Gadolinium (Gd)-based contrast agents (GBCAs) are chemicals injected intravenously during magnetic resonance imaging to enhance the diagnostic yield. Repeated use of GBCAs causes their deposition in the brain. Such deposition may affect various neuronal cells, including astrocytes. In this study, we examined the effect of GBCAs  on astrocyte migration, which is critical for formation of neurons during development and maintaining brain homeostasis. All GBCAs increased cell migration and adhesion with increased actin remodelling. Knockdown of integrin αvβ3 by RNAi or exposure to integrin αvβ3 inhibitor reduced astrocyte migration. GBCAs increased phosphorylation of downstream factors of αvβ3, such as FAK, ERK1/2, and Akt. The phosphorylation of all these factors were reduced by RNAi or integrin αvβ3 inhibitor. GBCAs also increased the phosphorylation of their downstream factor, Rac1/cdc42, belonging to the RhoGTPases family. Coexposure to the selective RhoGTPases inhibitors, decreased the effects of GBCAs on cell migration. These findings indicate that GBCAs exert their action via integrin αvβ3 to activate the signaling pathway, resulting in increased astrocyte migration. Thus, the findings of the study suggest that it is important to avoid the repeated use of GBCAs to prevent adverse side effects in the brain, particularly during development. Their role in brain development, maintenance of homeostasis, and as defence mechanism following brain injury have been well documented6. In the early neurogenic phase, astrocytes are formed from either the embryonic radial glia in the ventricular zone (VZ) or progenitor cells in the subventricular zone (SVZ)8. Immature astrocytes migrate to the cortical layers where they undergo additional round proliferation before exiting the cell cycle for maturation10. During brain development, astrocytes play an important role in controlling neurogenesis , neuronal migration, axonal growth, synapse formation and function, maintenance of the blood–brain barrier, and controlling homeostasis8. Thus, astrocyte migration from the VZ or SVZ is the critical first step in the regulation of brain development by this cell type.Astrocytes are the most abundant cell type in the central nervous system (CNS) and cover nearly half of the adult mammalian brain volume11. These processes require changes in cell–cell and/or cell-extracellular matrix (ECM) adhesion properties in which cell adhesion molecules are involved in binding with other cells or ECMs12. Integrins are a group of heterodimeric transmembrane proteins and they play a vital role during astrocyte proliferation and migration12. The activation of integrins leads to activation of the downstream intracellular signaling pathways through focal adhesion kinase (FAK), Src, Rac, and Rho, which jointly define the size, duration, and strength of adhesion13.Cell migration is determined by the polarization, coordinated regulation of actin cytoskeletal elements, and adhesive structures15. Integrin αvβ3 is upregulated in astrocytes during brain development and following brain injury. Binding of the integrin αvβ3 with their ligands activates FAK that functions as a receptor-proximal regulator of cell motility16. FAK activity and downstream signaling can promote changes in actin and microtubule structures. FAK can influence the activity of RhoGTPases', such as Cdc42, Rac, and RhoA, via direct interaction with, or phosphorylation of protein activators or inhibitors of RhoGTPases17. Activated integrin αvβ3 induces the cell to polarize, extend filopodia and lamellipodia to the leading front, and form focal adhesions, which consist of a layer made up of bundles of actin microfilaments called stress fibers15. These changes in turn activate astrocyte adhesion with other cells, facilitating their spreading, and migration. Thus, it is reasonable to speculate that disturbances in the integrin αvβ3 signaling pathway could impair astrocyte migration.Among integrins, integrin αvβ3 plays an important role during cell migration and is highly expressed in astrocytes3+) is extremely toxic. Therefore, it is chelated with an organic ligand molecule to produce highly soluble non-toxic complexes18. GBCAs are classified as linear or macrocyclic, based on their chemical compositions, and ionic or non-ionic based on their ion charge19. Following administration, GBCA is deposited in several organs, including the brain20. This deposition may affect not only neurons but also astrocytes23.Gadolinium-based contrast agents (GBCA) are intravenous drugs used in magnetic resonance imaging (MRI) to enhance the diagnostic yield. In the free form, Gadolinium ion , and Akt, a direct target for integrin αvβ3, leading to increased cell proliferation and enhanced focal adhesion29, indicating that Gd3+ may be at least in part dissociated from GBCA and induced such effect. However, Gd depositions in the brain consists of both soluble and insoluble forms34. The insoluble form may be Gd3+ that was released from GBCA and bound with organic or inorganic anions, whereas soluble forms consist of Gd-binding macromolecules and low molecular weight Gd complexes that might be the intact GBCAs. Based on these findings, we considered it important to examine the effect of GBCA on the integrin αvβ3 signaling pathway, which is responsible for astrocyte migration.Both GdThe final objectives of our study is to clarify the mechanisms of various GBCAs toxicity on brain development and function. The clarification may help to select appropriate GBCA and its exposure protocol to avoid adverse effect by repeated exposure during brain development and/or regeneration. For such a purpose, we examined the effects of the four types of GBCA  Fig.  in migra8. In order for astrocytes to move and migrate through a complex three-dimensional (3D) environment, not only do their chemical and physical properties play an important role, but these cells must also quickly adapt in the short term to the physical properties of their surroundings. The 2D  migration with flat plastic and 3D migration under the physical constraints of the surrounding cell and ECM has been used to elucidate the detailed mechanism of chemical and physical properties during astrocyte migration in cultured cells35. Therefore, we examined the effect of GBCAs in 2D and 3D cell migration using wound healing and cell invasion assays, respectively.Interaction of astrocytes with its surrounding structures is crucial for physiological tissue organization and performing its functions during development and pathological conditions during brain injury38 and the concentration of GBCA that was found in the waterwaste and sea water ranges from 0.1 to 100 nM40. Therefore, we used the doses ranging from 1 to 100 nM in the present study.Before we conducted this study, we have examined the cytotoxic effect of both gadolinium and GBCAs by MTS cell proliferation assay (range 0.1 nM–100 µM). We found that GBCAs exposure starting 10 µM or gadolinium exposure starting 100 nM reduced the cell viability of astrocytes  receptors, leading to aberrant Purkinje cell development41. A focal adhesion complex is a structural unit composed of a high density of proteins involved in relaying the information flow from outside the cell to the inside. The extent of physical attachment depends on the nature of signaling the cell receives. Quantifying the amount of cell attachment should be proportional to the number of attached focal adhesion sites formed42. We thus examined focal adhesion of astrocytes by focal adhesion assay after exposure to GBCAs. The representative photomicrographs of attached astrocytes stained with cresyl violet are shown  index after visualization of F-actin with Phalloidin-iFluor 594 reagent. The CFS index was determined based on F-actin cytoskeletal reorganization for each cellInteraction of integrin αvβ3 with their ligands results in activation of downstream signaling pathways, promoting changes in protein expression levels or the phosphorylation process. Activation of integrin αvβ3 can induce FAK, ERK1/2, and Akt phosphorylation signaling to activate these signaling cascades, some of which may be involved in inducing F-actin rearrangement. To examine the downstream targets of GBCAs-activated integrin αvβ3, we performed immunocytochemistry and Western blot analysis to measure phosphorylation of FAK, ERK1/2, and Akt in U87MG cells. Representative photomicrograph images of cell immunofluorescence with p-FAK (Y397), pERK1/2, or pAkt, and Phalloidin-iFlour 594 or 488 reagent are shown  were significantly decreased by knockdown of integrin αv, β3, or αvβ3 Fig. A–J. We a17. Thus, we examined the effects of GBCAs on RhoGTPases signaling using Western blot, wound healing, cell migration, and focal adhesion assays. Western blot analysis showed that GBCA-induced phosphorylation levels of Rac1/Cdc42 (S71) were significantly reduced after knockdown of integrin αv, β3, or αvβ3, and coexposure with integrin inhibitor cRGDfV , which is a cyclic RGD peptide and a specific antagonist to integrin αvβ3, only partially blocked GBCAs-induced cell migration. These results indicate the involvement of other factors than integrin αvβ3, such as other integrins containing αv or β3 subunit. While the inhibition of FAK, PI3K, or ERK1/2 phosphorylation suppressed the effect of GBCAs-induced 2D migration of astrocytes  cascade16. GBCAs increased phosphorylation levels of FAK (Y397), FAK (Y925), and ERK1/2 (T202/Y204). GBCA–increased phosphorylation levels of FAK(Y397) and FAK (Y925) were significantly reduced after knockdown of integrin αvβ3 or coexposure with cRGDfV. However, GBCA–induced increased phosphorylation levels of ERK1/2 (T202/Y204) that is located downstream of FAK (Y925) only reduced after knockdown of integrin β3 subunit or coexposure with cRGDfV, but not by knockdown of integrin αv. Thus, we hypothesized that the increase in phosphorylation levels of ERK1/2 (T202/Y204) might have occurred independently from FAK (Y925) phosphorylation through the activation of other integrins. This could also be a possible reason why inhibition of ERK 1/2 phosphorylation only partially inhibited GBCA–activated 3D migration  after knockdown of integrin αvβ3  alters the polymerization or stabilization of actin and microtubule filaments. Rac1 and Cdc42 can also control the cellular directionality of migrating cells when activated by the mechanical stretch of E–cadherin and P–cadherin. Rac1 signaling can relay directional information from the external environment to the actin cytoskeleton via E–cadherin mediated mechano-sensing so that the cell can migrate toward the target50. The laser ablation ICP–MS showed the deposition of linear GBCAs  highly deposited in DCN and granular layer of the cerebellar cortex, whereas the macrocyclic GBCAs  distributed throughout the cerebellar without any region–specific accumulation and exhibited low Gd concentrations51. Intracerebroventricular injection of gadodiamide or gadopentate dimeglumine showed the hypertrophy and hyperplasia of astrocytes and Bergmann glia, incoordination in walking several hours after injection, and also development of ataxia and seizure53. Again, these results support the previous hypothesis that Gd deposition may affect various subsets of cells in the brain, including glial cells. However, the mechanisms of GBCA toxicity has not yet been clarified. Our present study has shown the possibility that GBCA exposure disrupts integrin αvβ3–activated intracellular signal transduction pathway in the astrocytes and disrupts their function.Recently, in vivo and in vitro model have been used to examine GBCA deposition effects on various subsets of the cells in several regions of the brain. Transmission electron microscopy showed that gadodiamide deposited in the DCN, choroid plexus, and granular layer of cerebellar cortex, whereas gadobenate deposited in the DCN and choroid plexus of the cerebellumIn conclusion, our results have shown that exposure to GBCAs accelerated the migration of astrocytes mainly via the integrin αvβ3 signaling pathway and subsequent activation of FAK/PI3K/Akt/RhoGTPase signaling pathway, which induces F-actin rearrangement Fig. F. The ac54 were followed in the preparation of this paper. All experiments performed in this study were carried out in accordance with the approved guidelines and regulations.All animal experiment protocol in the present study was ethically reviewed and approved by the Animal Care, and Experimentation Committee, Gunma University . All mice were treated according to Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of Japan. All efforts were made to minimize animal suffering and the number of animals used. The ARRIVE guidelinesOmniscan, Magnescope, Magnevist, and Gadovist Fig.  were obt55 and U87MG human astrocytoma56 clonal cells were obtained from ATCC  and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) at 37 °C with 5% CO2. It should be noted that, although it would be identical to use the same subset of cells to examine the effect of GBCAs, there is a limitation to use primary cultured astrocytes. Wound healing assay cannot be performed using primary cultured astrocytes because the migration speed is very slow. Thus, we had to use C6 cells. On the other hand, to examine the signal transduction pathway, a Western blot for phosphoproteins needs to be conducted. However, most antibodies were generated against human proteins. Thus, we had to use U87MG cells. We have confirmed that GBCAs increased cell invasion of all three cell types, indicating that the effect of GBCA on these cells is comparable . Briefly, postnatal day 1, mouse cerebral cortices were dissected and digested with 2.5% trypsin  in Hank's balanced salt solution (Wako) for 30 min with continuous shaking at 37 °C. Cells were resuspended in an astrocyte culture medium , and 10–15 million cells were plated on 10-cm dishes coated with Collagen I . Cells were incubated at 37 °C in a CO2 incubator. On day 3 in vitro (DIV3), the astrocyte culture medium was replaced with phosphate-buffered saline (PBS). Dishes were then shaken by hand for 30–60 s until only the adherent monolayer of astrocytes was left. The PBS was then replaced with a fresh astrocyte culture medium. Astrocytes were harvested on DIV7 using 0.25% trypsin 1 mM disodium EDTA (Wako) and then plated on 12 or 24 well dishes. Cells were used for cell invasion assay or F-actin staining.Primary culture of mouse cerebral cortex astrocytes was prepared as previously described44. C6 cells were plated in a 24-well plates and cultured until confluent. Prior to making a scratch, cells were serum-starved in FBS-free DMEM for 6 h. A wound was created by scratching the monolayer with a 200-µL pipette tip. Floating cells were washed away using PBS. Serum-free DMEM and/or GBCAs, cyclo (Ala-Arg-Gly-Asp-3-Aminomethylbenzoyl), cRGDfV, PF573228, LY294002, U0126, Rhosin, CASIN, ML-141, and/or EHT-1846 were added to the wells and incubated for a further 24 h. At 0 and 24 h, live-cell staining was performed using Cellstain-Hoechst 33,258 solution  according to the manufacturer's protocol. Images of the scratched area were taken at 0 and 24 h in the same position. The cells were then visualized using a fluorescence microscope . Cell migration was determined at the edges of the wound, and the percentage migration was determined as the ratio between migrated distance and initial distance of the wound.The in vitro wound healing (scratch) assay have been described in previous study® Matrigel® Matrix according to the manufacturer's instructions . In brief, astrocytes were seeded at a density of 1 × 105 /mL in serum-free DMEM in the upper chamber. The lower chamber was filled with serum-free DMEM and/or GBCAs, cyclo (Ala-Arg-Gly-Asp-3-Aminomethylbenzoyl), cRGDfV, PF573228, LY294002, U0126, Rhosin, CASIN, ML-141, and/or EHT-1846. After 16–18 h of incubation, noninvading cells in the upper chamber were removed with a sterile cotton swab. The filters from the inserts were fixed with 4% paraformaldehyde (PFA) and stained with DAPI. The cells were then inspected using a laser confocal scanning microscope . The number of invading cells on the lower surface of the filter was counted.In vitro invasion assays were performed using a 24-well Millicel hanging cell culture insert and a Corning5 /mL in serum-free DMEM in the 24-well plate. Wells were filled with serum-free DMEM and/or GBCAs, cyclo (Ala-Arg-Gly-Asp-3-Aminomethylbenzoyl), cRGDfV, PF573228, LY294002, U0126, Rhosin, CASIN, ML-141, and/or EHT-1846. After 2 h of incubation, non-adherent cells were removed together with medium, and the adherent cells were washed with PBS. The cells were fixed with 4% paraformaldehyde (PFA) and stained with cresyl violet, then visualized using a fluorescence microscope (Keyence BZ9000). The number of adherent cells on the well were counted with Fiji ImageJ (NIH).Astrocytes or C6 cells were plated in 35 mm dish until 80% of confluence. Prior to seeding, cells were serum-starved in FBS-free DMEM for 6 h. In brief, astrocytes were seeded at a density of 2 × 1057. Cultured cells were exposed to GBCA for 30 min, then rinsed three times with PBS, fixed with 4% PFA, and blocked with 2% FBS. Cells were then incubated with rabbit monoclonal anti-phospho-FAK (Y397) XP , anti-phospho-Akt (S473) (D9E) XP , or anti-phospho-p44/42 MAPK (ERK1/2) (T202/Y204)  antibodies, followed by CytoPainter Phalloidin-iFluor 594 or 488 reagents (Abcam) and donkey anti-rabbit IgG (H + L) secondary antibodies, Alexa Fluor® 488 or 594 conjugate . Cell nuclei were also stained with DAPI. The cells were then inspected using a laser confocal scanning microscope .The immunocytochemistry for phosphorylation protein and F-action formation were described in previous studiesySuperPrep cell lysis and RT kit for qPCR reagent  according to the manufacturer's instructions. qRT-PCR was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO) as per the manufacturer's instructions and using a StepOne RT-PCR System (Thermo Fisher Scientific). The list of primers used in this study is listed in Supplementary Fig. 4  or β3 (ITGB3) , or negative control RNAs , using lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. The list of siRNA sequences used in this study is listed in Supplementary Fig. 4 . Brieflyy Fig. 4 . qRT-PCRCultured cells were homogenized in RIPA buffer  and protease inhibitors . Protein concentration was measured using the Bradford protein assay (Bio-Rad) according to the manufacturer's instructions. After boiling for 5 min, protein samples (5 µg) were subjected to 5%–20% SDS–polyacrylamide Supersep Ace (Wako) gel electrophoresis, and the separated products were transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20, followed by overnight incubation with the appropriate diluted primary antibodies for p-FAK (Y397) or (Y925) , FAK, p-ERK1/2 (T202/Y204) , ERK1/2 , p-Akt (S473) , Akt , p-Rac1/Cdc42 (S71) , Rac1/Cdc42 , Talin-1 , Vinculin , α-Actinin , Paxillin , Cortactin , and GAPDH . After washing with Tris-buffered saline containing 0.1% Tween 20, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibody  for one h at room temperature and detected using an ECL detection system (Wako). GAPDH was once again used as loading control.www.graphpad.com) or IBM SPSS Statistics 27 for Windows. All p-values < 0.05 were considered statistically significant.Data are expressed as mean ± SEM of three individual experiments performed in triplicate. One-way or two-way ANOVA followed by post hoc Bonferroni's and Turkey multiple comparison tests were performed using GraphPad Prism version 9.1.1 (GraphPad Software, San Diego, USA, Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Information 10.


Pseudozyma aphidis can activate microbe-associated molecular pattern (MAMP)-associated genes, it does not activate MAMP-triggered callose deposition and can, moreover, suppress such deposition triggered by Flg22 or chitin. While MAMP-associated gene activation by P. aphidis was not dependent on salicylic acid, jasmonic acid, or ethylene signaling, suppression of MAMP-triggered callose deposition required the salicylic acid and jasmonic acid signaling factors JAR1-1 and E3 ubiquitin ligase COI1 yet did not rely on EIN2, NPR1, or the transcription factor JIN1/MYC2. We also demonstrated the ability of P. aphidis, known to be an epiphytic yeast-like organism, to penetrate the stomata and establish within plant tissues, as do endophytes. These results thus demonstrate the potential of P. aphidis to suppress MAMP-elicited defenses in order to establish on and within host plant tissues.Beneficial microorganisms need to overcome the plant defense system to establish on or within plant tissues. Like pathogens, beneficial microbes can manipulate a plant’s immunity pathways, first by suppressing and hiding to establish on the host and then by inducing resistance to protect the plant. In the current study, we demonstrated that although IMPORTANCE Our study demonstrates the ability of P. aphidis to penetrate into plant tissues, where it avoids and overcomes plant defense systems in order to establish and subsequently protect the plant. In the environment, plants encounter a variety of different microbes. Beneficial and pathogenic microbes that successfully invade a plant mostly have the ability to manipulate the plant’s immune system to reprogram the host response and metabolism. On the other hand, plants have developed a multilayered defense system that includes preexisting structural barriers, as well as induced immune defense. Induced defense depends on the recognition of conserved microbial structures known as microbe-associated molecular patterns (MAMPs), such as bacterial flagella, or major components of fungal cell walls, such as chitin , 2. MAMP3–6–10–14–22–Trichoderma asperellum, for example, is an effective biocontrol agent for a number of soilborne pathogens of cucumber. Specifically, T. asperellum infection modulates the expression of genes involved in JA/ET-signaling pathways recruited for inducing systemic resistance  being seen  and reactive oxygen species (ROS) accumulation in the pathogen , we could demonstrate that P. aphidis cells are in close proximity to the cuticle plant surface and also to the stomata apparatuses , a cutin analogue, as sole carbon source. In our previous work, we demonstrated by halo assay that P. aphidis secretes cellulase that degrades carboxymethylcellulose -tagged P. aphidis isolate demonstrated how P. aphidis can penetrate into the cell through open stomata and establish within plant tissues  upon establishment on Arabidopsis plants. We used a promoter:GUS-transgenic line assay in which upregulation of MAMPs can be detected by GUS expression  analysis, demonstrating that the genes most activated above basal levels were MYB51 and At5g25260 , JA (coi1), or ethylene pathways (ein2 or sid2) .We considered whether t5g25260 . We coulP. aphidis, we tested whether P. aphidis, normally capable of inducing systemic resistance and MAMP-triggered genes, activated callose deposition in leaves. Intriguingly, inoculation with live P. aphidis did not activate such callose deposition  in the wild-type (WT) Columbia-0 (Col-0) or the ein2-1, sid2, npr1-1, or coi1-1 mutant backgrounds , with each plate containing up to 10 seeds in 50 mL seedling Murashige and Skoog (MS) growth medium agar .ium agar  and wereP. aphidis isolate L12 was maintained in solid culture on potato dextrose agar  at 26°C and transferred to fresh medium monthly. Liquid cultures were maintained in potato dextrose broth  for 3 to 7 days at 26°C on a rotary shaker set at 150 rpm. We obtained 108 cells/mL after 3 days in liquid culture. Cells were centrifuged, suspended in water or MS growth medium, and applied to plants.P. aphidis was maintained on PDA or PDB (Difco) with 70 mg/L hygromycin B solution  at 26°C. Liquid cultures were maintained on a rotary shaker at 150 rpm. We obtained 108 cells/mL after 3 days in liquid culture, which we spread (3 to 4 µL) onto the center of each leaf for induction of plant leaves.GFP-tagged Pseudomonas fluorescens WCS417r was maintained on King’s B medium supplemented with 50 μg/mL rifampicin  at 28°C on a rotary shaker at 150 rpm. P. fluorescens was applied onto plants as reported elsewhere (lsewhere .P. aphidis (108 cells/mL) applied at the plate center. Number of cells was recorded 7 days post inoculation. To assay for cutinase activity, TWA was supplemented with 1% TCL  suspended in 100% acetone or with 0.25% acetone. Plates were inoculated with 4 µL of P. aphidis (108 cells/mL) applied at the center of the plate. Plates were recorder 7 days post inoculation.Growth on cellulose requires cellulase activity. To assay for such cellulase activity, tap water agar (TWA) was covered with a cellulose membrane and inoculated with 4 µL of Arabidopsis WT and mutant seedlings (10 to 24 days old) grown in MS growth medium or on MS growth medium agar plates were treated with P. aphidis (1 × 108 spores/mL), P. fluorescens , 1 µM Flg22 , or 100 to 500 µg/mL chitin , as stated, for 18 h to 4 days before analysis. Chitin solution was prepared as described previously (4[Fe(CN)6], 1.25 mM K3[Fe(CN)6], 0.5 mM X-Gluc, and 1.25% Triton X-100} was poured into each well. The plants were vacuum-infiltrated for 10 min and then incubated at 37°C overnight, covered with aluminum foil. Tissues were destained with 100% ethanol overnight and placed in 70% ethanol before digital pictures were taken.GUS reporter lines seedlings (24 day-old) grown on MS agar plates were transferred to 6-well microplates 18 h after treatment for GUS staining, performed as described previously . Briefly2HPO4 (pH 9.5) and 0.01% aniline blue (Sigma-Aldrich) for several hours. The leaves were mounted on slides with 50% glycerol, and callose was observed immediately using a fluorescence microscope (Nikon Eclipse 80i) under UV light . Pictures were taken with a Nikon DS-oiMc camera and analyzed by NIS elements BR3.10 software. For callose counting, pictures from three independent biological repeats were used, with callose being counted in four areas of each picture.Following treatment, 10-day-old seedlings grown in MS medium in 12-well microtiter plates were fixed in a 3:1 ethanol:acetic acid solution for several hours. The fixative was changed several times to ensure both thorough fixing and clearing of the tissues, which is essential for good callose detection in the leaves. The seedlings were rehydrated in 70% ethanol for 2 h, 50% ethanol for an additional 2 h, and water overnight. After two or three water washes, the seedlings were treated with 10% NaOH and held at 37°C for 1 to 2 h to render the tissues transparent. After three or four water washes, the seedlings were incubated in 150 mM KArabidopsis plants and from plants 24 to 72 h posttreatment with 108P. aphidis spores/mL using a Qiagen RNeasy kit  according to the manufacturer’s instructions or using the LogSpin method  in 50% glycerol. Tissues sample were observed with a TCS SP8 confocal laser scanning microscope (Leica) and GFP was excited using a 488 nm laser to obtain maximum emission 500 nm, while chloroplasts were excited using a 488 nm laser to obtain maximum emission at 700 nm. To distinguish between chloroplast autofluorescence and GFP signals, propidium iodide was excited using a 514 nm laser to obtain maximum emission at 610 nm, using ×10 NA or ×40 water NA 1.1 objective lenses. Images were analyzed with the LAF AF computer program (Leica), using an image resolution of 1,024 by 1,024. The experiment was repeated three times.t tests were performed only when the data were normally distributed and sample variances were equal. Otherwise, a Mann-Whitney rank sum test was performed. Significance was accepted at a P value of <0.05 and is noted in figure or table legends. All experiments shown are representative of at least three independent repeats presenting the same pattern of results.


Bosmina is a globally distributed zooplankton that adapts to a eutrophic environment. In this study, we cultivated monoclonal Bosmina fatalis and determined its complete mitochondrial gene sequence using the Illumina HiSeq 4000 platform. It was 15,209 bp in length, including 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes, with the A + T content (69.2%) significantly higher than the G + C content (30.9%). All 22 typical tRNA genes had a classical cloverleaf structure, except for tRNAIle. The complete mitochondrial genome of nine other cladoceran species was used to reconstruct a phylogenetic tree, showing that B. fatalis has a closer relationship with Daphnia than other cladocerans. Bosmina is a filter-feeding zooplankton distributed worldwide   method and the Kimura2-parameter model based on 13 PCGs implemented in the MEGA version 7.0 , G (15.2%), T (37.0%), and C (15.7%) with A + T biased composition.The total length of the mitochondrial genome was 15,209 bp (GenBank accession no. MW770308), including 13 PCGs, 22 tRNA genes, and2 rRNA genes (16S rRNA and 12S rRNA). Among them, 23 genes were encoded by the J chain, and the other 14 genes were encoded by the N chain. The mitochondrial genome of All 22 tRNA genes ranged from 63 bp to 72 bp in size, except for tRNAIle (36 bp), showing the typical cloverleaf secondary structures except for tRNAIle, the 16 srRNA and 12 srRNA which were separated by tRNAVal, were 1381 bp and 755 bp in length, respectively.B. fatalis among 10 cladoceran species with complete mitochondrial genomes is shown in B. fatalis has a closer relationship with Daphnia than other cladocerans.The placement of


Angiogenesis, including the growth of new capillary blood vessels from existing ones and the malignant tumors cells formed vasculogenic mimicry, is quite important for the tumor metastasis. Anti-angiogenesis is one of the significant therapies in tumor treatment, while the clinical angiogenesis inhibitors usually exhibit endothelial cells dysfunction and drug resistance. Bisether (BTDE), a marine algae-derived bromophenol compound, has shown various biological activities, however, its anti-angiogenesis function remains unknown. The present study illustrated that BTDE had anti-angiogenesis effect in vitro through inhibiting human umbilical vein endothelial cells migration, invasion, tube formation, and the activity of matrix metalloproteinases 9 (MMP9), and in vivo BTDE also blocked intersegmental vessel formation in zebrafish embryos. Moreover, BTDE inhibited the migration, invasion, and vasculogenic mimicry formation of lung cancer cell A549. All these results indicated that BTDE could be used as a potential candidate in anti-angiogenesis for the treatment of cancer. Angiogenesis, the growth of new capillary blood vessels from existing ones and capillary venules, involves vascular endothelial cell proliferation, migration, matrix degradation, and branching to form new tubes . It has Besides the classical angiogenesis, Maniotis et al. firstly propose the concept of vasculogenic mimicry, which is a spontaneous and endothelial cell-independent tube-forming procedure . VasculoRhodomela confervoides, shows anti-angiogenesis properties by targeting multiple receptor tyrosine kinases [L. nana and Rhodomela confervoides, exhibits anti-angiogenesis effect both in vivo and in vitro through acting on VEGF signaling pathway [Symphyocladia latiuscula [Marine compounds are reported to have anticancer therapeutic and prophylactic activities ,19,20,21 kinases . Another pathway . Bis was provided by School of Medicine and Pharmacy. Antibodies against HIF-1α were purchased from Affinity , against β-catenin were purchased from Santa Cruz Biotechnology , against VEGF, GAPDH, Tubulin were purchased from HuaAn Biotechnology . Antibodies against AKT, p-AKT, ERK, p-ERK were purchased from Cell Signaling Technology . Transwell inserts (8 μm) were purchased from Corning company . Matrigel was the product of BD company .2 at 37 °C.Human umbilical vein endothelial cells line HUVECs was from American Type Culture Collection , human lung cancer cell lines A549 and H1975 were from Shanghai Cell Bank , Chinese Academy of Science. HUVECs was cultured in Dulbecco’s modified Eagle’s medium . Human lung cancer cell lines A549 and H1975 were kept in RPMI-1640 medium (Gibco-BRL). All mediums were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were cultivated in a humidified incubator containing 5% COThe proliferation effects of BTDE on differentiated HUVECs, A549, and H1975 cells were determined by 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. In brief, cells were plated in 96-well plate overnight to adhere, then 0−20 μM BTDE were administrated and incubated for 36 or 48 h. Total of 5 mg/mL MTT solution (20 μL per well) was added and incubated for another 4 h at 37 °C, discarding the supernatant and using dimethyl sulfoxide (DMSO) to dissolve products for 10 min at 37 °C. Microplate reader  was used to measure the 96-well plate at 570 nm, and the cell viability (%) was calculated by OD values.HUVECs, A549, and H1975 were plated in 96-well plate and cultivated to reach 90% confluence. Then scratch-wounds were made by 10 μL pipette tip. After washing twice with PBS, the cells were treated with fresh DMEM or RPMI-1640 (1% FBS) with 0−10 μM BTDE. After incubation for 36 or 72 h, images of wound in each well were recorded by inverted microscope . Image J was used to measure the area of each wound, and the migration rate was calculated as follows:Transwell chamber with 8 μm pore size was used to evaluate the migration of cells. Briefly, differentiated HUVECs, A549, and H1975 cells suspended in 1% FBS medium with 0−10 μM BTDE were seeded into the upper chamber of transwell 24-well plates. Then the lower chamber was added with complete medium and different 0−10 μM BTDE. After treatment for 24 h, the chambers were fixed with methanol, stained with 0.1% crystal violet, and the upper surface of the membrane containing non-migrating cells was gently wiped off with cotton swab. Random visual fields were calculated using inverted microscope , chambers were decolorized with 33% acetic acid, then each group of decolorizing solutions was transferred to a new 96-well plate. A microplate reader  was used to measure OD values of the plate at 570 nm.To investigate the invasion ability of HUVECs and A549, transwell invasion assay was conducted similarly with transwell migration assay except that the upper side of the chambers was spread with diluted matrigel (200 μg/mL).The ability of HUVECs or A549 to form capillary-like structures when treated with 0−10 μM BTDE was measured on matrigel. Briefly, pre-cooled matrigel was layered in 96-well plate and allowed to solidify at 37 °C for 45 min. HUVECs or A549 that has been treated with BTDE for 24 h was seeded on matrigel, after 20 h incubation for HUVECs or 30 h for A549, tube structure was recorded by inverted microscope  from randomly chosen fields. For investigating the effect of BTDE on performed vascular tubes, same concentrations of BTDE were added with HUVECs or A549 after tubes had already formed for 8 or 6 h, and incubated for another 6 h for HUVECs and 20 h for A549. Total length of tubes was measured by Image J software .For intersegmental vessel formation assay, Tg (flk1: EGFP) zebrafish embryos were generated by natural pairwise mating. Healthy, hatched zebrafish were picked out at 16 h post-fertilization and distributed into a 24-well plate (10 embryos per well). Embryos were treated with 0−10 μM BTDE for 24 h at 28.5 °C and then observed for intersegmental blood vessel (ISV) under inverted fluorescence microscope . Vessel growth was measured by Image J software.For toxicity assay, zebrafish embryos were picked out at 4 h post-fertilization and distributed into a 24-well plate (about 17 embryos per well). Embryos were treated with 0−20 μM BTDE for 24 h at 28.5 °C and then observed for morphologic changes under stereo microscope . The deformity and mortality rates were recorded.2/1 μM ZnCl2, pH 7.6), and washed twice for 40 min each time in rinse buffer , then incubated 48 h at 37 °C in renaturation solution containing 50 mM Tris/0.15 M NaCl/5 mM CaCl2/1 μM ZnCl2 and 0.02% Brij-35, pH 7.6). Gels were finally stained with 0.05% Coomassie Blue R250 for 30 min and decolorized with decolorizing liquid (10% acetic acid and 30% methanol) until negative staining bands appear. Gels were recorded by Bio-Red Gel Imaging Analysis System .Gelatin zymography assay was used to determine the activity of MMP9. HUVECs was treated with different concentrations of BTDE in serum free DMEM for 24 h, then culture supernatants were collected and centrifuged at 1200 rpm for 5 min, and then 12,000 rpm for 5 min to remove cellular components. Proteins existed in supernatants and were separated by 8% SDS-PAGE containing 1 mg/mL gelatin under non-reducing condition and then subjected to electrophoresis. Gels were washed twice for 40 min each time in washing buffer (2.5% Triton X-100/50 mM Tris/5 mM CaClHUVECs and A549 were treated with different concentrations of BTDE (0−10 μM) for 24 h. Cells were centrifuged, then washed twice with PBS and lysed with loading buffer for 45 min in 4 °C. Then, the cell lysate was boiled for 10 min and stored at −80 °C. Protein samples were resolved by 8–10% SDS-PAGE, transferred to nitrocellulose filter membranes . The nitrocellulose filter membranes were blocked with 5% skimmed milk and then incubated with the primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with HRP-secondary antibody at 25 °C for 1 h. Finally, the image was detected by Tanon 5200 .p < 0.05 were considered statistically significant.Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test, and values were expressed as mean ± SD. Differences of The present study illustrated for the first time that BTDE inhibited HUVECs migration, invasion, tube formation, and the activity of MMP9 in vitro. In zebrafish embryo, BTDE also restrained the growth of zebrafish embryo intersegmental blood vessel. In addition, BTDE suppressed A549, H1975 migration and A549 invasion, as well as the vasculogenic mimicry of A549 cells. These results definitely revealed that marine bromophenol compound BTDE was a potential agent for future cancer therapy due to its valid anti-angiogenesis effect.


Vitis rupestris B38 × ‘Horizon’ and ‘Horizon’ × Illinois 547-1, for 2 and 3 years, respectively. The acylation ratio followed a bimodal and skewed distribution in both families, with repeatability estimates larger than 0.84. Quantitative trait locus (QTL) mapping with amplicon-based markers (rhAmpSeq) identified a strong QTL from ‘Horizon’ on chromosome 3, near 15.85 Mb in both families and across years, explaining up to 85.2% of the phenotypic variance. Multiple candidate genes were identified in the 14.85–17.95 Mb interval, in particular, three copies of a gene encoding an acetyl-CoA-benzylalcohol acetyltransferase-like protein within the two most strongly associated markers. Additional population-specific QTLs were found in chromosomes 9, 10, 15, and 16; however, no candidate genes were described. The rhAmpSeq markers reported here, which were previously shown to be highly transferable among the Vitis genus, could be immediately implemented in current grapevine breeding efforts to control the degree of anthocyanin acylation and improve the quality of grapes and their products.Hydroxycinnamylated anthocyanins (or simply ‘acylated anthocyanins’) increase color stability in grape products, such as wine. Several genes that are relevant for anthocyanin acylation in grapes have been previously described; however, control of the degree of acylation in grapes is complicated by the lack of genetic markers quantitatively associated with this trait. To characterize the genetic basis of anthocyanin acylation in grapevine, we analyzed the acylation ratio in two closely related biparental families,  Vitis spp.), where they contribute to the coloration of red/black grape berries and resulting products such as juices and wines [V. vinifera cultivars are derivatives of hydroxycinnamic acids  [Anthocyanin pigments are widespread in the plant kingdom, including grapevines (nd wines . Anthocynd wines . Anthocycosides) . These hcosides) , purportV. vinifera cultivars reported that the acylated anthocyanin fraction  varied from 4.2% to 53.5% [Vitis accessions [The proportion of anthocyanins that exist in acylated forms can vary widely among grape cultivars. For example, a survey of 50 Spanish cessions . DNA marcessions ,8 and cocessions ,10,11.Vitis.The biosynthetic pathway of anthocyanins in grapes and other plants is well understood , and sevVitis biparental populations, V. rupestris B38 × ‘Horizon’ and ‘Horizon’ × Illinois 547-1. These populations segregated for the degree of anthocyanin acylation during 2 and 3 consecutive years, respectively. A set of 2000 amplicon-based markers (rhAmpSeq) were applied to find a stable major QTL on chromosome 3. An analysis of genomic regions harboring this marker-trait association revealed acyltransferase candidate genes regulating the presence/absence of anthocyanin acylation in grapes. Moreover, our highly transferable rhAmpSeq markers linked to anthocyanin acylation could be rapidly adopted in current breeding programs for the selection of grape materials with improved fruit quality.To identify novel markers for anthocyanin acylation, we quantified the anthocyanin acylation ratios  in two genetically related 1 grapevine families, V. rupestris B38 × ‘Horizon’ and ‘Horizon’ × Illinois 547-1 , were grown without replication  in Geneva, New York. ‘Horizon’ (white grape cultivar) is a complex hybrid and includes ancestry from V. vinifera, V. labrusca, V. aestivalis, and V. rupestris [V. rupestris B38 × V. cinerea B9; therefore, V. rupestris B38 (the only parent accumulating anthocyanins) is a parent in one family and a grandparent in the other. Fruit sampling and analysis were conducted in 2012 and 2013 for V. rupestris B38 × ‘Horizon’, and in 2011–2013 for ‘Horizon’ × Illinois 547-1. The progeny consisted of 118 and 116 vines in V. rupestris B38 × ‘Horizon’ and 93, 162, and 142 vines in ‘Horizon’ × Illinois 547-1, in each year, respectively. For each genotype, 150 g of fruit were sampled at maturity, based on fruit total soluble solid content (°Brix). TSS means and standard deviation at maturity in the families were 22.7 ± 1.9 and 18.4 ± 1.4 in 2012 and 2013 in V. rupestris B38 × ‘Horizon’ and 17.1 ± 2, 21.4 ± 2.3, and 20.1 ± 2.1 in 2011–2013 in ‘Horizon’ × Illinois 547-1. In addition, in 2013, ‘Horizon’ × Illinois 547-1 fruits were analyzed at an earlier phenological stage, i.e., mean TSS level of 16.5 ± 2.1, to study the effect of ripening stage on the degree of acylation. Sampled fruit was transported in cooler boxes and stored at −20 °C before shipping to the Cornell Ithaca campus for phenotyping.Two Fupestris . Illinoig). An anthocyanin fraction was then isolated by solid-phase extraction (SPE) fractionation based on the protocol of Manns et al. 2012. HPLC analyses were performed on an Agilent Model 1260 Infinity series consisting of an in-line vacuum degasser, autosampler, binary pump, diode array detector, and thermostatted column compartment controlled via Chemstation software . All solvents, including methanol, acetonitrile, ethyl acetate, and formic acid , were high-performance liquid chromatography (HPLC) grade. Anthocyanin HPLC analyses were based on the method described by Manns et al. 2012. Separations were performed on a Kinetex C18 column  fitted with a KrudKatcher guard filter . Anthocyanin identification was based on commercial standards where possible and otherwise based on characterized color extracts, retention times, and UV absorption characteristics  [Frozen berry samples were destemmed, partially thawed, and homogenized in a 250 mL stainless steel Waring blender  at medium speed for 30 s and then at high speed for 30 s. A 10 g portion of berry slurry was transferred to a 15 mL centrifuge tube and frozen at −20 °C. Prior to analysis, samples were thawed, equilibrated to room temperature, and centrifuged  . Quantif2 = (σG2)/(σG2 + σε2/n), where σG2 and σε2 are the variances of the genetic and residuals variance, respectively, and n is the number of experiments over the years, respectively.Broad-sense heritability was estimated for each population according to REML (restricted maximum likelihood) variance components using the following equation: HVitis genome was conducted as described in the work of [https://github.com/avinashkarn/analyze_amplicon/blob/master/analyze_amplicon.pl; (accessed on 2 April 2021)), which was further converted to a variant calling format (VCF) file using the haplotype_to_VCF.pl perl script ).DNA from individuals was extracted using modified Qiagen DNeasy columns with 2.7% PVP-40 added to the extraction buffer, as previously described . rhAmpSe work of ,20. rhAmMultidimensional scaling (MDS) and Mendelian error detection were performed as quality controls  to identV. rupestris B38 × ‘Horizon’ and ‘Horizon’ × Illinois 547-1, 2 test and monomorphism (non-segregating); (3) SeparateChromosomes2 module was used to split the marker into linkage groups (LG); (4) OrderMarkers2 module was used to order the markers within each LG using 20 iterations per group, computing parental (sex-specific) and sex-averaged genetic distances. The marker order of the genetic maps was evaluated for the consistency, genome organization, and structural variation by correlating with their physical coordinates on the V. vinifera PN40024 reference genome . Finall−4. Consistently with the observed binomial distribution on anthocyanin acylation suggesting a single major gene, we tested normal and binomial single-QTL models on acylation ratios and total acylated anthocyanins using the function scanone. Significance thresholds were defined based on 1000 permutation tests at α = 0.05. The percentage of variance explained in the context of a full additive model was calculated using fitqtl.Sex-averaged genetic maps were used for QTL mapping in the R package qtl . The genV. rupestris B38 × ‘Horizon’  and ‘Horizon’ × Illinois 547-1  populations, respectively. The acylation ratio was computed as the ratio of acylated anthocyanins relative to the total anthocyanin concentration. Total anthocyanins and acylated anthocyanins were poorly correlated , as were total anthocyanins and acylation ratio . However, acylated anthocyanins and acylation ratio were strongly correlated . Total anthocyanins and acylated anthocyanins showed a skewed normal distribution, whereas the acylation ratio showed a bimodal distribution, more clearly defined in the V. rupestris B38 × ‘Horizon’ population  within each population  and continuous (quantitative) single-QTL models. For binary single-QTL models, acylation ratio variables were converted to binary traits (0′s and 1′s), according to the requirements of r/qtl, based on k-means clustering analysis. A large QTL on chromosome 3  was consistently identified across populations and evaluation years. A stronger signal was observed when using a normal model compared to a binary model B. AdditiA large QTL on the arm of chromosome 3 was consistently found across different genetic backgrounds and years B. SeveraV. vinifera reference genome was inspected on the URGI database (https://urgi.versailles.inra.fr/Species/Vitis/Annotations (accessed on 6 June 2021)) (https://integrape.eu/resources/genes-genomes/genome-accessions/ (accessed on 26 November 2021)) lists not three but four genes encoding an acetyl-CoA-benzylalcohol acetyltransferase-like protein, which could be explored in further fine-mapping studies.The 14.85–17.95 Mb region on chromosome 3 of the 12X.v2 PN40024 e 2021)) B,C. In te 2021)) B. WithinAnthocyanins serve a dual role in the food industry as natural pigments and as health-promoting dietary constituents purported to have multiple beneficial properties ,10,11. AV. rupestris B38 × ‘Horizon’ and ‘Horizon’ × Illinois 547-1). These ranges for acylation ratios were within previously observed ranges in both V. vinifera and wild Vitis accessions [In this study, the portion of acylated anthocyanins that existed in acylated form  followed a clear binomial distribution within each population (cessions . AcylateO-glucoside-6-O-acyltransferase (3AT) described by Rinaldo et al. [3AT expression increases in the skins of most red berries (but not white) during ripening in both V. vinifera [Vitis [Using highly transferable rhAmpSeq markers, we mapped a significant association between the anthocyanin acylation ratio and a genomic region harboring three copies of a gene encoding acetyl-CoA-benzylalcohol acetyltransferase-like protein. Previously, a weaker QTL (based on a single year of data from the ‘Horizon’ × Illinois 547-1 population genotyped with GBS) located nearby was detected using total acylated anthocyanins, emphasizing the advantage in normalizing acylated anthocyanin concentration against total anthocyanins . Acetyl-o et al. , in whicvinifera ,14 and wa [Vitis .3AT is a likely candidate for controlling acylation ratios in grapes. Notably, both extreme alleles producing high acylation in the V. rupestris B38 × ‘Horizon’ and ‘Horizon’ × Illinois 547-1 populations came from ‘Horizon’, which produces white fruit. This cultivar resulted from a cross between ‘Seyval blanc’ and ‘Schuyler’, and the latter was a hybrid of ‘Primitivo’ (a red wine variety) and ‘Ontario’ (white-fruited). White-fruited grapes result from loss-of-function mutations of VvMYBA protein [Vv3AT [VvMYBA loss-of-function, white-fruited genotypes can form a valuable genetic source of highly functional Vv3AT alleles.Mapping population studies in other plant families, e.g., maize, have also reported that anthocyanin acyltransferases are important for controlling acylation ratios . Thus, 3 protein ,32, a man [Vv3AT . TherefoV. vinifera mapping populations did not detect major QTLs for acylation ratio, nor a QTL on LG 3 associated with Vv3AT. A thorough study of anthocyanin profiles in a ‘Syrah’ × Pinot noir population yielded only minor QTLs for anthocyanin acylation on LGs 3, 7, 8, 12, and 18. The authors speculate that their inability to detect a major QTL may be due to low segregation of the acylated ratio trait [Vv3AT in Pinot noir [Vv3AT copies, the resulting population would be heterozygous. A separate study on an F1 V. vinifera mapping population (‘Red Globe’ × ‘Muscat Hamburg’) also identified weak QTLs for acylation ratio [VvAT1, VvAT9) as well as genes involved in ion transport. We did not detect these same QTL, although we did detect a minor QTL in one year and one population  on Chr 9. The interval for this QTL contains a 5-O-glucosyltransferase (5GT) responsible for the production of anthocyanin 3,5-diglucosides in wild Vitis species. However, the relationship between diglucosides and acylated anthocyanins is unclear.Interestingly, two previous studies on io trait . Productnot noir  may reflon ratio . Again, V. rupestris B38 × ‘Horizon’ [Vitis spp., these markers have not been widely implemented. Highly transferable molecular markers are essential for the implementation of marker-assisted selection strategies, especially in grapes where the use of wild relative species in breeding programs is fairly common [Vitis species.The previous report with one harvest year on ‘Horizon’ × Illinois 547-1 used GBS to develop a haploblock panel of thirteen amplicon sequencing SNP markers to predict acylated ratios in Horizon’ . Howevery common ,34. Unliy common  since th


Additionally, venous-occlusion DOS (VO-DOS) and frequency-domain DOS (FD-DOS) were used to measure muscle blood flow (F) and tissue oxygen saturation (StO2), and resting calf muscle oxygen extraction fraction (OEF) was calculated from MRO2, F, and blood hemoglobin. Lastly, the venous/arterial ratio (γ) of blood monitored by FD-DOS was calculated from OEF and StO2. PAD patients who experience claudication (n = 28) were randomly assigned to exercise and control groups. Patients in the exercise group received 3 months of supervised exercise training. Optical measurements were obtained at baseline and at 3 months in both groups. Resting MRO2, OEF, and F, respectively, increased by 30%  (p < 0.001), 17%  (p = 0.003), and 7%  (p = 0.11), after exercise training (median (interquartile range)). The pre-exercise γ was 0.76 ; it decreased by 12%  after exercise training (p = 0.011). Improvement in exercise performance was associated with a correlative increase in resting OEF .Exercise training can mitigate symptoms of claudication  in patients with peripheral artery disease (PAD). One adaptive response enabling this improvement is enhanced muscle oxygen metabolism. To explore this issue, we used arterial-occlusion diffuse optical spectroscopy (AO-DOS) to measure the effects of exercise training on the metabolic rate of oxygen (MRO Peripheral artery disease (PAD) is a common manifestation of the progressive stenosis of peripheral arteries. More than 200 million people have PAD worldwide, and the prevalence of PAD is ≥20% in individuals over the age of 80 years . Approxi2), the level of anaerobic metabolism needed to supply the total energy load from walking is lower.Claudication is triggered by limb ischemia that arises from large artery stenosis, vascular dysfunction, inflammation, and other factors ,9. Numer2, and in particular, whether these adaptations improve resting muscle MRO2. To date, direct blood flow measurements with optical diffuse correlation spectroscopy have provided evidence that exercise training increases the peak calf muscle blood flow reached during walking [2 levels during exercise. Another way to sustain higher MRO2 levels are adaptations that yield greater oxygen extraction per unit of blood delivered [2, blood flow (F), and oxygen extraction fraction (OEF).Thus, it is of interest to ascertain what underlying adaptations from exercise training enable improvements in MRO walking ; similar walking ,18,19,20elivered . Herein,2 and F. Using Fick’s law [2, F, and blood hemoglobin (C) measurements. The exercise training effects on resting calf muscle tissue oxygen saturation (StO2), total hemoglobin concentration (THC), and blood volume fraction (fBV = THC/C) were also determined from FD-DOS and C measurements.Specifically, we incorporate the techniques of arterial-occlusion and venous-occlusion diffuse optical spectroscopy  into a randomized clinical study to ascertain the effects of a 3-month supervised exercise-training program on resting calf muscle MROck’s law ,22, we c2 enable the direct calculation of the ratio of venous and arterial blood monitored by FD-DOS (γ). In contrast to pulse oximetry, the StO2 measurement is a weighted average of arterial and venous oxygen saturations [2 = (1 − γ) SaO2 + γ SvO2. The γ ratio thus informs the interpretation of absolute StO2 levels. Furthermore, a change in γ will result in a change in StO2, even if SaO2 and SvO2 are constant. One study has measured γ for FD-DOS cerebral oximetry in children with congenital heart disease [Importantly, our knowledge of OEF and StOurations : StO2 =  disease . To our We hypothesize that exercise-training increases resting muscle oxygen metabolism and that these increases are driven by enhanced calf muscle oxygen extraction per unit of blood delivered. We also hypothesize that γ remains constant before and after exercise training.The exercise and control groups were well-matched, i.e., in terms of baseline demographics and performance variables . The exe2), blood flow (F), oxygen extraction fraction (OEF); calculated from MRO2 and F data, tissue oxygen saturation (StO2), and total hemoglobin concentration (THC) across both the exercise and control group populations are reported in BV), venous/arterial ratio of blood monitored by FD-DOS , the recovery half-time of deoxy-hemoglobin (Hb)  for Hb to return to baseline after arterial occlusion, calf muscle tissue reduced scattering coefficients , and the differential pathlengths (L) of light transport through the calf muscle , i.e., the average 3-month/baseline ratio was 1.99 . There was no accompanying change, however, in the ankle brachial index, i.e., the average 3-month/baseline ratio of 1.01  was not different from unity (p = 0.50).  s and 0.63 .) For patients in the control group, the corresponding changes in peak walking time and ankle brachial index were −0.91  and 1.00 , respectively; these changes were not significant.  s and 0.63 .)The peak walking time for patients in the exercise group (patients that received 3 months of supervised exercise training) significantly increased after exercise training  and 1.02  for the exercise and control groups, respectively, were not different from unity, and for the FD-DOS THC metric, the average 3-month/baseline THC ratios of 1.08  and 1.00  for the exercise and control groups were not different from unity. The recovery half-time of Hb was also not altered by exercise training  and 0.92 , which were both not different from unity). Additionally, the thickness of the near-surface tissue layer overlaying the muscle was not altered by exercise training  and 0.99 , which were both not different from unity).2  of 1.30  across patients in the exercise group was higher than unity (p < 0.001), while the corresponding average rF of 1.07  was not statistically different from unity (p = 0.11). For patients in the control group, no differences were observed between the 3-month and baseline MRO2 and F measurements. Further, the median rMRO2 was higher in the exercise group than it was in the control group (p = 0.006), while there was no difference in median rF between the exercise and control groups (p = 0.78).The optical AO-DOS and VO-DOS results support our hypothesis that exercise training increases resting calf muscle metabolic rate of oxygen a but doep = 0.003), while the average rOEF of 1.02  across patients in the control group was not different from unity (p = 0.81). rOEF in the exercise group was higher than rOEF in the control group (p = 0.046).Additionally, exercise training did not alter resting calf muscle tissue oxygen saturation and blood volume fraction c,d, but p = 0.011)). The decrease in γ suggests that a partial redistribution of calf muscle blood volume from veins to arterioles could have occurred, and the effect may be an explanation for why OEF increased, but StO2 remained unchanged after exercise training (see p = 0.24), but there was also no difference in the average rγ between the exercise and control groups (p = 0.22).Finally, our results do not support the hypothesis that the venous/arterial ratio of blood monitored by FD-DOS is the same before and after exercise training. The ratio actually decreased after exercise training f; the avning see . Of note2, OEF, and γ with improvement in exercise performance ), but was correlated with rOEF ). There was no correlation between rPWT and rγ ). Additionally, in another secondary analysis, there was no correlation between rMRO2 and rγ ).In a secondary analysis, we used linear regression to assess the correlations of changes in MROformance . The rel2 and OEF in PAD patients. This result, in combination with previous observations of larger fractional increases (relative to resting levels) in MRO2 and OEF during walking after exercise training [2 and OEF levels occur during walking. These enhancements likely delay the onset of limb ischemia caused by the energy load from walking, which may in turn delay the onset of claudication. We also measured the venous/arterial ratio of blood monitored by FD-DOS in calf muscle , and we found that exercise training decreased γ.We found that exercise training increases the resting calf muscle MROtraining , indicat2 levels are enabled by increases in muscle blood flow and/or by increases in muscle oxygen extraction per unit of blood delivered [Higher MROelivered ,24. Fromelivered ,18,19,20elivered .2) and the calf muscle oxygen extraction fraction (OEF) [2 filterability due to decreased blood viscosity [2 changes. A future study with a larger sample size is warranted to confirm this finding. Possibly, adaptations that increase OEF may have other benefits not related to MRO2 that enhance exercise performance.The amount of oxygen extracted per unit blood delivered is the product of the arterial blood oxygen concentration (CaOon (OEF) . Our resiscosity ,7,8. Ind2 was not altered by exercise training, despite its relation to OEF. Our observation of a decreased γ coefficient after exercise training (see Equation (4)) suggests a possible explanation for increases in OEF that are not accompanied by a reduction in StO2. StO2 can vary when OEF changes, but it will also vary if the relative arteriole and venous contributions to the StO2 signal change [2. We did not, however, observe a significant correlation between MRO2 and γ changes.Surprisingly, measured StOl change ,23, i.e.2 measurement would push StO2 higher, thereby counteracting the effects on StO2 from decreases in capillary and venous saturations caused by increased OEF. To our knowledge, this type of mechanism has not been reported in the literature. Note, however, the magnitude of the γ decrease that we measure is less than the magnitude of the OEF increase, which suggests that the γ change is only a partial explanation of the measured StO2 behavior.Since we found that the overall calf muscle blood volume fraction was not altered by exercise training, this drop in venous blood volume would be compensated by increased arteriolar and capillary blood volume . A larger arterial contribution to the StOOur measurements did not demonstrate a significant effect from exercise training on the recovery half-time of deoxy-hemoglobin after arterial occlusion. Higher maximal calf blood flow during reperfusion reduces the recovery time, but higher calf oxygen extraction during arterial occlusion increases the recovery time . We specOur average pre-exercise resting calf muscle oxygen metabolism measurements of 2.74  µmol/100 mL/min  mL/100 g/min) are roughly consistent with previous measurements of resting calf muscle oxygen metabolism in PAD patients  mL/100 g/min , 0.10  are consistent with previously reported VO-DOS calf muscle measurements in healthy adults, i.e., 0.88 ± 0.35 mL/100 mL/min , but low2 of 66.2 ± 7.5% (mean ± SD) is within the range of FD-DOS calf muscle measurements reported in the literature for PAD patients, e.g., 68 ± 6% [Our average pre-exercise StO 68 ± 6%  and 56 ± 68 ± 6% , as well 68 ± 6% , 55.5 ±  68 ± 6% , 62 ± 5% 68 ± 6% , 66.2 ±  68 ± 6% , 72.0 ±  68 ± 6% . Our ave 68 ± 6% , and in  68 ± 6% , 48 ± 18 68 ± 6% . Finally 68 ± 6% . It is a 68 ± 6% .2, where rSaO2 is the three-month/baseline ratio of the actual arterial oxygen saturations).The present study has limitations. First, the sample size was small, and we may be underpowered to detect increases in resting blood flow from exercise training. The median resting blood flow increase of 7% from exercise training b may proBV calculations assumed that the systemically measured blood hemoglobin concentration is the same as the blood hemoglobin concentration in the tissue volume sampled by the optical techniques. If blood hemoglobin is lower in the calf muscle microvasculature than in the systemic measurements, then the actual F and fbv will be higher than the calculated values. We anticipate this source of error, however, should not alter our conclusions about relative F and fBV changes from exercise, especially given our observation of constant systemic hemoglobin. Nevertheless, future work is needed to investigate whether blood hemoglobin in the calf muscle changes with exercise.Third, our F and fFourth, variance in the adipose/skin thickness above the muscle can influence the absolute optical measurements ,30,36. OIn this study, we carried out additional measurements on a subset of patients enrolled in a randomized control trial conducted at the University of Pennsylvania. The details of the trial’s study design are described elsewhere . SubjectSubjects randomized to the exercise group performed three 60 min supervised exercise training sessions each week for a period of 3 months at the vascular laboratory of the University of Pennsylvania. As described elsewhere , during 2) and blood flow (F) in the most symptomatic leg were noninvasively measured at baseline and at 3 months with the optical techniques of AO-DOS and VO-DOS (2), total hemoglobin concentration (THC), and multispectral tissue reduced scattering coefficients  for λ = 685, 785, and 830 nm wavelengths) in resting muscle using frequency-domain DOS. As described in BV), and venous/arterial ratio of blood monitored by FD-DOS (γ). The recovery half-time for deoxy-hemoglobin  to return to baseline after arterial occlusion was also measured  resting calf muscle metabolic rate of oxygen (MROd VO-DOS . We addi2 and THC (a muscle water volume fraction of 70% was assumed [s’ (685 nm), µs’ (785 nm), µs’ (830 nm)).Optical measurements were performed using a custom-built three-wavelength  frequency-domain diffuse optical spectroscopy instrument described elsewhere . First,  assumed ). MultisFor the arterial-occlusion and venous-occlusion DOS measurements, a second MRI compatible optical probe was secured to the same location above the calf flexor. Of note, these measurements were carried out in a 3T MRI scanner (the MRI data is outside the scope of this manuscript). A MRI fiducial was placed above the center of the probe to mark the probe’s position on the anatomical image for measurement of the skin and adipose tissue thickness. The amplitude and phase of multispectral intensity modulated light was monitored before/during/after a 5 min arterial occlusion and a 1 min venous occlusion at 2.5 cm source-detector distance (1 measurement was acquired every 8 s). The venous occlusion was initiated 10 min after the completion of arterial occlusion . Arteria2) and deoxy-hemoglobin (Hb) from changes in the multispectral amplitude data [The semi-infinite modified Beer–Lambert approach was adopted to derive changes in calf muscle oxy-hemoglobin  was measured in the calf by calculating the rate of conversion of oxy-hemoglobin to deoxy-hemoglobin during arterial occlusion [Resting metabolic rate of oxygen  was measured from the rate of increase in total hemoglobin during venous occlusion ,47:F = , mL blood/100 mL/min, and g/(dL blood), respectively.Here, the units of MRO2 to determine the venous/arterial of blood monitored by FD-DOS (γ). More precisely, StO2 measurements reflect a mixture of arterial, capillary, and venous blood, i.e., StO2 = ka SaO2 + kc ScO2 + kv SvO2 [2, ScO2, and SvO2 are the arterial, capillary, and venous oxygen saturations, and ka, kc, and kv are the respective weight of each compartment’s contribution to the total blood volume sampled by FD-DOS (ka + kc + kv = 1). Assuming that plasma oxygen concentration is negligible compared to bound oxygen concentration, OEF = (SaO2 − SvO2)/SaO2, or in terms of StO2, OEF = (SaO2 − StO2)/(γSaO2), where γ ≡ kv + kc kw is the FD-DOS venous/arterial ratio [w arises from making a simplification that represents ScO2 as a weighted average of SaO2 and SvO2, i.e., ScO2 = (1 − kw) SaO2 + kw SvO2. Assuming again that SaO2 = 1,We then used the OEF calculated from Equation (3) and the measured StO kv SvO2 . SaO2, Sal ratio . Here, tBV) in the muscle tissue by taking the ratio of THC and C, i.e., fBV = THC/C .Finally, we calculated the blood volume fraction . All statistical tests were two-sided, and n = 28) to estimate the associations between the optical metrics that significantly changed from exercise training  and improvement in exercise performance. Improvement in exercise performance was defined as the relative 3-month/baseline ratio for PWT . The Pearson correlations (R) between the relative 3-month/baseline ratios of the optical metrics and rPWT were computed, as well as the slopes and 95% confidence intervals of the best-fit lines. Finally, to estimate whether a redistribution in resting calf muscle blood volume is associated with increased oxygen metabolism, we performed an identical linear regression analysis to compare MRO2 and γ changes.For our secondary analyses, we used linear regression analyses across our entire cohort (In a randomized study of 28 PAD patients with intermittent claudication, we employed optical techniques to demonstrate increased resting calf muscle oxygen metabolism levels from exercise training, which appear to be predominantly driven by increases in resting calf muscle oxygen extraction per unit of blood delivered. Exercise training did not alter the calf muscle blood volume fraction, though our results suggest a partial redistribution of calf muscle blood volume from veins to arterioles. Improvement in peak walking time on the treadmill was associated with a correlative increase in resting calf muscle oxygen extraction fraction. Future work is needed to better understand the underlying exercise-induced adaptations that increase resting muscle oxygen extraction.


Punica granatum L.) is a rich source of polyphenolic components. The purpose of this study was to characterize the phenolic composition and flavonoids and anthocyanin content of different parts (peel and aril) of the Sefri variety of pomegranate. Our results showed that Peel extract was richer in these compounds than that of the Arils, especially in Punicalagin (A and B). DPPH free radical scavenging, reducing power (FRAP), β-carotene bleaching, and hydrogen peroxide scavenging assays revealed a greater dose-dependent activity of pomegranate peel phenolic extract (PPPE) compared to pomegranate aril phenolic extract (PAPE). PPPE was also more potent than PAPE concerning its ability to inhibit conjugated diene formation and to reduce α-tocopherol disappearance induced by CuSO4-mediated LDL peroxidation. Interestingly, both extracts (PPPE and PAPE) significantly inhibited lipid peroxidation and the formation of reactive oxygen species (ROS) in stressed J82 human bladder cancer cells. These results reflect the protective effects that this Moroccan variety of pomegranate can provide against the development of metabolic disorder, cancer, atherosclerosis, and cardiovascular disease. Given these properties, further studies should be undertaken to investigate possible applications of Sefri pomegranate extracts in the fields of food preservation and health supplements.Natural antioxidants products are widely distributed in food and medicinal plants. These natural antioxidants, especially polyphenols, exhibit a wide range of biological activities including anti-cancer, anti-inflammatory, and anti-atherosclerosis activities. Pomegranate ( Pomegranate peel, seeds, juice, and arils are a rich source of several valuable bioactive compounds with considerable nutritional, antioxidant, and other beneficial properties ,2,3. PeeMorocco’s annual pomegranate production exceeds 58,000 tones from a total area of 5000 hectares ,23 and hSefri pomegranates, notably the anti-oxidative and physicochemical properties of pomegranate peel phenolic extract (PPPE) and pomegranate aril phenolic extract (PAPE) and evaluated their effects on atherosclerosis and bladder cancer cells.In this study we investigated several beneficial health properties of various bioactive compounds from LDL oxidation is considered to be a hallmark of early atherogenesis. Nutritional antioxidants such as phenolic compounds can markedly inhibit LDL oxidative damage by reducing free radicals generated during oxidative metabolism, preserving endogenous antioxidants in LDL (vitamin E and carotenoids), chelating transition metal ions, and modulating the oxidative state of the arterial cell wall. These properties act to inhibit cell-mediated oxidation of LDL and increase serum paraoxonase (PON1) activity .Polyphenols and/or their derivatives are used to treat cancer. Cancer initiation may be modulated by an increase in ROS levels, which can damage DNA and stimulate pro-oncogenic signaling . OxidatiIn the Beni Mellal-Khenifra area, all parts of the pomegranate, especially peel, are used for health remedies to treat diarrhea, ulcers, nasal bleeding, and inflammation. Moreover, this fruit is highly consumed by patients with vascular disorders. This variety may have a more powerful health potential regard to its possible richness in phenolic compounds.To the best of our knowledge, and even though various pomegranates are cultivated in different regions of Morocco, only a few studies have focused on the chemical composition and properties of the Sefri variety of pomegranate and even fewer on its biological properties, particularly in the prevention of diseases linked to oxidative stress such as cardiovascular disease and cancer. From this perspective, we conducted this study firstly to analyze the chemical composition of phenolic compounds from peels and arils of this variety. At the biological level, we took a particular interest in studying the relationship between antioxidant activity of the Sefri cultivar and its impact in preventing lipid peroxidation in human LDLs  and in m2-4H2O, ethylene-di-amine-tetra-acetic acid (EDTA), butylated hydroxytoluene (BHT), ferrozine ascorbate, H2O2, potassium ferricyanide, phosphate buffer, ferric chloride, gallic acid, catechin, Folin-Ciocalteu reagent, sodium nitrite, aluminium chloride, glacial acetic acid, acetonitrile, and formic acid, which were obtained from Sigma-Aldrich Chemical Co. . Ethanol, methanol, trichloroacetic acid (TCA), sodium carbonate, and sodium hydroxide were purchased from Fisher Scientific . Synergi 4 μm Hydro-RP 80A columns (250 mm × 4.6 mm × 5 μm) and C18 5 μm columns (250 mm × 3.0 mm) were purchased from Phenomenex .All chemicals were of analytical reagent grade and were purchased from Sigma-Aldrich Chemical Co. , except for Trolox, FeClPomegranate fruit was harvested from Sefri pomegranate trees . The authenticity of the variety was confirmed by Dr Abbas Younes, taxonomist, and a voucher specimen was conserved for further reference at our laboratory herbarium . The fruit was washed and hand peeled. Arils were squeezed using a commercial blender to obtain pomegranate molasses. Air-dried pomegranate peels were ground to a fine powder, which was stored in a freezer.Peel powder and pomegranate aril molasses were macerated by sonication in 70% methanol/0.1% acetic acid for 48 h in the dark. The hydroalcoholic extracts were centrifuged for 10 min, and the solids were removed by vacuum filtration through a Whatman filter Grade GF 10. The supernatants were concentrated under vacuum and were then freeze-dried and stored at −80 °C until used.w/v Na2CO3 was added. Following a 2-h incubation at room temperature in the dark, the absorbance was read at 760 nm using a UV-6300PC double beam spectrophotometer . TPC was determined using the gallic acid calibration curve and was expressed as milligrams of gallic acid equivalents per gram of dry matter. All samples were analyzed in triplicate.Total phenolic content (TPC) was determined using the modified Folin-Ciocalteu method . Gallic Total flavonoid content (TFC) was quantified using the method described by Woisky et al., with minor modifications . BrieflyTotal anthocyanin content (TAC) was determined by the pH differential method as described by Sellappan et al.  using twTAC was calculated as cyanidin-3-glucoside equivalents (2):−1·mol−1).MW: molecular weight of cyanidin-3-glucoside (449.2 g/mol). DF: dilution factor. ε: Molar extinction coefficient  column and pre-column , were used. The chromatogram was monitored at 220–400 nm), with the spectra recorded continuously throughout the elution. The following two eluents were used: A, double distilled water containing 0.2% acetic acid (pH 3.0) and B, acetonitrile. The flow rate was 1 mL/min), and the gradient was optimized as follows: 5 min (0–5%) B; 5–10 min (5–13%) B; 10–13 min (13–18%) B; 13–20 min (18%) B; 20–23 min (18–25%) B; 23–35 min (25%) B; 35–40 min (25–30%) B; 40–41 min (40–85%) B; 41–50 min (85–90%); 50–54 min (90–100%); and B; 54–60 min (100–5%) B. The samples were filtered through 0.2-mm PTFE filters, and 10 µL of the sample were injected at a stable column temperature of 30 °C. The absorption wavelengths used to detect the polyphenols ranged from 200 to 400 nm. Peak areas were quantified using a calibration curve obtained using gallic acid, α and β punicalagin, and ellagic acid as external standards. For this purpose, calibration curves were prepared for each analytical standard. Linearity, the limits of detection and quantification, are cited in An HPLC system composed of an autosampler (SIL-HTc), a degasser (DGU-14A), a column oven (CTO-10AS), and a diode array detector (SPD-M10A) , as well as an Inertsil0 is the absorbance of the control reaction  and AS is the absorbance of the test compound. Ascorbic acid was used as a positive control.The DPPH  assay uses the capacity of the DPPH radical to scavenge, as a measure of the antioxidant activity that prevents lipid peroxidation. The free-radical scavenging activities of PPPE and PAPE were evaluated using the DPPH assay based on the method of Zhang et al. , with sl50) was calculated from the plotted graph of scavenging activity against the concentrations of each extract. All experiments were performed in triplicate.The half-maximal extract concentration  prepared in 50 mM phosphate buffer (pH 7.4) was determined by measuring the absorption at 230 nm. The absorption of the assay mixture, which contained 500 µL of different concentrations of PPPE, PAPE, or a standard ascorbic acid solution (0–200 µg/mL) together with 1 mL of H2O2 was determined after 30 min against a blank solution containing phosphate buffer without H2O2. The percentage of H2O2 scavenged was calculated using the following formula:The ability of PPPE and PAPE to scavenge Hh et al. . The con3Fe(CN). The mixtures were shaken well and were incubated at 50 °C for 20 min, following which 1 mL of 10% TCA was added. They were then centrifuged at 3000 rpm for 10 min. The supernatants (1.5 mL) were mixed with 1.5 mL of distilled water and 0.1 mL of 0.1% FeCl3. The absorbance was read at 700 nm. Ascorbic acid was used as a reference standard. The total antioxidant activity (TAA) determined by FRAP was expressed as mg of ascorbic acid equivalents per gram of dry matter (mg AAE/g dm).Ferric antioxidant power was determined using the potassium ferricyanide-ferric chloride assay . A 500 µ0 and A°0 are the absorbances measured at t = 0 min of the incubation of the test sample and control, respectively, and (A180) and (A°180) are the absorbances measured in the sample and control, respectively, after a 180-min incubation. The assay was carried out in triplicate.The antioxidant activities of PPPE and PAPE were evaluated using the β-carotene-linoleic acid (BCB) assay as per the method of Jayaprakasha et al. . β-carot−2 M, pH 7). Protein concentrations were measured using commercial assay kits  according to the manufacture’s protocol and were expressed as LDL total protein concentration.LDL (low density lipoproteins) were isolated from fasting human heparinized plasma using the method of Sattler et al. . BrieflyThe present study was conducted according to the guidelines set out in the Declaration of Helsinki. The protocol was approved by the Ethics Committee of the Sherbrooke University Institute of Geriatrics (# 2009/19). Written informed consent was obtained from all subjects.LDLs were oxidized using transition metal ions as oxidizing agents . BrieflyLipid peroxidation was evaluated by measuring conjugated diene formation and the disappearance of vitamin E (α-tocopherol). Oxidized LDL levels, alone or in the presence of PPPE or PAGE, were continuously monitored at 234 nm as previously described by Berrougui et al. 2006 , to measThe endogenous LDL content of vitamin E was assayed by measuring the α-tocopherol content following 2-h oxidation of LDL in the presence or absence of 0.2 mg/mL of PPPE or PAPE using reverse-phase HPLC and electrochemical  and UV (at 292 nm) detection as previously described . α-Tocop6 cells) for 30 min at RT. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (ab96879) at 1/500 dilution for 30 min at RT. Flow cytometry data were collected on a CytoFLEX instrument  and analyzed using FlowJo 10.2 software .For intracellular staining, Fu5AH hepatic cells were fixed, permeabilized using staining Kit (abcam) and stained with anti-PON1 antibody  human bladder cancer cell line (batch # 70002468) was purchased from the American Type Culture Collection  via Cederlane® company . The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in a humidified 95% air/5% CO2 atmosphere at 37 °C. Cells were trypsinized using 0.05% EDTA-0.02% trypsin. The Fu5AH (rat hepatom cell line) were kindly provided by Dr J. Genest’s laboratory . Fu5AH cells were maintained in minimal essential medium containing 5% bovine serum and antibiotics . Ru. Ru47]. It is well documented that pomegranates, especially peels, are rich in ellagitannins, which can make up 66% of the total polyphenols . We quan50 values . Ascorbic acid (IC50 = 6.61 µg/mL) was used as a positive control and exhibited 1.89-fold higher activity than PPPE and 3.27-fold higher activity than PAPE. The IC50 values obtained for DPPH in our study were much lower than those reported by Ali et al., who obtained an IC50 of 14.75 µg/mL for a peel extract and 128.27 µg/mL for an aril extract [50 ranging from 42.71 to 65.55 µg/mL) than the Moroccan Sefri variety. However, another study conducted by Guo et al. [50 of 0.032 µg/mL, which is much lower than our results. These differences could be explained by the growing conditions or the analytical methods used. The DPPH scavenging activity of pomegranate fruit is associated with their total phenolic, anthocyanin, and flavonoid contents, as shown in DPPH radical scavenging activity was assayed using increasing concentrations of PPPE and PAPE, with ascorbate as an internal standard (0 to 0.4 mg/mL). Our results  showed t extract , indicato et al. , showed 2O2 free radicals (IC50 = 19.96 ± 0.02 µg/mL) than PAPE (IC50 = 37.06 ± 0.05 µg/mL (p < 0.001). In terms of antioxidant activity (AA%) at the same concentration, ascorbate exhibited the highest AA% followed by PPPE and then PAPE. At 200 ug/mL, the AA% was 98.21%, 87.8%, and 64.12%, respectively, for ascorbic acid, PPPE, and PAPE. H2O2 scavenging activity was likely affected by the concentration of phenolic compounds. Since phenolic compounds are powerful chain-breaking antioxidants, they may accelerate the decomposition of H2O2 to H2O and oxygen [2O2 is highly reactive and contributes to the formation of transition metal ion-dependent OH radical-mediated oxidative DNA, protein, and lipid damage [Our results also showed that PPPE has a better capacity to scavenge Hd oxygen . H2O2 isd damage .2O2 assays did not confirm the antioxidant activities of the pomegranate extracts. Given this, FRAP and BCB assays were conducted to provide a reliable assessment of the antioxidant properties of pomegranate compounds.The specificity and sensitivity of the DPPH and Hp < 0.001) (Our results from the FRAP assay showed that PPPE has a higher capacity to reduce the ferric tripyridyl-triazine complex (Fe(III)-TPTZ) to a ferrous complex (Fe(II)-TPTZ) due to the electron-donating abilities of its rich phenolic compounds as expressed by TAA (374.83 ± 16.85 mg AAE/g dw) than PAPE  . A similp < 0.001). This is consistent with a study carried out by Singh et al. [We evaluated the antioxidant potential of PPPE and PAPE for lipid peroxidation using the β-carotene/linoleic acid bleaching assay, which is based on a decrease in the color of β-carotene following its reaction with radicals generated when linoleic acid is oxidized. h et al. , who alsh et al.  reportedh et al.  reportedh et al. . Our resp < 0.0001), and that PPPE was 8% more efficient than PAPE . This effect was confirmed by measuring the α-tocopherol content of ox-LDL in the presence or absence of PPPE or PAPE. As shown in p < 0.001, for non-ox-LDL and Ox-LDL, respectively). However, the phenolic pomegranate extracts significantly prevented α-tocopherol degradation. The results in p < 0.01, and 2.81 ± 0.17, p < 0.05, respectively, compared to the Ox-LDL values). These results are in agreement with those obtained by Aviram et al. [To gain more insight into the antioxidant mechanism of pomegranate phenolic-rich extracts, we analyzed the ability of PPPE and PAPE to inhibit copper-induced LDL oxidation, prevent the disappearance of vitamin E (α-tocopherol), and promote PON1 activity. The copper-induced LDL oxidation (ox-LDL) results showed that both PPPE and PAPE significantly inhibit conjugated diene formation  (p ˂ 0.05) as well as PON1 protein expression    than PAPctively) . These rCancer cells grow better in oxidative stress conditions as this increases their survival potential via various pathways that induce redox signaling activation, that in turn may lead to the suppression of tumor guardian and suppressor genes , the act2O2 control by 32.04% and 37.95%, respectively (p ˂ 0.001). On the other hand, 100 µg/mL of PAPE caused no significant decrease in intracellular ROS levels whereases 200 µg/mL of PAPE reduced intracellular ROS formation by 27.83% (p < 0.001). However, in terms of an in vitro antioxidant effect, PPPE reduced intracellular ROS formation 10% more than PAPE. These results are in agreement with those reported by Rosenblat et al., who showed that pomegranate polyphenols, especially punicalagin and gallic acid, markedly reduce ROS formation in J774 cells [In the present study, we examined the effects of PPPE and PAPE on ROS production and lipid peroxidation in J82 human bladder cancer cells. Our results showed that both PPPE and PAPE induce a significant decrease in the ROS content of J82 cells in a dose-dependent manner compared to the control A. PPPE (74 cells .p ˂ 0.01), respectively, compared to the control. PAPE (100 and 200 µg/mL) caused a much lower reduction in TBARS of 17.32% (p < 0.05) and 23.4% (p < 0.01), respectively, than PPPE. Park et al. [Lipid peroxidation, which is another approach for evaluating oxidative damage, was quantified by measuring TBARS B. The twk et al.  showed tk et al.  reportedk et al.  showed tFruits are a rich source of vitamins, minerals, and biologically active compounds. However, they are often consumed without the peels although some fruit peels are rich in polyphenolic compounds, flavonoids, ascorbic acid, and other biologically active compounds that have a positive effect on health. Our results show that pomegranate peel and aril extracts increase serum PON1 activity, which may delay the development and progression of atherosclerosis. We also show that pomegranate extracts attenuate ROS production and lipid peroxidation in J82 human bladder cancer cells. These results indicate that pomegranate extracts from peel and arils protect against oxidative stress and exhibit anticarcinogenic activity against J82 cells. Finally, pomegranates may be a promising source of cancer-preventing agents due to their high phenolic content. However, pomegranate peel extract exhibited a better potential effect than aril extract.


To assess the maximal lymph nodal diameter on the 8th edition American Joint Committee on Cancer staging system of nasopharyngeal carcinoma (NPC).This study extracted NPC patients between 2004 and 2016 in the Surveillance, Epidemiology, and End Results database. Included patients were divided into 3 groups: ≤3 cm, >3–6 cm, and >6 cm based on the maximal lymph nodal diameter. Cumulative survival curves of 5-year overall survival (OS) and cancer-specific survival (CSS) were calculated using the Kaplan-Meier method between the 3 groups.P = .240) and CSS  of ≤3 cm and >3–6 cm groups were not different. In contrast, the 5-year OS and CSS were different between >6 cm and ≤3 cm groups, and between >6 cm and >3–6 cm groups. The stratified hazard ratio of OS and CSS was 1.75  and 1.77  for the >6 cm group in the multivariate regression analysis.The 5-year OS (64.0% vs 59.3%, It is reasonable that the maximal lymph nodal diameter with >6 cm is classified as stage N3 of the 8th edition American Joint Committee on Cancer staging system for NPC. An accurate N stage is crucial to formulate treatment plans and evaluate prognosis. The 8th edition American Joint Committee on Cancer (AJCC) staging system of NPC was proposed. In the 8th edition staging system, the maximal lymph nodal diameter on N stage is divided into 2 groups: >6 cm and ≤6 cm. The maximal lymph nodal diameter with >6 cm is classified as stage N3. Metastatic lymph node with a size >3–6 cm, which was defined as stage N2 in the 2008 Chinese edition staging system is excluded in the 8th AJCC edition. This raises a question of whether survival rates among groups of ≤3 cm, >3–6 cm, and >6 cm are different. Thus, we conducted this retrospective cohort study to assess the maximal lymph nodal diameter on N stage of the 8th edition staging system using data from the Surveillance, Epidemiology, and End Results (SEER) database.Distant metastasis is the main failure pattern for nasopharyngeal carcinoma (NPC).22.1This study extracted NPC cases from 2004 to 2016 in the SEER database. Patients were included when they met the following criteria:(1)pathologically confirmed NPC;(2)definite data of maximal lymph nodal diameter could be extracted;(3)World Health Organization (WHO) type I, II, or III.,4 included patients were divided into 3 groups: ≤3 cm, >3–6 cm, and > 6 cm based on the maximal lymph nodal diameter.Patients with unknown clinical information were excluded. Variables of age, race, sex, WHO classification, tumor grade, radiotherapy, and chemotherapy were extracted. According to the 2008 Chinese edition and the 8th AJCC edition of NPC,2.2 Categorical variables of age, race, sex, tumor histology, tumor grade, radiotherapy, and chemotherapy were analyzed by using the χ2 test or Fisher exact test.Age was transformed to a categorical variable according to a previous study.Cumulative survival curves of 5-year overall survival (OS) and cancer-specific survival (CSS) were calculated using the Kaplan-Meier method. Differences between survival curves were compared using the log-rank test. The hazard ratios (HRs) and 95% confidence intervals (CIs) for OS and CSS were estimated with the use of a stratified Cox regression model, with the stratification factors of age, race, sex, WHO classification, tumor grade, radiotherapy, and chemotherapy.http://www.R-project.org). P values were two-tailed. Values of P < .05 were considered statistically significant.All statistical analyses were performed using SPSS Statistics Version 26.0 software  and R software version 4.0.3  in the multivariate regression analysis .The 5-year OS of the ≤3 cm, >3–6 cm, and >6 cm groups was 64.0%, 59.3%, and 41.5%, respectively Fig. . OS was 3.3P = .004) in the multivariate regression analysis .The 5-year CSS of the ≤3 cm, >3–6 cm, and >6 cm groups was 71.8%, 67.0%, and 49.0%, respectively ,6–8 The small sample size of lymph nodal size with >6 cm group might significantly reduce the statistical power of the analysis.The proportion of lymph nodal size with >6 cm is small, which was less than 4.5%.(2),7,11 The lymph nodal diameter in cross-sectional imaging might not be the maximal diameter.The N classification of AJCC staging system defines lymph nodal size by the largest dimension, irrespective of the measurement plane. This measurement was considered the best surrogate of gross lymph nodal volume. However, several studies defined the maximal lymph nodal diameter based on cross-sectional imaging. Second, the maximal lymph nodal diameter is mainly defined based on magnetic resonance imaging. The optimal cut-off value of metastatic lymph nodes size as N3 criteria needs to assess using a more reliable algorithm. Third, the tumor volume,14 and metabolic tumor volume–18 may be better factors than the metastatic lymph nodes size in representing the tumor burden. These factors might replace the maximal lymph nodal diameter in further staging systems.Moreover, the efficacy of metastatic lymph nodes size with >6 cm as N3 criteria should be further identified. First, maximal lymph nodal diameter >6 cm as N3 criteria was established in the 6th edition AJCC staging system. The maximal lymph nodal diameter of the 6th and 7th edition AJCC staging system is mainly based on palpation. The measurement differs among clinicians.,6–8According to our study, it was reasonable that metastatic lymph nodes with a size >3–6 cm was not classified as stage N2 in the 8th edition AJCC staging system. The Cox regression analysis showed that metastatic lymph nodes with a size >3–6 cm was not an independent prognostic factor for CSS and OS. Similarly, several studies revealed that metastatic lymph nodes with a size >3–6 cm failed to achieve an independent prognostic factor in survivals.,2 N stage was the most important predictive factor of distant failure. Thus, this study could not assess the distant-metastasis free survival among groups of ≤3 cm, >3–6 cm, and >6 cm. Whether the worse OS and CSS with metastatic lymph nodes size >6 cm were due to distant failure or not was still unknown. In further, more studies are needed to assess the association between metastatic lymph nodes size and distant failure.This study had a limitation. Data of distant metastasis could not be extracted due to the limitations of SEER database. It was reported that the major failure pattern of NPC was distant metastasis.In conclusion, it is reasonable that lymph nodal size with >6 cm is classified as stage N3 of the 8th edition AJCC staging system for NPC.Conceptualization: Shi-Ting Huang, Xin-Bin Pan.Data curation: Shi-Ting Huang.Formal analysis: Shi-Ting Huang, Ling Li.Methodology: Song Qu, Xiao-Dong Zhu.Software: Song Qu.Validation: Ling Li, Kai-Hua Chen, Xiao-Dong Zhu.Writing – original draft: Xin-Bin Pan.Writing – review & editing: Xin-Bin Pan.