On day 4 of each trial, participants completed a 120-min treadmill run at 70% of maximal oxygen consumption (V̇o2 max). Blood was taken at baseline (BASE), immediately after exercise (EE), after 60 (R1) and 120 (R2) min of recovery, and on three follow-up days (FU1-FU3). Markers of bone resorption [COOH-terminal telopeptide region of collagen type 1 (β-CTX)] and formation [NH2-terminal propeptides of procollagen type 1 (P1NP)] were measured, along with osteocalcin (OC), parathyroid hormone (PTH), albumin-adjusted calcium (ACa), phosphate, glucagon-like peptide-2 (GLP-2), interleukin-6 (IL-6), insulin, cortisol, leptin, and osteoprotogerin (OPG). Area under the curve was calculated in terms of the immediate  and short-term  responses to exercise. β-CTX, P1NP, and IL-6 responses to exercise were significantly lower in the immediate postexercise period with CHO feeding compared with PBO , although there was no difference in the short-term response . No other variable was significantly affected by CHO feeding during exercise. We conclude that CHO feeding during exercise attenuated the β-CTX and P1NP responses in the hours but not days following exercise, indicating an acute effect of CHO feeding on bone turnover.Bone resorption is increased after running, with no change in bone formation. Feeding during exercise might attenuate this increase, preventing associated problems for bone. This study investigated the immediate and short-term bone metabolic responses to carbohydrate (CHO) feeding during treadmill running. Ten men completed two 7-day trials, once being fed CHO (8% glucose immediately before, every 20 min during, and immediately after exercise at a rate of 0.7 g CHO·kg body mass We also measured markers associated with feeding, exercise, and bone to explore possible mediating and mechanistic factors.o2 max) 53.0 ± 6.4 ml·kg−1·min−1] provided informed consent and completed medical history questionnaires. The institutional research ethics committee approved the study in accordance with the Declaration of Helsinki.Ten healthy, physically active men [means ± SD: age 24 ± 3 yr, height 1.75 ± 0.08 m, body mass 72.9 ± 7.5 kg, body fat percentage 14.3 ± 2.4%, maximal oxygen consumption  or CHO ingestion during 120 min of treadmill running at 70% of V̇tabolism .Height and body mass were recorded before body fat analysis was conducted by bioelectrical impedance . Preliminary testing also involved the assessment of the cardiorespiratory responses to running. Participants first performed a submaximal test to establish the relationship between running speed and oxygen consumption during level running, which was completed at a 0% gradient from a gentle starting speed. The speed of the treadmill was increased by 1 km/h at 3-min intervals for at least 15 min (5 stages). Expired air  was collected into Douglas bags during the last minute of each stage to determine oxygen consumption. Oxygen consumption at each stage of the submaximal test was plotted against the running speed at that stage so that the relationship between running speed and oxygen consumption could be determined.o2 max was calculated. With the data from the submaximal running test, the running speed that elicited 70% V̇o2 max at a 0% gradient was determined.The maximum oxygen uptake test was then performed after a 15-min recovery period, consisting of a continuous incremental uphill running test at constant speed until volitional exhaustion. Running speed for this test was determined from the results of the submaximal test. The gradient of the treadmill was increased by 1% at the end of each minute from a starting gradient of 0%. Once maximum oxygen uptake was determined, the oxygen consumption representing 70% V̇Participants adhered to their normal diet and refrained from exercise or strenuous physical exertion. Participants recorded their dietary intake and were asked about lifestyle activity , with diet and sleep patterns being replicated between trials.o2 max. Further blood samples were withdrawn immediately after exercise (EE) (1100) and after 60 (R1) (1200) and 120 (R2) (1300) min of recovery. Heart rate (HR) and rating of perceived exertion  were recorded before exercise and at 10-min intervals throughout exercise for participant monitoring purposes.Participants attended the laboratory (0800) after an overnight fast (since 2000 the previous evening) and remained fasted until the final blood sample was drawn. On arrival, nude body mass was determined and participants then rested in a semirecumbent position and were fitted with a heart rate monitor . At 0830, a resting blood sample (BASE) was withdrawn from a prominent forearm vein. Participants then completed 120 min of running on a treadmill  at 70% V̇−1·h−1. The total amount of glucose ingested was 102.1 ± 10.6 g in a total solution volume of 1,276 ± 132 ml. These totals were divided equally over seven mean ingestions of 14.6 ± 1.5 g of glucose in 182 ± 19 ml of solution. During the PBO trial, participants ingested equal volumes of a taste-matched flavored water drink containing no CHO.During the CHO trial, participants ingested an 8% glucose solution immediately before, every 20 min during, and immediately after exercise at a rate of 0.7 g CHO·kg body massUpon completion of exercise, nude body mass was determined and participants consumed water equal to 150% of the body mass lost over the subsequent 120 min. Participants were instructed not to perform any further exercise.Participants attended the laboratory (0800) after an overnight fast (from 2000 the previous evening) and rested for 30 min, after which a blood sample was withdrawn (0830) from a prominent forearm vein. During this time, participants continued to record their diet and maintained dietary control and refrained from all exercise.g and 4°C. After centrifugation, plasma was dispensed into Eppendorf tubes and stored at −80°C for subsequent analyses of COOH-terminal telopeptide region of collagen type 1 (β-CTX), NH2-terminal propeptides of procollagen type 1 (P1NP), osteoprotogerin (OPG), osteocalcin (OC), parathyroid hormone (PTH), leptin, glucagon-like peptide-2 (GLP-2), and interleukin-6 (IL-6). Prior to storage, measurements of plasma glucose and lactate were performed .Fifteen milliliters of venous blood was dispensed into three 5-ml tubes lined with ethylenediaminetetraacetic acid (EDTA) and centrifuged immediately for 10 min at 2,000 β-CTX, P1NP, OC, and PTH were measured by electrochemiluminescent immunoassay (ECLIA) on a Modular Analytics E170 analyzer . Interassay coefficient of variation (CV) for β-CTX was <3% between 0.2 and 1.5 μg/l, with sensitivity of 0.01 μg/l. P1NP interassay CV was <3% between 20 and 600 μg/l, with sensitivity of 8 μg/l. OC interassay CV was <5% between 2 and 200 μg/l, with sensitivity of 0.6 μg/l. PTH interassay CV was <4% between 1 and 30 pmol/l, with sensitivity of 0.8 pmol/l. OPG was measured with an enzyme-linked immunosorbent assay (ELISA) supplied by Immuno Diagnostic Systems , with an interassay CV of <8% across the range 1–30 pmol/l and sensitivity of 0.14 pmol/l. Leptin was measured by ELISA supplied by IDS, having an interassay CV of <8% across the range 3–50 μg/l and sensitivity of 1 μg/l. GLP-2 was measured by ELISA (Yanaihara Institute), with an interassay CV of 1.1–11.1% across the range 3.1–33.4 ng/ml and detection limit of 0.5 ng/ml. IL-6 was measured by ELISA , with an interassay CV of <10% across the range 0.15–10 pg/ml and detection limit of 0.039 pg/ml.g and 4°C. Resultant serum was dispensed into Eppendorf tubes and stored at −80°C for the subsequent analysis of cortisol, insulin, calcium, albumin, and phosphate (PO4).The remaining 5 ml of venous blood was dispensed into a serum tube and allowed to clot at room temperature for 60 min before being centrifuged for 10 min at 2,000 4 were measured with standard commercial assays supplied by Roche Diagnostics performed on the Roche Modular E170. The range of measurement in serum is 0.05–5.00 mmol/l for calcium, 10–70 g/l for albumin, and 0.10–6.46 mmol/l for PO4.Cortisol was measured with an ECLIA on the Roche Modular E170, with an interassay CV of <6% between 16 and 1,750 nmol/l and sensitivity of 8 nmol/l. Insulin was measured by ECLIA on a Cobas e601 , having an interassay CV of <6.1% across the range 44–505 pmol/l and sensitivity of 1.8 pmol/l. Calcium, albumin, and POP ≤ 0.05. Data were analyzed with SPSS V20. Effects of exercise were assessed on the PBO trial data with a one-way ANOVA for normally distributed data and a Friedman's ANOVA for nonnormally distributed data. Within-exercise variables were analyzed with a repeated-measures ANOVA. The area under the curve (AUC) with respect to BASE was calculated for all biochemistry markers from the percent change data .P = 0.473) or HR (P = 0.869), but, as expected, both increased over the duration of the exercise bout (P < 0.001). There was also no interaction between condition and time for either RPE (P = 0.847) or HR (P = 0.170). Blood glucose concentrations (P = 0.002) at EE (6.3 ± 0.6 mmol/l) compared with BASE (4.9 ± 0.2 mmol/l). Blood glucose concentrations were significantly (P < 0.001) higher in the CHO trial than in the PBO trial at EE. Blood lactate concentrations (P = 0.002) before returning back toward BASE during recovery.There were no significant differences between PBO and CHO for RPE  higher than at BASE (63.1 ± 30.5 ng/ml) in the PBO trial . AUC anaP = 0.343; P = 0.786; OC concentrations were not affected by exercise in the PBO trial  or by CHP < 0.001) by 87% at EE (6.6 ± 2.0 pmol/l) compared with BASE (3.7 ± 1.2 pmol/l) in the PBO trial. Thereafter, concentrations returned toward BASE levels and were slightly lower than BASE at R1 (3.6 ± 1.2 pmol/l) and R2 (3.3 ± 1.3 pmol/l). PO4 was increased by 21% at EE (1.4 ± 0.1 mmol/l) because of the exercise bout (P < 0.001) and then returned to BASE values (1.1 ± 0.1 mmol/l) by R1 (1.1 ± 0.2 mmol/l). No exercise effect was shown for albumin-adjusted calcium (ACa) in the PBO trial (P = 0.871). There were no immediate or short-term effects of CHO feeding on calcium metabolism markers (PTH increased ( markers .P < 0.001), IL-6 (P < 0.001), and leptin (P < 0.01), but there were no significant effects of exercise on cortisol, OPG, or GLP-2. CHO feeding significantly attenuated the elevation in IL-6 concentrations (223%) seen immediately after exercise in the PBO trial (P = 0.036), although these differences did not persist over the follow-up days (P = 0.327). There were no other immediate or short-term effects of CHO feeding on any of the remaining modulators of bone metabolism measured  were shown for insulin (measured .1) CHO feeding during exercise attenuated the β-CTX and P1NP responses in the hours but not days following exercise and 2) IL-6 responded in a similar manner to bone turnover after CHO feeding during exercise.Our main findings were that The reduction in bone resorption with CHO feeding during strenuous exercise suggests a potential strategy for athletes and those performing arduous occupational training  to minimize increased bone resorption resulting from such exercise . From thr = 0.74, P < 0.05) existing between the immediate responses of IL-6 and β-CTX in the CHO trial. The ingestion of CHO before and during endurance exercise attenuates the rise in circulating IL-6 associated with exercise  is consistent with the amount and rate typically recommended for endurance exercise benefits .This work was funded by the No conflicts of interest, financial or otherwise, are declared by the author(s).Author contributions: C.S., W.D.F., and J.P.G. conception and design of research; C.S., I.V., and T.W.J. performed experiments; C.S., I.V., R.M.J., and J.C.T. analyzed data; C.S., I.V., T.W.J., and R.M.J. interpreted results of experiments; C.S. drafted manuscript; C.S., I.V., T.W.J., R.M.J., W.D.F., and J.P.G. edited and revised manuscript; C.S., I.V., T.W.J., R.M.J., J.C.T., W.D.F., and J.P.G. approved final version of manuscript; R.M.J. prepared figures.


Agrobacterium infection is a key factor for the high level of genetic transformation efficiency. The objective of this study is to evaluate the plant factors related to transformation in cotyledonary nodes during the Agrobacterium infection process. This study selected three genotypes  with high transformation efficiency, which presented better susceptibility to Agrobacterium infection, and three low transformation efficiency genotypes , which showed a relatively weak susceptibility. Gibberellin (GA) levels and soybean GA20ox2 and CYP707A2 transcripts of high-efficiency genotypes increased and were higher than those of low-efficiency genotypes; however, the opposite performance was shown in abscisic acid (ABA). Higher zeatin riboside (ZR) content and DNA quantity, and relatively higher expression of soybean IPT5, CYCD3 and CYCA3 were obtained in high-efficiency genotypes. High-efficiency genotypes had low methyl jasmonate (MeJA) content, polyphenol oxidase (PPO) and peroxidase (POD) activity, and relatively lower expression of soybean OPR3, PPO1 and PRX71. GA and ZR were positive plant factors for Agrobacterium-mediated soybean transformation by facilitating germination and growth, and increasing the number of cells in DNA synthesis cycle, respectively; MeJA, PPO, POD and ABA were negative plant factors by inducing defence reactions and repressing germination and growth, respectively.The susceptibility of soybean genotype to  Agrobacterium infection [Over the past decade, genetically modified soybean has continued to be the predominant commercialized biotech crop, reaching 75.4 million hectares  in 2011 . HoweverAgrobacterium-mediated transformation is proved to be a superior soybean transformation method over other transformation systems because it offers significant advantages including easier manipulation, lower transgene copy number and greater transgene stability, which frequently result in a higher transformation success rate [Agrobacterium-mediated transgenic soybean line, foreign genes (contained within T-DNA) are delivered from Agrobacterium into plant host cells and eventually integrated into the host genome. This infection process is a very critical early step of the whole transformation process beginning with recognition of plant signals by Agrobacterium, followed by Agrobacterium attachment to the wounded plant tissue and the survival of imported T-DNA from the host defense system [Agrobacterium infection is necessary for ensuring a high level of soybean transformation efficiency, which depends on both plant genotypes [Agrobacterium strains [Agrobacterium events during infection [Agrobacterium virulence genes [Agrobacterium infection process, more efforts are required to investigate details on the delicate plant cellular response during Agrobacterium infection.Most plant transformation systems are lengthy processes comprised of multiple complicated steps. ess rate . To genee system . Therefoenotypes ,8 and Ag strains ,10. Compnfection ,12,13, onfection ,16,17,18ce genes ,20,21. AAgrobacterium-mediated method in 1988 [Agrobacterium [Since the successful transformation of the cultivated soybean by  in 1988 , a lot o in 1988 ,24,25. Iacterium ,23,26,27Agrobacterium-mediated transformation, the addition of phenolic compounds [l-cysteine, thiol compounds and dithiothreitol) appeared to improve T-DNA delivery by inhibiting the activity of plant pathogen-response and wound-response enzymes, such as polyphenol oxidase (PPO) and peroxidase (POD) [Agrobacterium infection. In plant tissue culture, adding phytohormones usually caused changes in endogenous hormones. Endogenous hormones played essential roles in regulating plant growth, development, and stress responses. Gibberellin (GA) is known to induce the germination process and promote degradation of storage material in seeds [Agrobacterium-mediated transformation systems, MeJA may participates in this process. Other phytohormones such as cytokinin were required to induce cell division and growth in plant tissue cultures. Villemont et al. [Petunia mesophyll cycling cells with no phytohormone treatments could not be transformed. Thus, efficient Agrobacterium transformation might occur at a particular stage of the plant cell cycle. The previous researches implied that germination and growth, cell division and defense response status of explant tissues might be a crucial effect on the transformation.Studies optimizing ompounds , antioxiompounds ,30 and pompounds ,32 partlompounds . These ase (POD) , but didin seeds , providiin seeds ,36, but in seeds . Methyl in seeds . Bacterit et al.  investigAgrobacterium [Agrobacterium infection process as based on the screened soybean cultivars with contrasting transformation efficiencies. Our data suggest that GA and ZR play a positive role on Agrobacterium-mediated soybean transformation; however, ABA, MeJA, PPO and POD have a negative effect. Furthermore, these factors participated in germination and growth, cell division and defense response in cotyledonary nodes.Although a lot of progress has been made in soybean transformation, transgenic efficiency still requires improvement. Most screened cultivars had a shortcoming on agronomic traits. Genotype-dependency of the soybean transformation significantly limited its application of elite and commercially valuable cultivars. It took a long time for breeding a commercial transgenic soybean after getting a transgenic plant in the laboratory. If researchers can get high transformation efficiency at any time from elite and commercially valuable cultivars, the transgenic breeding efficiency would be improved greatly. As soybean genotypes display a variation in susceptibility to acterium , which p−1 phosphinothricin on shoot induction medium for four weeks, resistant explants to phosphinothricin produced multiple observable buds  expression and BAR gene accumulation of cotyledonary nodes during co-cultivation period. Major GUS staining tissues were around cotyledonary nodes, which were easily accessible to the Agrobacterium. According to the stained  and 4 DAC, suggesting that Agrobacterium was more acceptable by high-efficiency genotypes. These results indicate that high-efficiency genotypes are more susceptible to Agrobacterium infection.To determine the susceptibility of screened soybean genotypes to  stained E and uns stained F, transiGA20ox2 encodes an enzyme required for biosynthesis of GA [GA20ox2 transcripts increased in General, Williams 82, Shennong 9 and Bert at 1 DAC  (Agrobacterium infection caused a significant increase in MeJA levels and GmOPR3 expression of low-efficiency genotypes.To study the responses of genotypes to wounding and ene OPR3  were inv1.5-fold . In addi1.5-fold A. A soybwo-fold) B. The rePPO1 [PRX71 [PPO1 and PRX71 increased more significantly in General, Liaodou 16 and Kottman  and three low-efficiency genotypes  from T0 plants  agar (pH 7.0)) containing 25 mg·L−1 rifampicin and 50 mg·L−1 kanamycin and incubated at 28 °C until colony formation. Fifty milliliters liquid YEP medium containing antibiotics (same as above but without agar) was inoculated with a single colony and shaken at 28 °C, 180 rpm until the OD600 reached 0.8, −70 °C glycerol stocks. Before explant inoculation, 30 µL of A. tumefaciens glycerol stock was added to 5 mL liquid YEP medium containing antibiotics for 24 h at 28 °C, 180 rpm. Subsequently, 30 µL of the 5 mL starter culture was transferred to a 100 mL YEP culture, and grew overnight to OD600 = 0.8 at 28 °C, 180 rpm. The A. tumefaciens culture was centrifuged for 10 min at 3500 rpm and the pellet cell was resuspended in 1/10 Gamborg’s B5 medium with 3% (w/v) sucrose, 3.9 g·L−1 4-Morpholineethanesulfonic acid (MES), filter-sterilized 0.25 mg·L−1 gibberellin A3 (GA3), 1.67 mg·L−1 6-benzylaminopurine (6-BA), 400 mg·L−1l-Cysteine (l-Cys), 154.2 mg·L−1dl-Dithiothreitol (dl-DTT) and 200 µm As, pH 5.4.For our experiment, the binary plasmid, pCAMBIA3301-1 with w/v) sucrose and 0.7% (w/v) agar, pH 5.8, under dark for 16 h, at 25 °C (Agrobacterium suspension in the shaker (126 rpm) for 30 min (w/v) agar. Co-cultivation plates were incubated at 24 °C for 5 days in the dark (Soybean seeds were surface sterilized using chlorine gas made by mixing in 3.5 mL of 12 N HCl and 100 mL bleach (5.25% sodium hypochlorite) for 12 h A. Sterilat 25 °C B. The seat 25 °C C. Explanr 30 min D. Thereathe dark E.w/v) sucrose, 0.59 g·L−1 MES, pH 5.7 and filter-sterilized Gamborg B5 vitamins, 500 mg·L−1 carbenicillin, 1.67 mg·L−1 6-BA were added after autoclaving. Explants were subsequently removed majority of the hypocotyl approximate 3–5 mm below the cotyledonary node and cultured on shoot induction medium (same as above but with 0.8% (w/v) agar and 5 mg·L−1 phosphinothricin) with the hypocotyl embedded in the medium and the cotyledonary node region facing upwards. The medium was changed every two weeks (w/v) sucrose, 0.3% (w/v) phytagel, 0.59 g·L−1 MES, pH 5.7 and filter-sterilized 50 mg·L−1l-Asparagine (l-Asp), 50 mg·L−1l-Glutamine (l-Glu), 0.5 mg·L−1 GA3, 0.1 mg·L−1 indole-3-acetic acid, 1 mg·L−1 zeatin, 250 mg·L−1 ticarcillin (Tic), 100 mg·L−1 cefotaxime (Cef), and 5 mg·L−1 phosphinothricin were added after autoclaving. The medium was changed every two weeks (w/v) sucrose, 0.3% (w/v) phytagel, 0.59 g·L−1 MES, pH 5.6 and filter-sterilized 2 mg·L−1 indolebutyric acid, 50 mg·L−1l-Asp, 50 mg·L−1l-Glu, 250 mg·L−1 Tic, 100 mg·L−1 Cef were added after autoclaving  were used to determinate genetically modified plants containing the phosphinothricin 2EDTA (pH 8.0), 0.8% Triton-X, 0.5 mM K4Fe(CN)6, 0.5 mM K3Fe(CN)6, 1 mM X-gluc ) overnight at 37 °C to verify T-DNA transfer. After staining, the explants were washed in 70% ethanol and assessed blue-staining areas.After 5 days of co-cultivation, explants were first washed using sterile distilled water, then excised into cotyledonary node and immediately incubated with GUS histochemical stain (100 mM phosphate buffer (pH 7.0), 10 mM NaBAR gene accumulation during co-cultivation period. Explants were washed 5 times using sterile distilled water and surface dried with a paper towel, then excised into cotyledonary node (approximately 5 mm above and below junction of the cotyledon and the hypocotyl) (BAR gene coding region was amplified using a pair of primers: 5′-GCACCATCGTCAACCACTA-3′ and 5′-TCAGCAGGTGGGTGTAGAG-3′. The amplified products were isolated by electrophoresis on a 1% (w/v) agarose gel and photographed with the gel imaging system.The PCR analysis was conducted to determine pocotyl) A, immediet al. [−1 butylated hydroxytoluene as antioxidant) in darkness period at 4 °C overnight. The extract centrifuged at 12,000 rpm for 15 min at 4 °C. For purification, the supernatant was passed through C18 Sep-Pak cartridges . The eluate was dried down by pure N2 at 35 °C, then dissolved in 2 mL of phosphate-buffered saline (PBS) (pH 7.5) containing 0.1% Tween-20 (v/v) and 0.1% (w/v) gelatin. Free GA, ABA, ZR and MeJA were quantified by Enzyme-linked Immunosorbent Assay (ELISA) Kit  following the protocol described by Yang et al. [et al. [The extraction and purification of endogenous hormones used the method modified by Wang et al. . Frozen g et al. . Calcula [et al. .Extract of enzymes was prepared at 4 °C using the procedure with slight modifications as suggested by Sharma and Singh . Frozen et al. [410·min−1·mg−1 protein.PPO activity was assayed by the modification of the technique described by Concellón et al. . The ass2O2. The increase in absorbance at 470 nm was measured using spectrophotometer. Levels of enzyme activity were expressed as ΔA470·min−1·mg−1 protein.POD was measured according to the method of Reuveni . The reaGA20ox2, CYP707A2, IPT5, OPR3) encode key enzymes involved in hormone metabolism [PPO1, PRX71) encode PPO and POD, respectively [CYCD3, CYCA3) work in relation to cell cycle regulation [Arabidopsis or Medicago, but have not been investigated in soybean. We obtained soybean genes that were homologous with Arabidopsis or Medicago. The information of selected genes can be found in EF1α gene was used as a calibrator. The following thermal cycle conditions were used: 95 °C for 30 s, followed by 35 cycles of 95 °C for 5 s, 58 °C for 20 s, and 72 °C for 20 s. The qRT-PCR experiments were performed with three biological replications, and each biological replication was measured in three technical replications. Following the PCR, a melting curve analysis was performed. Threshold cycle was used for relative quantification of the input target number. Relative fold difference (N) is the number of infected target gene transcript copies relative to the uninfected control gene transcript copies and is calculated as follows:Ct = ΔCt of the infected  samples minus ΔCt of the uninfected control (0 HAI) samples, and ΔCt is the difference in threshold cycles for the target gene and the EF1α internal reference.There are three groups of selected genes used in this study. The first group of genes , fluorescence of individual nuclei was measured to carry out DNA semiquantitative analysis. Average optical density was used as a measurement of DNA content standards, which is calculated as follows:4′-6-Diamidino-2-phenylindole (DAPI) is utilized extensively in detecting and quantifying of DNA. Fluorescence of DAPI-stained DNA is proportional to DNA quantity. In this experiment, the cotyledonary node samples were fixed in FAA  before embedded in Steedman’s wax , and theThe assays of endogenous hormones, enzymes and genes were determined in three biological replications and each biological replication was measured in triplicate, the means and standard deviations were calculated. The significance analysis was performed by Duncan’s new multiple-range test in DPS v7.05 statistical software .Agrobacterium. There is a tangible and complex link between the plant factors that participate in the response mechanism of Agrobacterium-plant interaction. We suggest that in cotyledonary nodes GA and ZR are positive plant factors for Agrobacterium-mediated soybean transformation by facilitating germination and growth and increasing the number of cells that in DNA synthesis cycle, respectively; whereas ABA, MeJA, PPO and POD are negative plant factors for Agrobacterium-mediated soybean transformation by repressing germination and growth, and inducing defence reactions, respectively.Our results indicate that soybean genotype-dependency is mainly caused by genotypes’ susceptibility to


Escherichia coli, and to a less extent in Bacillus subtilis. However, metabolic flux of the high SA-producing strains has not been explored. In this study, we constructed with genetic manipulation and further determined metabolic flux with 13C-labeling test of high SA-producing B. subtilis strains.Shikimic acid (SA) is a key chiral starting molecule for the synthesis of the neuramidase inhibitor GS4104 against viral influenza. Microbial production of SA has been extensively investigated in B. subtilis 1A474 had a mutation in SA kinase gene (aroI) and accumulated 1.5 g/L of SA. Overexpression of plasmid-encoded aroA, aroB, aroC or aroD in B. subtilis revealed that aroD had the most significantly positive effects on SA production. Simultaneous overexpression of genes for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroA) and SA dehydrogenase (aroD) in B. subtilis BSSA/pSAAroA/pDGSAAroD resulted in SA production of 3.2 g/L. 13C-Metabolic flux assay (MFA) on the two strains BSSA/pHCMC04/pDG148-stu and BSSA/pSAAroA/pDGSAAroD indicated the carbon flux from glucose to SA increased to 4.6% in BSSA/pSAAroA/pDGSAAroD from 1.9% in strain BSSA/pHCMC04/pDG148-stu. The carbon flux through tricarboxylic acid cycle significantly reduced, while responses of the pentose phosphate pathway and the glycolysis to high SA production were rather weak, in the strain BSSA/pSAAroA/pDGSAAroD. Based on the results from MFA, two potential targets for further optimization of SA production were identified. Experiments on genetic deletion of phosphoenoylpyruvate kinase gene confirmed its positive influence on SA production, while the overexpression of the transketolase gene did not lead to increase in SA production.B. subtilis, aroD exerted most significant influence on SA accumulation. Overexpression of plasmid-encoded aroA and aroD doubled SA production than its parent strain. MFA revealed metabolic flux redistribution among phosphate pentose pathway, glycolysis, TCA cycle in the low and high SA-producing B. subtilis strains. The high SA producing strain BSSA/pSAAroA/pDGSAAroD had increased carbon flux into shikimate pathway and reduced flux into TCA cycle.Of the genes involved in shikimate pathway in  Illicium plant. However, microbial fermentation as an alternative process for SA production has attracted more and more interests.Shikimic acid , is a key chiral starting material for the synthesis of the antiviral neuraminidase inhibitor GS4104 . It is aEscherichia coli, for SA accumulation  glucose or 20% [U-13C] glucose and 80% unlabeled glucose. The mass isotopomer patterns in cellular amino acids were analyzed by GC-MS  was mutated in both strains and de nova synthesis of these amino acids from glucose was blocked. We measured the 13C label state of extracellular shikimate by using LC-MS  increased its flux by 2.4 folds (from 1.9% in BSSA/pHCMC04/pDG148-stu to 4.6% in BSSA/pSAAroA/pDGSAAroD) in strain BSSA47407 . However, the net flux of reaction encoded by tkt decreased from 4.6% in the parent strain BSSA/pHCMC04/pDG148-stu to 1.3% in the high SA-producing strain BSSA/pSAAroA/pDGSAAroD, suggesting that this reaction was severely disturbed and might be interesting to see how the SA synthesis would be influenced if tkt were overexpressed.As shown in Figure AroA/pDGSAAroD. Two reactions were considered in this study for generation of PEP: 1) The enolase  catalyzes glycerate-2-phosphate into PEP  in the SA-producing BSSA/pSAAroA/pDGSAAroD was still high (159%). For the purpose to increase supply of PEP to shikimate synthesis, knockout of this pyk might be a choice to increase SA production.PEP is one of the initial substrates for shikimate synthesis. In almost all known microorganisms, the DAHP synthase catalyzes the condensation of PEP and E4P, which is the first reaction of the shikimate pathway. It is reasonable to consider any changes of PEP generation and consumption in the high SA-producing BSSA/pSAB. subtilis. For the purpose to increase carbon flux to PEP, efforts were made to overexpress tkt in BSSA/pSAAroA/pDGSAAroD. However, the resulting strain BSSA/Ωtkt::pSAtkt did not show significant changes in SA production  at the Ohio State University (http://www.bgsc.org/). Both B. subtilis and E. coli were routinely cultivated in Luria Bertani (LB) broth at 37°C. When needed, antibiotics were included at the following concentrations, 15 μg/mL of erythromycin, chloramphenicol of 5 μg/mL, ampicillin of 100 μg/mL and neomycin of 30 μg/mL.The bacterial strains, plasmids, and primers used in this study are listed in Table 600) using a UV/visible spectrophotometer. Cellular dry weights were determined with duplicate samples of culture broth, by precipitation of cells and washed with distilled water and lyophilization.Cell growth was monitored by optical density measurements at 600 nm ; KH2PO4 (6); Urea (6); sucrose (80); MgSO4 · 7H2O (1); FeSO4 (0.01); MnSO4 (0.01); Yeast Extract (4). For induction of the target gene overexpression in recombinant B. subtilis strains, IPTG at final concentration of 0.05 mM and xylose at final concentration of 5 g/L were added into the fermentation broth.s et al.  by deletTM) to remove cells, and the supernatant was applied for HPLC analysis. The HPLC was run with mixture of solution A  and solution B (methanol) as eluant and was operated at a flow rate of 0.35 mL/min. SA and 3-dehydroshikimic acid were detected at 215 nm with adiode array detector. The following gradient was used: at 0–7.5 min, 95% of solution A and 5% of solution B; at 7.5-8.0 min, 100% of solution B; at 8.0-15 min, 100% of solution B; 15.0-15.5 min, 95% of solution A and 5% of solution B; 15.5-22.5 min, 95% of solution A and 5% of solution B. Standard SA and 3-hydroskimic acid were eluted at 5.411 and 6.241 min, respectively, under these conditions.SA and 3-dehydroskimic acid accumulation in culture supernatant was determined with an HPLC system (Agilent 1200 series) equipped with a SB C18 column (4.6 mm × 250 mm × 5 μm). Five ml samples of fermentation broth were centrifuged at 13,000 rpm for 5 min on Microfuge® 18 centrifuge .Plasmid and chromosomal DNAs were isolated using the Plasmid Minspin HP Kit and the SiMax™ Genome DNA Kit, respectively. Purification of DNA fragments from agarose gels was done with the TIANgel Midi Purification Kit. Restriction enzymes, ligases and other DNA-manipulating enzymes were used according to their manufacturer’s instructions.aro genes involving shikimate pathway, i.e., aroA , aroB (NP_390151.1), aroC (NP_390189.1), and aroD (NP_390444.2) were PCR amplified from genomic DNA of B. subtilis strain 168 using primers listed in Table SpeI and XmaI and then cloned into the same digested pHCMC04 -labeled isotope isomer  or as a mixture of 20% (w/w) [U-13C]  and 80% (w/w) unlabeled glucose. B. subtilis strains pre-cultivated overnight in 5 mL of M9 minimal medium supplemented with 5 g/L unlabeled glucose, and the precultures (2 mL) were used as inoculum for 13C-labeling experiments. The 13C-labeling experiments were performed in a 250-mL flask that contained 50 mL of M9 minimal medium, which was supplemented with either 20:80 (w/w) mixture of [U-13C] glucose:unlabeled glucose or 100% [1-13C] glucose. Temperature and the shaking rate of the incubator were held constant at 37°C and 200 rpm, respectively. When cells reached exponential phase, aliquots of 0.5 mL was aseptically collected from the culture at intervals of 0.5 h. Measure the OD600 on a spectrometer to monitor the growth of the cells. For extracellular metabolite (shikimic acid and 3-dehydroshikimic acid) analysis by HPLC, culture samples were centrifuged for 5 min at 4°C and 20,000 g to remove the cells. The specific growth rate (μ) was defined as the increase in cell biomass concentration per unit time. The specific glucose consumption rate (qglu) was calculated during the exponential growth phase as the differential change in glucose with time (t) normalized to the biomass concentration. The specific SA production rate (qsa), and the specific 3-dehydroshikicmic acid production rate (qdhs) were calculated during the exponential growth phase as the differential change in SA, and 3-dehydroshikicmic acid with time (t) normalized to the biomass concentration, respectively.The chemically defined M9 minimal medium  was appl13C-labeled biomass for the GC-MS measurement followed standard protocols [Preparation of cellular hydrolysate from rotocols . For GC-13C-labeling states of SA and 3-dehydroshikimic acid. SA and 3-dehydroshikimic acid was eluted with mixture of 0.1% formic acid in water (solution C) and acetonitrile (solution D) at ratio of 98:2 (solution C: solution D) and at a flow rate of 0.15 mL/min. MS data was collected in negative ion mode. The capillary voltage was set to 3500 kV, and the fragmentor voltage at 90 V. The drying gas temperature was maintained at 350°C with a flow rate of 11 L/min and a nebulizer pressure of 40 psi.Agilent 1290 UHPLC system equipped with ZORBAX 300SB-C18 Column (2.1 mm × 100 mm × 1.8 μm) and Agilent 6530 Q-TOF mass spectrometer fitted with an electrospray ionization (ESI) source were used to measure 13C MFA was a well-established method, and details concerning the general modeling framework of classical stationary 13C labeling experiments could be found elsewhere [13C labeling, several flux ratios, including (1) Pyr originating from malate, (2) PEP from OAA, (3) OAA from PEP, (4) PEP from transketolase, (5) PEP from the PP pathway, (6) pentose-5-phosphate from Glu-6-P, (7) OAA from the glyoxylate shunt, (8) erythrose-4-phosphate from pentose-5-phosphate, (9) Ser from Gly, (10) Gly from Ser, and (11) PEP and E4P from shikimic acid were calculated from the MDVM of precursors  cycle and the glyoxylate shunt. The reactions catalyzed by PEP carboxykinase (v10), pyruvate carboxylase (v11), and malic enzyme (v12) were also included. The networks of active pathways identified by flux ratio analysis were used for flux quantification. The following enzyme reactions were considered reversible: phosphoglucose isomerase (v2), transketolase (v5 and v7), transaldolase (v6), the sequence of glycolytic reactions leading from triose 3-phosphate to PEP (v8) and malate dehydrogenase (v17). The metabolic reactions considered as shown in the Additional file For quantification of carbon fluxes in ykinase v0, pyruvaAll the above data, including 1) the stoichiometric reaction matrix, (2) the flux ratios derived from metabolic flux ratio analysis  of 15 proteinogenic amino acids and shikimic acid from Bacillus subtilis BSSA/pHCMC04/pDG148-stu (a) and Bacillus subtilis BSSA BSSA/pSAAroA/pDGSAAroD (b) during exponential growth phase. Appendix S1. Stoichiometric reactions implemented in the central metabolic network of Bacillus subtilis. Mass isotopomer distribution of TBDMS-derivatized protein-bound amino acids and shikimic acid (corrected). Click here for file


Zanthoxylum heitzii is a spice used to prepare several dishes and to treat tumors, syphilis, malaria, cardiac palpitations, urogenital infections in the west region of Cameroon, but the antitumor mechanisms and chemical composition are not yet investigated.Zanthoxyllum heitzii (Rutaceae) on apoptosis in human promyelocytic cells, their mechanisms and the chemical composition. The 3--2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the fifty percent inhibition (IC50) concentration of the cell lines after treatment. The effect on morphology was observed using a light or fluorescence microscopy. The rate of apoptosis and the cell cycle were measured using flow cytometry (FCM). The phytochemical analysis of the extract was carried with HPLC/MS methods.This study was aimed to determine the antiproliferative effects of four extracts from the fruits and barks of Z. heitzii but in different quantities. Syringic acid and Myricetin concentrations were between 17-21 fold higher in the fruits than the stem bark. Rhamnetin (393.35 μg/mL) and Oleuropein (63.10 μg/mL) were identified only in the stem barks of Z. heitzii. Among the four extracts tested for cytotoxicity properties, only the methanol extract of fruits and barks significantly inhibited cell proliferation of HL-60 cells with IC50 value of 20 μg/mL and 12 μg/mL respectively. HL-60 cells treated with Z. heitzii extracts significantly produced reactive oxygen species (ROS) with concurrent loss of mitochondrial membrane potential (MMP). Modifications in the DNA distribution and enhanced of G1/G0 phase cell cycle arrest were observed in a concentration dependent manner.The phytochemical analysis of the extracts indicated the presence of four known polyphenols  in both fruits and barks of Z. heitzii plant exert inhibitory effect on HL-60 cells through the reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and cell cycle destabilization.Polyphenols from  Phenols are compounds possessing one or more aromatic rings with one or more hydroxyl groups. They are broadly distributed in the plant kingdom and are the most abundant secondary metabolites of plants, with more than 8,000 phenolic structures currently known, ranging from simple molecules (phenolic acids) to highly polymerized substances (tannins). The beneficial effects of dietary polyphenols on human health have been widely assumed to act through various biological effects such as free radical scavenging, metal chelation, modulation of enzymatic activity and altering signal transduction pathways , 2. PhytZanthoxylum heitzii (Aubrev. & Pellegr) or Fagara heitzii is a plant from Rutaceae family , ρ-coumeric acid (Rt 3.95 min), vanillic acid (Rt 3.79 min), gallic acid (Rt 1.89 min), caffeic acid (Rt 3.72 min), ferulic acid (Rt 3.99 min)), apigenin (Rt 4.83 min), naringenin (Rt 4.85 min), luteolin (Rt 4.43 min), epicatechin (Rt 3.67 min), quercetin (Rt 4.42 min), carnosic acid (Rt. 8.55 min), chlorogenic acid (Rt 3.59 min), rosmarinic acid (Rt 3.97 min), apigenin 7-glucoside (Rt 3.89 min), oleuropein (Rt 3.969 min), amentoflavone (Rt 5.16 min), naringin (Rt 3.83 min), rutin hydrate (Rt 3.69 min), hesperidin (Rt 3.85 min). Calibration concentrations were 1, 4, 5 and 20 ppm except one, apigenin 7-glucoside, was 0.9, 1.8, 4.5, 9, and 18 ppm and injection volume were 5 μL for all standards.p< 0.05 was considered to be statistically significant.All of the data were presented as the mean ± standard deviation (SD). The viability experiments were done in triplicates and each data point represents the average of at least 3 independent experiments. Three independent experiments were performed for other assays and one of them was chosen as results to post in this study. Statistical analyses (two group comparisons) were performed using the Student’s t-test.


Cells were treated with UVB (100 mJ/cm2) and simultaneously with EA (5 μM in 0.1% DMSO) or RA (2.7 μM in 0.5% DMSO). Moreover, these substances were added to the UVB-irradiated cells 1 h or 6 h before harvesting, depending on the established UVB-induced cytokine expression peak. Cytokine gene expression was examined using quantitative real time polymerase chain reaction. RA produced a significant reduction in UVB-induced expression of IL-6, IL-8, MCP-1, and TNF-α when applied at the same time as irradiation. EA showed milder effects compared with RA, except for TNF-α. Both substances decreased IL-6 expression, also when applied 5 h after irradiation, and always produced a significant increase in UVB-induced IL-10 expression. Our findings suggest that EA and RA are able to prevent and/or limit the UVB-induced inflammatory cascade, through a reduction in proinflammatory mediators and the enhancement of IL-10, with its protective function.Ultraviolet radiation (UV) induces an increase in multiple cutaneous inflammatory mediators. Ellagic acid (EA) and rosmarinic acid (RA) are natural anti-inflammatory and immunomodulatory compounds found in many plants, fruits, and nuts. We assessed the ability of EA and RA to modulate IL-1 Similar to EA, RA exhibits many bioactivities, mainly antioxidant and anti-inflammatory in nature, and also antiviral and antibacterial activities , 6. Its ignaling –8.Both EA and RA have been shown to be valuable defences against noxious substances, whether they are toxic chemicals or ultraviolet radiations that affect our skin daily.EA is able to prevent collagen degradation by blocking matrix metalloproteinase production in UV-B-exposed fibroblasts and to diminish production of proinflammatory cytokines and skin infiltrating macrophages . Hence,  in vitro study, we investigated the ability of EA and RA to modulate the effects of UVB irradiation in immortalized keratinocytes.In thisμg/mL streptomycin, GIBCO, Grand Island, NY). Cells were cultured in a humidified incubator at 37°C with 5% CO2, until they reached a confluence of about 80%.Spontaneous immortalized keratinocytes (HaCaT cells) were grown in Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum , 2 mM L-glutamine , and antibiotics , emitting UVB light between 290 and 320 nm, with a peak emission of 300 nm, were used. The intensity of UVB irradiation, measured with a UV meter  was 0.8 mW/cmμM for EA and 2.7; 13.7; 27.5; 55 μM for RA) were prepared in dimethylsulfoxide  and diluted in DMEM so that the final concentration of DMSO in the medium was 0.1% for EA and 0.5% for RA.EA and RA were supplied by Rottapharm Madaus Dermo-Cosmetic R & D Division . Multiple concentrations  or RA (2.7 μM in 0.5% DMSO) for 6 h and 24 h. mRNA was extracted at 6 and 24 h, respectively, in line with each cytokine expression peak  and mRNA was extracted at 24 h , HaCaT cells were separately incubated with EA  according to the manufacturer's instructions. cDNA was prepared using the Transcriptor High Fidelity cDNA Synthesis Kit . Quantitative real time polymerase chain reaction  was performed to confirm differences in the expression levels of IL-1Cycling conditions were as follows: denaturation (95°C for 10 min), amplification and quantitation  repeated 40 times (38 times for IL-10 and 30 times for 18S), melting curve program (65–95°C with a heating rate of 0.1°C/s and continuous fluorescence measurement), and cooling step (40°C for 30 s).t-test, otherwise with Wilcoxon. Values of P < 0.05 were considered significant. Data are expressed as means ± SD of three independent experiments, each performed in duplicate.All statistical analyses were performed using GraphPad Prism 4.0 . Data that passed the normality test were analysed with a two-tailed μM in 0.1% DMSO and RA 2.7 μM in 0.5% DMSO were used in our experimental settings, since, from preliminary tests, cell viability was very similar to the control . In relation to IL-8, EA induced a slight, nonstatistically significant increase in UV-induced gene expression. On the other hand, the 40-fold increase in TNF-α mRNA expression induced by UVB alone was strongly downregulated by RA (P < 0.01) and nearly reset by EA , the expression of IL-6 induced by UVB showed a significant reduction following RA . Both EA Figures .α, IL-6, IL-8, MCP-1, and IL-10, in accordance with previous studies illustrating beneficial properties of EA and RA [α expression. TNF-α is involved in the initiation and promotion of the inflammatory pathway following UVB: it exerts pleiotropic effects, including modulation of cell adhesion molecules, promotion of apoptosis, and activation of lymphocytes [α expression.In this study, using immortalized human keratinocytes, we proved that EA and RA are able to modulate the expression of inflammatory mediators, such as TNF-A and RA , 12. UsiWe found that EA and, to a more efficient extent, RA decreased expression of IL-6, a multifunctional cytokine induced in the early reaction to UVB. This corroborates the findings of Vostálová et al. , RA but β expression, EA and RA did not significantly affect the moderate increase in UVB-induced IL-1β expression. IL-1β is involved in inflammatory pathways triggered by many noxious stimuli such as infections or UVB irradiation [β production in hairless mice after UVB [ In vivo models are probably more reliable for studying IL-1β after UV stimulation.With regard to IL-1adiation . Only onfter UVB . In vivoγ and granzymes [α [α could be explained by the different pathways activated by common cellular targets involved in the inflammatory cascade. For example, nuclear factor kappa B (NF-κB), which is required for transcription of many proinflammatory mediators such as TNF-α [κB inhibition [α exerted by EA and RA in our experiments seems to have an independent pattern, unrelated to IL-10.On the other hand, IL-10 gene expression after UVB seemed to be potentiated by both EA and RA. In particular, RA seemed to be more effective after a prolonged incubation period 24 h), inducing the highest IL-10 increase when added immediately after irradiation. EA produced a milder effect compared with RA after incubation for 1, 6, or 24 hours. IL-10 is a cytokine expressed by most T cells including regulatory T cells, antigen-presenting cells, macrophages, and epithelial cells. In recent years, it has been demonstrated that IL-10, which has traditionally been thought to contribute to the immunosuppressive milieu, is required for efficient immunosurveillance of the initiation and progression of skin tumours. More specifically, IL-10 induces infiltration and activation of intratumoural-specific cytotoxic CD8 + T cells, expressing interferon-ranzymes . Furtherranzymes , 17. In ranzymes . They nozymes [α , 12. Indzymes [α , who fouzymes [α –21, coulas TNF-α , is inhias TNF-α , 23. Int h, induchibition , the dowOur study reinforces the idea that EA and RA have multifaceted properties, including the ability to prevent and/or limit UVB-induced damage, increasing cell viability and decreasing the inflammatory response, through a reduction in multiple proinflammatory mediators and enhancement of the protective IL-10.


PLoS Biology, volume 1, issue 1:In The Roles of APC and Axin Derived from Experimental and Theoretical Analysis of the Wnt PathwayEthan Lee, Adrian Salic, Roland Krüger, Reinhart Heinrich, Marc W. KirschnerDOI: 10.1371/journal.pbio.0000010fluxes and flux appeared without the fl.Table 1: In the legend, the words coefficients appeared without the fi. In the table, some numbers in the section “Binding, dissociation” were marked with a ± sign that should have been a ∓.Table 3: In the legend, the word coefficients appeared without the fi.Table 4: In the legend, the word Please see the corrected legends and table below.Table 1. Numeric Values of Input Quantities of the Model for the Reference Statev12 = 0.42 nM · min−1 (rate of β-catenin synthesis), v14 = 8.2 · 10−5 · nM · min−1 (rate of axin synthesis), k4 = 0.27 min−1, k5 = 0.13 min−1, k6 = 9.1 · 10−2 nM−1 · min−1, k−6 = 0.91 · nM−1 · min−1, k9 = 210 min−1, k10 = 210 min−1, k11 · 0.42 min−1, k13 = 2.6 · 10−4 min−1, k15 = 0.17 · min−1. See Table S2, found at http://dx.doi.org/10.1371/journal.pbio.0000010.t002, for more precise numbers used in the calculations.The data are grouped into concentrations of pathway components, dissociation constants of protein complexes, concentration ratios, fluxes and flux ratios, and characteristic times of selected processes. Experimental evidence for these data is discussed in the text. From these data, the following rates and rate constants are calculated: Bold: Measured values, Italics: Estimated values.DOI: 10.1371/journal.pbio.0000010.t001Table 4. Concentration Control Coefficients for the Total Concentrations of β-Catenin and Axin Relative to Changes in the Concentrations of Pathway ComponentsThe control coefficients were obtained by numerical determination of the response to a change of total concentrations by 1%. Coefficients are given for the reference state and for the standard stimulated state.DOI: 10.1371/journal.pbio.0000010.t004The full text XML and HTML versions of the article have been corrected online. This correction note may be found at DOI: 10.1371/journal.pbio.0020089.


PLoS Biology, volume 2, issue 12.In 10.1371/journal.pbio.00203809 beads per g of cells” contains an error. The correct amount of beads is 8 × 108, i.e., ten times less.In Materials and Methods, the sentence “The magnetic beads were added to the extract to a ratio of about 8 ×10Published February 15, 2005


Through optimizing the TE-G/KMnO4 ratio in the redox reaction between carbon and KMnO4, flower-like MnO2/G microspheres (MnO2/G-10) are obtained with 83.2% MnO2 and 16.8% residual graphene. Meanwhile, corresponding MnO2/rGO composites are prepared by using rGO as raw materials. Serving as a working electrode in a three-electrode system, MnO2/G-10 composite displays a specific capacitance of 500 F g−1 at 1 A g−1, outstanding rate performance, and capacitance retention of 85.3% for 5,000 cycles. The performance is much better than that of optimized MnO2/rGO composite. We ascribe this to the high carbon fraction in TE-G resulting in a high fraction of MnO2 in composite, and the oxygen-containing groups in rGO reduce the resulting MnO2 fraction in the composite. The superior electrochemical performance of MnO2/G-10 is dependent on the hierarchical porous structure constructed by MnO2 nanosheet arrays and the residual graphene layer in the composite. In addition, a supercapacitor assembled by TE-G negative electrode and MnO2/G positive electrode also exhibits superior performance. In consideration of the low cost of raw materials, the MnO2/G composite exhibits great application potential in the field of supercapacitors.To enhance the electrochemical performance of MnO Yang et al. , herein, TE-G was adopted as raw materials, and a hydrothermal reaction was performed to fabricate MnO4) were obtained from Tianjin Damao Chemical Reagent Factory.Potassium chloride (KCl), Expandable graphite , and potassium permanganate .Thermally-exfoliated graphite (TE-G) was synthesized according to our previous work . Specifi4 was put into deionized water (80 ml) and stirred for 30 min to produce a uniform solution. Meanwhile, different amounts of TE-G powders were put into the KMnO4 solution and stirred for 30 min, and then, the mixture was put into a stainless-steel autoclave. The hydrothermal reaction was conducted at 180°C for 15 h. The production was filtered, rinsed repeatedly by deionized water, and dried at 60°C for 12 h to obtain MnO2/G composites. The redox reaction equation of C and KMnO4 can be described as: 4MnO4− + 3C + H2O → 4MnO2 + CO32− + 2HCO3−. According to the equation, the theoretical mass ratio of KMnO4 and C can be calculated as 1/17.7. Therefore, to change the MnO2 fraction in the MnO2/G composite, the KMnO4/TE-G mass ratio was set as 5, 10, and 20, and the resulting composites were coded as MnO2/G-5, MnO2/G-10, and MnO2/G-20. In addition, the hydrothermal reaction of TE-G in deionized water and in KCl solution was carried out under the same condition, and the resulting samples were coded as TE-G-H2O and TE-G-KCl, respectively. The rGO was used to prepare MnO2/rGO composites. The ratio of KMnO4/rGO was kept the same as the ratio of KMnO4/TE-G composites, and the sample was named MnO2/rGO-5, MnO2/rGO-10, and MnO2/rGO-20.In a typical synthesis, 1.0 g KMnOThe field-emission scanning electron microscopy  and transmission electron microscopy  were used to observe the morphologies of samples. The crystallographic feature was performed by X-ray diffraction  with Cu Kα radiation source. X-ray photoelectron spectra (XPS) were recorded by using a Thermo Scientific K-Alpha XPS spectrometer. The working voltage was 12 kV, and the X-Ray source was Al Kα. Pore size distribution and the specific surface area were tested by using the SSA-7000 device, according to the BJH model and BET method.2) was used to support the slurry and served as the working electrode. The platinum sheet was acted as the counter electrode, and the saturated calomel electrode (SCE) was used as the reference electrode. A CHI 660E electrochemical workstation (Shanghai Chenhua Co. Ltd.) was used to test electrochemical impedance spectroscopy (EIS), galvanostatic charge/discharge curves (GCD), cycling stability, and cyclic voltammetry (CV) curves. Asymmetric supercapacitor (ASCs) devices were assembled by using TE-G as the negative electrode and MnO2/G composite as a positive electrode with 6 M KOH electrolyte. The separator was glass fiber filter paper. In the ASCs device, Formula I (m+/m−=(C−×ΔV−/(C+×ΔV+) can be applied to obtain the ratio of positive/negative electrode material. Formula II /acetylene black/active materials were weighed at the ratio of 5:10:85, and dissolved in N-methyl-2-pyrrolidone (NMP) to prepare a slurry. Foam nickel  was heated at 500°C to prepare TE-G. Under a high temperature, the intercalation agent in EG expands and violently decomposes, resulting in a large amount of gas spilling and forming micropores, mesopores, and macropores. As shown in 4 reacts with C to generate MnO2, in which, each single-layer of graphene in TE-G reacts with KMnO4 and is then wrapped by abundant MnO2 nanosheets. The loading of MnO2 thick nanosheets on graphene layers leads to the exfoliation of TE-G. From the inset SEM image, after the redox reaction, graphene nanosheets were wrapped by MnO2 nanosheet arrays in different directions to produce flower-like microspheres. Compared with rGO, the consumption of graphene nanosheets and the generation of thick MnO2 layers lead to the delamination of TE-G. In order to confirm the advantage of MnO2/G composite, corresponding MnO2/rGO composites were prepared, and the microstructure and electrochemical performance were investigated.2/G composites samples were characterized by using TEM and SEM. From 2/G samples, under a low ratio of KMnO4/TE-G, a few graphene sheets in TE-G participate in the redox reaction with KMnO4. Hence, a few MnO2 nanosheets are generated on the graphene surface  facet of δ-MnO2 , and residual graphene nanosheet still exists in the MnO2/G composite.Elemental mapping and EDS were conducted to verify residual graphene in MnOnosheets  keep thenosheets . The disnosheets , which r−1 is attributed to the O-H vibration of GO or rGO. The peaks of ∼1,633 cm−1 and 1,313 cm−1 correspond to the stretching and bending vibration of C-O, respectively. Compared with GO or rGO, there are a few oxygen-containing groups in TE-G, which is conducive to the redox reaction between TE-G and KMnO4, and a high fraction of carbon would consume a large amount of KMnO4 and generate many more MnO2 nanosheets. To verify the high fraction of MnO2 in MnO2/G composites, the TG curves of MnO2/rGO-5 and MnO2/rGO-20 were measured to obtain the content of MnO2 in MnO2/rGO composites. As given in 2/rGO-5 and MnO2/rGO-20 are 57.7 and 67.0%, respectively. Therefore, the fraction of MnO2 can be calculated as 63.6 and 73.8%, respectively. The result shows that the MnO2 content of MnO2/rGO is much lower than that of the corresponding MnO2/G composite. The reason can be ascribed to the low C fraction in rGO, resulting in fewer MnO2 nanosheets.Fourier transform infra-red (FTIR) was provided in 4, four peaks can be observed at 12.2°, 24.7°, 36.6°, and 65.6°, these peaks correspond to the (001), (002), (100), and (110) facets of δ-MnO2 (JCPDS # 80–1098)  peak of carbon (TE-G) at 26.4° disappears, which is assigned to the loading of MnO2 thick nanosheets on graphene layers leads to the exfoliation of TE-G. This phenomenon indicates the consumption of TE-G and results in a low fraction of carbon in MnO2/G composites. Compared with MnO2/G composite, hydrothermally-treated TE-G samples under deionized water or KCl both show a sharp diffraction peak at 26.4°  . When th in MnO2 . From th in MnO2 , the 2p  element . Hence, 2 adsorption-desorption isotherms curves and pore distribution of different samples according to the BJH model and BET method. TE-G exhibits a high adsorption capacity and a big specific surface area at low pressure. The specific surface area of TE-G is 1055.7 m2 g−1, and a big specific surface area facilitates the infiltration and stripping of TE-G. When increased the amount of KMnO4, abundant MnO2 nanosheets loading on graphene surface decreases the specific surface area. The specific surfaces of MnO2/G-5, MnO2/G-10, and MnO2/G-20 are 252.3 m2 g−1, 76.1 m2 g−1, and 39.4 m2 g−1. From 2 on graphene decreases the fraction of micropores and mesopores. However, the macroporous structure of MnO2/G composite would accelerate the charge transfer and ion diffusion, further improving the electrochemical performance.2/G composites at 20 mV s−1. The CV curve of TE-G displays a quasi-rectangular shape, reflecting the EDLC characteristic of TE-G. When introducing MnO2, the resulting MnO2/G composites show two pseudocapacitive peaks of MnO2 corresponding to the faradic redox reaction of MnO2. The faradic redox reaction mechanism of MnO2 is verified as the valence shift between Mn4+/Mn3+ and Mn3+/Mn2+  originate from the faradic redox reactions related to Mn3+↔ Mn2+ + e− . On the basis of the equation of SC = I·Δt/(mV), the specific capacitance would be obtained. From −1 at 1 A g−1. When hybridizing with MnO2, MnO2/G composites show high specific capacitances. Among these composites, the MnO2/G-10 sample has the maximum specific capacitance of 500 F g−1 at 1 A g−1. Even operated at 10 A g−1, the capacitance is 314 F g−1, which is assigned to a large number of MnO2 nanosheets with high capacitance content loaded to the surface of graphene. In comparison with MnO2/G-10, the MnO2/G-20 composite has a capacitance of 158 F g−1, because of the stacked MnO2 aggregation on graphene  and charge transfer resistance (R2), respectively. ZView software was used to obtain the fitting curves (solid line) in 2/G-5, MnO2/G-10, and MnO2/G-20 are 0.484, 0.480, 0.213, and 0.217 Ω, respectively. In addition, the R2 values are 0.341, 0.669, 0.332 and 0.379 Ω, respectively. Therefore, the MnO2/G-10 composite exhibits the minimum value of R1 and R2 among these samples, which indicates the minimum internal resistance and charge transfer resistance. The reason can be explained as the residual graphene in composite enhances the electronic conductivity. Moreover, hierarchical porous flower spheres of MnO2 promote the fast transfer of charges/ions, which facilitate the pseudocapacitive reaction of MnO2 in the electrolyte. Unfortunately, abundant MnO2 aggregated clusters impede the rapid transfer of charges/ions, increase the internal resistance, which leads to the poor electrochemical performance of KMnO4/G-20 composite.2/G composite, the electrochemical performance of MnO2/rGO composites are given in 2/rGO composites show the pseudocapacitive feature of MnO2, the area of MnO2/rGO composites enclosed by the CV curve is much smaller than that of the MnO2/G-10 composite. In addition, the maximum discharge time of MnO2/rGO-20 is 64.7 s, the specific capacitance can be calculated as 129.4 F g−1, much lower than that of the MnO2/G composite. The reason is the less carbon fraction in rGO limits the redox reaction with KMnO4, resulting in less MnO2 nanosheets loading on rGO  has a low capacitance retention rate of 85.3%. Particularly, the specific capacitance of our MnO2/G composite is 500 F g−1, much higher than reported samples. Therefore, the MnO2/G composite with flower spheres structure displays an excellent electrochemical performance, which can be ascribed to two aspects. First of all, a large amount of MnO2 nanosheets arrays loading on graphene constructs a homogeneous hierarchical porous structure, which promotes the transport of electrons and ions, and reduces the charge transfer resistance. Moreover, the special microstructure facilitates the interface contact between MnO2 nanosheets and electrolyte and releases a high specific capacitance. Secondly, TE-G is composed of stacking graphene layers, which facilitates the redox reaction between KMnO4 and C, resulting in a high fraction of MnO2 in composite, which increases the pseudocapacitance. Moreover, the residual graphene layer in composite improves the conductivity of electrode material and decreases the internal resistance, which enables an outstanding rate capability and cycling performance.To testify the outstanding performance of MnO.3 F g−1 . The max.3 F g−1 . Althoug2/G-10 positive electrode. From −1 at 0.5 A g−1. The capacitance reduces to 78.8 F g−1 at a large current density of 5 A g−1, indicating an excellent rate capability. −1 at the power density of 250.0 W kg−1. With an increase of power density, the energy density drops to 10.94 Wh kg−1 (2500 W kg−1), further reflecting the excellent power/energy combination. −1, the capacitance retention rate is 98.4% after 5,000 cycles, reflecting the superior cycling stability. Therefore, flower-like MnO2/G microspheres exhibit outstanding performance in ASC.In addition, we assembled an asymmetric supercapacitor (ASC) with TE-G negative electrode and MnO2, a KMnO4-assisted hydrothermal method was adopted to achieve the exfoliation of TE-G and the loading of MnO2 nanosheets. Through changing the ratio of TE-G and KMnO4, flower-like MnO2/G microspheres (MnO2/G-10) were fabricated containing 83.2% MnO2 and 16.8% residual graphene layer. To confirm the advantage of TE-G reactants, corresponding MnO2/rGO composites were prepared by using rGO as raw materials. When tested in a three-electrode system, the MnO2/G-10 sample displays a maximum specific capacitance of 500 F g−1, an outstanding rate of performance, and a high capacitance retention rate . The performance is much better than that of the optimized MnO2/rGO composite. The reason can be explained as the high carbon fraction in TE-G resulting in a high fraction of MnO2 in flower-like MnO2/G microspheres, and the oxygen-containing groups in rGO reduce the effective redox reaction between KMnO4 and carbon. The superior electrochemical performance of MnO2/G-10 is related to the hierarchical porous structure constructed by MnO2 nanosheet arrays and conductive graphene in the composite. Moreover, the ASC consisted of MnO2/G positive electrode and TE-G negative electrode has a capacitance of 100.8 F g−1 at 0.5A g−1, with a high capacitance retention of 98.6% for 5,000 cycles. The energy density is 14.0 Wh kg−1 at the power density of 250.0 W kg−1. In consideration of the low cost of raw materials, the MnO2/G composite shows great application potential in the supercapacitors field.To hybridize thermally-exfoliated graphite (TE-G) and MnO


Therefore, we postulate that HET may be useful against inflammation-induced anxiety-like behavior.Hochuekkito (HET) is a Kampo medicine used to treat postoperative and post-illness general malaise and decreased motivation. HET is known to regulate immunity and modulate inflammation. However, the precise mechanism and effects of HET on inflammation-induced central nervous system disorders remain unclear. This study aimed to assess the effect of HET on inflammation-induced anxiety-like behavior and the mechanism underlying anxiety-like behavior induced by lipopolysaccharide (LPS). Institute of Cancer Research mice were treated with LPS , a bacterial endotoxin, to induce systemic inflammation. The mice were administered HET  once a day for 2 weeks before LPS treatment. The light-dark box test and the hole-board test were performed 24 h after the LPS injection to evaluate the effects of HET on anxiety-like behaviors. Serum samples were obtained at 2, 5, and 24 h after LPS injection, and interleukin-6 (IL-6) levels in serum were measured. Human and mouse macrophage cells  were used to investigate the effect of HET on LPS-induced IL-6 secretion. The repeated administration of HET prevented anxiety-like behavior and decreased serum IL-6 levels in LPS-treated mice. HET significantly suppressed LPS-induced IL-6 secretion in RAW264.7 and THP-1 cells. Similarly, glycyrrhizin, one of the chemical constituents of HET, suppressed LPS-induced anxiety-like behaviors. Our study revealed that HET ameliorated LPS-induced anxiety-like behavior and inhibited IL-6 release  Inflammation is the body’s initial response to infection and tissue injury and has been shown to play an important role in the development of neurodegenerative diseases and psychiatric disorders, such as anxiety, bipolar disorder, schizophrenia, and depression . The serThe light-dark test and the hole-board test is an anxiogenic challenge for measuring the response of an animal model to an unfamiliar environment and has been used to examine the effects of benzodiazepine anxiolytics, such as diazepam, on their emotional behavior . The typIt was reported that 92.7% of Japanese family physicians have prescribed Kampo medicine . Kampo mThis study was conducted in accordance with the recommendations of the Guide for Animal Experiments of the Advanced Science Research Center, Okayama University. The animal study protocol was approved by the Animal Care Use Committee of the Advanced Science Research Center, Okayama University (OKU-2020565). A total of 234 male Institute of Cancer Research (ICR) mice weighing 29–35 g were purchased from Charles River Laboratories . The mice were housed in an air-conditioned room (23 ± 1°C with approximately 60% humidity) in groups of five per cage under a constant light-dark cycle (lights on from 08:00–20:00) and fed standard laboratory food and tap water.Escherichia coli O127:B8; Sigma-Aldrich, St. Louis, MO, United States) was dissolved in saline solution and administered by intraperitoneal (ip) injection at a dose of 1 ml/kg. Control mice were administered the vehicle. HET (Lot No. 20160041010) was provided by Tsumura & Co. . HET was used in the form of an extract powder made of the following raw materials: 4.0 parts Japanese Pharmacopoeia (JP) Astragalus root , 4.0 parts JP Atractylodes lancea rhizome , 4.0 parts JP Ginseng , 3.0 parts JP Japanese angelica root , 2.0 parts JP Bupleurum root , 2.0 parts JP Jujube , 2.0 parts JP Citrus unshiu peel , 1.5 parts JP Glycyrrhiza , 1.0 parts JP Cimicifuga rhizome , and 0.5 parts JP ginger . The plants were identified by their external morphology and marker compounds as per the Japanese pharmacopeia and company standards. The extract quality was standardized based on good manufacturing practices, as defined by the Ministry of Health, Labour, and Welfare of Japan. The 10 herbs were boiled in purified water at 95°C for 1 h. The liquid extract solution was filtered from the non-soluble waste and reduced to a concentrate. The concentrate was spray-dried to produce the extract powder. A three-dimensional high-performance liquid chromatogram of HET provided by Tsumura & Co. is presented in in vivo and doses were set based on a previous report (LPS (from s report .We assessed the preventive effect of the repeated administration of HET and glycyrrhizin on the LPS-induced anxiety-like behaviors in mice using the hole-board test and light-dark test. HET (0.1–1.0 g/kg) and glycyrrhizin (10–30 mg/kg) was administered orally (po) once a day for 2 weeks until the day before the experiments. Diazepam (0.1–1.0 mg/kg) was administered intraperitoneally (ip) 30 min before the experiments. The animals were then intraperitoneally injected with LPS (300 µg/kg) 1 h after the final HET and glycyrrhizin administration. The hole-board and light-dark box tests were performed 24 h after the LPS injection. Serum samples from mice were obtained 2, 5, and 24 h after the injection. The experiments were conducted between 10:00 and 16:00.The hole-board test apparatus consisted of a gray box (50 × 50 × 50 cm) with 16 (4 × 4) equidistant holes 3 cm in diameter on the floor. The number of head dips registered in a 10 min session was recorded by an observer blinded to the experimental treatment. In addition, the locomotor activity of the mice was recorded by an overhead charged-coupled device camera using LimeLight video tracking .The light-dark box consisted of a light zone (20 × 20 × 25 cm) and a dark zone , separated by a partition with a single opening (5 × 8 cm) to allow the mice to pass from one zone to the other. Illumination in the light zone was 500 lx, while that in the dark zone was prevented by covering with a lid. A mouse was placed in the center of the light zone at the beginning of the test and the total time it spent in the light zone was recorded for 10 min.The human acute myeloid leukemia cell line THP-1 was obtained from the RIKEN BioResource Center , and the mouse macrophage cell line RAW264.7, was obtained from the European Collection of Authenticated Cell Cultures . THP-1 cells were maintained in RPMI1640 medium  supplemented with 10% fetal bovine serum (FBS) and 10 mg/ml of 1% (v/v) penicillin-streptomycin . RAW264.7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% FBS and 10 mg/ml of 1% (v/v) penicillin-streptomycin.4 cells/cm2 into 24 well plates. Phorbol 12-myristate 13-acetate (PMA) at a concentration of 200 nM was added to induce differentiation of the cells into macrophages and incubated for 24 h at 37°C in humidified air supplemented with 5% CO2. The cells were then exposed to LPS (1.0 µg/ml) and HET  and cultured for another 24 h. The culture supernatant was collected and the concentration of IL-6 secreted in it was measured. Likewise, the RAW264.7 cells were seeded at a density of 5 × 103 cells/cm2 into 24 well plates and incubated for 24 h. The cells were exposed to LPS (1.0 µg/ml) and HET  for 24 h. The concentration of IL-6 in the culture supernatant was determined.THP-1 cells were seeded at a density of 2 × 10IL-6 concentrations in serum and culture medium were determined by enzyme-linked immunosorbent assay (ELISA) using the mouse IL-6 antibody pair or human IL-6 antibody pair  according to the manufacturer’s instructions. The assay range for human IL-6 was 2–200 pg/ml. The assay range for mouse IL-6 was 4–500 pg/ml.p < 0.05 was considered as statistically significant.Values were expressed as mean ± standard error of mean (SEM). The data were analyzed by Student’s t-test or one way and two-way analysis of variance (ANOVA), followed by Dunnett’s test or Tukey-Kramer test  to determine differences among the groups. p < 0.01]  = 3.49, p = 0.08; LPS×HET: F  = 2.75, p = 0.12]  = 10.02,  < 0.01] . Additio < 0.05] . In cont = 0.12] .p < 0.01]  = 10.28, p < 0.01; LPS×HET: F  = 1.07, p = 0.32]  = 7.33,  < 0.01] . HET did = 0.39] . In cont = 0.32] . None ofp < 0.05; Light-dark test: F  = 2.14, p = 0.13]  = 3.74, p = 0.08; LPS×HET: F  = 0.11, p = 0.75]; Light-dark test: LPS: F  = 35.73, p < 0.01; HET: F  = 10.07, p < 0.01; LPS×HET: F  = 0.12, p = 0.73] (Glycyrrhizin (30 mg/kg) significantly increased the number of head dips and the time spent in light in naïve mice  . In cont = 0.73] .t-value = 2.964, p < 0.01).LPS-treatment caused a significant increase in IL-6 serum concentrations after 2 and 5 h . In all p < 0.05] and THP-1 (p < 0.01)] cells.THP-1 and RAW264.7 cells were used to investigate the effect of HET on LPS-induced IL-6 secretion by macrophages. Exposure of HET at concentrations of 0, 10, 50, 100, and 300 μg/ml did not affect the proliferation of THP-1 cells or RAW264.7 cells (data not shown). LPS exposure at a concentration of 1.0 µg/ml significantly increased IL-6 secretion by THP-1 and RAW264.7 cells. HET significantly suppressed LPS-induced increased IL-6 secretion by RAW264.7  [F  receptor and serotonergic receptor. Therefore, several crude drugs composed HET may contribute to the anxiolytic-like effects observed in this study.Regarding the active components responsible for the effects of HET, several major chemical components have been previously identified . In thisToll-like receptors (TLRs) are pattern recognition receptors localized on the cell surface of macrophages and other innate immune cells and play an important role in host defense responses . LPS, a In conclusion, we demonstrated that HET ameliorated anxiogenic-like behavior under inflammatory conditions induced by LPS using two different behavioral models. Additionally, it was revealed that glycyrrhizin, one of the chemical constituents of HET, suppressed LPS-induced anxiety-like behavior. We also showed that HET decreased IL-6 expression in LPS-induced mice and LPS-exposed macrophages. In conclusion, our study demonstrated that HET inhibits cytokine release, suggesting that the effect of HET on inflammation-induced anxiety-like behavior involves an anti-inflammatory action through IL-6 suppression in the early stages of inflammation.


Chaetoceros calcitrans. The samples were submitted to an in vitro digestion protocol, and the compounds were determined by HPLC-PDA-MS/MS. A total of 13 compounds were identified in all tests. After in vitro digestion, the relative bioaccessibility of carotenoids and chlorophylls ranged from 4 to 58%. The qualitative profile of carotenoids reflected the initial sample, with all-E-zeaxanthin (57.2%) being the most bioaccessible compound, followed by all-E-neochrome (31.26%), the latter being reported for the first time in the micellar fraction. On the other hand, among the chlorophylls only pheophytin a (15.01%) was bioaccessible. Furthermore, a chlorophyll derivative (Hydroxypheophytin a’) was formed after in vitro digestion. Considering all compounds, xanthophylls (12.03%) and chlorophylls (12.22%) were significantly (p < 0.05) more bioaccessible than carotenes (11.22%). Finally, the considerable individual bioaccessibilities found, especially for zeaxanthin, demonstrate the bioactive potential of this bioresource. However, the large reduction in the totality of compounds after in vitro digestion suggests that additional technological strategies should be explored in the future to increase the efficiency of micellarization and enhance its bioactive effects.This study aimed to investigate the bioaccessibility of carotenoids and chlorophylls from the biomass of microalgae  As the world’s population becomes more aware of health and global sustainability issues, the potential for microalgae-based processes and products to contribute to solutions to these issues is becoming evident. These microorganisms have been considered potential bioresources to meet the population’s growing needs for a supply of healthier, natural, and sustainable food products, especially fine chemical compounds with potential health-promoting effects ,2.2 [Chaetoceros calcitrans, belonging to the Chaetocerotaceae family, is a diatom that contains large amounts of natural antioxidants such as carotenoids and chlorophylls [Microalgae are acknowledged as one of the most promising renewable resources on the planet. They have some highly relevant characteristics, such as their rapid growth rate and the ability to survive in adverse conditions and substantially contribute to the capture of atmospheric CO2 ,4. In ad2 . Among trophylls ,7.Carotenoids and chlorophylls constitute groups of large and complex compounds ubiquitous in microalgae species ,9. Many However, for bioactive compounds to exert some activity at the biological level, these molecules must be bioaccessible for intestinal uptake and subsequent systemic distribution in the human body ,17. ThusBioaccessibility is dependent on the degree of release, solubilization, and incorporation of intracellular compounds in mixed-bile-salt micelles . For micChaetoceros calcitrans, following an in vitro digestion protocol.Considering these aspects of bioaccessibility, the objective of this work was to evaluate the bioaccessibility of carotenoids and chlorophylls of ultrasonicated biomass of C. calcitrans  and the micellar fraction after digestion, are shown in The microalgae cell wall is the first barrier to the effective use of bioactive compounds from this promising group of microorganisms. However, previous studies have demonstrated the efficiency of using ultrasound to increase micellar incorporation of microalgae compounds such as obliquus ,21,22. T−1, of which all-E-echinenone (116.65 ± 1.52 µg·g−1) was the most abundant, followed by all-E-β-carotene (55.44 ± 0.25 µg·g−1). On the other hand, the initial chlorophyll extract presented five compounds, making a total of 3944.16 ± 41.56 µg·g−1; among these, pheophytin a (3257.52 ± 40.23 µg·g−1) was the most abundant.From a quantitative point of view, the initial extract of carotenoids showed seven compounds, totaling 239.88 ± 1.82 µg·gIn general, after digestion simulation, the qualitative profile of carotenoids reflected that of the initial sample. On the other hand, only one of the chlorophylls in the initial extract was identified after digestion. Among the chlorophylls, the derivated compound identified as hydroxypheophytin a’ was detected only in the micellar fraction.Possibly, this chlorophyll derivative was generated due to in vitro digestion conditions ,27, as tE-β-carotene (8.45 ± 0.15 µg·g−1) and all-E-equinenone (7.91 ± 0.10 µg·g−1) remained the major carotenoids, followed by all-E-zeaxanthin (7.10 ± 0.10 µg·g−1), all-E-neochrome (2.33 ± 0.5 µg·g−1), all-E-lutein (1.72 ± 0.03 µg·g−1) and 15Z-echinenone (0.74 ± 0.03 µg·g−1).A significant reduction of all compounds was observed in the micellar fraction after in vitro digestion. All-E-β-carotene in detriment to other xanthophylls: (i) The high content of unsaturated fatty acids in the biomass of C. calcitrans is one possible cause [Two hypotheses (i and ii)can be considered to explain the higher micellar content of all-le cause , as sevele cause ,32,33,34le cause ,20,35.−1) are approximately twice the total carotenes content (8.45 ± 0.15 µg·g−1).Although many factors need to be evaluated, a convergence in the literature towards greater micellar incorporation of xanthophylls is evident ,37. This−1) remained the majority compound. According to the literature [Referring to the micellarized chlorophyll fraction, pheophytin a , followed by all-E-neochrome (31.26% ± 0.42), all-E-β-carotene (15.24% ± 0.20), all-E-lutein (14.36% ± 0.33), all-E-echinenone (6.78% ± 0.01) and 15Z-echinenone (4.59% ± 0.16), while the only bioaccessible chlorophyll was pheophytin a (15.01% ± 0.20).In terms of relative bioaccessibility (%) of ompounds a,b, therompounds a. Among These bioaccessible compounds perform essential physiological and pharmacological activities which improve human health, well-being and nutritional status. These molecules are excellent antioxidants, reduce oxidative stress, benefit cardiovascular health. They also help prevent obesity, diabetes, some types of cancer, and neurological sequelae. In addition, some compounds such as β-carotene act precisely as vitamin A precursors, and zeaxanthin and lutein act as eye health regulators ,40.E-zeaxanthin (57.29%) from C. calcitrans was superior to the findings for sonicated biomass from Phaeadactylum tricornutum (29%) [Nannochloropsis sp. (<15%) [S. obliquus (9%) [Scenedesmus bijuga (6%) [P. tricornutum (17%) [E-β-carotene (15.24%) exceeded the values found in the sonicated biomass of Chlorella vulgaris (12%), Chlamydomonas reinhardtii (<10%) [S. obliquus (3%), S. bijuga (8%), and Chlorella sorokiniana (13%). Likewise, all-E-lutein (14.36%) surpassed the bioaccessibility found for sonicated biomass of S. obliquus (12%), S. bijuga (3%) and C. sorokiniana (6%) [Comparatively, the relative bioaccessibility of all-um (29%) , Nannoch. (<15%) , S. obliuus (9%) , Scenedeuga (6%) , and a dum (17%) . In addii (<10%) , S. obliana (6%) ,23.P. tricornutum up to 5 times higher than those established in this work [C. vulgaris was found to be approximately 2-fold higher [E-equinenone (6.78%) was similar to that found for sonicated biomass of S. obliquus (6%) [Z isomer (4.59%) and all-E-neochrome (31.26%).On the other hand, a study found bioaccessibility values of β-carotene of his work . Likewisd higher . The biouus (6%) . HoweverE-neochrome has been reported, a compound whose bioactive properties remain neglected, despite its remarkable structure  [The relative bioaccessibility of pheophytin a (15.01%) was higher than that reported for sonicated biomass of s (~10%) . Comparis (~10%) .p > 0.05) from the bioaccessible total carotenes. According to the literature, xanthophylls are generally more bioaccessible than carotenes due to their lower hydrophobicity [Highlighting the totality of compounds, hobicity ,37.C. calcitrans (12.22%) is four times greater than that of the sonicated biomass of S. obliquus (3%), for example, and is within the range determined for edible algae [When compared to different sources, the total bioaccessible chlorophyll of the sonicated biomass of trans 12.% is fourle algae . On the trans 12.% is fourle algae .As already demonstrated for different matrices, including microalgae, the transfer of carotenoids and chlorophylls to the micellar fraction can be influenced by numerous factors, especially the location in the matrix and the effect of constituents such as proteins, fatty acids, soluble fibers and minerals ,46,47,48Finally, when comparing the initial totality of compounds, both classes of pigments were reduced by more than 80% after mimicking digestion. This fact leads us to consider exploring alternatives to increase the micellarization efficiency and enhance its bioactive effects in vivo. The use of emulsions as a vehicle is an attractive option, mainly due to the increase in stability and incorporation of structures with non-polar characteristics in the micellar phase ,21,49. IE-β-carotene, all-E-lutein, and chlorophyll a (with purities ranging from 95.0% to 99.9%), were purchased from Sigma-Aldrich . All solvents for extraction and chromatography analysis were purchased from Merck . The α-amylase (A3176), pepsin (P7000), pancreatin (P1750), lipase (L3126) and bile (B8631) were purchased from Sigma-Aldrich .The standards all-Chaetoceros calcitrans (CCMP1315) were used in the experiments. Stock cultures were propagated and maintained in BG-11 medium (Braun-Grunow medium) [−2·s−1 and a photoperiod of 12 h.Axenic cultures of  medium) . The inc−1, isothermal reactor operating at a temperature of 26 °C, luminous intensity of 25 μmol.m−2.s−1, continuous aeration of 1 VVM (with air enriched with 3% CO2) and photoperiod of 24:0 h light/dark. The biomass was separated from the BG-11 medium by centrifugation , and the supernatant was discarded. The paste obtained after centrifugation was frozen at −18 °C for 24 h, and freeze-dried for 24 h at −50 °C above −175 μm Hg. The samples were stored under refrigeration until the analysis.The biomass productions were made in phototrophic conditions. The cultivations were performed in a bubble column photobioreactor under a batch regime fed on 2.0 L of BG-11 medium. The experimental conditions were as follows: initial cell concentration of 100 mg·L−1, CaCl2 6 mmol·L−1, KCl 5 mmol·L−1) and were subjected to 15 min of an ultrasonic probe  to break the cell wall . The ul−1 of α-amylase, followed by incubation at 37 °C, 10 min, 7.5× g in a shaking incubator . Before starting the gastric phase, the pH was adjusted to 2.5 with HCl 1 mol L−1 followed by 2 mL of pepsin . The total volume was adjusted to 40 mL, and the solution was incubated for 1 h, 37 °C, 7.5× g . After this step, the pH was increased to 6.0 with 1M NaHCO3 and the intestinal phase started with a bile solution , 4000 U.mL−1 of pancreatin and 1000 U.mL−1 of lipase. The pH was adjusted to 6.5 and the total volume to 50 mL, the incubation occurred for 2 h at 37 °C and 7.5× g . After completion of the in vitro digestion, the solution was centrifuged at 8000× g, 60 min at 4 °C . The supernatant containing the mixed micelles was collected, covered with nitrogen gas, frozen at −40 °C and lyophilized for further extraction of pigments. The pigments bioaccessibility was calculated as the ratio between carotenoid content in the micellar fraction (supernatant) and original content in the C. calcitrans Equation (1).The samples were submitted to an in vitro simulated digestion model, according to the protocol adapted from INFOGEST  and modiC. calcitrans carotenoids and chlorophylls was extracted according to the literature [g. In addition, the carotenoids extract was saponified for 16 h with 10 g 100 mL−1 methanolic KOH at room temperature, and the alkali was removed by washing with distilled water. All extracts were concentrated in a rotary evaporator, placed in N2 atmosphere, and kept at −40 °C in the dark until analyzed.The original content of terature . The freThe micellarized pigments were extracted according to an adapted protocol . The lyo−1, the injection volume was 20 μL, the column temperature was maintained at 29 °C, the UV-Vis spectra were acquired between 220 and 700 nm, and the chromatograms were processed at 450 nm.The pigments were analyzed by high performance liquid chromatography HPLC  equipped with binary pumps (model LC-20AD), online degasser, and automatic injector . The chromatograph with photodiode array detection (PDA) (model SPD-M20A) was connected in series to an atmospheric pressure chemical ionization (APCI) source , and a mass spectrometer Shimadzu 8040 triple quadrupole. The pigments separation was performed on a C30 YMC column  . HPLC-PDA analysis was performed according to Rodrigues et al. . Prior t2): 3.0 L.min−1; drying gas flow (N2): 5.0 L.min−1; collision-induced dissociation (CID) gas: 23 kPa (argon); event time: 0.5 s. To improve identification quality, MS/MS was used simultaneously in SIM (Select Ion Monitoring) and MRM (Multiple Reaction Monitoring) modes.The MS/MS analysis was conducted according to Giuffrida et al.  with ada+) and MS/MS fragments), compared with data available in the literature [E-lutein, all-E-β-carotene end chlorophyll analytical curves were used to quantify the xanthophylls, carotenes and chlorophylls, respectively.The identification was performed according to the following combined information: elution order on C30 HPLC column, co-chromatography with authentic standards, UV-Vis spectrum, and mass characteristics  and differences between more than two variables were assessed by a one-way ANOVA followed by Tukey’s test (p < 0.05).The statistical analysis was performed using GraphPad Prism 5.0 software . Differences between the two variables were detected by Student’s C. calcitrans for the first time. The relative bioaccessibility of sonicated biomass varied over a wide range (4–58%). The qualitative profile of bioaccessible carotenoids reflected the initial sample, with all-E-zeaxanthin (57.29%) being the major compound, followed by all-E-neochrome (reported for the first time in the micellar fraction). In contrast, pheophytin a (15.01%) was the only bioaccessible chlorophyll. Additionally, a chlorophyll derivative (hydroxypheophytin a’) was detected only in the micellar fraction. Considering all classes, xanthophylls (12.03%) and chlorophylls (12.22%) were significantly more bioaccessible than carotenes (11.28%). Although the considerable bioaccessibility of individual compounds is evidence for the bioactive potential of this source, the reduction of approximately 80% in the content of the compounds after in vitro digestion suggests that additional strategies to increase the micellarization efficiency are required in the future.This study investigated the bioaccessibility of carotenoids and chlorophylls from the diatom