One-hour post-dose, the curcuminoids plasma concentration was 40% higher in participants consuming the MAG-OM3 compared to the other formulations. Thus, in a young population, plasma curcuminoids 24 h pharmacokinetics and its increase shortly after the single dose intake were higher when provided with MAG-OM3 than rice oil.There is a growing interest for curcuminoids in the general population and the scientific research community. Curcuminoids, derived from turmeric spice, are lipophiles and therefore have a low solubility in water which hence have a low bioavailability in the human plasma. To circumvent this issue, a natural product developed by Biodroga Nutraceuticals combined curcuminoids with omega-3 fatty acids (OM3) esterified in monoglycerides (MAG). The objective was to perform a 24 h pharmacokinetics in humans receiving a single dose of curcuminoid formulated by three different means, and to compare their plasma curcuminoids concentration. Sixteen males and fifteen females tested three formulations: 400 mg of curcuminoids powder extract, 400 mg of curcuminoids in rice oil and 400 mg of curcuminoids with 1 g MAG-OM3. Blood samples were collected at 0, 1, 2, 3, 4, 5, 6, 8, 10 and 24 h post dose intake. Plasma samples were analyzed by ultra high-performance liquid chromatography with a triple quadrupole mass spectrometer (UPLC-MS/MS). Twenty-four hours after a single dose intake, the total plasma curcuminoids area under the curve (AUC) reached 166.8 ± 17.8 ng/mL*h, 134.0 ± 12.7 ng/mL*h and 163.1 ± 15.3 ng/mL*h when curcuminoids were provided with MAG-OM3, with rice oil or in powder, respectively. The C Enthusiasm and interest towards the consumption of curcuminoids is growing in the general population and in the scientific community. Curcuminoids are natural polyphenols extract representing 1–6% of the commonly known turmeric spice (Curcuma longa) and are composed of curcumin (60–77%), demethoxycurcumin (DMC) 17–27%) and bisdemethoxycurcumin (BDMC) 3–15%) [7% and bi–15% 7% , antibacOne major limitation with the use of curcuminoids is that they are lipophiles, hence they have a low oral absorption and plasma bioavailability even when provided at dosages of 1000 mg ,9,10,11.In a previous pharmacokinetic study conducted by our group, we compared plasma concentrations of omega-3 fatty acids (OM3) esterified in monoacylglycerol (MAG), ethyl esters or triglycerides . The stuTherefore, to take advantage of the amphipathic characteristics of MAG-OM3, we hypothesized that curcuminoids with MAG-OM3s will, over a 24 h pharmacokinetics, increase plasma curcuminoids more than when provided formulated in powder or with rice oil, a neutral oil.The objective of this study was to compare the plasma concentration of curcuminoids over 24 h when it was provided as a single oral dose with MAG-OM3s, rice oil  or provided in powder.https://clinicaltrials.gov with the registration number: NCT04382014 and could be accessed 5 June 2020.The study was a triple-blind, randomized, crossover clinical trial and it was conducted at the Research Center on Aging, Centre Intégré Universitaire de Santé et des Services Sociaux. The Research Ethics Committee of the CIUSSSE-CHUS approved the study protocol (reference no 2020-3569). This study is registered on 2; have normal to moderately high lipidemia and using a contraceptive method during the study for women of childbearing age.Recruitment was conducted between July 2021 and January 2022. Inclusion criteria were males and females between 18 and 50 years old; not taking any curcuminoids or OM3 supplements 6 months prior the study and during the study; have a body mass index (BMI) between 18.5 and 34.9 kg/mExclusion criteria were allergy to fish or seafood; under a specific diet like vegetarian or vegan; performing more than 180 min/week of intense physical activity or more than 210 min/week of moderate intensity, having an history of current or past alcohol or drug abuse; smoking tobacco or marijuana; being or been an elite athlete; suffering from malnutrition; taking any anticoagulant or medication affecting fat absorption and lipid metabolism; taking any medication interacting with curcumin such as angiotensin II blockers, beta-blockers, calcium channels blockers, etc. Moreover, pregnant, or breastfeeding women; women with menopause or pre-menopause; anyone with a systemic disease, or having a liver, kidney or thyroid disease were excluded. Other exclusion criteria include having a cardiac event in within 6 months prior starting the study; having an history of thrombosis or hemorrhagic diathesis; having a gastrointestinal disease, a neurodegenerative disease, a genetic disorder, a psychiatric history, or a neurological disorder; having any major surgery in the last year or having donated blood in the last 30 days.Participants corresponding to these inclusion/exclusion criteria were thereafter invited for an on-site screening visit. They had to be fasted for 12 h prior the screening visits and not consume alcohol 48 h prior the visit. All participants provided informed written consent before any procedure was conducted. After obtaining their written consent, the following measures were collected: weight, height, blood pressure, pulse, and a fasted blood sample.Fasting glucose, total cholesterol, triglycerides, cholesterol high density lipoproteins (HDL-C), cholesterol low density lipoproteins (LDL-C) and glycated hemoglobin (HbA1c) were quantified in the fasted blood samples. Blood biochemistry was performed at the Centre Hospitalier Universitaire de Sherbrooke clinical laboratory.The participants tested three different supplements produced by Biodroga Nutraceuticals: curcuminoids extract emulsified in 760 mg of an OM3 oil esterified in MAG providing 350 mg of eicosapentaenoic acid and 152 mg docosahexaenoic acid (NPN 80107639), curcuminoids extract combined with rice oil (NPN 80102058) and curcuminoids extract in powder with 23% microcrystalline cellulose and 0.2% maltodextrin (NPN 80102057). Each capsule contained 200 mg of curcuminoids extract. Curcuminoids single dose supplementation in humans is considered safe and well tolerated without any serious adverse effects [Curcuminoids content of capsules containing only powder, rice oil or MAG-OM3 were analyzed using the whole weighted content. Briefly, for powder capsules, whole weighted content was added to 25 mL of reagent alcohol, vortexed for 30 s and sonicated for 30 min. For capsules containing rice or MAG-OM3, 25 mg of the capsule content was added to 25 mL of reagent alcohol, vortexed for 30 s and sonicated for 30 min. The mixtures from both extraction procedure was thereafter similar. Mixtures remained at room temperature for 5 min, 10 µL was added to 50 mL of methanol and vortexed for 15 s. 1 mL of the supernatant was filtered using 0.22 µm filter made of polytetrafluoroethylene. One hundred µL of the solution was transferred with 150 µL of internal standard composed of Curcumin-d6 as mentioned in the extraction from plasma, vortexed and 200 µL of the solution was transferred into vials for analysis. Quantification was made by ultra high-performance liquid chromatography with a triple quadrupole mass spectrometer (UPLC-MS/MS) as described below. The fatty acid profile of capsules was also performed as previously described Chevalier et al.  Curcuminhttps://www.randomizer.org/. Participants, research nurse and experimenter were blinded to supplement allocation. On each study visit, participants were fasted for 8 h prior the visit and avoided drinking alcohol 24 h prior their appointment. A catheter was installed in the participant’s forearm by the research nurse. After collecting a first fasted blood sample of 5 mL (T0), participants were provided a low-fat standardized breakfast of 410 kcal  composed of a banana, two toasts with jam, a protein milk and a coffee or a tea. Two capsules were orally administrated to the participants while they were taking their breakfast. The capsules were not all identical in shape and color, therefore participants were blindfolded to remain blinded. After the supplement intake, 5 mL of blood was collected in EDTA vacutainer tubes at the following timepoints: 1, 2, 3, 4, 5, 6, 8, 10, and 24 h (10 samples per treatment per participant). Samples were stored on ice before being centrifuged at 1700 g for 10 min at 4 °C. Plasmas were aliquoted and stored at −80 °C until analysis. Each plasma sample was coded with a random number to keep the personnel blinded to the participant’s number, its treatment and the timepoint at which the blood sample was collected. This procedure enables to keep the personnel blinded when performing biochemical analysis thereafter.The study design is depicted in During the pharmacokinetic day other meals such as a lunch  composed of one serving of chow mein, baby carrots, an apple, a yogurt, a juice and a fig bar, dinner , composed of one serving of General Tao, one slice of bread, one mini cucumber, one apple sauce, a protein-enriched milk, a tomato juice and a granola bar were provided. An evening snack  was composed of one granola bar and one package of gummy fruits was also provided at the end of the day and before participants leave the clinical room for the evening. Participant were instructed to refrain from eating other food in the evening and until a last blood sample collection the day after (T24 h). Menus and amount of food consumed for each participant was monitored and identical at each study day. Drinking water was recommended throughout the study days. Lunch was provided 4 h after the supplement intake whereas dinner and snack were provided 9 h and 10 h after the supplement intake.Helix pomatia  diluted in phosphate buffer 0.1 M, pH = 6.86 were added. The tubes were vortexed for 1 min and incubated at 37 °C for 1 h to hydrolyze the phase-2 conjugates of curcuminoids [g at room temperature for 6 min. The superior organic phase was then transferred, and the process was repeated a second time. The combined organic phase was evaporated under nitrogen stream. The curcuminoids extract was reconstituted in 100 µL of methanol, centrifuge at 5000 g for 5 min to remove any particles, and 50 µL of the supernatant was then transferred to HPLC vials.Curcuminoids were extracted from plasma samples according to the method of Cuomo et al.  Briefly,uminoids ,21. Then20  with 0.1% formic acid and mobile phase B was composed of Acetonitrile with 0.1% formic acid. The gradient elution program was as follows: 0–1.5 min (0–0% B), 1.5–1.6 min (0–95%B), 1.6–4.5 min (95–95% B), 4.5–4.6 min (95–0%B) and 4.6–6 min (0–0%B). The flow rate was 0.5 mL/min, and the total run time was 6 minThe samples were stabilised at 4 °C in the autosampler of the UPLC-MS/MS for 30 min prior its injection (4 µL). Calibration curve was made fresh every day of analysis, and quality controls were introduced at the beginning and at the end of the sequence. The calibration curve uses six concentrations ranging from 0 to 150 ng/mL made in a blank plasma matrix using a mixture of the three standard stock solutions of curcumin, BDMC and DMC . The low and high control quality were at concentration of 8 ng/mL and 200 ng/mL. Analyses of curcumin, BDMC and DMC levels were performed on a Waters Acquity UPLC H-Class system coupled to Waters Xevo TQ-S Micro tandem mass spectrometer with ESI source in positive mode . Curcuminoids were separated on a Waters Acquity UPLC BEH C18 column  coupled with a Waters Acquity UPLC BEH C18 VanGuard pre-column  at 40 °C. Mobile phase A was composed of Acetonitrile: Hm/z 369.1 → 177.1); DMC (339.1→ 147.0), BDMC (309.0 → 147.1) and curcumin-d6 (375.1 → 180.1) with a dwell time of 0.025 s. Mass Lynx software (Waters corporation) was used to generate the calibration curves by plotting the peak area ratio of curcumin, BDMC and DMC to internal standard (curcumin-d6). Each curcuminoid generated a calibration curve daily. Correlation coefficients for all calibration curves reached R2 > 0.99. The concentrations of curcuminoids of the plasma samples were calculated using a linear regression using the 1/x weighting factor.Nitrogen desolvation was set at 600 °C with a flow rate of 1000 L/h, the nitrogen cone gas was set at 80 L/h and the argon collision gas was set at 5 psi. The source temperature was at 150 °C and the capillary voltage was set at 1.0 kV. The cone voltage was 22 V for curcumin, 28 V for curcumin-d6, 34 V for DMC and 22 V for BDMC and the collision energy was at 22 eV. Quantification was performed in multiple reaction monitoring mode (MRM) with the following transition monitored: curcumin (max) and the time to reach it (Tmax). The AUC 0–24 h was used to calculate our sample size since it is more evaluated in the pharmacokinetic studies than AUC 0–6 h. Using the website https://clincalc.com/stats/samplesize.aspx [The primary outcomes of this study were area under the curve between 0 h and 24 h (AUC 0–24 h), between 0 h and 6 h (AUC 0–6 h), the maximal concentration , and the dependent variable was treatment (supplements) expressed in curcuminoids concentration (ng/mL). Multiple comparisons were performed with the Tukey test if the ANOVA result was significant. The AUC, from 0 h to 24 h, and 0 h to 6 h were calculated using the trapezoid rule and generated the Cmax and Tmax in GraphPad. AUC, Cmax, and Tmax were not normally distributed, and the Friedman test was used to compare groups. If significant, then the Dunn’s test was used for multiple comparison. To account for multiple testing, results were considered significant when p < 0.025 in the post hoc analysis. To compare results from males and females, a two-way ANOVA was performed to compare treatments, AUC, Cmax and Tmax. The independent variable was sex, and the dependent variables were AUC, Cmax and Tmax.Statistical analyses were performed using GraphPad Prism version 9.3.1 for Windows . The anthropometric characteristics were analyzed using an unpaired t test if data were normally distributed and if not, a Mann–Whitney non-parametric was used. Seventy-eight persons were contacted or manifested their interest to participate to the study . They we2. The results of blood profiles were all within clinical normal range. There were no statistical differences between males and females’ anthropometric characteristics.Participants’ anthropometric characteristics are described in max and Tmax pharmacokinetics parameters for total curcuminoids upon receiving the three curcuminoids formulations. Total curcuminoids 0–24 h AUC was 24% higher when provided with MAG-OM3 compared to rice oil (p = 0.0297) (p = 0.0230) , AUC (0–6 h), C 0.0297) B. The 0– 0.0230) . The maxPharmacokinetics parameters of secondary outcomes are described in Plasma concentration of total curcuminoids, curcumin, DMC and BDMC 1 h after the single dose intake were significantly higher when provided with MAG-OM3 compared to when they were provided in powder . Twenty-p = 0.0337), DMC (p = 0.0113) and BDMC (p = 0.0063) were at least 40% higher when provided with MAG-OM3 compared to the rice oil formulation  can be analyzed together.max and Tmax were not different between treatments. Hence, our hypothesis is partially rejected. After the 24 h follow-up, plasma curcuminoids returned close to baseline no matter the supplement. In previous pharmacokinetic studies evaluating different formulations, the 0–24 h AUC range between 95.3 ± 4.6 ng/mL*h and 4474.7 ± 1675.2 ng/mL*h [max of 22.4 ± 1.9 ng/mL and a Tmax of 2.4 ± 0.4 h vs. To MAG-OM3 . Additionally, micronized curcumin [max of 15.3 ± 8.9 ng/mL and a Tmax of 8.8 ± 6.4 h. Nonetheless, other alternatives with superior plasma concentrations are also available like Meriva [max = 209.9 ± 54.9 ng/mL and a Tmax of 3.8 ± 0.6 h and NovaSol [max of 1189.1 ± 518.7 ng/mL and a Tmax of 1.1 ± 0.4 h. However, these alternatives are mostly synthetics, and some have potential adverse effect such as potential implication in renal and liver toxicity caused by tween which should be carefully considered [Twenty-four hours after the single dose intake of curcuminoids with MAG-OM3, rice oil and powder, the total curcuminoids in the plasma 0–24 h and 0–6 h AUC were higher when provided with MAG-OM3 compared to when provided with rice oil although C ng/mL*h ,23,24 an ng/mL*h ,26 which ng/mL*h  a formulnsidered ,29,30.max was to nondetectable to 9.0 ± 2.8 ng/mL with doses ranging from 30 mg to 2000 mg [max = 15.7 ± 1.6 ng/mL suggesting that the excipients in the formulation might improve curcuminoids bioavailability.This study also generated an unexpected result with the curcuminoid powder since its 0–24 h AUC plasma curcuminoids was high compared to powders used in other studies even when provided at high doses ,31. Here 2000 mg . Here, 4p < 0.0001), BDMC (p < 0.0001), DMC (p < 0.0001) and total curcuminoids (p < 0.0001) plasma concentration levels were significantly higher when consumed with MAG-OM3 compared to other formulations. Although, Cmax of curcumin, BDMC, DMC and total curcuminoids did not differed between treatments, the timing of these concentrations differed between treatments. This result support that MAG-OM3 are good carriers of curcuminoids in the early phase of the postprandial transit, hence delivering these compounds at a faster rate than other formulation. MAGs are directly absorbed by enterocytes without needing to be hydrolyzed by pancreatic lipases [Our result also supports that one hour after the single dose intake, curcumin  and females (n = 15) were balanced whereas most other studies were done only on males ,51,52 orIn terms of limitations, this pharmacokinetic study tested only one dose of the products and nor efficacy, nor their pharmacodynamics were tested, hence limiting the extend to which these results can be transferred to the general population. This study was also limited to healthy males and females between 18 and 50 years old without severe health conditions. Additionally, the standardized low-fat meal is not representative of the usual North American diet and limit transfer of these data to the general population.In conclusion, participants consuming curcuminoids with MAG-OM3 had higher plasma curcuminoids concentration over 24 h compared to when curcuminoids was provided with rice oil. Moreover, in postprandial, especially 1 h after the single dose intake, curcuminoids with MAG-OM3 increased more curcuminoids plasma concentrations compared to other formulations supporting that curcuminoids were available faster than the other formulations.


Tafasitamab is an Fc-modified monoclonal antibody that binds to CD19, a cell-surface antigen that is broadly expressed on various types of B-cell non-Hodgkin’s lymphoma (NHL). Antibody-dependent cellular cytotoxicity (ADCC), a key mode of action of tafasitamab, is mediated through the binding of tafasitamab’s Fc region to FcγRIIIa receptors on immune effector cells and results in antitumor activity. Despite the proven clinical activity of tafasitamab in combination with lenalidomide in the treatment of diffuse large B-cell lymphoma (DLBCL), a higher number of immune cells in cancer patients may improve the activity of tafasitamab. Here, we characterized two ex vivo-expanded FcγRIIIa receptor—expressing cell types—γδ T and MG4101 natural killer (NK) cells—as effector cells for tafasitamab in vitro, and found that in the presence of these cells tafasitamab was able to induce ADCC against a range of NHL cell lines and patient-derived cells. We also explored the concept of effector cell supplementation during tafasitamab treatment in vivo by coadministering MG4101 NK cells in Raji and Ramos xenograft models of NHL. Combination treatment of tafasitamab and allogeneic MG4101 NK cells in these models demonstrated a survival benefit compared with tafasitamab or MG4101 monotherapy . In conclusion, adoptive cell transfer of ex vivo-expanded allogeneic NK or autologous γδ T cells in combination with tafasitamab treatment may potentially be a promising novel approach to increase the number of immune effector cells and enhance the antitumor effect of tafasitamab. Tafasitamab MOR208) is an Fc-modified, humanized monoclonal antibody that binds to the human B-cell surface antigen CD19 08 is an . TafasitTafasitamab has two amino acid substitutions in the Fc region S239D/I332E) that increase its affinity for Fcγ receptors, including FcγRIIIa (CD16) 39D/I332E. The ADC-expanded allogeneic NK cells (MG4101) reported stable disease in 8 of 17 evaluable patients (47.1%) and a promising safety profile [Several clinical trials have reported that low numbers of NK cells in peripheral blood adversely affect the treatment outcomes of patients with lymphomas undergoing immunotherapy , 7. Furt profile . Similarcells MG41 reporte profile –15.To explore this concept for tafasitamab, we confirmed the ability of ex vivo-expanded γδ T or allogeneic NK cells (MG4101) to mediate tafasitamab-induced ADCC against lymphoma cells in vitro. In addition, we investigated the potential of adoptive cell therapy in combination with tafasitamab by supplementing tafasitamab treatment with MG4101 ex vivo-expanded NK cells in disseminated tumor mouse models of NHL.Tafasitamab and XmAb5603, an anti-CD19 analogue of tafasitamab with a wild-type IgG1 Fc region, were provided by MorphoSys AG. As controls, either a polyclonal IgG1 isotype control (Sigma I5154) or an IgG control (Sigma I4506) were used.γδ T cells were generated by zoledronate and interleukin-2 stimulation and culture of peripheral blood mononuclear cells (PBMCs) from different healthy donors for 9–12 days as described previously .PBMCs were isolated from healthy donors by leukapheresis. NK cells were enriched, expanded, and cryopreserved as described previously .Primary human leukemia and lymphoma cells were isolated from heparinized peripheral blood or bone marrow biopsies of patients recruited at the Nuremberg University Hospital, Germany.Jeko-1, Mino, Daudi, U-2932, Raji, and Ramos cell lines were obtained from the German Collection of Microorganisms and Cell Cultures or the American Type Culture Collection and cultured in standard medium.T) with antibody and effector cells (E) for 4 h at 37 °C and E/T ratios ranging from 0.7:1 to 20:1, cells were harvested, stained with TO-PRO-3, and evaluated by flow cytometry. Cell lysis at each E/T ratio was calculated using the following formula: ADCC with γδ T cells was determined by flow cytometry as described previously . After iE/T ratio of 3:1. After incubation for 2 h, plates were centrifuged and calcein release was quantified using a fluorescence microplate reader. As a control, maximal lysis (release) was induced with 1% Triton X-100. The percentage of ADCC-mediated cell lysis was measured by calcein release from target cells and calculated using the following formula: For quantification of ADCC, target cells were labeled with calcein-AM. Monoclonal antibodies (tafasitamab or control IgG) were added to the target cells and incubated with MG4101 NK effector cells at an 5 Raji cells/mouse were intravenously (i.v.) injected into the tail vein of CB17/Icr-Prkdcscid/CrlCrlj (CB-17 SCID) mice on Day 0 and cryopreserved MG4101 NK cells (2 × 107 cells/mouse) or freezing medium (vehicle control) were administered i.v. on Days 1, 3, 6, 8, and 10. Tafasitamab was administered subcutaneously (s.c.) once on Day 1 at a dose of 2.5 μg/kg. In the disseminated Ramos model, 1 × 106 Ramos cells/mouse were injected i.v. on Day 0 and MG4101 NK cells were administered i.v. on Days 4, 7, 11, 14, 18, and 21. Tafasitamab was administered i.v. on Days 3, 6, 10, 13, 17, and 20 at a dose of 10 mg/kg. Mice were monitored daily for tumor-associated morbidity, mortality, and hind limb paraplegia.For the disseminated Raji model, 1 × 10All animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience & Biotechnology.t test. Increased life span (%) in mouse models of survival was calculated using the following formula: P value < 0.05 was considered statistically significant for all experiments.Statistical significance in ADCC experiments was calculated using a two-tailed paired E/T ratio-dependent manner  cell lines in an ner Fig. a, b. Furner Fig. c, d.Fig.E/T ratio–dependent manner, XmAb5603 showed no substantial increase in cell death compared with background levels  to mediate tafasitamab-induced ADCC was evaluated in vitro using Raji and Ramos cell lines. Compared with the IgG isotype control, tafasitamab induced increased ADCC against both Raji and Ramos cell lines Fig. .Fig. 3AlNext, tafasitamab in combination with MG4101 immune cell supplementation was evaluated in two disseminated BL survival models in severe combined immunodeficiency (SCID) mice. First, in a preventive Raji model, a single subcutaneous low dose of 2.5 μg/kg tafasitamab was given on Day 1 after tumor inoculation, and MG4101 cells were administered i.v. on Days 1, 3, 6, 8, and 10. Combination treatment resulted in a greater increase in lifespan (ILS) than tafasitamab and MG4101 monotherapies  and antibody-dependent cellular phagocytosis (ADCP).In January 2020, MorphoSys and Incyte entered into a collaboration and licensing agreement to further develop and commercialize tafasitamab globally. Following accelerated approval by the U.S. Food and Drug Administration in July 2020, tafasitamab is being co-commercialized by MorphoSys and Incyte in the United States. Incyte has exclusive commercialization rights outside the United States.XmAb® is a trademark of Xencor, Inc.


SERK, WOX9, BBM, FUS3, CUC, and WUS were characterized as the molecular markers for tree peony SE. To our knowledge, this is the first study of the SE of tree peony using transcriptome sequencing. These results will improve our understanding of the molecular mechanisms that underly SE in tree peony and will benefit the propagation and genetic engineering of this plant.Low propagation rate is the primary problem that limits industry development of tree peony. In this study, a highly efficient regeneration system for tree peony using somatic embryogenesis (SE) was established. The transcriptomes of zygotic embryo explants (S0), non-embryonic callus (S1), embryonic callus (S2), somatic embryos (S3), and regenerated shoots (S4) were analyzed to determine the regulatory mechanisms that underlie SE in tree peony. The differentially expressed genes (DEGs) were identified in the pairwise comparisons of S1-vs-S2 and S1-vs-S3, respectively. The enriched DEGs were primarily involved in hormone signal transduction, stress response and the nucleus (epigenetic modifications). The results indicated that cell division, particularly asymmetric cell division, was enhanced in S3. Moreover, the genes implicated in cell fate determination played central roles in S3. Hormone signal pathways work in concert with epigenetic modifications and stress responses to regulate SE.  Paeonia Sect. Moutan DC.) is an important ornamental plant that is native to China. The plant is also known for its edible and medicinal values. Cortex Moutan is a famous Chinese traditional medicine listed in the Chinese Pharmacopeia , SI2 (WPM + 4.0 mg·L−1 phytagel + 30 g·L−1 sucrose + 0.5 mg·L−1 6-BA + 1.0 mg·L−1 GA3), SI3 (WPM + 4.0 mg·L−1 phytagel + 30 g·L−1 sucrose + 1.0 mg·L−1 6-BA + 0.5 mg·L−1 GA3), and SI4 (WPM + 4.0 mg·L−1 phytagel + 30 g·L−1 sucrose + 1.0 mg·L−1 6-BA + 1.0 mg·L−1 GA3) for shoot development. When the shoots had grown to 3–4 cm, each was separated and cultured on root induction medium, RI1 (WPM + 4.0 mg·L−1 phytagel + 30 g·L−1 sucrose + 3.0 mg·L−1 6-BA + 3.0 mg·L−1 IBA + 2.0 mg·L−1 caffeic acid), RI2 , RI3  at 4 °C for 15 days, followed by a poikilothermic (22 °C for 16 h and 18 °C for 8 h) condition for 45 days to induce roots. The ratio of explants with callus induction, embryonic callus induction, shoot induction, and root induction were recorded after 14 days, 2 months, 4 months, and 6 months of culture, respectively. The number of shoots per explant was also determined after 4 months of culture. Samples at different culture stages including S0 , S1 , S2 , S3 , and S4 (the regenerated shoots after 4 months of culture) were also collected for histological and transcriptomic analyses. The former samples were stored in 2.5% glutaraldehyde and incubated at 4 °C for 8 h, while the latter samples were immediately frozen in liquid nitrogen and stored at −80 °C until use.4) for 2 h at 4 °C. Subsequently, they were dehydrated in a series of ethanol  and then dried, gold coated, and photographed with a SU-8010  scanning electron microscope.All the induction stages were recorded and photographed using a Leica Stereo Microscope . For scanning electron microscopy (SEM) observation, the samples in 2.0% glutaraldehyde were washed with 0.1 M phosphate buffer  and fixed by 1% osmic acid  was extracted using Trizol extraction kit  according to the manufacturer’s instructions. Two embryos, calli or shoots were used in each sample. After digestion with RNase-free recombinant DNase I, the quality and quantity of the total RNA were determined by NanoDrop spectrophotometer . High-quality RNA from the five samples were used for cDNA library construction and BGISEQ-500 RNA-Seq. The cDNA Library preparation was performed following the Illumina manufacturer’s instructions. The double-stranded cDNA of the above five samples was sequenced using an Illumina HiSeq™ 4000 (4 × 100 bp read length) platform at the Beijing Genomics Institute Company, Shenzhen, China. All transcription sequencing data raw files has been deposited at the NCBI Sequence Read Archive under the BioProject ID PRJNA864612.https://busco.ezlab.org/, accessed on 1 August 2021). The longest open reading frame (ORF) per transcript contig was identified using TransDecoder. The sequence similarities and ORFs were predicted and identified by BLASTP, ORF finder, and TransDecoder software (v.5.1) . The Unigenes were annotated and functionally classified by conducting a BLAST search against seven databases, including NR (NCBI non-redundant protein sequences), NT (NCBI non-redundant nucleotide sequences), Pfam (the prediction of protein structure domain), Kyoto Encyclopedia of Genes and Genomes (KEGG), Eukaryotic Orthologous Groups of proteins/Clusters of Orthologous Groups of proteins (KOG/COG), KEGG Orthology (KO), and Gene Ontology (GO).The quality of raw reads from each library was assessed using the FASTQC program and Trimmomatic software (0.39) to filter the reads and remove adapter sequences. The clean reads were then assembled de novo using Trinity to obtain reference transcriptome unigenes for annotation and DGE analysis. The quality of the assembled transcripts was evaluated using the Benchmarking Universal Single-Copy Orthologs (BUSCO) database  ≤ 0.001, and log2(Fold change) ≥2 with fragments per kilobase million (FPKM) of >1 in at least one sample. GO and KEGG enrichment analyses were performed using Fisher’s exact test for the elucidation of the biological functions of the genes. The heatmap of DEGs was constructed using Heml software (Version 1.0) .The differentially expressed genes (DEGs) were analyzed using DESeq2. The false discovery rate (FDR) is used as a correction in many applied multiple testing problems. The parameters were set at Important DEGs related to SE were selected to check their relative gene expression by qRT-PCR. RNA was extracted from the samples using a total RNA extraction kit  according to the manufacturer’s protocol. The quality and quantity of RNA were assessed with 1.2% agarose electrophoresis and a NanoDrop 2000c spectrophotometer , respectively. Primers were designed using Premier 5.0 software . The first-strand cDNA synthesis was performed using a FastQuant RT Kit  following the manufacturer’s instructions. The relative expression of the candidate genes was calculated using a double standard curve according to the CFX96 Real-Time system  by normalizing to the reference gene ACTIN according to Wang et al. (2020). All qRT-PCR reactions were performed in triplicate with at least three biological replicates. The primer information used in this experiment are shown in The experimental assays used to obtain all results were repeated at least three times. Results were presented as means ± the standard error (SE) and analyzed using one-way ANOVA, followed by Duncan’s multiple-range test. Statistical analysis was performed with SPSS 22.0 . SERK, WOX9, BBM, FUS3, CUC, and WUS were characterized as molecular markers for SE in tree peony. This study improves our understanding of the molecular mechanisms that underlie SE.A highly efficient regeneration system of tree peony via SE was established. Based on this regeneration system, the transcriptomes of five SE stages were analyzed. Totals of 32,176, 20,561, and 24,039 DEGs were identified in pairwise comparisons of S0-vs.-S1, S1-vs.-S2, and S1-vs.-S3, respectively. Functional characterizations of the DEGs based on GO and KEGG analyses are presented. A total of 224 DEGs were identified for their potential associations with SE and the regulatory pathways, including 26 decisive DEGs in SE and SAM construction, 21 DEGs annotated to cell division and expansion, seven DEGs annotated to cell fate determination, 37 DEGs annotated to auxin signaling pathway, 24 DEGs annotated to cytokinin signal pathway, 10 DEGs annotated to the ABA signaling pathway, 2 DEGs annotated to the GA signaling pathway, and 10 DEGs annotated to the ethylene signaling pathway, 54 DEGs annotated to the central histones of nucleosome and epigenetic modifications, and 31 DEGs annotated to stress responses. The genes involved in these processes were discussed in this study, which helps to elucidate their roles in SE. The temporal program for gene expression by qRT-PCR during SE was also analyzed, and the results confirmed the patterns of gene expression of the transcriptomes. Taken together, cell division, particularly asymmetric cell division was enhanced during SE. Moreover, the determination of cell fate and the genes related to meristem formation played central roles in the construction of SAM during somatic embryo formation. The hormone signaling pathways work in concert with epigenetic modifications and stress responses to regulate the induction of SE and the development of somatic embryos.


In this study, we explored the relationship between inflammatory adipokine levels and coronary artery disease (CAD). We collected subcutaneous adipose tissues(SAT), pericardial adipose tissues(PAT), and epicardial adipose tissues (EAT) and serum samples from 26 inpatients with CAD undergone coronary artery bypass grafting and 20 control inpatients without CAD. Serum inflammatory adipokines were measured by ELISA. Quantitative real-time PCR and western blot were used to measure gene and protein expression. Adipocyte morphology was assessed by H&E staining. Immunohistochemistry and immunofluorescence were used to measure endothelial and inflammatory markers. Serum pro- and anti-inflammatory adipokine levels were higher and lower, respectively, in the CAD group than those in the control group (P < 0.05). In CAD, the pro-inflammatory adipokine levels via ELISA in EAT and PAT were elevated. Pro-inflammatory adipokine mRNA expression was increased, while anti-inflammatory adipokine mRNA expression decreased, in CAD relative to NCAD in EAT and PAT rather than SAT. In EAT, adipocyte area and macrophage-specific staining were lower, while lymphatic vessel marker expression was higher in CAD. Additionally, the endothelial marker expression in EAT was higher than PAT in CAD. The three tissue types had different blood vessel amounts in CAD. The regulation and imbalance expression of the novel biomarkers, including inflammatory adipokine, macrophage infiltration, angiogenesis, and lymphangiogenesis in EAT and PAT, may be related to the pathogenesis of CAD. The serum levels of inflammatory adipokines may correlate to CAD, which requires large sample size studies to get further validation before clinic practice. However, the pathogenesis of CAD is still not clearly understood. Chronic inflammation is a key pathological mechanism of CAD that may increase aggravation of the disease2. Large numbers of pro- and anti-inflammatory adipokines promoting CAD development are secreted from epicardial adipose tissue (EAT)4. Previous studies have found that adipokines and inflammatory factors in EAT are associated with CAD, such as secreted frizzled-related protein 5, angiopoietin-like 46. Complement-Clq tumor necrosis factor (TNF)-related protein (CTRP)1/9, chitinase-3-like protein 1 (YKL-40), secreted frizzled-related protein 4 (SFRP-4), salusin-β, meteorin-like (Metrnl), and salusin-α are emerging adipokines potentially correlating with CAD. EAT promotes macrophage infiltration, which secrete inflammatory factors, via directly migrating from EAT to coronary arteries during the occurrence and development of CAD8. Local inflammation and ischemia induce the production of blood vessels and lymphatic vessels, and proinflammatory factors can promote the secretion of certain vascular lymphatic modulators, such as vascular endothelial growth factors (VEGFs)10. Preclinical studies in animal models of hypercholesterolemia show that adventitial angiogenesis promotes atherosclerotic plaque progression, and that lymphatic vessels also exist and develop in parallel with blood vessels11. The vascular and lymphatic endothelial chemical markers CD31, CD34, podoplanin (PDPN), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) are used to assess lymphatic vessel formation in tissues13. EAT, pericardial adipose tissue (PAT), subcutaneous adipose tissue (SAT) may play a different effects on myocardium due to the different biomolecules, genetic characteristics and anatomical location14. However, the relationship between the expression levels of the above factors in EAT, PAT, SAT and CAD remains unclear.To date, the incidence of coronary artery disease (CAD) remains high and the main cause of death worldwideIn this study, we investigated the expression of inflammatory adipokines and regulators of lymphatic vessel formation, as well as differences in neovascularization and lymphangiogenesis in EAT, PAT, and SAT, between CAD and non-CAD (NCAD) patients.15. Diabetes was defined as diabetes symptoms and random blood glucose ≥ 11.1 mmol/L, or fasting plasma glucose ≥ 7.0 mmol/L, or 2-h oral glucose tolerance test level ≥ 11.1 mmol/L, or no diabetes symptoms and at least twice blood glucose meets the above criteria16. Dyslipidemia was defined as serum total cholesterol ≥ 5.18 mmol/L, high-density lipoprotein cholesterol (HDL-C) ≤ 1.04 mmol/L, low-density lipoprotein cholesterol (LDL-C) ≥ 3.37 mmol/L, or triglyceride ≥ 1.7 mmol/L or previous diagnosis of dyslipidemia in medication17. In this study, all patients quit smoking less than 1 year. So, smoking was defined as current or prior smoking. Stroke was defined as a patient with a history of ischemic stroke.In this study, 26 inpatients with angiographic CAD who underwent coronary artery bypass grafting were enrolled in the CAD group from March to September 2019, and in the same period, 20 control inpatients with chronic valvular heart disease without coronary artery stenosis needing valve replacement were enrolled in the NCAD group. The inclusion criterion for CAD patients was the presence of three-vessel disease . The exclusion criteria were age > 80 years, acute myocardial infarction, active chronic inflammation disease, liver or renal failure, and pharmacological glucocorticoid or immunosuppressive therapy. The baseline characteristics of the demographic and clinical information were collected by the master’s degree students. The drugs including aspirin, β-R inhibitor, statin, ACEI/ARB patients use after discharge hospitalization. Hypertension was defined as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg at rest, or previously diagnosed as hypertension in antihypertensive therapyTen milliliters of fasting arterial blood was collected in a blood collection tube before heparinization of all patients, followed by centrifuging for 10 min at 3500 rpm, then the serum samples were collected and stored at -80 °C. EAT, PAT, SAT (500–600 mg each) were collected during the coronary artery bypass grafting or valve replacement surgery from the atrioventricular groove next to the right atrial appendage, the pericardial surface, and chest incision, respectively. Each adipose tissue sample was divided into three portions: two were frozen immediately at − 80 °C for RNA and protein extraction, the other was immersed in neutralized formalin for embedding tissue in paraffin blocks.The serum inflammatory adipocyte factor levels were measured using an ELISA kit  according to the manufacturer’s protocol. The pro-inflammatory adipocyte factors included tumor necrosis factor-α (TNF-α), CTRP1, YKL-40, SFRP-4, Salusin-β. The anti-inflammatory adipocyte factors included adiponectin (ADP), CTRP9, Metrnl, Salusin-α. Samples were tested in duplicate and experiments were repeated twice.Total RNA was extracted from EAT, PAT, SAT in liquid nitrogen using TRIzol reagent  according to the manufacturer’s instructions. The concentration and purity of extracted RNA were assessed by calculating the ratio of optical density at 260 nm and 280 nm. One microgram total RNA was used as a template to be converted into cDNA using a FastQuant RT Kit (with gDNase) (Tiangen Biotech). RT-qPCR analysis was conducted using the SuperReal PreMix Plus (Tiangen Biotech) to determine the gene expression. Every mRNA amplification reaction conditions were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 15 s. The primers for CTRP1, YKL-40, SFRP-4, CTRP9, Metrnl, vascular endothelial growth factor (VEGF)-C, VEGF-D, vascular endothelial growth factor receptor 3 (VEGFR-3), and GAPDH are shown in Table The EAT, PAT, and SAT samples were weighed and treated with Radio Immunoprecipitation Assay (RIPA) Lysis Buffer  (300 μL/10 mg) extract protein, incubated on ice for 30 min, followed by centrifugation at 12,000 g for 10 min at 4 °C, collected the supernatants. The protein concentrations were determined using a BCA protein assay kit . Afterward, samples (50 μg of protein) were separated by 12% SDS–polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene difluoride membrane. During the test of Western blotting, the blots were cut prior to hybridization with the antibodies, according to the molecular weight, determining the position of the target protein to complete the cut of polyvinylidene difluoride membrane. Next, the samples were blocked with PBS-Tween 20 (TBST) containing 5% non-fat dry milk for 1 h at 25 °C. The membranes were incubated with primary antibody anti-CTRP9 , anti-CTRP1 , anti-YKL-40 , overnight at 4 °C, with anti-GAPDH used as an internal control. Followed by incubation with secondary antibodies for 1 h. The membranes were detected with chemiluminescence solution A and B mixed at a 1:1 ratio, the bands’ density was calculated by densitometric analysis using Image J 1.6.0 software.Adipose tissue was fixed in 4% paraformaldehyde overnight, dehydrated, embedded in paraffin wax, and then cut into 5 μm-thick slices for H&E staining, immunohistochemistry, and immunofluorescence. The sections for H&E staining were subsequently deparaffined and rehydrated via a xylene and alcohol series before performing H&E staining according to standard histology procedures. The mean area of the adipocytes of each slide were measured using an automated image analysis system (ImageJ 1.6.0).The sections for immunohistochemistry were heated to 60 °C for 1 h, then dewaxed, rehydrated, and rinsed with PBS (Solarbio). Antigen retrieval was performed in sodium citrate buffer with a microwave oven for 10 min, then rinsed with PBS. Sections were incubated with primary antibodies CD68+ (Abcam), CD206+ , CD11c+ (Abcam), PDPN (Santa Cruz Biotechnology), LYVE-1 (Abcam) overnight at 4 °C in a moist chamber, then rinsed with PBS. The sections were incubated with secondary antibodies for 30 min at 37 °C, then rinsed three times with PBS; 50–100 μL DAB reagent was added. Images were taken at a Olympas confocal microscope (Japan). The AOD of each slide was calculated from three separate fields viewed at × 400 magnification with a Nikon fluorescence microscope (Japan).The sections for immunofluorescence were heated to 60 °C for 1 h, then dewaxed, rehydrated, and rinsed with PBS. Antigen retrieval was performed in sodium citrate buffer with a microwave oven for 15 min, then rinsed with PBS. Sections were incubated with primary antibodies CD31+ (Abcam), CD34+ (Santa Cruz Biotechnology), overnight at 4 °C in a moist chamber, then rinsed with PBS. Then, the sections were incubated with secondary antibodies for 1 h at room temperature, then 50–100 μL DAPI reagent was added. The images were measured using an automated image analysis system (ImageJ 1.6.0). The AOD of each slide was calculated from three separate fields viewed at × 400 magnification with a Nikon fluorescence microscope (Japan).Statistical analysis was performed using SPSS 19.0 . All tests were two-tailed, and the differences were considered statistically significant at P < 0.05. Normality of all continuous variables were tested by Kolmogorov–Smirnov, and reported as mean value and SD or median and the interquartile range (IQR), Mann–Whitney U test was used to compare the relationship between the CAD and NCAD groups. Whereas categorical variables were expressed as percentages, the differences between the two groups were tested with chi-square test. The Kruskal–Wallis H test was used for the comparison among the three groups, and the multiple comparison between the groups was used “all pairwise”, the test level was adjusted to P = 0.0083. Correlation analysis between variables was performed using Spearman’s rank correlation coefficient. Receiver operating characteristic curve was used to determine the diagnostic efficacy and the best diagnostic cut-off point for serum inflammatory factor level.This study was carried out in accordance with the World Medical Association’s Code of Ethics (Helsinki Declaration) and approved by the Institutional Review Boards of the Affiliated Hospital of Chengde Medical University . Written informed consent was obtained from each patient before enrollment.P > 0.05). Compared with the NCAD group, morbidity due to chest pain and hypertension were significantly higher in the CAD group (both P < 0.05), whereas average uric acid and blood urea nitrogen were lower in the CAD group relative to the NCAD group  (Table Patient baseline characteristics are shown in Table 5) Table .Table 2BP < 0.05), whereas levels of anti-inflammatory adipokines, including ADP, CTRP9, salusin-α, and Metrnl, were lower in the CAD group relative to the NCAD group  , 0.785 , 0.685 , 0.759 , and 0.832 , respectively, with the optimal diagnostic cut-off points at 99.03 ng/L, 49.06 ng/L, 7.72 ng/L, 47.71 ng/L, and 35.75 ng/L, respectively. The AUCs for the anti-inflammatory adipokines ADP, CTRP9, salusin-α, and Metrnl were 0.731 , 0.708 , 0.672 , and 0.776 , respectively, with the optimal diagnostic cut-off points at 417.4 ng/L, 18.89 ng/L, 71.76 ng/L, and 152 ng/L, respectively (Table The areas under the ROC curve (AUCs) for the proinflammatory adipokines TNF-α, CTRP1, salusin-β, YKL-40, and SFRP-4 were 0.764 [95% confidence interval (CI): 0.617–0.911; The proinflammatory adipokines CTRP1, salusin-β, YKL-40, and SFRP-4 were positively correlated with classic proinflammatory adipokines TNF-α . CTRP1 and salusin-β were positively correlated with SFRP-4 , and YKL-40 was positively correlated with CTRP1, salusin-β, and SFRP-4 . SFRP-4 was negatively correlated with ADP (r =  − 0.358). The anti-inflammatory adipokine Metrnl was positively correlated with CTRP9, salusin-α, and ADP  (Table P < 0.05), whereas CTRP9 levels in EAT and PAT was significantly reduced in the CAD group relative to the NCAD group (P < 0.05). However, CTRP1, YKL-40, and CTRP9 levels in SAT were not statistically different between the CAD and NCAD groups    Fig. . Evaluat05) Fig. .Figure 2CTRP1, YKL-40, and SFRP-4 were significantly higher in the CAD group relative to the NCAD group in EAT and PAT , although the difference was not statistically significant between the two groups in SAT . In EAT, mRNA levels of the anti-inflammatory adipokines CTRP9 and Metrnl were significantly decreased in the CAD group relative to the NCAD group (both P > 0.05). In PAT, CTRP9 mRNA level was significantly lower in the CAD group relative to the NCAD group (P < 0.05), and Metrnl mRNA level was not statistically different between the two groups (P > 0.05). Additionally, differences in CTRP9 and Metrnl mRNA levels were not statistically significant in SAT between the two groups (both P > 0.05). Moreover, mRNA levels of VEGF-C, VEGF-D, and VEGFR-3 were significantly higher in the CAD group relative to the NCAD group in EAT , and VEGF-C and VEGFR-3 levels were significantly higher in the CAD group relative to the NCAD group in PAT (both P < 0.05). Furthermore, mRNA levels of VEGF-C, VEGF-D, and VEGFR-3 in SAT were not statistically different between the two groups  . Additionally, CTRP1 level was positively correlated with SFRP-4, YKL-40 and Metrnl level; SFRP-4 level was positively correlated with YKL-40 level; and CTRP9 level was positively correlated with Metrnl level . As are shown in Table CTRP1 level was positively correlated with SFRP-4, VEGF-D, and VEGFR-3 levels; and SFRP-4 level was positively correlated with YKL-40 and VEGFR-3 levels .Correlations among adipocyte mRNA levels in EAT are shown in Table 2, 4180.7 μm2, 5545.9 μm2, respectively, whereas those in NCAD patients were 3046.5 μm2, 4115.2 μm2, and 4813.7 μm2, respectively. Adipocyte morphology in the three tissues differed, with the adipocyte area of EAT significantly smaller than that of PAT and SAT (both P < 0.05), whereas there was no statistical difference in the adipocyte areas of PAT and SAT in NCAD patients (P > 0.05) ; however, PAT macrophage infiltration was not statistically different between the CAD and NCAD groups (P > 0.05). We then determined differences in the M1 and M2 macrophage phenotypes by immunohistochemical staining for CD11c and CD206, respectively. We found that the number of CD11c+ macrophages was significantly increased and that of CD206+ macrophages significantly decreased in EAT from CAD patients relative to NCAD patients (both P < 0.05). In SAT, there was no statistical difference between CD11c+ and CD206+ macrophages between the CAD and NCAD groups (both P > 0.05). Additionally, calculation of the CD11c+/CD206+ ratio, which reflects macrophages shift into an inflammatory state, revealed that CD11c+/CD206+ macrophage ratio was significantly increased only in EAT in CAD patients when compared with NCAD patients (P < 0.05) , whereas their levels in PAT and SAT were not statistically different between the two groups (both P > 0.05). Additionally, PDPN level in CAD patients differed among the three tissue types, with levels in SAT lower than those in EAT and SAT ; however, there were no significant differences in LYVE-1 levels in CAD patients among the three tissue types (P > 0.05)  Fig. .Figure 6P < 0.05), although no statistical difference in PAT was observed between groups (both P > 0.05). In SAT, the number of CD34+ cells was higher in CAD patients relative to NCAD patients (P < 0.05), whereas the number CD31+ cells did not differ significantly between the CAD and NCAD groups (P > 0.05). The numbers of CD31+ and CD34+ cells in CAD patients differed among the three tissue types, with those in PAT significantly lower relative to EAT and SAT , although no significant difference was observed between SAT and PAT (P > 0.05)  Fig. .Figure 7P < 0.05), whereas those from NCAD patients did not differ significantly (P > 0.05). The number of blood vessels in EAT from CAD patients was the largest  were elevated in CAD patients, whereas those of anti-inflammatory adipokines  were lower relative to those in NCAD patients. Intimal atherosclerosis stimulates the secretion of proinflammatory adipokines and inhibits the release of anti-inflammatory adipokines, thereby affecting cardiac metabolism and inflammation and exerting crucial proinflammatory and pro-atherogenic effects in vivo24. Therefore, CTRP1, salusin-β, YKL-40, SFRP-4, CTRP9, salusin-α, and Metrnl may play important roles in CAD.Atherosclerosis is an imbalanced chronic inflammatory reaction in the arterial wall caused by certain stimuli and that produces a wide range of inflammatory molecules, which diffuse into the blood circulation and provide information regarding the existence and status of disease during its development25. Atherosclerosis entails a complex interaction of risk factors, some of which stimulate inflammation and promote the secretion of inflammatory adipokines26. In the present study, we found that mRNA and protein levels of proinflammatory factors positively correlated with other levels of proinflammatory factors, with the same correlations observed for anti-inflammatory factors. Previous studies demonstrated that inflammatory adipocytokines can be used as biomarkers of atherosclerosis27. In the present study, we showed that serum levels of CTRP1, salusin-β, YKL-40, SFRP-4, CTRP9, salusin-α, and Metrnl might represent CAD biomarkers according to ROC analysis.Previous studies show that adipocytokines affect each other, with proinflammatory adipokines promoting the secretion of other types of proinflammatory adipokines and inhibiting anti-inflammatory adipokine expression28. Activated M1 and M2 macrophages perform different functions by secreting either proinflammatory or anti-inflammatory factors29. Moreover, M1 macrophages exert strong microbicidal activities capable of inhibiting the proliferation of surrounding cells and damaging adjacent tissue, whereas M2 macrophages play an anti-inflammatory role by removing debris and promoting the proliferation of adjacent cells and tissue repair. The M1/M2 macrophage ratio reflects a shift in the state of local inflammation30. In the present study, the results showed macrophages in EAT from CAD patients were predominantly of the M1 phenotype associated with CAD.Additionally, we found that CD68+ M1 macrophages and the CD11c+/CD206+ ratio were significantly elevated in EAT from CAD patients relative to NCAD patients, whereas the number of M2 macrophages was significantly reduced. Macrophages are immunocompetent cells that response to local inflammation, and adipose tissue exhibiting chronic inflammation in CAD patients harbors abundant amounts of macrophages31 and CD34 a transmembrane phosphoglycoprotein on the surface of vascular endothelial progenitor cells, hematopoietic stem cells, and hematopoietic progenitor cells32. Inflammation is an important trigger of angiogenesis in ischemic tissues and can promote the secretion of pro-angiogenic factors and the proliferation of endothelial cells, with this also pro-angiogenic effect also accelerated by some inflammatory cytokines10. In the CAD group, we observed increased neovascularization which may be a useful pathological founding.LYVE-1 is the main receptor of hyaluronic acid in the endothelium of lymphatic vessels33, and PDPN is expressed in lymphatic endothelial cells and fibroblastic reticular cells in lymph nodes and capable of promoting lymphangiogenesis and lymphatic invasion34. In the present study, we found significantly elevated PDPN and LYVE-1 levels in EAT from CAD patients relative to NCAD patients. Inflammation is aggravated and lymphatic drainage is insufficient in atherosclerotic lesions, with various stimuli promoting the generation of lymphatic vessels35, which often occurs in parallel to angiogenesis. Ioannis et al.36 demonstrated increased lymphangiogenesis in adipose tissue around atherosclerotic plaques and accompanied by higher expression of lymphangiogenesis regulators. VEGF-C, VEGF-D, and VEGFR-3 regulate vacsular growth in endothelial tissue and promote the proliferation and migration of vascular endothelial cells, thereby playing an important role in lymphangiogenesis and angiogenesis37. In the present study, mRNA levels of VEGF-C, VEGF-D, and VEGFR-3 were higher in EAT, and VEGF-C and VEGFR-3 levels were higher in PAT from CAD patients relative to NCAD patients, with correlations also observed between VEGF-C, VEGF-D, and VEGFR-3 levels and the number of inflammatory adipocytes. These results suggest that neovascularization and lymphangiogenesis in EAT and the number of inflammatory adipokines related to CAD.Furthermore, we found that the number of blood vessels increased in EAT from CAD patients along with a significantly higher populations of CD31+ and CD34+ cells in EAT relative to those observed in NCAD patients. CD31 and CD34 are markers of angiogenesis, with CD31 a signaling-related glycoprotein abundant on vascular endothelial cellsCTRP9, Metrnl, VEGF-C, VEGF-D, and VEGFR-3, protein expression of YKL-40, and levels of CD31, CD34, PDPN, CD206 and the CD11c+/CD206+ ratio among the three adipose tissues. EAT and PAT constitute a unique paracrine visceral fat reservoir with unique anatomical, biomolecular, and genetic characteristics that regulates the production and metabolism of the adjacent myocardium38. However, the embryology and blood supply of EAT and PAT differ, which were observed in their metabolic and physiologic properties39. In the present study, the adipocyte areas were found significantly smaller in EAT relative to PAT and SAT in CAD patients and NCAD patients. Although EAT, PAT, and SAT constitute white adipose tissues, in addition to the different anatomical positions of the three tissues, their different effects on CAD are mainly due to their different physiological characteristics. EAT and PAT are visceral fat that provide energy to and protect the heart. Additionally, they secrete active substances under pathological conditions and become cardiotoxic, resulting in local inflammation and cardiac dysfunction.We observed differences in mRNA levels of First, the sample size was relatively small in our study, although we have searched Medline on the sample size of the former study to find some information of the sample size for the present design. Additionally, we have calculated the sample size and statistical power of all the factors in our study after some pre-study. Second, in our study, the blood and fat deposits tissues samples were collected during the hospitalization before the secondary prevention stragedy for CAD therapeutic treatment use. Therefore, we did not consider the impact of therapeutic treatment between CAD and the control groups. Third, the above defects of the study design can only lead to a relatively conservative conclusion, and we will try our best to go deep into some further researches on the topic.The regulation and imbalance expression of the novel biomarkers, including inflammatory adipokine, macrophage infiltration, angiogenesis, and lymphangiogenesis in EAT and PAT, may be related to the pathogenesis of CAD. The serum levels of inflammatory adipokines may correlate to CAD, which requires large sample size studies to get further validation before clinic practice.Supplementary Figures.


This study guides consumers to the safest red meat relating to Neu5Ac and Neu5Gc content, though the dog and cat meat are not conventional red meat globally.Sialic acids (Sias) are acidic monosaccharides and red meat is a notable dietary source of Sia for humans. Among the Sias, N-acetylneuraminic acid (Neu5Ac) and 2-keto-3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN) play multiple roles in immunity and brain cognition. On the other hand, N-glycolylneuraminic acid (Neu5Gc) is a non-human Sia capable of potentiating cancer and inflammation in the human body. However, their expression within the animal kingdom remains unknown. We determined Neu5Ac and KDN in skeletal muscle and organs across a range ( Sialic acids (Sias) are a diverse family of acidic, nine-carbon-backbone monosaccharides. They are ubiquitous in the animal kingdom and usually present as a conjugated sugar at the outermost (non-reducing) end of glycan chains of oligosaccharide structures of gangliosides, glycolipids and glycoproteins within the cell membrane and in secreted vertebrate glycoconjugates including those found in the human ,2,3,4. SHumans obtain their Sia Neu5Ac and Sia Neu5Gc from dietary sources. They also generate antibody responses against ingested Neu5Gc; thus, humans have circulating anti-Neu5Gc antibodies which are known as “xeno-auto-antigens”. As a result, Neu5Gc can facilitate the progression of cancer in humans . The canSome meats are consumed more than others around the world, with red meat providing important micro- and macronutrients for human health. Animal organ meat is also consumed world-wide providing a rich source of iron, zinc and vitamins A, B2 and D . Humans Ovis aries, n = 10), cattle , goat , pig , deer , horse , kangaroo , cat  and dog  were collected. The experimental protocol was approved by The Animal Care and Ethics Committee of Charles Sturt University, Wagga Wagga, NSW, Australia (SL100018).All samples were collected from the Preclinical Veterinary Laboratory of Charles Sturt University, NSW, Australia. About 10–50 g of tissue for each sample from sheep (n = 3), rumen, reticulum, omasum, abomasum, small intestine, large intestine and tongue were also collected from mature aged sheep. All these samples were stored at −20 °C pending analysis. Further details on the animals used in this study are provided in In case of sheep, muscle samples were obtained from 9 primal cuts, namely the leg chump, hind shank, short loin, eye of rack, tenderloin, fore shank, forequarter, neck muscle and peritoneal muscle. These tissues were used to determine if there were any differences in Neu5Ac and KDN concentrations from different muscles of the carcass that might help explain their possible functionality in metabolism. For the other animal species, muscle tissue was obtained from the thigh region. Heart, liver, lung, kidney, spleen and fat tissues were collected from each of the 9 species. Other edible organs, such as brain  was added slowly to the bottom layer, agitated for 2 min and centrifuged at 3000× g for 15 min at 4 °C. The 2 upper layers were combined, and 10 µL 1-butanol was added as an anti-foaming agent. Each sample was then freeze-dried  and re-dissolved in 200 µL chloroform:methanol . This solution was then sonicated  on ice for 15 s (5 s × 3 times) and retained for ganglioside Sia analysis. In order to estimate protein-conjugated Sia, 200 µL 0.05 M H2SO4 was added, sonicated for 15 s and then heated for 1 h at 80 °C. Maximum release of ganglioside—Conjugated Sia was achieved in 0.1 M trifluoroacetic acid  heated for 150 min at 80 °C [® Merck Millipore, Darmstadt, Germany) prior to quantitation of sialic acid using UHPLC.The preparation of brain sample for UHPLC analysis was undertaken using our published method . Brieflyat 80 °C . SubsequStandards of Neu5Ac and KDN were obtained from Sigma Aldrich, St. Louis, MO, USA. Ten mg of each dried standard (Neu5Ac—Cat no. A0812 and KDN—Cat no. 60714) was dissolved in 1 mL of deionised water to prepare 10 mg/mL stock solutions. Then, 2 µL 10 mg/mL Neu5Ac and 1 µL 10 mg/mL KDN was mixed in 0.997 µL deionised water. This solution was then serially diluted to prepare a set of standards.For quantification of both conjugated and free Sia, DMB  reagent was used. The DMB reagent was prepared according to the method of Inoue  as folloStandard curves for Neu5Ac were constructed in the concentration range of 0.004–1 ng/µL while, for KDN, standard curves were constructed in the concentration range of 0.002–0.5 ng/µL v/v/v) with flow rate of 1.5 mL/min for 3.5 min. The columns were washed with a mixture of methanol: water: acetic acid (80:20:0.05 v/v/v) for 1.4 min before equilibration for 4 min . Fluorescence was detected through emissions at 448 nm s and 373 nm excitation. All samples were analysed in duplicate with injection volume of 10 μL and concentrations were expressed as µg/g for each sample. The coefficient of variation (CV) for UHPLC analysis within the same day and within different days was 2–5%.The measurement of Neu5Ac and KDN was undertaken with our validated published method . In brieTotal protein concentration was assessed in each homogenate with the Pierce BCA protein assay kit . The concentrations of free and conjugated Sias were normalised to the protein content and expressed as μg Sia/g protein for all samples with the exception of fat tissue. All samples were assayed in duplicate.Fat content was assessed by extraction of samples in hexane using a Soxtec 2050 apparatus  according to AOAC 2003.06. In brief, 0.50–1.00 g of the freeze-dried ground sample was weighed and added to a cellulose thimble  and then placed in a beaker. The sample was then oven-dried  for a further 2 h at 105 °C to remove any additional moisture and the thimbles were then placed in the Soxtec extraction apparatus. The corresponding beakers were then rinsed with 70–90 mL of hexane into a pre-dried and pre-weighed aluminium cup containing small glass beads. The extraction proceeded at 155 °C with boiling for 20 min, rinsing for 40 min and recovery for 10 min. Upon completion, the aluminium cups were placed in a drying oven  at 105 °C for 20–30 min, and cooled in a desiccator , prior to weighing and calculating the fat content (%). The final concentration of free and conjugated Sias in fat tissue was normalised to the lipid concentration and expressed as μg Sia/g lipid.Results for total Sia, Neu5Ac and KDN in different organ tissues from nine species were analysed using linear mixed models. Species and sample name (muscle or organ) were used as the fixed effects, as well as their interaction to assess whether sample effects differed between species. Animal number was set as a random effect. Residual plots were used to ensure that the model assumptions were met for all models. With the exception of sheep, comparisons between organs within the same species were made by least significant differences (LSDs) when the overall F-test was significant. Comparisons between species for the same organ were also compared using LSDs. The analysis was conducted using GenStat statistical software (17th Edition).p < 0.001, p = 0.024, p < 0.001), except for the cat (p = 0.066)  .p = 0.023), but not with the free Neu5Ac (p = 0.784), for the species–sample interaction.The concentration of conjugated Neu5Ac (118.86–4007.80 µg/g protein) was 3–39 times higher than free Neu5Ac (4.04–448.09 µg/g protein) including that in fat (14.90–50.55 µg/g lipid for conjugated and 0.05–7.23 µg/g lipid for free), based on organ tissue and animal species. In the horse, the conjugated Neu5Ac in lung (4007.80 µg/g protein), spleen (417.38 µg/g protein) and fat tissue (14.90 µg/g lipid) were 55, 103 and 270 times higher than free Neu5Ac, respectively. Overall, conjugated Neu5Ac was 70–97% of total Neu5Ac. Surprisingly, free Neu5Ac (177.32 µg/g protein) in cat muscle was higher than conjugated Neu5Ac (118.86 µg/g protein). In dog muscle, however, the concentration difference of free and conjugated Neu5Ac was quite small (conjugated form 202.75 µg/g protein and free form 155.08 µg/g protein) . The conp = 0.003, p = 0.051, p = 0.003, p < 0.001, p = 0.263, p = 0.268, p = 0.136, Significant species differences for the total KDN concentration were observed for most of the organ tissues including the lung  was the predominant form in all tested tissues . The onlThe mean proportion of Neu5Ac, Neu5Gc  compared to those of other animals. Conversely, in kidney and muscle of cattle, the proportion of Neu5Ac (60%) was higher than that of Neu5Gc (40%). However, cattle lung, heart, liver and fat tissue contained lower proportions of Neu5Ac relative to Neu5Gc by 39%, 48%, 33% and 44%, respectively. The two female deer contained only Neu5Ac in all tested organs and were lacking any Neu5Gc except in the muscle [5% comparn = 10), the concentrations of conjugated and free forms of Neu5Ac and KDN were analysed in muscles from nine different commercial primal cuts in the body and eight additional organ tissues (p > 0.05). However, the difference was significant among eight additional organ tissues for both free and conjugated Neu5Ac and KDN  [Sia is found rarely as a free molecule . The bio2O2)  through 2O2) . The rea2O2) . Lipid h2O2) . Studies2O2) ,25.Although the biological functions of KDN are not fully documented, surprisingly high levels of free KDN are observed in many human cancers  and it hIn this study, we quantified the concentration of Neu5Ac and KDN in raw organ tissue only. Samraj, Pearce, Läubli, Crittenden, Bergfeld, Banda, Gregg, Bingman, Secrest and Diaz ,26 foundOur findings are consistent with the recent pig study reported by Ji, Wang, Chen, Yang, Zhang, Zhang, Troy and Wang  who asseRed meat has been an indispensable part of the human diet, and global meat consumption has increased by 58% over the 20 years to 2018 . Red meaWe have reported the proportion Neu5Ac, Neu5Gc and KDN in the organs and skeletal muscle across the selected range of animal species . TheoretThe similar concentrations of Sia Neu5Ac across nine different commercial ovine muscle meat cuts reported here have shown that higher valued cuts do not provide any additional nutritional advantage for the consumer. We also found Neu5Ac was the major form of Sia across the spectrum of organ tissues and animal species with the exception of the spleen of cattle (~85% Neu5Gc), pig (~75% Neu5Gc), horse (~75% Neu5Gc) and cat (~60% Neu5Gc). Therefore, these organs must be avoided for human consumption.Red meat is an excellent source of protein, iron, zinc, vitamin B12, niacin, and creatine, but is also the main source of Neu5Ac for human consumption. The concentration and proportion of Neu5Ac, Neu5Gc and KDN in red meat was both species and tissue specific. The results of this study will assist consumers in making a more informed decision on their choice of red meat based on concentrations of both health-giving Sia Neu5Ac and the pro-inflammatory Sia Neu5Gc.


Carbocatalysts, materials which are predominantly composed of carbon and catalyze the synthesis of organic or inorganic compounds, are promising alternatives to metal-based analogues. Even though current carbocatalysts have been successfully employed in a broad range of synthetic transformations, they suffer from a number of drawbacks in part due to their heterogeneous nature. For example, the insolubility of prototypical carbocatalysts, such as graphene oxide (GO), may restrict access to catalytically-active sites in a manner that limits performance and/or challenges optimization. Herein we describe the preparation and utilization of soluble asphaltene oxide (sAO), which is a novel material that is composed of oxidized polycyclic aromatic hydrocarbons and is soluble in a wide range of organic solvents as well as in aqueous media. sAO promotes an array of synthetically useful transformations, including esterifications, cyclizations, multicomponent reactions, and cationic polymerizations. In many cases, sAO was found to exhibit higher catalytic activities than its heterogeneous analogues and was repeatedly and conveniently recycled, features that were attributed to its ability to form homogeneous phases. Soluble carbocatalysts, materials which are predominantly composed of carbon and catalyze the synthesis of organic or inorganic compounds, are promising alternatives to metal-based analogues. For example, due to the its large particle size, GO can undergo rapid decomposition when exposed to high temperatures, light or microwaves.14 Although heterogeneity can facilitate purifications, the catalytically-active sites present on GO may be inaccessible due to its heterogeneous nature and, as such, performance may be intrinsically limited. While enhanced solvent dispersibility has been reported to increase the performance and material quality of GO,15,16 a fully soluble derivative has hitherto remained unknown.Due to increasing costs, high toxicity, and limited resources, alternatives to transition metal-based catalysts used to promote the preparation of organic and inorganic compounds have been widely studied. Among these, carbocatalysts, which are materials that are composed primarily of carbon, have attracted attention for use in a variety of synthetic applications.17 While the structure of asphaltene is reminiscent of a graphene sheet, it is relatively small.18 Subjecting asphaltene to a modified Hummers' method afforded asphaltene oxide (AO), which was found to exhibit catalytic activities similar to that of GO but under a wider range of conditions. Indeed, AO facilitated condensations, C–C cross couplings, etherifications and heterocyclizations, and the use of microwaves were found to promote some of these transformations. Building on these results, we posited that asphaltene may be converted to homogeneous carbocatalyst by enhancing the degree of oxidation. Our supposition was inspired by a previous report,19 which showed that exposing asphaltene to 70% nitric acid at elevated temperatures facilitates its removal from crude oil due to an enhanced solubility. Although this procedure was used in oil refinery applications, we hypothesized that the soluble product may function as a homogeneous carbocatalyst. Moreover, the enhanced solubility was expected to promote catalytic function and result in higher activities when compared to heterogeneous counterparts. Herein we report the synthesis and characterization of soluble asphaltene oxide (sAO) and describe the utility of this material in facilitating synthetic transformations, including relatively complex syntheses such as multicomponent reactions and cationic polymerizations.We recently reported a new carbocatalyst based on asphaltene.δ). NMR data were collected in CDCl3, DMSO-d6 or D2O and the residual solvent was referenced to 7.26, 2.50 and 4.79 ppm, respectively. The following abbreviations apply: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. FT-IR spectra were recorded using KBr pellets on a PerkinElmer Frontier MIR spectrometer. Elemental analyses were performed using a Thermo Scientific Flash 2000 Organic Elemental Analyzer that was calibrated with 2,5-bis(5-tert-butyl-benzoxazol-2-yl)thiophene (BBOT). Matrix-assisted laser desorption/ionization (MALDI) spectra were recorded on a Bruker Ultraflex III with 2,5-dihydroxybenzoic acid (DHB) as a matrix. THF solutions of sAO and DHB were combined and then evaporated prior to mass analysis. Polystyrene equivalent molecular weights and polydispersity index (Đ) values were measured by gel permeation chromatography (GPC) using a Malvern VISCOTEK GPCmax system. All samples were analyzed using THF as the eluent at a flow rate of 0.8 mL min−1. Atomic force microscopy measurements were performed using a Multimode 8 Nanoscope® V (Bruker). Samples were dissolved in THF (1.0 mg mL−1) and spin coated onto silica at 3000 rpm for 1 min. Mass spectrometry (MS) data were recorded on a Thermo LCQ Fleet Quadrupole Ion Trap Mass Spectrometer in Atmospheric Pressure Chemical Ionization (APCI) and Electrospray Ionization (ESI) mode (electrospray voltage 3.5 kV). Microwave reactions were conducted using an Anton Paar GmbH-Monowave 300 microwave reactor.All chemicals were purchased from Sigma Aldrich, TCI or Thermo Fisher Scientific Chemicals, and were used as received. Asphaltene  was kindly supplied by Korea Petroleum. Unless otherwise noted, all experiments were performed under ambient conditions. NMR spectra were recorded on a Bruker Ascend 400 MHz spectrometer. Chemical shifts are reported in parts per million (n-heptane (2.0 L) and then sonicated. The mixture was filtered through a 0.22 μm nylon membrane and washed with n-heptane (0.5 L × 3). The filtered asphaltene particles were collected, dried at 80 °C under vacuum for 1 day, and then directly used.Asphaltene (10.0 g) was mixed with 2, was generated during the reaction. Afterward, the mixture was slowly poured into deionized water (0.5 L). The resulting solution was filtered through a 0.22 μm nylon membrane to remove the unreacted asphaltene particles. The filtrate was collected and dried under vacuum (100 torr) at 100 °C. After removal of the residual water and nitric acid via distillation, the remaining solid products were collected and dried under vacuum at 50 °C for 2 days. The final product (2.1 g) was obtained as a brown powder.A 500 mL flask was charged with asphaltene (4.0 g), nitric acid (0.1 L), and a stir bar. The mixture was refluxed at 120 °C for 24 h. CAUTION: a brown gas, presumably NOn-heptane to remove the residual oil contaminants and then subjected to a modified version of a previously reported oxidation method.19 Briefly, asphaltene was refluxed in 60% nitric acid at 120 °C for 24 h, and then diluted with deionized water. After filtering the diluted solution to remove the insoluble particulates, the filtrate was dried by heating to 100 °C under low pressure and then placed in a vacuum oven that was set to 50 °C for two days. The product obtained from this procedure was a brown powder and found to be soluble in methanol (up to 200 mg mL−1), ethanol (140 mg mL−1), THF (200 mg mL−1), DMSO (80 mg mL−1), DMF (80 mg mL−1), acetone (200 mg mL−1) and water (100 mg mL−1) , sAO contained a relatively high oxygen content , as determined by elemental analysis (see νO–H = 3500–2800 cm−1), a strong carbonyl signal  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>OC = 1720 cm−1), and epoxide signals (νC–O = 1240 cm−1 and 1050 cm−1), and resembled a spectrum recorded for AO (see −1) was measured to be 2.7; for comparison, the pH of an AO suspension was measured to be 3.5 under similar conditions.Asphaltene was first washed with ysis see . The FT-r AO see . The pH via IR spectroscopy and elemental analysis  and the appearance of a new signal which was assigned to a nitrile group  scale" fill="currentColor" stroke="none"><path d="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>NC = 2230 cm−1). Introducing sAO to 3-cyanophenyl isocyanate resulted in a reduction in the intensity of the signal assigned to the hydroxyl group (νO–H = 3320 cm−1) and was accompanied by the formation of strong signals that were assigned to the nitrile (νNC = 2230 cm−1), amido (νN–H = 3090 cm−1) and carbamyl (νOC = 1590 cm−1) groups of the expected product. Finally, using methodology that was previously reported to form C–C bonds via the ring-opening of the epoxide groups on GO,21 sAO was treated with malononitrile under basic conditions. The disappearance of signals attributed to the epoxide groups (νC–O = 1230 cm−1) in the starting material was observed along with the formation a new νNC at 2180 cm−1. Elemental analysis of the products of the aforementioned reactions enabled quantification of the respective nitrogen contents and thus the corresponding functional groups: carboxylic acid (5.1 × 10−3 mol g−1), hydroxy (5.6 × 10−3 mol g−1) and epoxide (7.5 × 10−3 mol g−1). The concentrations of these groups were measured to be significantly higher than those measured for AO and GO (see Table S2vide infra) exhibited by sAO.As summarized in 1H NMR spectrum recorded for the material exhibited broad signals at δ 8.62–7.41 ppm and 2.31–1.00 ppm in D2O which were assigned to aromatic and alkyl chain protons, respectively , aromatic groups (δ 134.4–129.0 ppm) and alkyl chains (δ 40.4–16.1 ppm) were also recorded  .Since sAO was found to be acidic, initial efforts toward using sAO as a carbocatalyst began with esterification chemistry. Although inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid are commonly used to catalyze such transformations,17 Building on these results, we focused on employing sAO in microwave-assisted Fischer indole syntheses. Indole derivatives have been employed in numerous biological and pharmaceutical applications and the demand for these compounds continues to grow.23 As shown in We previously showed that asphaltene-based carbocatalysts feature an advantage over their graphene analogues in that they can be utilized in microwave-assisted chemistry.via extraction. To quantify the recyclability of sAO, an aqueous solution of the catalyst was collected after a Fischer indole synthesis and re-used without further purification. The starting reagents (phenylhydrazine and cyclohexanone) were added to a solution of the catalyst in water ([sAO]0 = 50 mg mL−1) and then irradiated with microwaves for 1 h at 150 °C. After extraction of the corresponding indole products with dichloromethane, the aqueous phase containing the catalyst was collected, introduced to a new bolus of starting reagents and irradiated again. Using this methodology, the catalyst solution was successfully and repeatedly used over multiple cycles to facilitate condensations with minimal changes catalytic activity. For example, an indole product was obtained in a 52% yield using a catalyst solution that was subjected to five (5) successive cycles; for comparison, a yield of 59% was obtained after the first cycle . The Biginelli reaction is a typical example of a MCR and has been used to obtain dihydropyrimidinone (DHPM) and other valuable heterocyclic products.via filtration after pouring the corresponding reaction mixtures into excess methanol to induce precipitation. Poly(n-butyl vinyl ether) was obtained in quantitative yield, and featured a number average molecular weight (Mn) of 9.8 kDa and a polydispersity index (Đ) of 1.3 as determined by gel permeation chromatography. Similarly, N-vinyl carbazole was quantitatively converted to its corresponding polymer (Mn = 11.1 kDa). Although the latter exhibited a Đ value of 5.1, broad polydispersity indices are often observed in cationic polymerizations due to the high reactivity of the cation during the propagation step.26 Reduced distributions may be achieved through further optimization . In a broader perspective, asphaltene-based catalysts can be expected to expand the realization of metal-free catalysts for facilitating synthetic reactions under heterogeneous and, now, homogeneous conditions, and thus potentially usher further improvements in catalytic activity as well as selectively.In summary, a homogeneous carbocatalyst was prepared through the oxidation of asphaltene. To the best of our knowledge, this is the first report of a soluble carbocatalyst that facilitates a broad range of synthetic transformations. Indeed, sAO effectively promoted esterifications, inter- and intramolecular condensations, multicomponent reactions and cationic polymerizations, and was successfully used in microwave reactors. Since sAO is soluble in water, it was found to be straightforward to separate and recycle There are no conflicts to declare.RA-010-D0RA01762K-s001