Brassica oleracea L. var. italica Plenck), green curly kale (Brassica oleracea var. sabellica L.), red mustard (Brassica juncea (L.) Czern.) and radish (Raphanus sativus L.) hydroponic microgreens. Macro-  and oligo-elements , ascorbic acid, total soluble polyphenols, total carotenoids, total anthocyanins, total isothiocyanates and total antioxidant capacity  were determined before and after the standardized simulated gastrointestinal digestion process. All microgreens provided relevant amounts of vitamin C (31–56 mg/100 g fresh weight) and total carotenoids (162–224 mg β-carotene/100 g dry weight). Mineral content was comparable to that normally found in hydroponic microgreens and the low potassium levels observed would allow their dietetic recommendation for patients with impaired kidney function. Both total soluble polyphenols and total isothiocyanates were the greatest contributors to the total antioxidant capacity after digestion  while macroelements showed an important bioaccessibility (34–90%). In general, radish and mustard presented the highest bioaccessibility of bioactive compounds and minerals. Overall, the four hydroponic Brassicaceae microgreens present a wide array of antioxidant bioactive compounds.Microgreens constitute an emerging class of fresh, healthy foods due to their nutritional composition. In this study the content of minerals and antioxidant bioactive compounds, and for the first time bioaccessibility, were evaluated in broccoli ( Microgreens are a new class of small, fresh, edible vegetables considered as a good nutritional source because of their high mineral and bioactive compound content. The meaning of microgreen refers to immature greens harvested at soil level between the first and third week after sowing, when the cotyledon is fully developed and the first true leaves have emerged ,2,3, beiThese new and young vegetables are a versatile, nutritive and sustainable crop from cultivation to consumption. They can be adapted to different agronomic practices to obtain a final product which is of high organoleptic and nutritional quality . LikewisIn addition, microgreens have been considered as healthy foods because of their general higher levels of phytochemicals with respect to their mature counterparts ,6,7,12. Brassica vegetables, at the mature stage, contain beneficial nutrients for human health [Brassicaceae family is one of the most widely grown to date [Brassicaceae microgreens is limited regarding the concentrations of the antioxidant bioactive compounds and minerals that were examined in this work. There are some studies on this subject in broccoli [It is known that n health , and avan health . Probabl to date . Neverthbroccoli ,18,19,20broccoli , mustardbroccoli ,23,24 anbroccoli ,26 microbroccoli , a prereBrassicaceae microgreens: broccoli, kale, mustard and radish.The aim of the present study was to evaluate the content and for the first time the bioaccessibility of the main antioxidant bioactive compounds , total antioxidant capacity, as well as macro-  and oligoelements  provided by the four studied hydroponic Brassicaceae family were evaluated in this study: broccoli , green curly kale (Brassica oleracea var. sabellica L.), red mustard (Brassica juncea (L.) Czern.) and radish (Raphanus sativus L.). Mustard and radish cultivar seeds were purchased from CN Seeds Ltd.  and kale and broccoli from Rocalba S.A.  and Intersemillas S.A. , respectively.Four microgreen species belonging to the −2 for broccoli and kale and 2.8 seeds cm−2 for mustard and radish. The sown substrates were moistened with water and introduced into a growth chamber at 18 °C and 90% relative humidity (RH) until the germination of the seeds. Then, they were moved into an unheated greenhouse where no artificial light treatment was applied. The incidence of natural light at this time of year provided a daily average of 18 °C and 61% RH. The following nutritive solution expressed as mmol/L for each component was applied daily: NO3− (5.3), H2PO4−2 (1.5), SO4−2 (4.4), HCO3− (0.5), Cl− (5.3), K+ (1.5), Ca+2 (6.3), Mg+2 (1.3) and Na+ (3.1). An average fertigation value of 20.4 l m−2 per day from June to September 2017 was recorded. No phytosanitary treatment was used.Microgreens were produced by the Agronomic Innovation Center (CIAM) of Grupo Alimentario Citrus Company  at the end of August 2017. A hydroponic system was created by placing substrates of pine tree fibers (12 cm × 12 cm × 0.4 cm) on plastic trays. Two seeding densities were selected: 3.8 seeds cmBrassicaceae microgreens [Nine days after seeding for radish and 7 days for broccoli, kale and mustard, the microgreens were transported in plastic trays (58 cm × 39 cm) from CIAM to the University of Valencia (UV). They were fertigated just before being moved in order to maintain good humidity conditions during the 30 min period of transportation. In our laboratory at the UV, a total of 40 trays were received . For each microgreen, approximately 400 g were harvested as close as possible to the root using sterilized scissors. Next, a pool was made to homogenize each microgreen sample, and then they were randomly divided into several replicates. Fresh microgreens were used immediately for ascorbic acid analysis and the rest of the collected samples were weighted inside aluminum containers before freezing at −80 °C. Frozen microgreens were lyophilized for 48 h  and maintained in a desiccator until constant weight to obtain dry weight (DW) percentage  in accordance with the range 3.9–8.1% described in previous studies on these rogreens . Next, s4)2CO3), sodium bicarbonate (NaHCO3) and calcium chloride dihydrate (CaCl2(H2O)2) were purchased from Sigma Chemical Co. . Potassium chloride (KCl), sodium chloride (NaCl), magnesium chloride hexahydrate (MgCl2(H2O)6) and potassium dihydrogen phosphate (KH2PO4) were supplied by Merck . Culture-grade water was obtained from B. Braun . Simulated Salivary Fluid (SSF), Simulated Gastric Fluid (SGF), Simulated Intestinal Fluid (SIF) and enzymatic activity assays were prepared according to Minekus et al. (2014). A water bath with orbital shaking  and centrifuge  were used to simulate the gastrointestinal digestion process.Pepsin , pancreatin , bile extract (porcine), ammonium carbonate ((NH2HPO4), potassium phosphate dibasic (K2HPO4), sodium phosphate dibasic (Na2HPO4), potassium chloride (KCl) and sodium acetate (C2H3NaO2) were obtained from Merck . Sodium carbonate (Na2CO3), potassium persulfate (K2S2O8), Folin-Ciocalteu reagent, 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2,2′-azobis-(2-amidinopropane) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), gallic acid and sulforaphane standard (≥90%) were purchased by Sigma Chemical Co. . Ethanol (96%), methanol and n-hexane (96%) were provided from Scharlau . Sodium fluorescein was obtained from Fluka Chemie AG  and 1,2-benzenedithiol (BDT) (96%) from Acros organics . Water was purified by a Milli-Q system .Glacial acetic acid, metaphosphoric acid, formic acid and L (+) ascorbic acid (≥99%) were supplied by Panreac Química . Sodium salt dihydrate 2,6-dichlorophenolindophenol (DCFI), Tris(hydroxymethyl)aminomethane, potassium phosphate monobasic (Na3) (65%) were purchased by Merck  while hydrochloric acid (37%) was obtained from Scharlau .Titrisol concentrated standards (1000 mg) of macro and oligoelements  and nitric acid  and 5 µL of 0.3M CaCl2 were added to the gastric solution obtained and agitated for 1 min. The pH was adjusted at 7.0 ± 0.1 with 6M NaOH and culture-grade water was added up to a final volume of 40 mL. The intestinal mixture was incubated again at 95 opm and 37 °C for 2 h and after this period the digested samples were cooled in an ice bath and centrifuged at 3100 g and 4 °C for 90 min to obtain the bioaccessible fraction (BF). The values of the blank of digestion obtained in each assay were subtracted from the values of the digested microgreen samples to remove possible interferences caused by digestive enzymes or simulated fluids, in order to avoid overestimation of results. The results of bioaccessibility were calculated as the ratio between the concentration of each bioactive compound in the BF and the initial concentration in microgreens. The results were expressed as percentage of bioaccessibility according the next Equation (1):Freeze-dried samples were rehydrated to their original moisture contents in order to be as close as possible to the edible fresh microgreen , and in w/v) metaphosphoric acid (20 mL) were homogenized in a Polytron  at 15,000 rpm for 1 min, centrifuged at 3000 g for 20 min and 4 °C, and filtered through Whatman nº 4 filter paper. BF samples were directly used for the titrimetric method. Both kind of samples were mixed 1:1 (v/v) with acetic acid—Metaphosphoric acid and the amount of acid ascorbic was measured using 2,6-DCFI. Concentration of AA was calculated by using L (+)-ascorbic acid standard solution (1 mg/mL). The results were expressed as mg AA/100 g fresh weight (FW).Total ascorbic acid (AA) was determined by the AOAC Official Method 967.21  and the v/v) acetone/Tris buffer solution (pH 7.4) and mixed overnight in darkness at room temperature. Afterwards, samples were centrifuged at 3100 g for 10 min and supernatants were diluted 1/6 (v/v) in acetone/Tris buffer solution before measuring absorbance at 470, 537, 647 and 663 nm. Carotenoid content was obtained by following the next Equation (2) and the results were expressed as mg of β-carotene/100 g DW.663 − 0.00697 A647 − 0.002228 A663Anthocyanin = 0.8173 A663 − 0.000897 A537 − 0.003046 A647Chla = 0.013773 A647 − 0.004305 A537 − 0.005507 A663Chlb = 0.024054 ATotal carotenoids were extracted as described by Sims and Gamon . Quantifg for 8 min. The supernatant was diluted 1/5 (v/v) in water and 100 µL was used for cyclocondensation reactions [The extraction of total isothiocyanates was performed as described by Torres-Contreras et al. . Freeze-eactions . BF sampw/w) acetic acid aqueous solution (4 mL) in a sonicator for 10 min. After centrifugation at 3100 g for 10 min at 4 °C and filtering through a Whatman nº 4 filter paper, the extract of microgreens or BF were diluted 1/5 (v/v) in two different buffer solutions (0.025 M potassium chloride pH 1 and 0.4 M sodium acetate pH 4.5). Absorbance of diluted samples in both buffers was measured at 520 and 700 nm. The anthocyanin concentration was calculated according to the following Equation (3), and the final results were expressed as mg of cyanidin-3-glucoside/100 g DW.A = (A520 − A700) pH1 − (A520 − A700) pH 4.5MW (molecular weight for cyanidin-3-glucoside) = 449.2 g/molDF (dilution factor) = 53 = factor for conversion from g to mg10ε = 26,900 molar extinction coefficientl = path length in cmAnthocyanin pigments were extracted according to Hanlon and Barnes  with somg for 5 min at 4 °C and the hexane phase was discarded). The washed methanolic extract was filtered using Whatman nº 4 filter paper and an aliquot of 100 µL of sample extract, BF or standard was mixed with 3 mL of 2% (w/v) sodium carbonate aqueous solution and 150 µL of 50% (v/v) Folin-Ciocalteu reagent. The mixture was incubated at room temperature in darkness for 1 h, and the absorbance at 765 nm was measured on a spectrophotometer . Quantification was achieved using a gallic acid external standard calibration curve in the range of 0–1000 mg/L. The results were expressed as gallic acid equivalent (GAE)/100 g DW.The total soluble polyphenols content was analyzed by the Folin-Ciocalteu method with some modifications , and extLyophilized microgreens were previously subjected to the same methanolic extraction process described above for total soluble polyphenols and BF were directly used.+ radical cation stock solution was generated by chemical reaction with 7 mM ABTS and 140 mM K2S2O8 overnight in darkness at room temperature. Next, it was diluted in ethanol until an absorbance of 0.700 ± 0.020 at 734 nm and 30 °C to obtain the ABTS+ working solution. The optimal dilution of the samples to obtain a percentage of absorbance inhibition of approximately 50% was 1/3 (v/v) in ethanol. At the same time, Trolox standard solutions were prepared in a range of 0 to 300 µM. The absorbance of 2 mL of ABTS+ working solution was considered the initial point of reaction (A0). Then, diluted samples or Trolox standards (100 µL) were added immediately and the absorbance were measured after 3 min (Af). All readings were carried out in a thermostatized UV–vis spectrophotometer. The percentages of absorbance inhibition were obtained from the following Equation (4): TEAC assay measures the reduction of the radical cation ABTS by antioxidant compounds, and the spectrophotometric method proposed by Cilla et al.  was used3 1420  with fluorescence filters for an excitation wavelength of 485 nm and an emission wavelength of 535 nm at 37 °C. The optimization of the assay parameters was required. Sodium fluorescein and freshly prepared AAPH solution were used at a final concentration of 0.015 and 120 mg/mL respectively. Samples were diluted 1/250 (v/v) and 20 µM Trolox was used as antioxidant standard. All of them were prepared with phosphate buffer . The final reaction consisted of 80 μL of fluorescein, 40 μL of AAPH and 80 μL of diluted sample, Trolox standard or phosphate buffer (blank) and the fluorescence was recorded every 5 min over 70 min . The results were calculated considering the differences of areas under the fluorescence decay curve (AUC) between the blank and the sample over time, and were expressed as µM Trolox Equivalents/100 g DW.The ORAC assay measures the capacity of the antioxidant compounds to scavenge peroxyl radicals; the fluorimetric method described by Cilla et al.  was used2O3) and cesium chloride (CsCl) at 0.1% (p/v) were added to samples and standards to eliminate possible chemical interferences of phosphate on calcium and to avoid potassium ionization, respectively.The main macroelements  and oligoelements  were evaluated according to Cilla et al. ,38. BrieMineral concentrations were determined by flame atomic spectrometry  and the quantification of minerals was calculated from their standard calibration curves (mg/L): K (0.25–2.5), Ca (0.125–5.0), Mg (0.125–1.0), Fe (0.0625–5.0) and Zn (0.0625–2.5). The results were expressed as mg of each element/100 g FW. In addition, a dried hay powder  was used to confirm the accuracy of the method. It was prepared and analyzed using the same procedure as that followed for the microgreen samples. The certified and experimental values were (mg/L) 640 ± 10 and 609 ± 4 for calcium, 145 ± 4 and 115 ± 1 for magnesium, 3380 ± 80 and 2850 ± 18 for potassium, 11.4 ± 0.0 and 13.4 ± 0.1 for iron and 3.2 ± 0.17 and 4.01 ± 0.10 for zinc, respectively. The coefficient of variation with regard to the precision for all minerals was in the range of 0.62–2.42%.p < 0.05, was used. All analyses were performed with the software Statgraphics Plus 5.1 .All analyses were carried out in triplicate in at least two independent experiments, and data were expressed as mean ± standard deviation. Experimental data were subjected to one-way analysis of variance (ANOVA) to determine significant differences among samples composition. Tukey’s multiple range test, at a significance level of The results of antioxidant bioactive compounds content in fresh microgreens and their bioaccessible fraction, as well as the bioaccessibility, are shown in p > 0.05) were found between broccoli, kale and mustard. For radish, lower (46–66 mg/100 g DW) and similar contents (85–200 mg/100 g DW) have been described [Brassicaceae microgreens analyzed showed extremely high total carotenoid concentrations compared to their mature counterparts (Brassicaceae family) [Regarding to total carotenoids content, the concentration ranged from 162 to 224 mg β-carotene/100 g DW. Radish microgreen showed the lowest value before the digestion process, and no statistically significant differences  .The total isothiocyanate concentration in microgreens ranged from 608 to 810 mg sulphoraphane/100 g DW . MustardBrassica microgreens with red to purple seed-leaves have been identified [Total anthocyanin content in microgreen samples were from 1.4 to 36.4 mg cyanidin-3-glucoside/100 g DW, following this increasing order: kale, radish, broccoli and mustard . The scaentified . Regardientified . In contentified  and 1480entified  have beeentified . In addientified . Differeentified  suggesteentified .Brassica microgreens revealed more complex profiles and a greater variability in the content of polyphenols in microgreens compared to mature plants [Brassicaceae family including radish and mustard. Lower values of BA were determined by HPLC in broccoli flavonoids (11%) and for total polyphenols in raw kale (15%) or in kale subjected to different culinary techniques (7%) [Total soluble polyphenol content in the microgreens varied from 1890 to 2416 mg GAE/100 g DW, with the highest value for kale and the lowest for mustard . Two none plants . As for e plants  using thues (7%) ,55,60. Tues (7%) . The meaThe results of total antioxidant capacity determined by TEAC and ORAC methods in microgreens, their bioaccessible fractions and the percentage retained in the BF are shown in Brassicaceae microgreens grown in peat moss substrate evaluated by Xiao et al. [The total content of mineral elements in the microgreens before and after gastrointestinal digestion and their BA are reported in o et al.  and expro et al.  and compo et al.  macro- ao et al. . As for o et al. . In geneo et al.  demonstro et al. , and couo et al. . Brassicaceae have been described in two different studies. Lucarini et al. [The highest BA for the three macroelements analyzed was found in mustard microgreens. In contrast, broccoli microgreens showed lower BA values for Ca and Mg. Although Fe and Zn could not be detected in the BF, a decrease in the amount of macroelements occurred after digestion process, high BA  was observed. This fact could be probably ascribed to the low content of ascorbic acid and high content in total soluble polyphenols in the BF  of broccoli, in contrast to mustard. There are no data in the literature about BA of mineral elements in microgreens. However, the values of Ca BA in conventional vegetables of i et al.  obtainedi et al.  showed 3Brassicaceae microgreens produced in this study could be considered as good sources of minerals and antioxidant phytochemicals in a balanced human diet. In particular, they contain relevant amounts of vitamin C, higher levels of total carotenoids than adult plants, mineral and antioxidant bioactive compound contents comparable to other hydroponic microgreens, and low K, making them suitable for patients with impaired kidney disease.In general, the four hydroponic In this study, bioaccessibility data for antioxidant bioactive compounds, total antioxidant capacity, and mineral elements in microgreens are provided for the first time. Radish and mustard showed the highest BF and BA values for antioxidant parameters, while broccoli and mustard provided the lowest and highest values for minerals, respectively. Despite the expected decrease in different compounds after the in vitro digestion process, the bioaccessible fractions of microgreens still contained remarkable total antioxidant capacities and bioactive compounds with potential beneficial local effects in the gastrointestinal tract. For future studies, determining the bioaccessibility of the antioxidant phytochemicals in more microgreen species, as well as their potential bioactivity in pre-clinical and human intervention studies, ought to be addressed.


Taken together, our results suggest that improvements in mitochondrial function with colder temperatures may contribute to energy conservation and enhance cellular viability in hypoxic brain.Therapeutic hypothermia is a strategy that reduces metabolic rate and brain damage during clinically-relevant hypoxic events. Mitochondrial respiration is compromised by hypoxia, with deleterious consequences for the mammalian brain; however, little is known about the effects of reduced temperature on mitochondrial metabolism. Therefore, we examined how mitochondrial function is impacted by temperature using high resolution respirometry to assess electron transport system (ETS) function in saponin-permeabilized mouse brain at 28 and 37°C. Respirometric analysis revealed that, at the colder temperature, ETS respiratory flux was ~ 40–75% lower relative to the physiological temperature in all respiratory states and for all fuel substrates tested. In whole brain tissue, the enzyme maximum respiratory rates for complexes I-V were similarly reduced by between 37–88%. Complexes II and V were particularly temperature-sensitive; a temperature-mediated decrease in complex II activity may support a switch to complex I mediated ATP-production, which is considerably more oxygen-efficient. Finally, the mitochondrial H Animals were not fasted prior to experimental trials.All protocols were performed with the approval of The University of British Columbia Animal Care Committee. BALB/c Mice [2EDTA; 1% fatty acid-free BSA, pH 7.4 at 4°C) and then minced over ice for 2–3 mins until the individual tissue pieces were uniformly smaller than grains of sand. The resulting homogenate was permeabilized with 4 mM saponin in homogenization buffer for 45 mins, as described previously [2-EGTA, 5.8 NaATP, 6.6 MgCl2, 20 imidazole, 20 taurine, 50 potassium 2-(N-morpholino)ethanesulfonic acid (K-MES), 15 Na-phosphocreatine, 0.5 dithiothreitol (DTT); pH 7.1, adjusted with 5 N KOH), rinsed for 2 mins on ice, and then re-suspended in BIOPS medium. This rinse procedure was repeated 3 times. Permeabilized cells were kept on ice until use and all mitochondria were assayed within 2 hrs of isolation.Animals were sacrificed by cervical dislocation and whole brains were extracted over ice and bisected laterally within 30 secs. One half of each brain was frozen in liquid nitrogen and stored at -80°C for enzyme analysis (see below); the other half was placed into ice-cold homogenization buffer . Respiratory flux through the ETS was measured in permeabilized brain cells using a substrate-uncoupler-inhibitor-titration (SUIT) protocol described previously [p-phenylene-diamine  and ascorbate (2 mM) were added to the chamber to assess respiratory flux through complex IV. State II respiration was used as a proxy for leak respiration because native ATPases prevent the establishment of steady state IV respiration in the saponin-permeabilized cell preparation. Respiration values were obtained from steady-state conditions following each chemical addition. Protein content was analyzed using the Bradford technique [Permeabilized brain mitochondrial respiration was measured with an Oroboros Oxygraph 2-k high-resolution respirometry system , as described previously . Two ideeviously , 16. Briechnique .+-motive force, in the presence of succinate and oligomycin, using tetraphenylphosphonium (TPP+), as described previously [+ ion selective electrode (Oroboros). Note that this methodology excludes any contribution of ΔpH and therefore resulting measurements of the H+-motive force are likely slight underestimates of the true value. Mitochondrial matrix volume was not measured because changes in this parameter have minimal impact on TPP-mediated measurements of ΔΨm [+ flux were determined by inhibiting the substrate oxidation component by stepwise addition of 0.5 or 1.0 μl aliquots of malonate (2.0 M stock) and measuring the effect on ΔΨm. After the final malonate addition, FCCP (1 μM) was added to uncouple mitochondria and determine the degree of electrode drift. Proton flux curves were fit using a two-parameter exponential growth curve. A limit of the permeabilized brain preparation is that it usually presents high non-specific binding, which may vary with temperature, and is therefore not well suited to determine accurate values of ΔΨm. Therefore, in the present study we focused on the comparison of mitochondrial conductance and kinetics at the two different temperatures and not the absolute values of membrane potential.Proton flux kinetics across the inner mitochondrial membrane were assessed by simultaneous measurement of oxygen consumption and Heviously  and usins of ΔΨm . We empls of ΔΨm , 28. Themax) were assessed using spectrophotometric biochemical assays from whole brain, as described previously [max was determined at either 28 or 37°C. Then, Q10 ratios were calculated from complex Vmax data using the formula Q10 = (R2/R1)10/T2-T1, where R was the Vmax of a given enzyme at a given temperature, and T represented the two experimental temperatures at which Vmax was assayed.Citrate synthase (CS) and ETS complex maximal activities . For respirometry data, significance was evaluated using a repeated-measures 2-way ANOVA to test for significant interactions between the two independent variables: (i) the substrate or inhibitor injected (e.g. ADP vs. succinate), and (ii) the experimental temperature. Bonferonni post hoc multiple comparisons tests were run on each of the dependent variables to compare the single point means of interest. For total FCCP-uncoupled respiration, non-mitochondrial oxygen consumption, and Vmax data, significance was assessed using a student’s t-test. P < 0.05 was considered to achieve statistical significance unless otherwise indicated.Statistical analysis was performed using commercial software . For all experiments, individual F3,30 = 15.0, p < 0.0001). Further analysis with Bonferroni posthoc tests revealed specific changes in state II respiration , and the activities of complexes I, II and IV, which were each ~ 60–65% lower in permeabilized brain at 28°C relative to at 37°C . As a result of this consistent downregulation of the ETS, total ETS capacity  was reduced by ~ 67% in 28°C (p = 0.032). Non-mitochondrial oxygen consumption was determined following injection of antimycin A and was not significantly different between experimental temperatures (10 ratios for each respiratory state. The Q10 ratios for each respiratory state were relatively similar and ranged from 2.61 ± 0.09 (State II respiration) to 3.92 ± 0.31 (State III respiration)  and at a clinically-relevant hypothermic temperature (28°C). In permeabilized brain treated at the hypothermic temperature, respiratory flux through the ETS was decreased relative to permeabilized brain treated at the physiological temperature . Specifiiration) .max of CS and complexes I-V of the ETS at our two experimental temperatures  = 2.473, p = 0.0483), II (t(3) = 7.913, p = 0.0005), III (t(3) = 3.848, p = 0.0183), IV (t(3) = 3.717, p = 0.0099), and V (t(3) = 12.36, p = 0.0001) were all decreased in the hypothermic experiment by 35, 73, 38, 55, and 88%, respectively . Specifically, at physiological temperatures, the maximum state II respiration rate of mouse permeabilized brain was nearly 3-fold greater than in the hypothermic temperature . However, the rates at which oxygen consumption decreased and Ψm discharged with malonate additions was not affected by assay temperature . As a result of these matched reductions in respiration rates across experimental temperatures with malonate additions, respiration rates in partially inhibited mitochondria at physiological temperature remained elevated several-fold higher than in equally inhibited samples from hypothermic experiments. For example, when H+ flux was considered at a common Ψm of 125 mV, H+ flux at 28°C was ~ ¼ that at 37°C , the Q10 of state II respiration is 2.4 [10 value of 3.9 for murine brain State III respiration, which falls reasonably between the reported Q10 values for State III-respiring muscle (~ 2.8) and liver (~ 6.5) mitochondria, isolated from summer active squirrels and measured between 25–37°C [We compare the functional characteristics of hypoxia-intolerant murine brain mitochondria between a physiological temperature (37°C) and a clinically-relevant therapeutic hypothermia temperature (28°C). We report that at 28°C, murine permeabilized brain has lower ETS respiration rates  and maxi 2.5–4.0 , Table 1n is 2.4 , which a 25–37°C .in vitro temperature coefficient for murine brain ETS respiratory flux (in FCCP-uncoupled permeabilized brain) of 3.3, indicating that this tissue is moderately temperature-sensitive. This sensitivity may be expected to confer significant metabolic rate reduction-related energy savings at the colder temperature, which likely contributes to enhanced neuroprotection during hypoxia or ischemia in therapeutic hypothermia-treated subjects. Indeed, reducing metabolic demand when oxygen is limiting, particularly in brain tissue, is a common strategy employed by hypoxia-tolerant and hypoxia-adapted species [10 of H+ leak at a common resting membrane potential (125 mV) is 4.4, indicating that murine brain H+ leak decreases faster than does respiration rate with decreasing temperatures. Greater reductions in H+ leak indicate enhanced mitochondrial efficiency and are expected to confer further metabolic savings in hypoxia or ischemia, thereby likely contributing to the neuroprotective effect of therapeutic hypothermia treatment.Overall, we report an  species , includi species . Moreovein vitro respiration measurements from permeabilized brain, ex vivo whole brain assays of enzyme Vmax’s reveal more varying thermal sensitivity in mitochondrial function (10’s 1.2–2.4), whereas complex II (4.2), and particularly, complex V (11.3) are highly sensitive to temperature changes. The greater temperature sensitivity  between our whole brain and permeabilized brain measures of complex V activity may be due to reduced manipulation and degradation of the whole brain sample relative to permeabilized brain samples, and thus the Vmax data is likely more representative of in vivo flux potential for each complex.Relative to function ; Table 2+/O ratio is more efficient for complex I respiration (~ 10) than for complex II respiration (~ 6) [This profile of temperature sensitivity would favour a switch towards CI-mediated ATP-production, which is considerably more oxygen-efficient than CII-mediated ATP production . Such a on (~ 6) . Maximizon (~ 6) , whereason (~ 6) , and thuin situ are ~ 10°C warmer than physiological temperatures and that Vmax activity is maximized at ~ 50°C [An important caveat of our study concerns the experimental temperatures employed. Specifically, a recent study has demonstrated that mitochondria t ~ 50°C . HoweverMitochondria are the nexus of oxygen consumption and metabolism in the cell, and also coordinate deleterious and neuroprotective responses to low oxygen stress . Our fin


Glycyrrhiza consists of about 30 species, amoung these, G. glabra is the source of several phenolic compounds, known as flavonoids, such as licoagrodin, licoagrochalcones, licoagroaurone and licochalcone C, kanzonol Y, glyinflanin B and glycyrdione A, which have shown various pharmacological activities, including antitumor, antiparasitic, antileishmanial, anti-ulcer and antioxidative effects. Among these compounds, licochalcone C was isolated but its biology has not been fully examined. In our study we reproduced an inflammatory state by treating THP-1 (human myelomonocytic leukaemia) cells with pro-inflammatory stimuli, such as LPS and IFN-γ and we investigated the possible antioxidant activity of licochalcone C at a concentration of 50 μM. Our results show that treatment with licochalcone C attenuates the LPS-IFN-γ-induced inflammatory response by significantly decreasing the expression and activity of iNOS via NFκB (nuclear factor kappa-B), by influencing extracellular O2− production, and by modulating the antioxidant network activity of SOD (superoxide dismutase), CAT  and GPx (glutathione peroxidase) activity. Based on these results we hypothesize that Licochalcone C has antioxidant properties since it reduces the production of superoxide radicals and consequently reduces the activity of iNOS.The genus  Leguminosae) is a perennial herb that grows in Southern Europe, central Russia, Turkey, Iraq and Iran. It has been used in traditional medicine for the roots of several Glycyrrhiza species since ancient times. The genus Glycyrrhiza consists of about 30 species, among these, G. glabra is the source for several phenolic compounds, known as flavonoids, such as licoagrodin, licoagrochalcones, licoagroaurone and licochalcone C, kanzonol Y, glyinflanin B and glycyrdione A, which shown various pharmacological activities, including antimutagenic, antiparasitic, antileishmanial, anti-ulcer and antioxidative effects. . Cell counts were used to calculate results as nanomoles O2− per 106 cells per minute.Production of Oritchard  FerricytSOD activity was determined as described by Sun and Zigman . The ass2O2 was monitored continuously at 240 nm. The assay mixture in a final volume of 3 mL contained 10 mM potassium phosphate buffer, 10 mM H2O2 and 1.5-11 μg of protein of enzymatic extract. CAT units were defined as 1 μmole H2O2 decomposed/min at 25 °C.CAT activity was measured spectrophotometrically . The decet al.  for 30 min at 4 °C. For preparation of nuclear extract the cells, resuspended in 10 mM Tris–HCl, pH 7.4, 10 mM NaCl, 2 mM MgCl2, 0.6% Triton X-100, 1.0 mM PMSF, 1 mg/mL leupeptin, and aprotinin, were incubated at room temperature for 2 min, then cooled on ice for 5 min. After five passages through a 22-G needle, MgCl2 concentration was adjusted to 5 mM. Nuclei were obtained by centrifuging the suspension at 1200g for 15 min and cytoplasmic fractions consisted of the postnuclear supernatants. Nuclei were then harvested in RIPA buffer .THP-1 cells were washed once in cold phosphate-buffered saline  using the Breadford method, were separated by electrophoresis in a 7.5% sodium dodecyl sulfate–polyacrylamide gel  and transferred at 4 °C to nitrocellulose membrane  in glycine–methanol buffer. Nitrocellulose was then blocked in Tris-buffered saline (TBS)–milk and incubated, overnight, with various primary antibodies: anti-human iNOS , monoclonal anti-3-nitrotyrosine  and anti-human NF-κB (Santa Cruz Biotechnology). The nitrocelluloses were then washed in TBS, incubated with a secondary antibody conjugated with alkaline phosphatase for 2 h, washed again, and developed in an alkaline buffer with nitroblue tetrazolium (NBT) as substrate . Antibody anti-β-Actin (Sigma), and Actin (Sigma), were used as an internal standard. The densitometric analysis of Western blots was performed using a computerized densitometric system .Semi-quantitative reverse-transcribed polymerase chain reaction (RT-PCR) was used to determine mRNA levels of the inducible Nitric Oxide Synthase. Total RNA was extracted using 1 mL ULTRASPEC-RNA , as recommended by the manufacturer. RNA was dissolved in diethyl pyrocarbonate (DEPC)-treated water and quantified spectrophotometrically at 260 nm.2, 10x PCR buffer in a final volume of 20 μL. Reverse transcription was performed at 42 °C for 1 hr followed by heat inactivation of reverse transcriptase at 92 °C for 10 min. 18S was amplified from the same amount of RNA to correct for variation of different samples. PCR amplification was performed using a Programmable Thermal Controller . The PCR solution contained 10 μL of first-strand cDNA, 4 μL 10x PCR buffer, 2 mM MgCl2. The following primer pairs were used: sense 5’-CGT AAA GAC CTC TAT GCC AA-3’ and antisense 5’-AGC CAT GCC AAA TGT CTC AT-3’ for iNOS, and 18S primers, sense 5’-TAC GGA GCA GCA AAT CCA C-3’ and antisense 5’-GAT CAA AGG ACT GCA GCC TG-3’, 2U Termophylus Acquaticus (Taq) DNA polymerase , and water to a final volume of 50 μL. These samples were overlaid with mineral oil and subjected to 35 cycles at 95 °C for 60 sec, 60 °C for 60 sec, and to one cycle at 72 °C for 7 min for iNOS and 40 cycles at 95 °C for 60 sec, 58 °C for 60 sec, and to one cycle at 72 °C for 7 min for 18S. PCR products were run on 2% agarose gel electrophoresis and photographed after ethidium bromide staining under UV light. Bands on the gel were scanned using a computerized densitometric system .First-strand cDNA was generated by adding RNA (1 μg) to a mixture containing 1 mM deoxy-nucleoside-triphosphates (d-NTP), 1U/μL RNase inhibitor, 2.5 U/μL moloney murine leukemia virus reverse transcriptase, 2.5 μM oligo-dt, 5 mM MgCl3H) arginine monohydrochloride 64 Ci/mM, 1 μCi/μL  and 50 μL NADPH 10 mM were added to each cell homogenate samples and incubated for 30 min at room temperature. After incubation, the reactions were stopped with 400 μL of stop-buffer  and added of the equilibrated resin into each sample. The equilibrated resin bound unreacted arginine. After centrifugation, the radioactivity corresponding to L-(3H)-citrulline was measured with liquid scintillation spectrometry. Calcium was omitted from these incubations to favour the determination of the calcium independent iNOS isoform.The measure of the conversion of L-arginine to L-citrulline is a standard assay method currently used to quantitative NOS activity. Briefly, 10 μL of radioactive arginine, L-. Probability of null hypothesis of <5% (p < 0.05) was considered statistically significant.et al. [2− production and modulation of antioxidant network activity of SOD, CAT and GPx in a biological model. Moreover, licocalchone C, by scavenging ROS, is responsible for the inhibition of iNOS via NFkB in THP-1 cell line. Decreasing NO production, significantly prevents the formation of ONOO−. Since both the NO and NF-kB are important inflammatory mediators, they play key roles in modulating the formation of various radical species during the oxidative action of licocalchone C, and may represent a new target of action against inflammatory diseases which have in common an alteration of the redox status.As many studies have demonstrated that the use of several compounds of plant origin show a multiplicity of pharmacological activities against a number of pathological conditions, in this study we isolated the structural isomer of licocalchone A, licocalchone C, from licorice, which in the literature was shown to exhibit various biological activities ,39,40,41et al.  we found


O-demethylation, with a high yield of 97%. Cellular accumulation and cytotoxicity of naringenin and 8-prenylnaringenin in normal (BJ) and cancer cells (U-118 MG) was also examined. Obtained data indicated that 8-prenylnaringenin exhibited higher cytotoxicity against used cell lines than naringenin, and the effect of both flavones was stronger in U-118 MG cells than in normal fibroblasts. The anticancer properties of 8PN correlated with its significantly greater (37%) accumulation in glioblastoma cells than in normal fibroblasts. Additionally, naringenin demonstrated higher selectivity for glioblastoma cells, as it was over six times more toxic for cancer than normal cells. Our results provide evidence that examined prenylated and non-prenylated flavanones have different biological activities against normal and cancer cell lines, and this property may be useful in designing new anticancer drugs for glioblastoma therapy.Gliomas are one of the most aggressive and treatment-resistant types of human brain cancer. Identification and evaluation of anticancer properties of compounds found in plants, such as naringenin (N) and 8-prenylnaringenin (8PN), are among the most promising applications in glioma therapy. The prenyl group seems to be crucial to the anticancer activity of flavones, since it may lead to enhanced cell membrane targeting and thus increased intracellular activity. It should be noted that 8PN content in hop cones is 10 to 100 times lower compared to other flavonoids, such as xanthohumol. In the study presented, we used a simple method for the synthesis of 8PN from isoxanthohumol— Humulus lupulus L., Cannabaceae), have encouraged researchers to evaluate their possible applications in therapies for various diseases. This activity is based on molecular mechanisms involving mainly inhibition of cyclooxygenase (COX), and lipooxygenase (LOX) − ion of m/z = 339.1235, in accordance with the mass of [C20H20O5 − H]− (339.1232). In the IR spectrum, there was no absorption band at 1275 cm−1, observed for the substrate (–OCH3 group).The analysis of the product spectral data . In the DEPT 135° spectrum of the product, there are no signals of C=O (δ = 198.6 ppm), C-8 (δ = 109.3 ppm), C-9 (δ = 165.9 ppm), C-10 (δ = 104.3 ppm), and C-1’ (δ = 132.1 ppm), which indicates that they are the quaternary carbon atoms, whereas the signals of methylene carbon atoms C-1” and C-3 (δ = 23.2 ppm and δ = 44.4 ppm) were negative. The absorption band in the UV spectrum was observed at λmax = 293.6 nm.In the low-field region of the Prenylated hop compounds may play an important role in therapy of brain tumors. The major hop chalcone xanthohumol reduced cell viability and induced apoptosis in the U87 glioma cells and T98G cells of human glioblastoma multiforme .Due to reports that substitution of the prenyl group increases the hydrophobicity of flavonoids and may led to its higher cellular accumulation  and in c50 for glioblastoma was found to be approximately 138 and 211 µM for 8-prenylnaringenin and naringenin, respectively, whereas in normal fibroblasts IC50 was 172 and 1090 µM for 8PN and N, respectively. It has to be pointed out that naringenin was more selective for glioblastoma cells as it was over 6 times more cytotoxic for cancer than normal cells, while higher inhibitory activity of 8-prenylnaringenin for both cell types was evident.Neutral red cell viability assays showed that both flavonoids exerted stronger inhibitory effects on the cancer cell lines as compared with normal cells . At conc50 from 2.2 to 178 µM) and very weak activity in normal human skin fibroblasts (142BR) (IC50 > 183 µM). Ayob et al. −; cald: 339.1232 [C20H20O5 − H]−. Anal. calcd. for C20H20O5: C 70.57; H 5.87. Found C 70.61; H 5.98. UV (MeOH) λmax: 338.7; 293.6 nm.8-Prenylnaringenin (yield 97%) m.p. = 192–193 °C lit. 193 °C  IR (KBr)Human glioblastoma U-118 MG cells (ATCC HTB-15) and normal human fibroblast BJ cells from a newborn male’s foreskin , Dulbecco’s Modified Eagle’s Medium (DMEM), Eagle’s minimum essential medium (EMEM), fetal bovine serum (FBS), and penicillin and streptomycin solution were obtained from the American Type Culture Collection . Tripsin-EDTA solution, phosphate-buffered saline (PBS) with and without magnesium and calcium ions, hydrocortisone, 0.33% neutral red solution (3-amino-m-dimethylamino-2-methyl-phenazine hydrochloride), and 0.4% trypan blue solution were provided by Sigma-Aldrich . DAPI  was purchased from Life Technologies . Chamber culture slides were obtained from Nunc , and all other cell culture sterile materials were purchased from Corning Incorporated .2, and at 95% humidity. Growth media were changed every 3 days and cells were passaged at 80% confluence after treatment with 0.25% trypsin-EDTA in PBS . Cell morphology was checked under a Nikon TE2000S Inverted Microscope  with phase contrast. Number and viability of cells were estimated by the trypan blue exclusion test using an Automatic Cell Counter TC20™ . All assays were performed in triplicate in three independent experiments.Glioblastoma cells were cultured in DMEM, and normal human skin fibroblasts in EMEM media, containing 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C, in 5% CO1H-NMR, 13C-NMR, DEPT 135°, COSY, HMQC, HMBC) of 8-prenylnarinenin were recorded at 500 MHz on a Bruker Ultra Shield TM Plus instrument in acetone-d6. UV spectra  were recorded in methanol. HR ESI-MS was taken on a Bruker micrOTOF-Q spectrometer. Elemental analysis was done with an EA 1108, Carlo Erba analyzer. IR spectra were measured on a Mattson IR 300 Thermo-Nicolet spectrophotometer .The NMR spectra  with a mixture of CH3Cl:MeOH in various ratios as developing systems. Compounds were detected by spraying the plates with 1% Ce(SO4)2 and 2% H3[P(Mo3O10)4] in 10% H2SO4, followed by heating to 120–200 °C. The product was separated by column chromatography using silica gel .Analytical TLC was carried out on silica gel G 60 F4 cells/well and allowed to attach for 24 h before treatment. Working solutions of naringenin or 8-prenylnaringenin (1–500 µM) were prepared in culture media. The DMSO concentration was adjusted to 0.1% in all samples, which had no significant effect on treated cell lines (not shown). After 24 h exposure to naringenin or 8-prenylnaringenin, media were replaced by 100 μL of NR solution (2% of the culture medium volume) and cells were incubated for 1 h (U-118 MG) or 2 h (BJ), followed by washing (with PBS). 100 μL/well of fixative  was added and plates were shaken until complete dye dissolution . Absorbance was measured at 540/620 nm with a microtiter plate reader  against a blank sample (fixative without cells).The cytotoxic effect of N and 8PN was estimated by neutral red assay (NR), which is based on the ability of normal cells to incorporate and bind neutral red within lysosomes. NR cell assay is more sensitive than other commonly applied tests  . Both ce4/well. After 24 h incubation, culture media were replaced with a 10 µM solution of naringenin or 8-prenylnaringenin and cells were incubated for 24 h, followed by rinsing with PBS (3 times). Cells were fixed with 3.7% formaldehyde solution, washed with PBS (3 times), and cell nuclei were stained with 300 nM DAPI in PBS for 15 min. The images were collected with an Olympus FV10i confocal microscope  under 60× magnification, using a 60× water immersion lens in blue (DAPI) and green (FITC) channel with the pinhole set to 1.0 confocal aperture in each channel . Excitation and emission for flavonoids were set to 490 and 520 nm, respectively, according to results from Mukai et al. [BJ and U-118 MG cells were seeded in 8-well chamber culture slides at a density of 4 × 10i et al. . ConfocaConfocal microscopy images were analyzed using ImageJ software . The total corrected cell fluorescence (TCCF) was measured using the procedure described by McCloy et al. . Each cep < 0.05 was considered as statistically significant. Calculations were performed using Statistica 12 software .To estimate the differences between flavonoid-treated and non-treated control samples, a statistical analysis was performed using the nonparametric Kruskal–Wallis test and paired Mann–Whitney U-test, to evaluate the differences between cells treated with naringenin and 8-prenylnaringenin. To assess differences between degree of N and 8PN accumulation in cells, Student’s t-test was executed. Our study revealed that isoxanthohumol derivative 8-prenylnaringenin was more cytotoxic against both used cell lines than its structural analogue naringenin, and that both flavones inhibited glioblastoma cells stronger than normal fibroblasts. The evident anticancer properties of 8PN correlated with its significantly greater accumulation in U-118 MG cells than in BJ cells. Naringenin had higher selectivity for glioblastoma cells, being over 6 times more toxic for cancer than normal cells.Evidence was provided that prenylated and non-prenylated isoflavones have different biological activities against normal and cancer cells, and this phenomenon may be useful in chemical synthesis to construct new, effective chemotherapeutic agents for glioblastoma.Our results indicate that the activity of 8PN is relatively moderate, and that it is not likely to become a therapy in itself but could serve as a starting point for lead optimization.To fully understand and assess the health effects of tested compounds, it is desirable to characterize their activity at the molecular level, and to compare their tissue distribution and subcellular localization in different cancer cell lines.


Perfusion MRI using an arterial spin labeling technique showed regional hypoperfusion in arcAβ compared to non-transgenic controls, which was alleviated by noscapine treatment. Similarly, measuring cerebral blood volume changes upon pharmacological stimulation using vessel dilator acetazolamide revealed recovery of regional impairment of cerebral vascular reactivity in arcAβ mice upon noscapine treatment. In addition, we assessed with immunohistochemistry beta-amyloid (Aβ) and inflammation levels in brain sections. Immunohistological stainings for Aβ deposition (6E10) and related microgliosis (Iba1) in the cortex and hippocampus were found comparable between noscapine-treated and untreated arcAβ mice. In addition, levels of soluble and insoluble Aβ38, Aβ40, Aβ42 were found to be similar in brain tissue homogenates of noscapine-treated and untreated arcAβ mice using electro-chemiluminescent based immunoassay. In summary, bradykinin receptors blockade recovered cerebral vascular dysfunction in a mouse model of cerebral amyloidosis. fMRI methods revealed the functional deficit in disease condition and were useful tools to monitor the treatment response.Functional magnetic resonance imaging (fMRI) techniques can be used to assess cerebrovascular dysfunction in Alzheimer’s disease, an important and early contributor to pathology. We hypothesized that bradykinin receptor inhibition alleviates the vascular dysfunction in a transgenic arcAβ mouse model of cerebral amyloidosis and that fMRI techniques can be used to monitor the treatment response. Transgenic arcAβ mice, and non-transgenic littermates of 14 months-of-age were either treated with the bradykinin receptors 1 and 2 blocker noscapine or received normal drinking water as control over 3 months ( Alzheimer’s disease (AD) is the most common form of dementia and represents a complex and multi-factorial disorder. Hallmarks of the disease are the accumulation of abnormal beta-amyloid (Aβ) and neurofibrillary tangles composed of hyperphosphorylated microtubule-associated tau protein, leading to neurodegeneration . MisfoldSeveral neuroimaging techniques have been developed to assess features of AD pathology in patients and animal models of the disease. The quest is to improve the diagnosis of the disease, to assess the disease stage, to select patients for treatment and to monitor the response to therapy. Among them, magnetic resonance imaging (MRI) has been used to detect degenerative changes in patients with AD . More re1R and B2R. Pharmacological antagonism or genetic deletion of B1R .All experiments were performed in accordance with the Swiss Federal Act on Animal Protection and were approved by the Cantonal Veterinary Office Zurich (Permit Number: 90-2016). All procedures fulfilled the ARRIVE guidelines on reporting animal experiments. ArcAβ transgenic mice, with human APP695 transgene containing the Swedish (K670N/M671L) and Arctic (E693G) mutation under the control of the prion protein promoter , were usg for 15 min. The blood : EDTA volume ratio was approximately 8:1. The plasma supernatants were collected as 50 μl aliquots, frozen and kept at -80°C, until use. The enzyme immunoassay  was used for bradykinin level measurement. A known concentration of biotinylated bradykinin was spiked into samples and standards, which were then added to wells. Here, biotinylated-bradykinin competes with the endogenous bradykinin in plasma for binding to the monoclonal anti-bradykinin antibody immobilized on the wall. After color development reaction, the intensity of the colorimetric signal was directly proportional to the amount of biotinylated bradykinin captured by the antibody, which inversely correlated to the amount of endogenous peptide in the sample or standard. A standard curve for the quantification of bradykinin concentration in the samples was generated. The lower detection limit of the assay was 1.4 ng/ml bradykinin.Six arcAβ and seven non-transgenic littermates (NTLs) of 17 months-of-age were used. Blood from the vena cava of the mice was collected into Eppendorf tubes filled with 50 μl 0.5 M ethylenediaminetetraacetic acid  and centrifuged at 4°C, 1000 A flowchart of the design for the noscapine treatment study is shown in All MRI scans were performed on a 7/16 small animal MR Pharmascan  equipped with an actively shielded gradient set of 760 mT/m with a 80 μs rise time and operated by a Paravision 6.0 software platform . Mice were anesthetized with an initial dose of 4% isoflurane  in oxygen/air (200/800 μl/min) mixture and anesthesia were maintained at 1.5% isoflurane in oxygen/air (100/400 μl/min) mixture. Mice were placed in prone position on a water-heated support to keep body temperature within 36.5 ± 0.5°C. Body temperature was monitored with a rectal temperature probe.n = 17).Perfusion MRI was performed using an arterial spin labeling technique as described previously . The Paxi.v. injection of contrast agent while the isoflurane level was reduced to 1.2%. T2-weighted anatomical reference images were acquired using a spin echo rapid acquisition with relaxation enhancement (RARE) sequence with the same anatomical geometry as RARE sequence at pre- and post-injection of vessel dilator acetazolamide. Fifteen minutes after injection of contrast agent, of gallanmine was injected i.v. (21 mg/kg body weight). Then, eight sequential pre-contrast agent scans at baseline signal intensity Spre (CBV0 image) were acquired using a RARE sequence: temporal resolution = 40 s, repetition time = 3333 ms, echo time eff = 81 ms, RARE factor = 32, field-of-view = 20 mm × 20 mm, imaging matrix = 133 × 103, slice thickness = 1 mm, 1.5 mm gap, resolution = 150 μm × 200 μm. FLASH sequence NR = 50 was used for ensuring the successful injection of the contrast agent. After 10 repetitions, iron oxide contrast agent Endorem  was administered i.v. A RARE sequence was started after 5 min after repetition = 100 and 8 averages, when the contrast agent concentration had reached steady state. After the 30th repetition (20 min), acetazolamide  was administered i.v. as a bolus. Additional 70 images were collected (46.7 min), yielding the image series S(t).Cerebral vascular reactivity was assessed, after a 1 week of recovery following perfusion MRI, by measuring cerebral blood volume (CBV) changes upon pharmacological stimulation using vessel dilator acetazolamide, as previously reported . The scai.p.). Mice were perfused using cold PBS (pH 7.4). Brains were removed from the skull. The left brain hemispheres were fixed in 4% paraformaldehyde  in PBS (pH 7.4) at 4°C and embedded in paraffin following routine procedures. The right hemispheres were snap frozen and stored at -80°C, until use.After MRI, mice were anesthetized under ketamine/xylazine/acepromazine maleate , NTL noscapine (n = 8), arcAβ (n = 7), and arcAβ noscapine (n = 4) mice. In brief, frozen brain tissues were homogenized using a glass Teflon homogenizer with homogenization buffer containing 100 mM Tris-HCl, 150 mM NaCl and complete EDTA-free protease inhibitor cocktail . The amount of buffer used was equivalent to 10-fold wet weight of the brain tissue. The brain homogenates were centrifuged at 100,000 g for 1 h and supernatants were collected Tris-buffered saline (TBS) fraction and pellets were homogenized again in homogenization buffer containing 2% of sodium dodecyl sulfate (SDS). Centrifugation was repeated and the supernatants (SDS-fraction) were again collected. The remaining pellet was suspended in 70% formic acid (FA), sonicated four times for 30 s at 30% power and again centrifuge at 100,000 g for 30 min. The supernatants were collected (FA-fraction), lyophilized and reconstituted in homogenization buffer. Aβ38, Aβ40, and Aβ42 levels were measured in the abovementioned brain homogenate fractions using an MSD 3plex multi-SPOT Aβ human kit . Plates were measured on an MSD SECTOR Imager 600 plate reader . The MSD DISCOVERY WORKBENCH software (Version 3.0.18)  with Data Analysis Toolbox was used to calculate sample concentrations by comparing them against a standard curve.Meso Scale Discovery (MSD) analysis was performed as described previously  in NTL  and Alexa488-ionized calcium-binding adapter molecule  immunostainings were done on 5 μm brain sections as previously described . Nuclei 2-weighted anatomical scan and the Paxino mouse brain atlas as reference  was calculated for each ROI. The baseline CBV and relative percentage changes of CBV versus baseline CBV (ΔCBV(t)) were computed on a pixel-by-pixel basis according to:Assessment of CVR was performed using Biomap 6 . The ROIs were defined in six brain regions . Baseline CBV (CBVCBV% (t), sqrt(Δ CBV% (t)) with time. ROIs of specific brain regions in the left and right hemispheres were merged into one ROI. 2-weighted anatomical scan and the Paxino mouse brain atlas as reference (t) and the late ΔCBV(%) of slices 90–100 (40–46.7 min) were compared between groups.Linear regression analysis was performed on the square root of the Δeference . The respost hoc analysis  was used. For comparison of age between groups, a one-way ANOVA with Tukey’s post hoc analysis was used. For comparison of bradykinin plasma level between NTLs and arcAβ mice, unpaired t-test was used after Shapiro–Wilk normality test. For group comparison of CBF, CBV0, late ΔCBV(%), slope of sqrt(ΔCBV%(t)) and immunostaining of Aβ and Iba1 in multiple brain regions, two-way ANOVA with Turkey’s post hoc analysis were used. For MSD measurement of Aβ level in different brain fractions of test–retest analysis of the CBF data of the same mice, a Pearson correlation analysis was performed. All data are present as mean ± standard deviation. Significance was set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.For comparison of the continuous numeric data such as weight, blood pressure before and after treatment, a two-way ANOVA with Tukey’s n = 8), NTL noscapine (n = 9), arcAβ (n = 9), and arcAβ noscapine (n = 11). One-way ANOVA showed that groups did not differ in terms of age among the groups (approximately 16.6 months-old after the treatment), F: F = 0.4467, p = 0.7212. And no differences between body weights before and after the treatment period were found between groups F = 0.104, p = 0.9574. As inhibitors of BRs can affect the angiotensin system  = 0.1594, p = 0.9229   .F = 2.316, p = 0.0351; reduced CBF in the cortex  and thalamus  in untreated arcAβ mice compared to untreated NTLs were observed (p = 0.0023) and thalamus  compared to untreated arcAβ mice. The test–retest results of two perfusion MRI measurements in the same mice are shown in r = 0.9491, p < 0.0001, n = 17) between test and re-test measures. Taken together, perfusion imaging revealed that noscapine treatment mitigated the hypoperfusion seen in arcAβ mice of this age.We first tested the effect of chronic oral noscapine treatment on cerebral perfusion of arcAβ mice. observed . Noscapit = 0 min) overlaid with corresponding T2-weighted MRI. No difference was observed in baseline CBV0 values among groups . These results suggest that B1R and B2R blockade with noscapine at this dosage and duration did not alter the amyloid load and degree of Aβ-associated microgliosis.Previous reports about the effect of Bhemistry . Two-waypost hoc analysis showed greater amount of Aβ38 , Aβ40 , and Aβ42  in FA fractions brain homogenates (n = 7) compared to NTLs (n = 6). No significant difference was detected in the level of Aβ38, Aβ40 and Aβ42 in TBS, SDS, and FA fractions between noscapine-treated arcAβ mice (n = 4) and untreated arcAβ mice (n = 7).We used MSD immunoassay to determine Aβ levels in three protein fractions from the brain hemispheres. Two-way ANOVA with Tukey’s ogenates  from arc1R and B2R antagonist noscapine ameliorated amyloidosis-associated cerebral hypoperfusion and vascular reactivity, but did not affect Aβ cerebral load and associated microgliosis.In the current study, we showed using fMRI techniques a pronounced vascular dysfunction in the arcAβ mouse model of cerebral amyloidosis. Chronic administration of B1R and B2R are expressed in a variety of cells including neurons, astrocytes, microglia, oligodendrocytes, and endothelial cells. Upregulation of brain BR levels was shown in rats  as well as vasoactive peptides of the renin-angiotensin system (1R and B2R (pA2 = 6.68)  . We deci = 6.68) .in vivo (1R improves CBF (We used established functional MRI protocols to assess the effects of noscapine treatment on cerebrovascular dysfunction in vivo . Cerebroin vivo . The braoves CBF . We haveoves CBF , which i1R significantly ameliorated sensory-evoked hemodynamic responses in the J20 mouse model (Cerebral vascular reactivity reflects the capacity of blood vessels to dilate and is an important indicator of the brain vascular reserve capacity. Dynamic measurement of CBV upon acetazolamide challenge is commonly used in humans and animals for quantification of CVR and has shown to be impaired in AD patients and models of cerebral amyloidosis . Acetazose model . As inhise model , we meas1R and B2R ameliorate Aβ-induced cerebrovascular dysfunction. The molecular mechanisms underlying cerebrovascular dysfunction in AD are not well-understood and it can only be speculated how noscapine treatment ameliorated hypoperfusion and reduced CVR in arcAβ mice. It has been suggested that blockade of BRs antagonized the production of vascular reactive oxygen species and inflammatory cytokines (The findings from CVR and CBF measurement clearly demonstrate that inhibition of Bytokines , which mytokines .In addition to functional vascular effects of noscapine treatment, we also assessed the effects on neuroinflammation and Aβ pathology. Gliosis is an early phenomenon both in mouse models of amyloidosis  and in p1R is involved in inflammation in the central nervous system mainly through the activation of microglia (Bicroglia  and astricroglia . Howevericroglia  and stimicroglia  of neuroicroglia . Using m1R and B2R on Aβ load have also been contradictory. B2R activation promoted α-secretase processing of amyloid precursor protein in skin fibroblasts from patients with AD, resulting in a lower production of Aβ (1R antagonist SSR240612 reduced Aβ deposition (1R antagonist R-715 treatment increased Aβ40 and fibrillar Aβ deposition in both transgenic mouse models of AD, Tg-SwDI (1R and B2R inhibition on cerebral Aβ load.Reports on the effect of Bon of Aβ . B1R antposition  in Tg-Sw Tg-SwDI  and 5 ×  Tg-SwDI . Aβ can  Tg-SwDI . In our 1R and B2R, as well as other factors like pharmacokinetics, biodistribution of the drug used, and the timing, dose and duration of the treatment. In our study, we found amelioration of cerebrovascular dysfunction with chronic oral noscapine treatment, while the treatment seemed to have no effects on cerebral Aβ load and microglia activation. In that context, fMRI could constitute a more sensitive and earlier indicator of treatment response than histological or biochemical assessment. It might be, however, conceivable that a different dose of noscapine or longer treatment periods might have also led to detectable changes in neuropathology. In addition, we started treatment in arcAβ mice at an advanced disease stage, which limited the potential effect scale on the different read-outs. Importantly, we have not used behavioral read-outs to assess effects of noscapine treatment on the cognitive deficit in arcAβ mice (The reported differences in the action of bradykinin receptor inhibitors might be due to the affinity of individual inhibitors to BcAβ mice  and thusLong-term oral noscapine treatment alleviates brain hypoperfusion and restores CVR in an animal model of amyloidosis as revealed by fMRI. Blocking BRs represents a potential target for treatment of AD-associated cerebrovascular dysfunction.http://doi.org/10.5281/zenodo.1209065.The datasets generated and/or analyzed during the current study are available in the repository RuN, LK, and JK conceived and designed the study and interpreted the results. RuN, DK, LL, and RW performed the experiments. RuN, RW, LL, LK, and JK analyzed the data. RuN and JK wrote the paper. All coauthors contributed constructively to the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arabidopsis thaliana, Eutrema salsugineum has been proposed as a model for studying salt resistance in plants. Here we surveyed 35 full-length AQP genes in E. salsugineum, which could be grouped into four subfamilies including 12 plasma membrane intrinsic proteins (PIPs), 11 tonoplast intrinsic proteins (TIPs), nine NOD-like intrinsic proteins (NIPs), and three small basic intrinsic proteins (SIPs) by phylogenetic analysis. EsAQPs were comprised of 237–323 amino acids, with a theoretical molecular weight (MW) of 24.31–31.80 kDa and an isoelectric point (pI) value of 4.73–10.49. Functional prediction based on the NPA motif, aromatic/arginine (ar/R) selectivity filter, Froger’s position and specificity-determining position suggested quite differences in substrate specificities of EsAQPs. EsAQPs exhibited global expressions in all organs as shown by gene expression profiles and should be play important roles in response to salt, cold and drought stresses. This study provides comprehensive bioinformation on AQPs in E. salsugineum, which would be helpful for gene function analysis for further studies.Aquaporins (AQPs) serve as water channel proteins and belong to major intrinsic proteins (MIPs) family, functioning in rapidly and selectively transporting water and other small solutes across biological membranes. Importantly, AQPs have been shown to play a critical role in abiotic stress response pathways of plants. As a species closely related to  AQP wase (H2O2) . Comparesculenta .Physcomitrella patens) and similar to bacterial glycerol channels (Selaginella moellendorffii) and moss  are the largest subfamily of plant AQPs. The most of the PIPs are commonly localized in the plasma membrane and are further divided into two phylogenetic groups PIP1 and PIP2. Tonoplast intrinsic proteins (TIPs) subfamily is usually localized in the tonoplast, which contain five classes TIP1, TIP2, TIP3, TIP4 and TIP5. NOD26-like intrinsic proteins (NIPs) named from NIP protein , were discovered in the plasma membrane of soybean cells . Small bchannels . Hybrid α-helical domains (TM1-TM6) linked by five loops (A-E), with both the N and C terminal having a cytoplasmic orientation. There are two highly conserved NPA (Asn-Pro-Ala) motifs in two half helices (HB and HE) of loopB and loopE at the center of the pore that have substrate selectivity  analysis was used. HMM profile of MIP (PF00230) was downloaded from Pfam protein family database (http://pfam.sanger.ac.uk/) and used as the query (P < 0.05) to search for AQP proteins in the E. salsugineum genome. To avoid missing potential AQP members, the NCBI BLAST tool was used to search E. salaugineum AQPs and known Arabidopsis AQP protein sequences as a query, and the top five aligned sequences were considered as candidates. After removing all of the redundant sequences, the sequences of putative EsAQP genes were loaded on relative chromosomes of E. salsugineum using the SnapGene tool. The map of the chromosome position of each EsAQP genes was drawn by MapInspect 1.0.The whole genome of http://topcons.cbr.su.se/pred/) and TMHMM (http://www.cbs.dtu.dk/services/TMHMM/). Protein subcellular localization of E. salsugineum AQPs was predicted in Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) and WoLF PSORT (http://www.genscript.com/wolf-psort.html). Functional predictions, such as NPA motifs, ar/R filters , Froger’s positions (P1-P5) and nine specificity-determining positions (SDP1-SDP9), were analyzed by the alignments with function known AQPs (EsAQP was illustrated with the Gene Structure Display Server 2.0 (http://gsds.cbi.pku.edu.cn/). The conserved motifs of EsAQP proteins were analyzed by MEME suite (http://meme-suite.org/).Multiple sequence alignments of putative AQP proteins were performed by ClustalW, and a phylogenetic tree was constructed using neighbor joining with MEGA 6.0 . The traown AQPs . The genE. salsugineum seeds  were provided by Prof. Hui Zhang . The seeds were plated on 1/2 MS medium and treated at 4 °C in dark for 7 days, then cultured in plant growth chamber with illumination of 150 µmol/m2/s, photoperiod 16/8 h of light/darkness at 25 °C and 60% relative humidity. After one week, the seedlings were transferred into a mixed medium with soil and vermiculite (3:1). Vernalization treatment for bolting was conducted in 4-week old seedlings at 4 °C for 4 weeks, and then they were moved back to the growth chamber until they grew flowers. Samples of roots, stems, leaves, flowers and siliques were collected, immediately frozen in liquid nitrogen and stored at −80 °C for further analysis.For abiotic stress assays, the 4-week old seedlings were exposed to 300 mM NaCl for 24 h as a salt stress condition, treated at 4 °C for 24 h as cold stress, and not irrigated until the soil moisture content was less than 20% for 7 days as drought stress. The aerial part of seedling was collected for further analysis.The total RNA was extracted using TRIzol reagent (Takara) following the manufacturer’s protocol. The quality of the RNA was determined using an ultraviolet spectrophotometer . After removing genomic DNA contamination with DNase I, cDNA was synthesized by using the PrimeScript™ RT Reagent Kit (Takara). Three biological replicates of cDNA samples were used for qRT-PCR analysis with three technical replicates.http://bioinfo.ut.ee/primer3-0.4.0/) and the reference gene was taken from 2O to a final volume of 20 µl. The PCR program was set as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Then, a melting curve was generated to analyze the specificity of each primer with a temperature shift from 60 to 95 °C. The fold changes of the EsAQP genes expression under abiotic stresses were calculated with the 2−ΔΔCt method, while the gene expressions level of EsAQP genes in each organ were calculated with the ΔCt method. The heat map of gene expression pattern was visualized using HemI software.Primers of EsPIP genes were designed using Primer 3.0 . Oocyte preparation, injection, and expression were performed as described by 0[d(V∕V 0)∕dt]∕[S0 × V w(Osmin–Osmout)]. V 0 and S0 are the initial volume and surface area of each individual oocyte, respectively; d(V/V0)/dt is the relative volume increase per unit time; Vw is the molar volume of water (18 cm3 mol−1); and Osmin–Osmout is the osmotic gradient between the inside and outside of the oocyte.The coding regions of EsPIP1;2 and EsPIP2;1 were subcloned into pCS107 vector using the restriction sites BamHI and EcoRI  was used. As a result, a total of 35 putative EsAQPs were identified for further analysis , most of our identified EsAQPs were matched to the existing annotations . The majority members of the EsTIP subfamily contained three exons with similar lengths, and the other members had two exons with similar lengths, except for EsTIP1;3, which had only one exon without intron. In the EsNIP subfamily, some members exhibited five exons with similar lengths, while others had four exons with varied lengths. All EsSIP subfamily genes displayed three exons with similar lengths. This description of exon-intron structure provides additional evidence to support the classification results  comprising 237–323 amino acids, had theoretical molecular weights (MW) of 24.31–31.80 kDa and isoelectric point (pI) values of 4.73–10.49 . The EsPNPA motifs, ar/R selectivity filters and Froger’s positions of AQP protein sequences play critical role in channel selectivity. The sequence alignment between AtAQPs and GhAQPs was carried out to analyze the conserved domains . The resE. salsugineum except for EsTIP5;1. Most of members in NIP subfamily had two motif 1, two motif 3, and two motif 6, except for EsNIP2;1 (lose one motif 1), EsNIP3;1 (lose one motif 6) and EsNIP5;1 (lose one motif 3). The two motif 6 might be used to distinguish EsNIPs with other EsAQPs. All EsSIP subfamily carried motif 3. Motif 8 appeared in EsSIP1s but not in EsSIP2;1, so it might be an specific trait of this group. This is a common phenomenon in plant SIP subfamily contains less motifs (MEME (Multiple EM for Motif Elicitation) is one of the most widely used tools for searching for novel “signals” in sets of biological sequences, include the discovery of new transcription factor binding sites and protein domains . Conservs motifs . Based oEsAQP genes in different organs, including root, stem, leaf, flower and silique, was analyzed by RT-qPCR. The results showed that 35 EsAQP genes were detected in all the organs , three EsNIPs  and EsSIP1;2 were highly expressed only in flower. Two EsTIPs  were expressed in silique with relative high abundance. Compared analysis of each EsAQP gene between different organs revealed that most EsAQP genes showed higher expression level in flower than in other organs.The expression of e organs . Almost EsAQP genes under salt, drought and cold stress by qRT-PCR. The results showed that most of the EsAQP genes were up-regulated under salt and cold stress but down-regulated under drought stress  (Selaginella moellendorffii) (Physcomitrella patens)    to fern dorffii)  and moss patens)  to higheine max) , the numT. salsuginea , each aquaporin subfamily are completely conserved in number, order and length of exons but varies in introns , with E replaced by D in SDP6. These results showed the conservation of plant PIPs in the transport of urea and hydrogen peroxide . EsTIP2;1 had an NH3-type SDPs, as confirmed in Arabidopsis TIP2;1 (3-type SDPs (T-L-G-T-A-S-H-P-A) with F/T replaced by G in SDP3. The NIP subfamily has low intrinsic water permeability and the ability to transport solutes like glycerol and ammonia , which were consistent with previous studies, especially with Arabidopsis AQP genes  and Arabidopsis  were up-regulated. Among these, the level of EsTIP3;2 was most significantly increased after drought treatment, which has low abundance in leaf , AtPIP2;1 (P43286), AtPIP2;4 (Q9FF53), AtTIP1;1 (P25818), AtTIP1;2 (Q41963), AtTIP1;3 (NP_192056), AtTIP2;1 (Q41951), AtTIP2;3 (Q9FGL2), AtTIP4;1 (O82316), AtTIP5;1 (NP_190328), AtNIP1;2 (Q8LFP7), AtNIP5;1 (NP_192776), AtNIP6;1 (NP_178191), CpNIP1 (CAD67694), GmNOD26 (P08995), HvPIP1;3 (BAA23745), HvPIP1;4 (BAF33068), HvPIP2;1 (BAA23744), NtAQP1 (O24662), NtTIPa (Q9XG70), OsNIP2;1 (Q6Z2T3), TaTIP2;1 (AAS19468), TaTIP2;2 (AAS19469), ZmPIP1;1 (Q41870), ZmPIP1;5 (Q9AR14).Multiple alignments were performed using ClustalX. The SDPs are highlighted in yellow, mismatch site highlighted in red and the representative sequences are marked in blue. The Genbank accession numbers: Click here for additional data file.