) than that of wheat straw (WS) (8.95 ± 0.95%) and berseem hay (BH) (3.04 ± 0.08%). A significant (p < 0.05) increase in protein solubilization was observed when WS and BH were supplemented with FR and FRL leaves at different proportions. The efficiency of microbial protein was significantly (p < 0.05) greater in diets consisting of WS and BH with supplementation of F. racemosa leaves in comparison to those supplemented with F. religiosa leaves. The overall conclusion is that the fractions extracted from F. religiosa showed greater inhibitory effects on rumen enzymes and recorded higher protein solubilization in comparison to the F. racemosa. Thus, PAs from F. religiosa are potential candidates to manipulate rumen enzymes activities for efficient utilization of protein and fiber in ruminants.Three proanthocyanidin fractions per species were sequentially extracted by 50% ( Ficus religiosa and F. racemosa are used for this purpose in subtropical agro-silvicultural systems in central India.Tree foliages are a source of protein, energy, and minerals for many herbivorous animals . Such foFicus species were assessed in our previous study [Crude protein 10.7–11.5%), neutral detergent fiber 42.5–49.3%), lignin 5.6–10.8%), and in vitro dry matter digestibility (37.9–55.8%) of leaves of these 2.5–49.3%%, neutra–10.8%, a2 extraction method and robotic solvent automation [The use and selection of appropriate solvent is equally important to obtain plant extracts. The amount and types of solvent are to be considered important parameters while extracting tannin-related compounds . Howevertomation ,9, wheretomation . UV analtomation . Howevertomation . Recent tomation .p < 05) reduced CH4 production and total methanogens and methanobacteriaceae [4 production without impairing in vitro organic matter digestibility (IVOMD) [4 is related to their chemical structure as phenols with two or more hydroxyl groups seemed to have higher efficiency than those containing only one. Structures diversity of CTs affects rumen in vitro CH4 production in sainfoin accessions. Procyanidin: Prodelphinidin (PD: PC) ratio is important and negatively associated with CH4 production [Studies on screening plant extracts have become an important research area for researchers globally to develop alternative feed additive to manipulate rumen ecology for reducing greenhouse gases . In animeriaceae . Tannins (IVOMD) . The abioduction . Globalloduction ,21. Howeoduction . In addioduction .F. religiosa and F. racemosa and to assess their effect on in vitro ruminal enzyme activities for better utilization in animal nutrition.In this study, the objective was to isolate and characterize PAs in a sequential extraction with series of solvents based on deferent polarity from tree foliage of v/v) acetone–water). The other fractions, FRL-50 (eluted by 50% (v/v) methanol–water), FRL-DW (eluted by distilled water), FR-70 (eluted by 70% (v/v) acetone–water), FR-50 (eluted by 50% (v/v) methanol–water), and FR-DW (eluted by distilled water), showed the presence of (+) catechin (I), (−) epicatechin (II), (+) gallocatechin (III), (−) epigallocatechin (IV) along with their -4-phloroglucinol adducts  gallocatechin, (−) epigallocatechin, (+) catechin, (−) epicatechin, and a similar order was followed by -4-phloroglucinol adducts with retention times (Rt) of 15.31–48.71 min. Similar trends for above compounds were observed in Vicia faba [Proanthocyanidins or condensed tannins are widely distributed within plants, particularly woody plants. These are mainly composed of (+) catechin or (−) epicatechin units, and these units attribute the properties to the condensed tannins. The basic structural unit of these phenolic polymers is flavan-3ol. All isolated fractions contained phenolic compounds except FRL-70 (eluted by 70% ( adducts . The (+)cia faba .Litchi chinensis [Saracaasoca [Ephedra sinica [The highest (0.524 ± 0.03) and lowest (0.098 ± 0.01) catechin was found in FR-DW and FR-70, respectively, whereas it was not detected in FRL-DW and FR-50 . A similhinensis , Saracaaacaasoca  and Ephea sinica . HPLC ana sinica . FurtherR:3S or 2R:3R in episomer and enantiomers such as ent-epicatechin (2S:3S). In R:3S and 2R:3R, respectively. In (+) gallocatechin and (−) epigallocatechin, one additional hydroxyl group attached at the 5′ position. The subunits of anthocyanin in F. racemosa and F. religiosa were predominated by cyanidin subunits with delphinidin in a ratio of 50:50 . There was a liner decrease in the activity of R-GPT and R-GOT with increase in their concentrations. The EC50 of TA for R-GOT was 20.54 ± 1.13 mg/mL and 17.54 ± 0.13 mg/mL for R-GOT of the protozoan and bacterial fractions, respectively. GPT activity also decreased (p < 0.05) with increasing concentration of TA, and its EC50 was 17.52 ± 0.70 mg/mL and 17.12 ± 0.15 mg/mL for protozoan and bacterial fractions, respectively. GA also lowered R-GOT and R-GPT in both in protozoan and bacterial fractions. The effect on R-GPT activity was nearly equal to that of TA for protozoan and bacterial fractions, whereas the GA was more significantly (p < 0.05) effective to R-GPT in the protozoan fraction (EC50 6.89 ± 0.12 mg/mL) than that of the bacterial fraction (EC50 15.52 ± 0.53 mg/mL). The FRL-50 and FRL-DW both (p < 0.05) inhibited R-GOT and R-GPT activities. FRL-50 had greater inhibition of R-GOT (protozoan fraction) activity than R-GOT  with EC50 4.72 ± 0.42 mg/mL and 7.12 ± 0.02 mg/mL, respectively. The EC50 of FRL-DW was 10.67 ± 1.12 mg/mL for R-GOT  and 7.69 ± 1.25 mg/mL for R-GOT , respectively. In the case of R-GPT activity, FRL-50 was more potent as evidenced from lower EC50 values of 5.44 ± 0.49 mg/mL and 5.28 ± 0.63 mg/mL for protozoal and bacterial fractions, respectively. The FRL-DW had a higher EC50 to inhibit R-GPT (13.59 ± 1.42 mg/mL and 16.69 ± 1.44 mg/mL) for protozoal and bacterial fractions, respectively. On the other hand, FR-70 and FR-50, polyphenolics from F. racemosa, inhibited R-GOT  more effectively with an EC50 17.17 ± 1.01 and 12.33 ± 1.26 mg/mL, respectively, but they were less active against the R-GOT bacterial fraction with relatively higher EC50 of 19.17 ± 1.00 and 16.33 ± 0.50 mg/mL, respectively. FR-50 exhibited significantly (p < 0.05) higher inhibition/reduction in GPT activity than FR-70 in both protozoal and bacterial fractions. The inhibition of R-cellulase activity was affected by the presence of phenolics in the rumen liquor (50) was 6.62 ± 0.56 mg/mL and 124.32 ± 1.63 mg/mL for GA and TA, respectively. The activity of ruminal cellulase was completely inhibited by the polyphenolics from F. religiosa. FR-50 showed greater potential (p < 0.05) to inhibit 50% of cellulase activity at 9.70 ± 0.54 mg/mL whereas FR-70 possessed an EC50 of 24.72 ± 1.25 mg/mL.The activities of ruminal glutamic oxaloacetic transaminase (R-GOT), glutamic pyruvic transaminase (R-GPT), and cellulase are shown . All then liquor . The celThe decrease in R-GOT, R-GPT, and R-cellulase in the presence of proanthocyanidins fractions could be related to the antimicrobial nature of these compounds or their metabolites produced during the fermentation process. Our studies revealed that presence of catechin, epicatechin, and other derivatives can reduce enzymatic activity. Similar results were shown in a study of male cow rumen fermentation by Oskoueian et al. . In the F. racemosa, there was a decrease of 20–30% and 18–20% in protease activity by FR-50 and FR-70 fractions, respectively. FR-DW was shown to decrease protease activity by 25–60%. Earlier studies conducted on bark extracts from different medical plants showed that isolated polycyclic phenolics inhibited protease activity from 22–56% [The presence of phenolics in the substrate reduced the oxidation rate of NADPH and NAD, resulting in reduced R-GDH activity in protozoal and bacterial fractions of the rumen liquor. The linear decrease in R-GDH activity was dependent of the quantity of TA and GA. The effect of TA on R-GDH activity was more pronounced on the protozoal fraction than in the bacterial fraction. There was decline of 12 units on the protozoal fraction, while in the bacterial fraction it declined by 7 units . On the m 22–56% .Onobrychis vicifolia CTs had profound effects on growth of five rumen bacteria. At the higher tannin protein ratio, inhibition in proteolysis occurs due to polyphenolic compounds adhering to the protein surfaces to cause interference with the interaction of the enzyme substrate [The interaction between PAs and proteolytic enzymes may inhibit proteolysis due to steric interference with the binding of the protease with susceptible sites on dietary protein. The degree of binding is dependent on the types of protein and types of PAs. The exact mode of action is not fully known but the reduction in proteolysis may be due to direct effects of PAs on microbial proteolytic enzymes or indirect effects on rumen metabolite concentration, which can inhibit proteolysis in some bacteria . In lineubstrate . Proanthubstrate , and thoubstrate .p < 0.05) than that of roughage WS (8.95 ± 0.95%) and berseem hay (3.04 ± 0.08%). Addition of FR and FRL to WS increased dietary protein solubilization to 16.61 ± 1.04% and 22.47 ± 1.37% at a ratio of 1:1, respectively. In case of FR, the solubilization again increased at 2:1 (28.58 ± 1.48) and then declined at 3:1 (15.29 ± 0.61). At 2:1 and 3:1 ratio of FRL, WS Rubisco solubilization declined to 22.47 ± 1.37% and 5.59 ± 0.34, respectively. This indicated a regulation in protein solubilization with certain quantities of PAs in the WS. Further supplementation of FRL leaves to BH in 1:1, 2:1, and 3:1 proportion gradually increased dietary protein to 14.47 ± 0.74, 17.80 ± 1.26%, and 25.90 ± 1.18%, respectively. For FR in addition to BH, the protein solubilization increased from 18.35 ± 1.48 at 1:1 to 21.70 ± 3.00% at 2:1 proportion and lowered at a 3:1 ratio (11.81 ± 1.01%). The determination of NRNAQ in rumen liquor after in vitro dietary protein solubilization from leaves and their addition with roughage WS and BH in combination of 1:1, 2:1, and 3:1 revealed that among the top feeds along with FR, it synthesized more microbial protein as evident by NRNAQ 0.22 ± 0.01 than FRL (0.21 ± 0.02). Indeed, the microbial synthesis of FR and FRL was higher (p < 0.05) to sole WS and BH as their NRNAQ was 0.16 ± 0.01 and 0.17 ± 0.01, respectively. The NRNAQ was low in FRL (0.19 ± 0.01) and FR (0.15 ± 0.01) at 1:1 with WS. Supplementation of top feed leaves to BH in 1:1 ratio showed a considerable increase in microbial protein synthesis relative to sole WS or BH as evidenced by NRNAQ 0.22 ± 0.01 and 0.26 ± 0.03 with FRL and FR, respectively. Increasing the level of top feed leaves in a ratio of 2:1 with WS the NRNAQ did not alter much. However, with FRL there was a slight decrease in NRNAQ (0.16 ± 0.01), whereas addition of FRL and FR to BH in a ratio of 2:1 lowered microbial protein synthesis to 0.17 ± 0.01 and 0.21 ± 0.01, respectively. Increase of top feeds in 3:1 ratio with WS declined NRNAQ as 0.18 ± 0.01 and 0.19 ± 0.01 and with berseem hay the NRNAQ was 0.17 ± 0.0 and 0.21 ± 0.02 for FRL and FR, respectively.Maximum solubilization of Rubisco in FRL (22.36 ± 1.24%) was followed by FR (17.26 ± 0.61%) . SolubilTrifolium repens and Lotus corniculatus solubilization with addition of phenolics (condensed tannins) has been reported [Medicago sativa) hay. Similarly, in our findings, the impact of addition of Ficus leaves to wheat straw and berseem hay increased microbial protein synthesis efficiency.The digestion of forage in the rumen occurs via a combination of solubilization and degradation. Solubilization is an important action and comprises the release of protein from the cell wall into ruminal fluid with chewing in the mouth. On the other hand, degradation is a catabolic process facilitated by microbial proteases resulting in peptides, amino acids, and ammonia . These treported . We obsereported  reportedreported  with sup256, Si gel .Catechin, epicatechin, Gallocatechin, epigallocatechin, catechin-4-phloroglucinol, epicatechin-4-phloroglucinol, gallocatechin-4-phloroglucinol, epigallocatechin-4-phloroglucinol, and Sephadex LH-20 were purchased from Sigma, USA. All other reagents and solvents used were of analytical grade. UV spectra were measured on a UNCAM UV/vis spectrophotometer . TLC, column chromatography, and paper chromatography were performed on precoated Si GFF. religiosa and F. Racemosa were collected from the Central Research Farm of ICAR-Indian Grassland and Fodder Research Institute and ICAR-Central Agro-Forestry Research Institute, Jhansi. These tree leaves are often used by the farmers for animal feeding. Collected samples were dried under shade on the cemented floor of the laboratory followed by drying in a hot air oven at 60 °C until the constant dry weight was achieved. Dried samples were powdered to pass through 1 mm sieve using electrical Willey mill. Powdered samples were stored in plastic containers (Tarson make) for further chemical analysis.The leaf samples from mature trees of Ficus religiosa (FRL) and F. racemosa (FR) (2 kg of each) were extracted with 70% acetone containing 0.1% ascorbic acid. The solvent was removed under vacuum in a rotatory evaporator at 40 °C. The aqueous phase was washed subsequently with chloroform, diethyl ether, and ethyl acetate. The remaining aqueous extract was diluted with an equal amount of methanol (MeOH) and charged over a pre-equilibrated Sephadex LH-20 (30 × 2.5 cm) column.  and paper chromatography (PC) profiling then tested against enzymes , the technique described by Mandel and Weber (1969) was followed . For intF. racemosa and F. religiosa and their combinations of 1:1, 2:1, and 3:1 with wheat straw (WS) and Berseem hay (BH) separately were incubated at 37 °C with 40 mL of strained rumen liquor from grazing sheep and 10 mL of CO2 saturated buffer  for 48 h in a full anaerobic environment and filtered through a G-1 crucible. The filtrate was centrifuged at 500× g for 15 min to remove the feed particles. The supernatant was then centrifuged at 20,000× g for 30 min at 4 °C. This process was repeated three times. The pellet was washed with saline and preserved for determination of microbial protein and RNA after lyophilization. The residue collected in the crucible was used for Rubisco determination by the Bradford method [Five hundred mg of green lyophilized and powdered tree leaves of d method  after prd method  and micrd method . The effp < 0.05 by post-hoc analysis with Tukey’s test.Microsoft Excel 2015 and R were used for statistical analysis. Analysis of variance (ANOVA) was performed in R to evaluate the measurements of enzymatic activities and means are considered as significantly different at Ficus tree species. The results indicated that isolated PAs inhibited the activities of rumen enzymes  and protein degradation in vitro. The mode of action of PAs is not fully known, but results revealed the relationship between proanthocynidins’ chemical structure and rumen enzymes’ activities, which may be utilized to manipulate the rumen fermentation, particularly the fiber (cellulose) and protein utilization . Further studies (in vivo) are required to better understand the relationship of the molecular structure of PAs with the ruminal enzymatic activities and microbial protein metabolism. In the future, the PAs rich plant extracts could be a novel feed supplement to enhance protein use efficiency by reducing methane and nitrogen emission in ruminants.Six fractions of PAs were extracted and quantified from two


Then, excess Ag is introduced to compensatefor the remaining intrinsic Pb vacancies. We find that excess Ag exhibitsa dynamic doping process with increasing temperatures, increasingboth the carrier concentration and carrier mobility throughout a widetemperature range; specifically, an ultrahigh carrier mobility ∼7300cm2 V–1 s–1 is obtainedfor Pb1.01Te + 0.002Ag at 300 K. Moreover, the dynamicdoping-induced high carrier concentration suppresses the bipolar thermalconductivity at high temperatures. The final step is using iodineto optimize the carrier concentration to ∼1019 cm–3. Ultimately, a maximum ZT valueof ∼1.5 and a large average ZTave value of ∼1.0 at 300–773 K are obtained for Pb1.01Te0.998I0.002 + 0.002Ag. These findingsdemonstrate that fine tuning of defects with <0.5% impurities canremarkably enhance carrier mobility and improve thermoelectric performance.High carrier mobility is critical to improving thermoelectricperformanceover a broad temperature range. However, traditional doping inevitablydeteriorates carrier mobility. Herein, we develop a strategy for finetuning of defects to improve carrier mobility. To begin, n-type PbTeis created by compensating for the intrinsic Pb vacancy in bare PbTe.Excess Pb The conversionefficiency depends on the dimensionless figure of merit ZT, ZT = S2σT/κ, where S is the Seebeck coefficient,σ is the electrical conductivity, T is theabsolute temperature in kelvin, and κ is the total thermal conductivity. In general, thermoelectricenhancement relies heavily on a large Seebeck coefficient, high electricalconductivity, and low thermal conductivity. Nevertheless, the abovethree thermoelectric parameters are governed primarily by the carrierconcentration and are not independently controllable, making themdifficult to be optimized simultaneously.9 Researchers have conducted in-depth and fruitfulresearch in upgrading thermoelectric performance via combined optimization of electrical and thermal transport. For electricalproperties, optimizing carrier concentration,12 increasingcarrier mobility,14 and modulating band structure19 are generally applied. For thermal properties, the traditional methodis declining the lattice thermal conductivity via hierarchical structures, involving atomic-scale defects,22 nanoscale defects,25 and microscale defects.27Thermoelectric materials can effectivelyutilize low-grade heatsources to generate electricity, such as industrial waste heat, automobileexhaust, solar energy, geothermal heat, p-type PbTe materialshave achieved extraordinary thermoelectric properties. Many prominent p-type PbTe systems have realized ZT >2.0, such as Pb0.98Na0.02Te,28 Pb0.98Na0.02Te–8%SrTe,29 Pb0.98Na0.02Te–6%MgTe,30 Pb0.95Na0.05Te–0.5%AgInSe2,31 and Pb0.075K0.025Te0.7S0.3.32 In contrast, their counterpart, n-type PbTe, shows lower ZT values because of the large energy offset (∼0.45eV) between the conduction bands, so further improvement of n-typePbTe is needed to match p-type PbTe. With time, we discovered severaloptimization methods that could bring about considerable intensificationin the thermoelectric performance of n-type PbTe, such as doping andband engineering.33 However, even thoughtraditional doping undoubtedly plays a significant role in increasingthe carrier concentration, dopants inevitably reduce carrier mobility.36 Band engineering, as another attractive and feasible method, facilitateselectrical performance by boosting the effective mass, while it isalso detrimental to the carrier mobility.38 In general, these methods are capable of enhancing the maximum ZT, whereas a major drawback is their tendency to decreasecarrier mobility, which limits the ZTave.39 To ensure good thermoelectric propertiesover a wide temperature range, comparable n-type PbTe systems withhigh carrier mobility must be developed.Among a variety of thermoelectric materials, PbTe is a representativethermoelectric material servicing at medium temperature. Numerousresearch studies have shown that 2 V–1 s–1 at 300 K in Pb1.01Te. Benefiting from the high carrier mobility, a maximum power factorof ∼31.3 μW cm–1 K–2 at 300 K is achieved in Pb1.01Te. Second, excess Ag issubsequently introduced to fill the remaining Pb vacancies. Resultsreveal that the comparatively low room-temperature carrier concentrationof ∼5.5 × 1017 cm–3 leadsto a high carrier mobility of ∼7300 cm2 V–1 s–1, resulting in a further increased power factorof ∼39.3 μW cm–1 K–2 at 300 K for Pb1.01Te + 0.002Ag. In particular, Ag atomsin the Pb1.01Te system undergo a dynamic doping processwith increasing temperatures, first compensating the Pb vacanciesand then entering more interstitial positions due to the temperature-dependentsolubility of Ag in the PbTe matrix,40 whichimproves the carrier concentration from ∼5.5 × 1017 cm–3 at 300 K to ∼8.5 × 1018 cm–3 at 873 K for Pb1.01Te+ 0.002Ag. Meanwhile, this unique dynamic doping behavior can effectivelydecouple the carrier concentration and carrier mobility across theentire temperature range and suppress bipolar diffusion at high temperatures.Last, 0.2% of I element (0.002I) is adopted to optimize the carrierconcentration while maintaining superior carrier mobility than othern-type PbTe systems with similar carrier concentrations. Meanwhile,the minimum lattice thermal conductivity can reach as low as ∼0.5W m–1 K–1 for Pb1.01Te0.998I0.002 + 0.002Ag. As a result, a maximum ZT of ∼1.5 at 773 K and a high average ZTave of ∼1.0 at 300–773 K are obtained forPb1.01Te0.998I0.002 + 0.002Ag.In this work, finetuning of defects in PbTe is successively carriedout to improve the carrier mobility. First, a small amount of excessPb (0.004–0.012) is introduced into PbTe. As Pb enters theintrinsic Pb vacancies, n-type PbTe with relatively low carrier concentrationis obtained. The dwindling vacancy defects improve carrier mobility,and the maximum carrier mobility reaches ∼3400 cm–4 Pa and putinto a furnace .After the temperature program finished, some silver ingots were obtained,which were then ground into powder for proceeding spark plasma sintering. Finally, the sintered cylindrical, smallpieces were cut into rectangles of 10 mm × 3 mm × 3 mm andslices of 8 mm × 8 mm × 1.5 mm to perform electrical andthermal performance tests. The electrical performance was measuredby Cryoall CTA  and Lake Shore 8400 Series  instruments. The thermal performance was tested by aNetzsch LFA 457 . The thermalconductivity, κ, was estimated by κ = D × Cp × ρ, where Cp is the specific heat capacity based on Debye’slaw and ρ is the sample density calculated by the ratio of massto volume. The optical band gap was measured by a Fourier transforminfrared spectrometer, IRAffinity-1S, based on the infrared diffusereflection method.All raw materials used in theexperiment were simple substanceswith more than 99.99% purity, including Pb, Te, Ag, and I. First,they were placed in quartz tubes according to the stoichiometric ratio.The quartz tubes were sealed below 10Supporting Information to create n-type PbTe, a modest quantityof Pb was first introduced, filling the intrinsic Pb vacancies, whichincreases carrier mobility and lowers thermal conductivity. Second,excess Ag is adopted for Pb1.01Te to fill the remainingPb vacancies and also occupy interstitial sites, allowing the Pb1.01Te + xAg system to achieve higher carrierconcentration, ultrahigh carrier mobility, and lower thermal conductivitysimultaneously across the entire temperature range. Third, the Iodineelement is adopted to enhance the carrier concentration, further improvingthe power factor and restraining the lattice thermal conductivityin the whole temperature range.In this study, the method offine tuning of defects is used inthe PbTe system to achieve superior electrical characteristics whilemaintaining a relatively low thermal conductivity. Figure S1a shows thepowder X-ray diffraction (XRD) patterns of Pbx1+Te ,and the corresponding lattice parameters are given in Figure S1b. All Pbx1+Te samples are NaCl-type cubic structures without extra diffractionpeaks detected, and the lattice parameters increase slightly withexcess Pb.x1+Te undergoes a p–n transitionat low temperatures, as shown in x ≥ 0.008, the Seebeck coefficient is completelynegative, resulting in n-type PbTe throughout the measuring temperaturerange. The combined behavior of electrical conductivity and Seebeckcoefficient indicates that the hole concentration in PbTe is suppressedby excess Pb. In –1 K–2 for PbTe to∼31.3 μW cm–1 K–2 for Pb1.01Te. In –1 K–1 causedby diminished lattice thermal conductivity, as shown in x1+Te shows higher electronic thermal conductivity, as shown in Figure S2b, because excess Pb fills the intrinsicPb vacancies, thereby creating weaker carrier scattering. Ultimately,an average ZT value of ∼0.5 for Pb1.01Te is obtained throughout the whole measuring temperature range,as shown in As can be seen in x1+Te, we carried out the Hall measurement.In 17 cm–3 in the n-type Pbx1+Te system is relatively low, not as outstandingas the common doping approaches, such as PbTe–Sb41 (∼5.2 × 1019 cm–3), PbTe–I42 (∼1.8 ×1019 cm–3), PbTe–Bi43 (∼2.0 × 1019 cm–3), etc. With the increasing Pb fraction, the carrierconcentration is gradually elevated, indicating that more and morePb enters Pb vacancies. Based on the inverse relationship betweencarrier concentration and carrier mobility, relatively low carrierconcentration brings about high carrier mobility since reduced Pbvacancies alleviate the charge carrier scattering, and the maximumcarrier mobility can reach ∼3400 cm2 V–1 s–1 for Pb1.01Te. x1+Te system, graphicallydepicting the phenomenon that excess Pb atoms occupy the intrinsicPb vacancies.To understand the electrical transport evolutionof Pb1.01Te + xAg (x =0–0.005) samples are performed, as shown in Figure S4a. All samples are NaCl cubic structures, and nopeaks of impurities are found. Likewise, the small amount of excessAg results in a slight expansion of the lattice parameters, as shownin Figure S4b.The powder X-ray diffraction (XRD) measurementsof Pb1.01Te + xAg system. 1.01Te + xAg is electron-dominatedn-type material. The introduction of Ag reduces the Seebeck coefficient,and the maximum absolute value decreases from ∼385.9 μVK–1 for Pb1.01Te to ∼249.0 μVK–1 for Pb1.01Te + 0.002Ag. The Pisarenkocurve in Figure S5 reveals that the effectivemass of the Pb1.01Te + xAg system remainsapproximately 0.22m0, indicating that the depressed Seebeckcoefficient of Pb1.01Te + xAg is attributedto the elevated carrier concentration rather than the reduced effectivemass. As a result, significantly incremental electrical conductivityand abated Seebeck coefficient further enhance the power factor comparedto Pb1.01Te. In particular, a superior room-temperaturepower factor of ∼39.3 μW cm–1 K–2 is acquired for Pb1.01Te + 0.002Ag, asshown in 1.01Te. 1.01Te + xAg at room temperature. Carrierconcentration increases as the Ag fraction rises; however, carriermobility is not impaired. Particularly, the maximum carrier mobilityis increased by more than twice from ∼3400 cm2 V–1 s–1 for Pb1.01Te to∼7300 cm2 V–1 s–1 for Pb1.01Te + 0.002Ag.1.01Te + xAg, we measured the temperature-dependentcarrier concentration, as shown in 1.01Te increase with increasing temperatures, asshown in Figure S6, which is derived fromthe low carrier concentration and thus pronounced bipolar effect athigh temperatures.44 However, Pb1.01Te + xAg systems still exhibit increasing carrierconcentration throughout the temperature range even though their bipolareffect is suppressed . We attribute the abnormal temperature-dependentbehavior of carrier concentration to Ag-induced dynamic doping.40 The dynamic doping process in Pb1.01Te + xAg is initiated by the multiple occupanciesof Ag atoms with increasing temperatures, and 1.01Te sample.It is noteworthy that the carrier mobilities of Pb1.01Te+ Ag at high temperatures are also higher than those of Pb1.01Te, which can be explained by the suppressed bipolar effect. As aresult, the carrier mobility of Pb1.01Te + Ag is greaterthan that of Pb1.01Te in the entire temperature range.To get an insight into the remarkable electricaltransport propertiesof Pb1.01Te (Pb-rich system), the formationenergy of AgPb (Ag-occupied Pb vacancies) is lower thanthat of VPb (Pb vacancies), indicating that the introductionof Ag reduces the Pb vacancies, thereby lowering the content of pointdefects and improving the carrier mobility. The formation energy ofAgi  is also very low in both Pb-richand Te-rich systems, demonstrating that Ag not only fills Pb vacanciesbut also occupies the interstitials in PbTe. The calculated resultsof defect formation energy well support the experimental results andverify the role of the Ag element in improving the electrical transportperformance of the Pb1.01Te system.To further reveal the role of Ag in PbTe, we utilizedfirst principlesto calculate various defects and their formation energy in the system,such as vacancies, interstitials, antisites, and Ag-filled intrinsicvacancies. 1.01Te + xAg. In ele = LσT  model). Because of the greater electrical conductivity,the Pb1.01Te + xAg system displays muchhigher electronic thermal conductivity than Pb1.01Te, asshown in lat calculated by κlat = κtot – κele asa function of temperature. In the high-temperature region, all ofthe Pb1.01Te + xAg samples exhibit lowerκlat than Pb1.01Te since the elevatedelectronic thermal conductivity of Pb1.01Te + xAg effectively suppresses the bipolar diffusion. Excess Ag depressesthe minimum lattice thermal conductivity from ∼1.2 Wm–1 K–1 for Pb1.01Te to ∼0.7 Wm–1 K–1 for Pb1.01Te + 0.005Ag.The solid black line in 30kB is theBoltzmann constant, ℏ is the Planck constant, M is the average atomic mass, V is theaverage atomic volume, θD is the Debye temperature,and γ is the Gruneisen constant. The κtot –κele values of Pb1.01Te + xAg samples coincide with the calculated values, indicating that thebipolar diffusion is suppressed in Pb1.01Te + xAg. Meanwhile, the enlarged band gap is responsible for restrainedbipolar diffusion due to the following formula46A is the constant and Eg is the band gap. Ag enlarges the band gapin tot – κele) – 1000 T–1 relationship is plotted in tot – κele values in the high-temperature regionare higher than the predicted values, indicating that bipolar diffusionoccurs in this region. According to 1.01Te + xAg system with thoseof the other n-type PbTe materials, we found that this system exhibitssuperior carrier mobility of ∼7300 cm2 V–1 s–1 at low carrier concentrations (∼1017 cm–3), as shown in ZT, as shown in 1.01Te to ∼0.6 for Pb1.01Te + 0.002Ag, andhigh-temperature ZT is significantly increased from∼0.5 for Pb1.01Te to ∼1.3 for Pb1.01Te + 0.004Ag.Comparing room-temperature carrier mobilityof the Pb1.01Te + 0.002Ag with the maximumaverage ZT, I doping is employed to increase thecarrier concentration, thereby improving the electrical performance. Figure S8 shows the phase identification of Pb1.01Tex1–Ix + 0.002Ag (x = 0–0.003). No newpeak is found in Figure S8a, and the samplesstill denote the NaCl structure. The lattice parameter decreases slightlyin Pb1.01Tex1–Ix + 0.002Ag, as shown in Figure S8b, because smaller I– (∼2.06Å) substitutes for larger Te2– (∼2.11Å). 1.01Tex1–Ix + 0.002Ag (x = 0–0.003). I doping can effectively improve theelectrical conductivity, as shown in –1 for Pb1.01Te + 0.002Ag to ∼3267.8 S cm–1 for Pb1.01Te0.998I0.002 + 0.002Ag.The Seebeck coefficients of all samples in 1.01Tex1–Ix + 0.002Ag decreases with increasing dopingamount of the I element since the more I content, the higher the carrierconcentration, as shown in Figure S9. Thehigh electrical conductivity and low Seebeck coefficient of the Pb1.01Tex1–Ix + 0.002Ag system indicate that the I element possessesextremely high doping efficiency in PbTe. The carrier concentrationcan be efficiently increased by doping with as little as 0.002I. –1 K–2 for Pb1.01Te + 0.002Agto ∼48.5 μW cm–1 K–2 for Pb1.01Te0.998I0.002 + 0.002Ag.The average power factor exceeds ∼30.0 μW cm–1 K–2 for Pb1.01Te0.998I0.002 + 0.002Ag, which is higher than those of other n-typePbTe systems , as shown in 1.01Tex1–Ix + 0.002Ag increases with increasing I content, primarily owing toan enhancement in electronic thermal conductivity. The lattice thermalconductivity decreases with increasing I content, as shown in –1 K–1 for Pb1.01Te0.998I0.002 + 0.002Ag.Based on Pb1.01Tex1–Ix + 0.002Ag system, the relationship betweencarrier concentration and the ratio of carrier mobility to latticethermal conductivity (μ/κlat) is plotted, asshown in 47 PbTe–Sb2Te3–Sb–Cu2Te,23 AgnPb100InTe100+2n,48 PbTe + Cu,49 PbTe–GeTe,50 PbTe–MnTe,51 PbTe–Ga52), the Pb1.01Tex1–Ix + 0.002Ag system possesseshigher μ/κlat values at lower carrier concentrations,indicating that a trace of Pb atoms (Pb-occupied Pb vacancies) andAg atoms (Ag-occupied Pb vacancies) can effectively regulate the intrinsicdefects in PbTe and reduce the scattering for charge carriers, protectingthe charge carrier transport while scattering phonon. Accordingly,owing to a better balance between electrons and phonons, the ZT value increases in the whole temperature range, and themaximum ZT value is enhanced from ∼1.2 forPb1.01Te + 0.002Ag to ∼1.5 for Pb1.01Te0.998I0.002 + 0.002Ag, as shown in ZT values and average ZTave valueswith various n-type PbTe materials, respectively. The large ZT values of Pb1.01Te0.998I0.002 + 0.002Ag in the whole temperature zone contribute to the excellentaverage ZTave value, reaching >1.0at300–773 K.To comprehensively evaluate the thermoelectricperformance of thePb2 V–1 s–1 at 300 K for Pb1.01Te. Then, theroom-temperature carrier mobility can reach as high as ∼7300cm2 V–1 s–1 for Pb1.01Te + 0.002Ag due to Ag-induced dynamic doping. Finally,Iodine doping dramatically increases the carrier concentration whilemaintaining superior carrier mobility compared to other PbTe systemswith only traditional dopants. The combination of high carrier mobilityand the suppressed bipolar effect enables the significant enhancementof thermoelectric performance with a high ZT ∼1.5at 773 K and average ZTave ∼1.0at 300–773 K for n-type Pb1.01Te0.998I0.002 + 0.002Ag. These findings provide insights intobalancing electron and phonon transport via finetuning of defects, which could be a promising aspect of co-optimizingthermal and electrical performance of materials with instinct defects.This work has provided a deeper insightinto designing fine-tuneddefects to improve the carrier mobility of n-type PbTe. In particular,by introducing a small amount of Pb, the reduced Pb vacancy improvesthe carrier mobility to ∼3400 cm


Reprocessing experiments showed that OBZ-BDB has remarkable reprocessability. The retention rates of the tensile strengths through three generations of reprocessing were 98%, 95%, and 84%, respectively. Meanwhile, OBZ-BDB cross-linked by boronic ester bonds had brilliant thermal properties. The Tg of the original OBZ-BDB was 224 °C. With the increase of the reprocessing generations, the Tgs basically remained unchanged.In order to obtain reprocessable polybenzoxazine thermosets with high heat resistance and mechanical strength retentions, network structures without irreversible parts were constructed via crosslinking benzoxazine oligomers using boronic ester cross-linkers. Firstly, the benzoxazine monomer containing carbon–carbon double bonds was synthesized via the Mannich reaction. After thermal ring-opening polymerization, the benzoxazine oligomer containing carbon–carbon double bonds (OBZ) was yielded. Through the thiol-ene click reaction of the OBZ and dithiol cross-linker bearing boronic ester bonds, the polybenzoxazine thermosets using boronic ester bonds as crosslinkages (OBZ-BDB) were successfully synthesized. The structures of OBZ and OBZ-BDB were characterized by SEC,  HoweverGenerally, dynamic covalent bonds can be classified into the dissociative type and the associative type according to their exchange mechanisms . In the Benzoxazine resin, as a new class of thermosetting polymer, has gained considerable attention in recent years on account of its brilliant heat resistance, excellent flame retardance, and high hydrophobicity ,14,15. Rg, dynamic boronic ester bonds were chosen to synthesize reprocessable benzoxazine resin (OBZ-BDB) with high Tg. In order to achieve the high retention of mechanical strength, benzoxazine oligomers were designed as the skeleton chain. It was further reacted with boronic ester cross-linkers to construct a purely reversible cross-linking network instead of a network with irreversible parts. Specifically, the benzoxazine monomer containing carbon–carbon double bonds was synthesized via the Mannich reaction. After thermal ring-opening polymerization, a benzoxazine oligomer bearing a carbon–carbon double bond was synthesized. Finally, we utilized the thiol-ene click reaction between the carbon–carbon double bonds of the benzoxazine oligomer and the thiols of boronic ester cross-linker to produce the reprocessable polybenzoxazine of OBZ-BDB.Considering that boronic ester bonds can dramatically improve the thermal stability due to their high bond energy , and thatert-butylphenol  and anisole (99%) were obtained from Shanghai Macklin Biochemical Co., Ltd. . Azobisisobutyronitrile  was obtained from J&K Chemical Ltd. . and purified by recrystallization from methanol. Toluene, n-hexane, methanol, and ethanol were purchased from Fuyu Fine Chemical Co., Ltd. . All of the other reagents and solvents were used as received.Allylamine (99.5%) was purchased from Zouping Mingyuan Import and Export Trade Co., Ltd. . Cardanol (98%) was obtained from Jinquan Chemical Co., Ltd. . Paraformaldehyde  and neutral aluminium oxide  were purchased from Sinopharm Chemical Reagent Co., Ltd. . Furthermore, 1-Thioglycerol (95%) was purchased from J&K Chemical Ltd. . Benzene-1,4-diboronic acid (98%) was supplied by Shanghai Bide Medical Technology Co., Ltd. . P-To a three-neck round flask, allylamine , paraformaldehyde , cardanol , and toluene (300 mL) were added and refluxed for 12 h. Then, neutral alumina was added and stirred for 0.5 h. The product was collected by filtration and evaporated to remove toluene. The solution was collected by filtration and dropped into cold methanol. Then, the lower phase was collected. After being dried in vacuum at 60 °C for 24 h, a yellowish liquid was obtained (yield 83%).1H NMR : 6.90–6.60 , 5.96–5.84 , 4.85 , 5.28–5.15 , 3.96 , 3.41–3.35 , 2.57–2.48 , and 1.64–1.51 .−1): 2926 and 2854 , 1505 (tri-substituted benzene ring mode), 1241 (C–O–C of the oxazine ring), and 968 (the oxazine ring mode).FT-IR  was added to BZ (15 g) and stirred at 100 °C for 5 min. The mixture was heated at 150 °C for 10 h. After cooling to room temperature, the crude product was dissolved in n-hexane and added dropwise into methanol for precipitation. After filtration and being dried in a vacuum at 50 °C for 12 h, an orange product was obtained (yield 70%).P-1H NMR : 7.20–6.45 (Ar–H), 6.10–5.88 (–CH2–CH=CH2), 5.28–5.11 (CH2=CH–), 4.06–3.54 (Ar–CH2–N), 3.41–3.35 (N–CH2–CH–), 2.51 (Ar–CH2–), 1.68–1.45 (Ar–CH2–CH2–CH2–), and 0.89 (CH2–CH2–CH2–CH2).−1): 2926 and 2854 , and 1483 (tetra-substituted benzene ring mode).FT-IR , 2.81 , 4.18 and 4.49 , 4.74 2), and 7.83 .−1): 2568 (–SH stretching vibration), 1219 (B–O stretching vibration), and 656 (B–O–B linkages within borate networks).FT-IR , BDB , and AIBN  were dissolved in 5 mL anisole to form the solution. Subsequently, the solution was poured into a Teflon mold. The sample was thermally cured according to the following temperature sequence: 75 °C for 6 h, 100 °C for 2 h, 120 °C for 2 h, and 140 °C for 2 h. Finally, it was dried under vacuum at 100 °C for 24 h.OBZ  with CDCl3 as the deuterium solvent.Nuclear magnetic resonance (NMR): −1 across the 4000–500 cm−1 range by the KBr pellet method.Fourier transform infrared spectroscopy (FT-IR): FT-IR spectra were obtained on a TENSOR 27 FTIR spectrometer  with a resolution of 4 cm−1) at 40 °C. Polystyrene standards were used for calibration.Size exclusion chromatography (SEC): The number average molecular weight and polydispersity index were measured by SEC using a 515 liquid chromatograph equipped with 3 Styragel columns and a refractive-index detector  in tetrahydrofuran  in shear mode. A specimen with dimensions of approximately 5.0 × 5.0 × 0.5 mm3 was tested at 1 Hz and 3 μm oscillation amplitude. The temperature was increased from 0 °C to 290 °C at a heating rate of 3 K·min−1.Dynamic mechanical analysis (DMA): DMA experiments were performed on a DMA/SDTA 861e instrument  under a nitrogen flow at a heating rate of 10 °C·min−1, from 30 °C to 800 °C.Thermo-gravimetric analysis (TGA): TGA was performed on a TGA/DSC STAR−1. Each sample was tested respectively for five specimens.Tensile tests: The tensile tests were performed on a testing machine UTM5105  at a crosshead speed of 2 mm·minReprocessing tests: The OBZ-BDB samples were ground into powder and fully filled into stainless steel mold. The dumbbell-shaped reprocessed samples were obtained by hot pressing under 16 MPa at 160 °C for 2 h. Each sample was tested respectively for five specimens.−1 and its polydispersity index is 3.50.1H NMR spectrum of OBZ (d). For comparison, allylamine (a), cardanol (b), and BZ monomer (c) are also listed. For BZ, the characteristic resonances at 4.85, 3.96, and 3.39 ppm are attributed to the protons of O–CH2–N (9), Ar–CH2–N (10), and N–CH2–CH=CH2 (2), respectively. The ratio of the integral areas of these resonance signals is 2:2:2, which is in good agreement with the theoretical value. This indicates the successful synthesis of the BZ monomer. For OBZ, the characteristic peaks of the oxazine ring at 4.85 and 3.96 ppm completely disappear. Meanwhile, a new characteristic peak corresponding to the Mannich bridge protons of –CH2–N(R)–CH2– (2) appears at 3.72 ppm, indicating the ring-opening polymerization of BZ. This is also confirmed in the FT-IR spectra of OBZ , CH2=CH-CH2-CH (b2), and Ar-CH2-CH2 (o), respectively. The calculated feed mass ratio of In order to construct an ideal network, the molar ratio of alkene to thiol is a crucial factor. If the crosslinking agent (BDB) is insufficient or excessive, some crosslinking points would not be formed. Therefore, we used an equal molar ratio to feed the alkene and thiol. The calculated molar content of CH−1 ascribed to –SH disappears after curing (see −1 assigned to the terminal vinyl bond obviously weakens. However, the peak intensity at 729 cm−1 assigned to the inner vinyl group in the cardanol side chain has little change. These results indicate that the click reaction mainly occurs in allyl double bonds and the terminal double bonds of the cardanol side chain.ring see b. MoreovWe think that the reprocessing mechanism of OBZ-BDB is shown as follows. During the hot-pressing process, the boronic esters at the interfaces between ground particles produce a metathesis reaction, generating some new cross-linking sites across interfaces. This causes ground particles to fuse with each other to form a whole. Therefore, the appearance of the samples is transparent and uniform.gs are listed in g of the original OBZ-BDB is 224 °C, indicating that OBZ-BDB has high heat-resistance. We think this is related to the high crosslinking density of OBZ-BDB. The segmental motions are frozen. The boronic ester bonds are embedded in a rigid polymer matrix, and their dynamic exchange reaction is inhibited. The OBZ-BDB exhibits high Tg. With the increase of reprocessing generations, no significant change in Tgs is observed, indicating that the reprocessed OBZ-BDB sample still has excellent heat resistance.d5, Td10, and char yield of the original OBZ-BDB remain at 325 °C, 341 °C, and 30%, suggesting that OBZ-BDB has favorable thermal stability. After three reprocessing generations, these parameters of OBZ-BDB can still remain at 312 °C, 328 °C, and 24%, indicating that the reprocessed OBZ-BDB also possesses good thermal stability.In conclusion, a reprocessable benzoxazine resin of OBZ-BDB cross-linked by boronic ester bonds was successfully synthesized via a thiol-ene click reaction between boronic ester crosslinker containing thiols and an OBZ oligomer containing double bonds.The hot-pressing temperature and time have an obvious influence on the tensile strength. The optimized reprocessing conditions are hot-pressing at 160 °C under 16 MPa for 2 h.The reprocessing tests confirm that the OBZ-BDB has remarkable reprocessability. The retention rates of the tensile strength through three generations of reprocessing are 98%, 95%, and 84%, respectively.g of the original OBZ-BDB is 224 °C. After three generations of reprocessing, no significant change in Tgs was observed.The OBZ-BDB has excellent heat resistance. The T


Increasing ultraviolet (UV) radiation is causing oxidative stress that accounts for growth and yield losses in the present era of climate change. Plant hormones are useful tools for minimizing UV-induced oxidative stress in plants, but their putative roles in protecting tomato development under UVC remain unknown. Therefore, we investigated the underlying mechanism of pre-and post-kinetin (Kn) treatments on tomato plants under UVC stress. The best dose of Kn was screened in the preliminary experiments, and this dose was tested in further experiments. UVC significantly decreases growth traits, photosynthetic pigments, protein content, and primary metabolites  but increases oxidative stress biomarkers  and proline content. Treatment of pre-and post-kinetin spraying to tomato plants decreases UVC-induced oxidative stress by restoring the primary and secondary metabolites’  status and upregulating the antioxidant defense systems . Thus, the application of Kn in optimum doses and through different modes can be used to alleviate UVC-induced negative impacts in tomato plants. Plants, being sessile creatures, cannot avoid exposure to ecologically induced alterations that seriously impact their growth and performance  are signaling agents that are widely applied for controlling growth and development activities in crop plants and alleviating the effects of abiotic and biotic stress  is among the largest commercially consumed vegetable crops across the globe with high nutrition value  plants were purchased from Mecca TRADE Co. as Asia Seed co. Lid product, Korea. The cultivars were selected based on their importance as vigor hybrids. It is the most popular and widely cultivated in Upper Egypt, with little physiological history in the literature under biotic/abiotic stresses. Before sowing, tomato seeds were sterilized with HgCl2 (0.1%) for 5 min and then rinsed vigorously with distilled water three times. Next, the seeds were sowed in trays filled with 100% peat moss. Then, the trays were kept in a greenhouse at 25 ºC. The trays were fertilized with a water-soluble fertilizer  at a rate of 150 mg/L. The seedlings (35 days) were then transplanted to pots (30 cm diameter) filled with 1 kg of clay soil. The experiment was conducted under natural conditions in the Botanical Farm of Botany and Microbiology Department, Faculty of Science, Assiut University, Egypt . Three seedlings of similar lengths and sizes were remained per pot to be used throughout the experiments. Each experimental pot was given a 250-mL full-strength nutrient solution . Group 2 was subjected to UVC radiation only-the third group containing five sub-groups (Kn application). Tomato plants (35 days) were subjected to UVC stress using UVC lamps  for 20 min. The plants have been arranged 40 cm away from the UVC lamp placed at the top of the plants.The third group containing five sub-groups was sprayed with different concentrations of Kn  a day before the exposure to UVC. Then, the plants were sprayed with 20 ml of the corresponding concentration. The plants were exposed to radiation within three days from 11:12 am. Pots were kept under natural humidity, temperature, and sunlight conditions at the Botanical Farm, Botany and Microbiology Department, Faculty of Science, Assiut University, Assiut, Egypt. The experiment was carried out with three replicates for each treatment and was fully randomized. One week after applying the last UVC dose, the plants were harvested to determine the best Kn concentration. Morphological traits (as lengths and weights) of the harvested plants were chosen for this purpose.The tomato plants were divided into four groups, as shown in Table Fifteen days after treatment (DAT), morphological characteristics of all treatments have been documented. Five roots were harvested and taken to the laboratory to measure various growth parameters, including plant height, fresh and dry weights, sprinkles and roots, and 72 h after shade drying at room temperature.a, b, and carotenoids contents were determined using the method of / Chl a content of intact fresh leaves) × 100.−1 FW using an extinction coefficient of 33.000 mol−1 cm−1.To determine anthocyanin levels, 0.5 g of the leaf sample was soaked in 3 mL of acidified methanol (1% v/v HCl) for 12 h in darkness at 4 °C with occasional shaking. The mixture was then centrifuged for 10 min at 14,000 × g at 4 °C. The absorption of the supernatant was read at 530 nm by a spectrophotometer  content was analyzed in Heath and Packer  techniquLeaf samples were extracted in 5% TCA and centrifuged at 11,500 × g for 15 min. The supernatant was mixed with 10 mM phosphate buffer (pH 7.0) and 1 M KI, and absorbance was recorded at 390 nm  was determined by extracting in K-phosphate buffer and using the detailed method of Yang et al.  concentration was estimated following Halliwell et al. , 100 µM FeCl3, 104 µM EDTA, 1 mM H2O2, and 100 µM ascorbate in final 1 mL. After incubation at 37 °C for 1 h, optical density was recorded at 532 nm.Hydroxyl radicals  activity was assayed by adopting Doderer et al.  method. Total soluble sugar was estimated according to the modified method of , and the mixture was completed to 5 mL with distilled water. Finally, the developed color was measured spectrophotometrically at 510 nm using quercetin as a standard curve.Zou et al.  method w−1 dry weight.Total phenolic content was estimated using the method described by Kofalvi and Nassuth , with mi2O2 was monitored at 240 nm for 3 min. Nakano and Asada . For calculation, an extinction coefficient of 2.8 mM−1 cm−1 was used. Glutathione peroxidase  was followed by Flohé and Günzler  leaves were extracted in 100 mM phosphate buffer (pH 7.8) containing PVP and EDTA. The homogenate was centrifuged at 15.000 × g for 10 min. The supernatant was used for assaying enzyme activity. The superoxide dismutase activity  was assayed by Misra and Fridovich . The abind Asada  method w Günzler  method. −1 cm−1 was used. The activity of soluble (SPO) and ionic (IPO) peroxidases (EC 1.11.1.7) was measured following the extraction methods and procedures of Ghanati et al. (2O2. The change in absorbance was modified to a unit with an extinction coefficient of 26.6 mM−1 cm−1.GST (EC 2.5.1.18) was assayed by recording the absorbance at 340 nm for 2 min in a 3-mL assay mixture containing 100 mM phosphate buffer (pH 6.5), GSH (1.5 mM), 1-chloro-2, 4-dinitrobenzene (1 mM), and enzyme extract. For calculation extinction, a coefficient of 9.6 mMi et al. . The act−1 protein−1 min. Polyphenol oxidase  activity was detected by Lavid et al.  activity was experienced according to Havir and Hanson . The enzd et al.  protocol2 and suspended in 2 mL of 5% TCA. Next, the homogenate was centrifuged at 10,000 × g for 15 min at 5 °C. Next, AsA extraction solution was mixed with 10% TCA, which was vigorously shaken and then placed in an ice bath for 5 min. Next, 0.5 ml of the extract was diluted to 2.0 mL using distilled water, and 0.2 mL of diluted Folin-Ciocaiteu reagent was added to the previous mixture, and the absorbance of the blue color developed was measured after 10 min at 760 nm. Finally, the AsA content was calculated using a standard curve of ascorbic acid.Ascorbic acid (AsA) was determined according to Jagota and Dani . First, For determination of reduced glutathione (GSH), fresh (100 mg) leaf tissue was macerated in phosphate buffer (pH 8.0) followed by centrifugation for 15 min at 3000 × g. Five hundred milliliters of supernatant and 500 µL of 5,5-dithiobis-2-nitrobenzoic acid were mixed and read at 412 nm after 10 min. The standard curve of GSH was used for the calculation Ellman .α-tocopherol content in the extracts was calculated using α-tocopherol as a standard curve.The α-tocopherol was measured based on , the whole data were analyzed statistically by analysis of variance (ANOVA), both one and two-way at 0.05 level. Duncan’s Multiple Range test (DMRTs) was used to further compare between subgroups at p ˂ 0.05. Multivariate analysis of variance (MANOVA) was applied to all physiological trait data. Minitab Software performed principal component analysis (PCA).Data were handled and presented in tables and figures as well as checked for normality using Shapiro–Wilk normality testing. Accordingly, data were parametric (p ˂ 0.001***) decrease in shoot length (42.0%), root length (35.65%), shoot FW (46.25%), root FW (26.53%), shoot DW (33.91%), and root DW by 45.0% as compared to control plants according to DMRTs, where, means with different letters are significantly different. Treatment of UVC-stressed plants with different Kn concentrations  increased various growth characters. The shoot length (SL) significantly (p < 0.05) increased by 13.82 and 17.42% for 0.05 and 0.1 mM Kn application. The highest increase was noticed at the level of 1 mM Kn, where SL increased by 30.81%, whereas, 14.00 and 11.01% increase was detected for 2 and 3 mM Kn spraying level. Root length also showed a maximum increase of 30.88% for 1 mM Kn treatment. The other doses 0.05, 0.1, and 2 mM Kn increased RL by 12.47, 0.1 by 17.65, and 11.14, respectively. The 3 mM Kn treatment remained ineffective in increasing the RL of the stressed plants. The highest increase of 50.65% was noticed by 1 mM Kn treatment in the FW of the shoot. The levels of 0.05 mM Kn increased the shoot FW by 27.11%, 0.1 mM Kn by 33.40%, 2 mM Kn by 37.68%, and 3 mM Kn by 38.44% compared to UVC -exposed plants. Root FW showed a non-significant trend under all Kn doses. Taking into consideration DW of the shoot, the level of 0.05 mM Kn caused a significant increase of 27.61%, 0.1 mM Kn, and 2 mM Kn each by 37.70%; however, the highest growth of 61.80% was noticed by 1 mM Kn treatment. Root DW was maximally (53.19%) increased by 1 mM Kn supply. A significant increase by 31.25% by 0.05 mM Kn, 33.33% by 0.1 mM Kn, 43.58% by 2 mM Kn, and 29.03% by 3 mM of Kn doses was observed compared to alone UVC-treated plants  increase in SFW by 27.75 and 29.42%, respectively, was noticed for plants that received Kn after UVC treatment as compared to alone 20- and 40-min UVC radiation  effect of Kn or UVC, or interaction between both treatments on growth parameters, e.g., SFW, RFW, SDW, and RDW (Table Two-way ANOVA presented in Table DW Table .The RFW decreased with increasing (20 to 40 min) duration of UVC radiation by 45.23 and 73.02%, respectively, compared to control plants. On the other hand, Kn application before UVC treatment significantly increased RFW by 55.82 and 64.76%, compared to increments by 44.16 and 35.71%, respectively, for plants that received Kn after UVC exposure compared to alone 20- and 40-min UVC radiation Fig. .The SDW was significantly decreased by increasing the duration of UVC radiation (20 to 40 min) by 29.77 and 51.14%, respectively, compared to control plants. The Kn application before and after UVC radiation increased SDW by 60.25 and 57.04%, as well as 38.87 and 28.08%, respectively in SDW compared to alone 20- and 40-min UVC radiation Fig. .The 20 to 40 min UVC radiation decreased RDW by 43.66 and 54.92%, respectively, compared to the control group. Kn before UVC treatment supply caused a significant increase in RDW by 60.00 and 60.97%, respectively, concerning 20- and 40-min UVC radiation. An increase of 48.05 and 48.38% was noticed in RDW when Kn was given after UVC radiation compared to plants that received UVC radiation for 20 and 40 min Fig. .Chl a and b contents by 29.72%, 67.56%, and 19.56% and 56.52%, respectively, concerning control plants. On the contrary, Kn application before and after UVC radiation increased Chl a by 58.73% and 73.52% as well as 61.76 and 68.96%, respectively, concerning 20- and 40-min treatments. Likely, the Chl b was stimulated by 30.18 and 51.21, as well as 42.18 and 37.50%, respectively, by Kn application before and after UVC exposure relative to 20- and 40-min UVC  on photosynthetic pigments and chlorophyll stability index (CSI) Table  as analyUVC Fig. , B.Fig. Carotenoids and CSI also showed a significant decreasing trend in response to UVC exposure for 20 to 40 min by 12.04% and 33.73% as well as 21.27% and 28.94%, respectively, compared to control plants. The Kn application before UVC radiation increased carotenoids by 77.25 and 54.91%, respectively, concerning alone 20- and 40-min UVC radiation. An increase of 54.93% and 68.20%, respectively, was observed after Kn-treated plants compared to 20- and 40-min UVC treatments. The CSI was increased by 18.37, 79.44, 9.41, and 19.52%, respectively, by Kn application before and after UVC exposure concerning their alone treatments Fig. , D.2O2, OH●─, and O2●─ in tomato plants treated with Kn in the absence or presence of UVC stress are presented in  increase in anthocyanins and flavonoids by 29.43, 52.73, 38.50, and 28.16%, as well as 23.66 and 27.87%, respectively as compared to their alone 20- and 40-min UVC treatment , flavonoids (22.08 and 37.17%), and phenolics (44.68 and 64.60%) by UVC stress Fig. . Before ent Fig. .The application of 20- and 40-min UVC treatments induced a significant partial deactivation of SOD and CAT activities by 31.29 and 21.99% as well as 68.37 and 11.31%, respectively, over the control plants Fig. , B. HoweIn APX activity, the doses of 20- and 40-min UVC treatments caused a significant increase and decreased by 38.97 and 46.10%, respectively, compared to control plants Fig. . On the The ionic and soluble POX activities were elevated significantly by 59.02 and 53.07 as well as 83.89 and 81.67% for exposure durations 20 and 40 min of UVC treatments, respectively, compared to control plants. For Kn application before UVC treatment, ionic peroxidase maximized by 53.59, 68.44, and that of soluble peroxidase by 56.47 and 68.62% respectively, and in plants of Kn after UVC treatments, ionic POX increased by 32.78, 25.55, and soluble POX by 52.79 and 53.19%; respectively over their alone 20- and 40-min UVC treatments Fig. , F.A significant decrease of 31.88 and 48.28% in GST activity was observed by the 20- and 40-min UVC treatments. Nevertheless, GST activity increased significantly by 43.93, 50.92, and 25.37, 40.31% respectively in Kn before and after UVC treatments compared to their alone 20- and 40-min UVC treatments Fig. .Fig. 6A–The 20-min exposure time of UVC imposed a significant stimulation of PAL activity by 15.35, but a higher duration of 40 min decreased its activity by 38.52% concerning the control plants Fig. . The Kn Plants treated with 20- and 40-min UVC treatments significantly increased the PPO activity by 17.72 and 74.47% compared to control plants. However, a declining trend was noticed when plants were given Kn spraying before UVC treatment by 32.44, 51.41%, and 22.54, 19.31% respectively compared to their alone 20- and 40-min UVC treatment Fig. .Plants exposed to 20- and 40-min UVC recorded a significant increase and decrease of AsA content by 7.94 and 13.58%, respectively, over the control plants. While, the plants treated with Kn before UVC increased by 52.00 and 38.16%, respectively, compared to 20- and 40-min treatments. The Kn after UVC treatment 20 min increased the AsA content by 9.94 but decreased by 1.70% at 40 min compared to the 20- and 40-min UVC treatments Fig. .Fig. 7A-The GSH in tomato plants was significantly increased in response to 20- and 40-min UVC treatments by 26.13 and 27.26% over the control. However, exposure to Kn before UVC resulted in a significant increase of 27.13 and 35.38%, respectively, concerning 20- and 40-min UVC treatments. Nonetheless, the Kn after UVC treatments increased the GSH by 26.55 and 23.37%, respectively, over the 20- and 40-min UVC treatment Fig. .The α-tocopherol was increased by 20- and 40-min UVC treatments by 10.48 and 5.61% concerning control. The Kn before UVC treatments increased the α-tocopherol by 45.28 and 47.15%%, respectively, compared to the 20- and 40-min UVC treatments. The Kn after UVC treatments also showed significant increments by 41.25 and 9.92%, respectively, concerning 20- and 40-min UVC treatment Fig. .Tomato plants exposed to 20- and 40-min UVC showed a significant increment in proline content by 52.21 and 69.39% over the control. On the other hand, plants treated with Kn before UVC treatment showed a decrease of 51.95 and 58.36%, respectively, compared to 20- and 40-min treatments. Also, the application of Kn after UVC decreased proline by 26.10 and 56.54% respectively over the alone 20- and 40-min UVC treatments Fig. .The lignin content of tomato plants exposed to 20- and 40-min UVC showed a significant increase of 63.02 and 82.24% over the control. On the other hand, plants treated with Kn before UVC treatments decreased lignin content by 50.92 and 64.72%, respectively, compared to the 20- and 40-min treatments. Also, Kn after UVC treatments decreased lignin by 20.84 and 11.94%, respectively, compared to the alone 20- and 40-min UVC treatments Fig. .A PCA was constructed to know the amount of data variability and interaction of various variables. Figure Solanum lycopersicum  and CSI of tomato seedlings were found to be decreased under UVC exposures  mutants of Arabidopsis thaliana. The significant improvement in non-enzymatic antioxidants along with proline content in Kn-treated plants suggested that these non-enzymatic antioxidants and proline content upregulated by Kn supported plants to regulate the oxidative stress under UVC toxicity as indicated by better growth performance and reduced ROS content in Kn-treated than non-Kn ones . Interestingly, Kn application increased the secondary metabolites on the one hand and triggered an increase in enzymes’ activity (PAL) on the other hand. A similar effect of Kn on increasing anthocyanins, flavonoids, phenols, and PAL activity has been observed previously in tomatoes under UV stress (Singh et al.  Staiger . In the  Staiger . Also, t Staiger . A secon Staiger E. The hiA final validation in testing Kn treatments’ effectiveness in alleviating UVC stress damages in the present study was documented from PCA analysis. According to the PCA loading plot Fig. , growth,The present study revealed that UV radiations caused oxidative stress that decreased growth and induced alterations in physio-biochemical attributes. In contrast, the UVC mediated decrement in growth traits, photosynthetic pigments, protein content, and primary metabolites was overcome by Kn application. The application of Kn at selected doses minimized the UV-induced oxidative stress and protected tomato plants under UVC. The application of pre-and post-Kn treatment also decreased UVC-induced stress by boosting the primary and secondary metabolites’ status and elevating the plant antioxidant gadgets. Thus, it was concluded that Kn could increase growth and yield through different modes of applications and alleviate UVC-induced ill effects in tomato plants.Ultrastructural changes of different organs under UVC stress should be documented.The correlation of endogenous hormones under UVC should be studied extensively.Genes controlling ROS production under UVC stress.Gene expression of PAL and other secondary metabolites under UVC-kinetin conditioning.Further studies on the interactive effect of UVC and kinetin or other PGR should be done on other crop plants.Induction of senescing by UVC and its recovery by kinetin.Although the present study provides new information on the mechanistic role of kinetin under UVC stress, many points should be taken into consideration for further studies: