Video game play has been linked to a range of cognitive advantages, and investigations in this domain have predominantly utilized cross-sectional designs or long-term training paradigms. Nevertheless, the specific effects of engaging in a single bout of video game play remain poorly understood. Consequently, the objective of this study is to examine the influence of a single session of mobile action video game (MAVG) play on attentional networks among college students.Seventy-two nonvideo game players were assigned randomly into an MAVG and a control game group. Participants in the MAVG group engaged in a 60-minute session of an action video game played on mobile phones, while the control group played a mobile card game for the same duration. All participants completed the attentional network test (ANT), which assesses alerting, orienting, and executive control network efficiencies, before and after the intervention.p < 0.05); the control game group did not. Neither executive control network efficiency nor orienting network efficiency were found to be improved by the intervention.The MAVG group had significantly improved alerting network efficiency following the intervention, compared to before (The present data demonstrated that a single bout of MAVG play can improve alerting network efficiency selectively in young-adult college students. MAVGs may be useful for promoting attentional function with the advantages of being accessible virtually any time and anywhere. Video games have become an integral part of many people’s daily lives. Some 2.6 billion people, about a third of the world’s population, play video games . Video gMobile devices, such as mobile phones, tablets, and other small mobile devices, enable people to engage in gaming in short bouts in their spare minutes in daily life. For example, college students often play mobile video games during class breaks and workers often play during their commutes. In China, there were some 516 million mobile video game (MVG) users in 2020 and more than 99 percent of video game players use mobile devices . AlthougIt is imperative to acknowledge that long-term training may not be a viable option for all individuals. Notably, not everyone may be inclined to cultivate a gaming habit. Furthermore, it is worth noting that cross-sectional studies can elucidate correlations but are inherently limited in establishing causal relationships. Exiting studies have yielded controversial conclusions regarding the short-term training effect on cognition . TherefoAttention, a basic function that facilitates interactions with one’s environment by enabling one to focus selectively on relevant information over other information, is a core process of cognition that regulates other cognitive systems. Video games have been shown to have positive effects on multiple dimensions of attention, such as attentional control , visual In the present study, we conducted a comparative analysis between a single session of mobile action video game (MAVG) play and mobile card game play, which aligns with the prevailing approach employed in the majority of intervention studies within this field . Our stuN = 36, 17 females; mean age = 22.28, SD = 1.80).Undergraduate students were recruited by posting recruitment notices around the Yangzhou University campus. Each participant was given the Edinburgh Handedness Inventory and given video game experience and health questionnaires. There were four inclusion criteria: (1) played video games no more than 2 h per week during the past 6 months ; (2) rigThe experimental procedure adhered to the Declaration of Helsinki and was approved by the Ethical Committee of Yangzhou University (YXYLL-2023-083). Upon arrival at the laboratory, each participant signed a written consent form.The ANT, a validated instrument developed by A target stimulus consisting of five horizontally arranged Flanker arrows or lines was then presented. The visual angle for the entire set of Flanker arrows or lines is 6.8°, while that of the center arrow is 1.3°. In the spatial cue condition, the location of the target stimulus (above or below the fixation cross site) was consistent with the cue. Target consistency with the center arrow determined the cue condition. In the consistent and inconsistent conditions, the other four arrows pointed or did not point in the same direction as the central arrow, respectively. In the neutral condition, there were lines, instead of arrows, with no directional information. The target stimulus screen was closed upon the participant responding or after 1,700 ms if there was no response. The participants were asked to press the key “1” on a numeric keyboard if the arrow pointed left, and press the key “3” if it pointed right. They were instructed to press the key as quickly and accurately as possible. The ANT comprised four blocks, each consisting of 48 trials. In each block, the trials presented distinctive combinations of cue conditions, flanker conditions, arrow orientations, and target display positions. A visual overview of the task is shown in Participants in the MAVG group were given a brief tutorial on how to play the game, practiced operating the Honor of Kings game  for 5 min, and then played the game for 60 min on mobile phones. The duration of the single intervention in this study was determined by the prolonged battles in the game “Honor of Kings,” and referencing previous studies .Participants in the control group engaged in a 60-minute session of Happy Poker on mobile phones, a widely recognized card game developed by Tencent Games. Although this game shares several characteristics with the experimental game, such as enjoyment and engagement, it does not involve the same cognitive constructs as the experimental game. The inclusion of an active control group was crucial to address concerns associated with non-specific placebo-based effects, as emphasized by One week before the experiment, participants who met the recruitment criteria were contacted individually by telephone to confirm their video game play experience; they were informed of the time and place of the experiment if they met the requirements. Participants were asked to abstain from playing MAVGs and from consuming caffeine-containing substances or alcohol for 24 h prior to the experiment.Upon their arrival at laboratory, the participants signed a consent form were given game instructions. After confirming that they understood the instructions, the eligible participants began the ANT separately in a quiet and dimly lit after understanding the instructions. A practice session with 24 trials preceded the experiment. Participants were required to obtain 80% accuracy in the practice session before commencing with the experiment. They were allowed to repeat the practice block again if they did not achieve 80% accuracy in the first attempt.Upon successful completion of the practice session, participants were allowed to start the experimental task, . Between the blocks, they could rest and then initiate the next block by pressing any key. After finishing, they were assigned randomly to either the MAVG or control game group for the intervention session after a short break. Both groups’ participants completed post-intervention ANT 10 min after the intervention in order to minimize any transient effect of heightened arousal levels .The present study adopted a 2  × 2 (TIME: Pre- and Post-intervention) mixed-factor design. GROUP was a between-subjects variable while TIME was a within-subject variables. The dependent variables included reaction times (RTs), accuracy in the attentional network test, and the efficiency of each attentional networks.The first statistical analysis in examining these networks consisted of conducting a 2 (GROUP) × 2 (TIME) × 3 (FLANKER TYPE) × 4 (CUE TYPE) mixed factors analysis of variance (ANOVA) on RTs and accuracy data, respectively. FLANKER TYPE  and CUE TYPE  were within-subject variables.p <  0.05.Then, efficiency of each attentional networks  was subjected to a 2 (TIME) × 2 (GROUP) repeated-measures ANOVA. Alerting, orienting, and executive control efficiencies were computed as No-cue RT minus Double-cue RT, Central-cue RT minus Spatial-cue RT, and Incongruent-flanker RT minus Congruent-flanker RT, respectively. Due to the three distinct large-scale neural networks underlying different aspects of attention , the thrRaw data used in this article can be found at p = 0.55, amount of time spent playing video games per week in the past 6 months, t (70) = 1.53, p = 0.13. Furthermore, there were no differences between the groups in terms of basic response times, which were calculated based on response times across all conditions, t (70) = 1.28, p = 0.21.The MAVG and control game groups were confirmed to be similar with respect to age, t (70) = 0.60, F  = 319.54, p < 0.01, ηp2 = 0.82) and FLANKER TYPE  = 1,345.52 p < 0.01, ηp2 = 0.95). Post hoc comparisons using the least significant difference (LSD) method indicated that participants exhibited significantly faster response times in the spatial cue condition  compared to the no cue , center cue , and double cue  conditions. Regarding FLANKER TYPE, participants responded significantly faster in the congruent condition  compared to the incongruent  and neutral  conditions.For the RT analysis, all incorrect trials or trials that were three SDs from the individual mean were excluded. An ANOVA revealed significant main effects of CUE TYPE  = 9.17, p < 0.01, ηp2 = 0.12). We conducted a FLANKER by CUE ANOVA, contrasting congruent, incongruent, and neutral conditions. Interaction analysis revealed that participants response significant faster in the congruent trails than in the incongruent trails in the no cue  = 631.93, p < 0.01, ηp2 = 0.82), central cue  = 942.75, p < 0.01, ηp2 = 0.87), double cue  = 860.36, p < 0.01, ηp2 = 0.86), and spatial cue  = 633.42, p < 0.01, ηp2 = 0.82) conditions. Significant differences between incongruent and neutral trials were also found in the no cue  = 697.52, p < 0.01, ηp2 = 0.83), central cue  = 903.45, p < 0.01, ηp2 = 0.86), double cue  = 842.41, p < 0.01, ηp2 = 0.86), and spatial cue  = 538.24, p < 0.01, ηp2 = 0.79) conditions. Specifically, the response speed of participants significantly faster in the neutral trials compared to the incongruent in all cue conditions. Congruent trials differed significantly from neutral trials only in the center cue condition  = 4.04, p = 0.046, ηp2 = 0.03), the response speed in the congruent trails was significant faster than the neutral trails in the center cue condition. No significant differences were observed in the no-cue  = 0.03, p = 0.87, ηp2 < 0.01), double cue  = 2.16, p = 0.14, ηp2 = 0.02), and spatial cue  = 0.68, p = 0.41, ηp2 < 0.01) conditions.Furthermore, we observed a significant interaction between FLANKER TYPE and CUE TYPE  = 3.95, p = 0.02, ηp2 = 0.05). A TIME by FLANKER ANOVA comparing pre- and post-intervention tests showed that RTs were significantly shorter after the intervention under the incongruent condition  = 9.26, p < 0.01, ηp2 = 0.03), with no significant differences observed in the congruent  = 1.11, p = 0.29, ηp2 = 0.04) and neutral  = 2.32, p = 0.13, ηp2 < 0.01) conditions. No other main effects or interactions reached statistical significance. Descriptive data for these conditions can be found in Additionally, we detected significant TIME × FLANKER TYPE interactions  = 9.97, p < 0.01, ηp2 = 0.13) and FLANKER TYPE  =149.08, p < 0.01, ηp2 = 0.68), but not of GROUP  = 0.69, p = 0.41, ηp2 = 0.01) or TIME  = 0.24, p = 0.63, ηp2 < 0.01). Subsequent post hoc comparisons employing the least significant difference (LSD) method for CUE TYPE revealed that response accuracy in the no cue condition was significantly lower than in the center cue (p = 0.04) and spatial cue (p < 0.01) conditions. Notably, response accuracy in the spatial cue condition was significantly higher than in the center cue (p < 0.01) and double cue conditions (p <0.01). No significant differences were observed between the no cue and double cue conditions or between the center cue and double cue conditions. Regarding FLANKER TYPE, participants exhibited significantly higher accuracy in the congruent and neutral conditions compared to the incongruent condition (p < 0.01), while no significant difference was found between the congruent and neutral conditions (p = 0.21).Mean accuracy values for the groups are reported with SDs in F  = 6.55, p < 0.01, ηp2 = 0.86). We conducted a FLANKER by CUE ANOVA, contrasting congruent, incongruent, and neutral conditions. Interaction analysis demonstrated that the accuracy of congruent trials significantly higher than incongruent trails in the no cue  = 79.65, p < 0.01, ηp2 = 0.36), central cue  = 112.20, p < 0.01, ηp2 = 0.44), double cue  = 119.25, p < 0.01, ηp2 = 0.46), and spatial cue  = 52.64, p < 0.01, ηp2 = 0.27) conditions. Additionally, he accuracy was significantly higher in the neutral trials compared to the incongruent in the no cue  = 84.20, p < 0.01, ηp2 = 0.37), central cue  = 108.47, p < 0.01, ηp2 = 0.43), double cue  = 94.61, p < 0.01, ηp2 = 0.40), and spatial cue  = 81.49, p < 0.01, ηp2 = 0.36) conditions. However, no significant difference between congruent and neutral trials was found in no cue  = 0.77, p = 0.38, ηp2 <0.01), central cue  = 1.91, p = 0.17, ηp2 = 0.43), double cue  =1.68, p = 0.20, ηp2 = 0.40) and spatial cue  = 0.95, p = 0.33, ηp2 = 0.36) conditions. No other interactions reached significant level.Furthermore, the ANOVA for accuracy revealed a significant CUE TYPE × FLANKER TYPE interaction  = 5.39, p <  0.05, ηp2 = 0.07) and GROUP  = 5.97, p = 0.02, ηp2 = 0.08). Post-hoc comparisons indicated that the alerting network efficiency of the MAVG group  was significantly higher than that of the control group . Furthermore, the post-intervention alerting network efficiency scores  were significantly higher than the scores obtained in the pre-intervention assessment . A significant TIME × GROUP interaction  = 3.94, p < 0.05, ηp2 = 0.06) was observed for alerting network efficiency. To provide a more detailed analysis of alerting network efficiency in response to this interaction, we conducted a GROUP by TIME ANOVA, comparing the MAVG and control groups. No significant between-group differences were found in the pre-intervention test  = 0.27, p = 0.61, ηp2 < 0.01). However, significant between-group differences emerged in the post-intervention test  = 7.80, p < 0.01, ηp2 = 0.08). The alerting network efficiency of the MAVG group  was significantly higher than that of the control group  in the post-intervention test  = 0.003, p = 0.96, ηp2 < 0.01), GROUP  = 0.49, p = 0.49, ηp2 < 0.01) and no significant TIME × GROUP interaction  = 1.61, p = 0.21, ηp2 = 0.02).In contrast, an ANOVA for orienting network efficiency yielded no significant main effect of TIME  = 5.00, p = 0.03, ηp2 = 0.07). Post-intervention executive network efficiency scores  were significantly lower than the pre-intervention scores . However, no significant main effect of GROUP  = 2.25, p = 0.14, ηp2 = 0.03) and no significant TIME × GROUP interaction  < 0.01, p > 0.99, Finally, an ANOVA for executive network efficiency showed a significant main effect of TIME  play on attentional networks. The findings revealed that a single 60-min bout playing MAVG could improve attentional networks, particularly of the alerting network, in a sample of young-adult college students generally considered to be at the peak of their cognitive functions . This rei.e., ≥10 h of gameplay), necessitating rigorous and extended training sessions (Interestingly, our MAVG did not exhibit improvement in the efficiencies of the orienting and executive networks, which was inconsistent with previous reports . There asessions . In prevsessions . The effsessions . Playingsessions .The present results suggest the MAVGs may, in addition to being a leisure activity, be useful for promoting attentional function with the advantages of being accessible virtually any time and anywhere. Alerting network deficiencies have been demonstrated in patients with ADHD and in HIV-infected patients . The preThere were several limitations in the present study. First, we observed a higher level of accuracy in the ANT compared to prior studies . While tThe present study has contributed evidence supporting the potential benefits of a single bout MAVG on attentional network function. Our observations revealed that a single 60-minute MAVG session resulted in a discernible enhancement in the efficiency of the alerting network, while other networks remained unaffected. These findings suggest that MAVGs may serve not only as a leisure activity but also as a practical means to enhance attentional function. Importantly, the accessibility of MAVGs at virtually any time and location further underscores their utility in promoting attentional capabilities.10.7717/peerj.16409/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.16409/supp-2Supplemental Information 2Click here for additional data file.


The results showed that PC possessed anti-EV71 activities in different cell lines with low toxicity. PC can block both the adsorption and entry processes of EV71 via directly binding to virus VP1 protein. PC may competitively interfere with the binding of VP1 to its receptor SCARB2. PC can also regulate three different MAPK signaling pathways to reduce EV71 infection and attenuate virus induced inflammatory responses. Importantly, intramuscular therapy of EV71-infected mice with PC markedly improved their survival and attenuated the severe clinical symptoms. Therefore, the natural compound PC has potential to be developed into a novel anti-EV71 agent targeting viral VP1 protein and MAPK pathways.Proanthocyanidins (PC), a natural flavonoid compound, was reported to possess a variety of pharmacological activities such as anti-tumor and anti-viral effects. In this study, the anti-Enterovirus 71 (EV71) activities and mechanisms of PC were investigated both  EV71 is one of the main pathogens causing hand, foot and mouth disease (HFMD) in infants, which affects the lives of millions of people, especially in the Asia Pacific region (Enterovirus 71 (EV71), a non-enveloped RNA virus, belongs to the enterovirus genus of small RNA virus family  belong to polyphenols, which widely exist in a variety of plants, and usually have many pharmacological activities, such as anti-oxidant, anti-inflammatory, anti-tumor, and anti-Alzheimer's disease effects . Dai et in vitro and in vivo in this study. The results indicated that PC possessed anti-EV71 activities in vitro with low toxicity. PC can interfere with virus entry by acting on VP1 protein, and inhibit inflammatory responses via acting on MAPK pathways. Moreover, intramuscular administration of PC markedly attenuated the severe clinical symptoms and prevents death of EV71-infected mice. Thus, PC merits further studies as a novel entry inhibitor for EV71 in the future.To further expand the anti-viral applications of PC, the anti-EV71 effects and mechanisms of PC were explored both 22.1Proanthocyanidins  was obtained from Macklin Biochemical Co., Ltd. . Guanidine Hydrochloride was bought from sigma . Human embryonic rhabdomyosarcoma (RD) and Vero cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) , penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in 5% CO2. EV71 strain BrCr-TR was obtained from the Wuhan Institute of Virology, Chinese Academy of Sciences.2.2The anti-EV71 activity was evaluated by the CPE inhibition assay . In brie2.3Inhibition of EV71 infection by PC was also measured by the plaque reduction assay as described previously . EV71  Tween 20 and 5% BSA overnight at 4 °C, the membranes were rinsed and incubated at 37 °C with antibodies against VP1 protein, cellular phosphorylated ERK, SAPK/JNK, p38MAPK, α-tubulin, GAPDH and β-actin proteins , respectively. The membranes were then washed and incubated with AP-labeled secondary antibodies  at RT for 2 h. Blots were developed with p-nitro blue tetrazolium chloride (NBT) and 5‑bromo-4‑chloro-3-indolyl phosphate toluidine (BCIP) solution at RT for 30 min. The relative densities of protein were analyzed by Image J (NIH) V.1.33 u (USA).2.6For pull down assay, the cell lysates of pCDNA3.1-SCARB2-Myc transfected 293T cells in the radioimmunoprecipitation assay (RIPA) buffer , and 1 mM EDTA) containing a proteinase inhibitor cocktail , were incubated with anti-myc antibody conjugated magnetic beads  overnight at 4 °C before washing the beads three times with PBS containing 0.1% Tween 20 (v/v) (PBST). Then the EV71 particles was incubated with or without PC  at 37 °C for one hour. After that, the EV71-PC mixture was added to SCARB2-myc coupled magnetic beads and incubated at 4 °C for 2 h. After washed thrice with PBST, the VP1 protein bound to the beads was analyzed by immunoblotting with an anti-VP1 antibody, and the SCARB2-myc on the beads was detected by anti-myc antibody.2.7SPR assays were conducted on a SPR biosensor instrument GE BiacoreT200 . EV71 VP1 protein (Abnova) was first immobilized onto the surface of a carboxymethylated dextran sensor chip (CM5) via amino group coupling. To assess real-time binding of PC to VP1 on CM5 chips, PC (50–3.125 μM) dissolved in DMSO was injected over the sensor chip surface within 2 min, followed by a 10 min wash with 1 × PBST buffer. The sensor chip surface was then regenerated by washing with NaOH (2 mM) for 30 s. All binding experiments were carried out at 25 °C with a constant flow rate of 2 μL/s PBS buffer. To correct for nonspecific binding and bulk refractive index change, a blank channel without VP1 was used and run simultaneously for each experiment. The changes in mass due to the binding response were recorded as resonance units (RUs).2.8http://www.biocheming.cn/). The crystal structure of EV71 VP1 protein (PDB code: 3VBH) was obtained from the protein data bank (http://www.rcsb.org), and processed in pyMOL software to export as PDBQT file for molecular docking. The 3D structure file of PC was obtained from PubChem database and imported into ChemBio3D Ultra 14.0 software for MM2 energy minimization before transformed into PDBQT file for molecular docking. The conformations with the lowest free energies of binding were selected as the best (probable) binding modes.Molecular docking was conducted in watvina software (2.9n = 10–12 per group) were intraperitoneally challenged with EV71 at a dose of 105 TCID50 per mice as reported , and maintained in a pathogen-free environment (23 ± 2 °C and 55% ± 5% humidity). All mice experiments were approved by the institutional animal care and use committee of the Ocean University of China . One breeding ICR mouse could give birth to 12–13 pups, which were kept in a single group. Three-day-old neonatal ICR mice (reported  followed2.10P < 0.05.All data are representative of at least three independent experiments. Data are presented as means ± standard deviations (SD). Statistical significance was calculated by GraphPad Prism 7.0 using one-way ANOVA analysis followed by post hoc Tukey's test. Statistical significance was considered to be 33.150 (50% Cytotoxicity Concentration) value of about 56.6 ± 2.7 μM.  A was rep 2.7 μM. B. In Ver± 1.4 μM .Fig. 1Inin vitro using plaque assay and CPE inhibition assay. Briefly, EV71 (MOI = 0.1) was pretreated with PC  at 37 °Cfor 1 h before infection. Then after virus adsorption for 1 h, the virus inoculum was removed and the media with PC at the indicated concentrations were added into cells. After 24 h incubation, the cell viability was measured by CPE inhibition assay and the viral titers in the culture media were determined by plaque assay. The results indicated that PC significantly reduced the virus titers of EV7 in Vero cells when used at the concentration > 1 μM (P < 0.05)  for PC was about 42.8, comparable to that of Guanidine hydrochloride (SI = 49.9)  C. CPE in = 49.9) . PC also = 49.9) . Moreove> 2.5 μM D. In add> 2.5 μM E and F, 3.2in vitro, the time-of-addition assay was performed as described previously (P < 0.01), suggesting that PC may have direct interaction with EV71 particles. The addition of PC during adsorption or post adsorption also possessed significant inhibition on EV71 multiplication (P < 0.05) (To explore the stage (s) at which PC exerted its inhibition actions eviously . Brieflyeviously A. At 24  < 0.05) B. Moreov < 0.05) C, consis < 0.05) B. Thus, Since PC may inhibit adsorption and post-adsorption processes of EV71, we further explored whether PC can directly block the binding (−2∼0 h p.i.) and entry of EV71 (0∼1 h p.i.) by using immunofluorescence assay. The results indicated that PC treatment (20 μM) during adsorption (−2∼0 h p.i.) significantly reduced the fluorescence of VP1 on cell surface, suggesting that PC can block the adsorption process of EV71 D and F. 3.3Scavenger receptor B2 (SCARB2) is reported to be the main functional entry receptor for EV71 in most cells , and VP1To further explore whether PC can directly bind to VP1 protein, the SPR assay was performed as reported previously . BrieflyIt has been shown that the 92–106, 212–225 and 280–293 amino acid sequences of VP1 protein are three main active sites for its binding to SCARB2 . To furt3.4P < 0.001) in virus control group (EV71) relative to the non-infected group (Mock)  A. Howevep (Mock) A. Among PC group B, indicaPC group C.Fig. 4TP < 0.01), and six DEGs related to inflammation and apoptosis were obtained in our study , and virus induced apoptosis related genes GADD45β, TXNIP, and MALAT1  were all significantly up-regulated in virus control group (EV71), and down-regulated after PC treatment (EV71+PC) (P < 0.01)  > 1) and down-regulation after PC treatment (log2FC(EV71/Mock) ≤ 0.5) in RD cells (ur study D. Among  < 0.01) D. Consid3.5Since the transcriptome analysis suggests that some DEGs involved in inflammatory response may be the main targets of PC after virus adsorption, we further explored if PC could influence the signaling pathways required for EV71 infection and virus induced inflammatory responses by western blotting. It has been reported that EV71 infection can induce the production of ROS to lead to the phosphorylation of p38MAPK, ERK and JNK, thereby promoting EV71 replication in host cells . Thus, wP < 0.001)  G-I. PC (< 0.001) H. Theref3.65 TCID50 per mice followed by intramuscular administration of drugs at 12, 24 and 48 h after exposure , the natural flavonoid compound, were reported to possess a variety of pharmacological activities . In thisFOS and JUN related to inflammatory response may be the targets of PC. Western blot assay verified that PC can truly down-regulate cellular MAPK pathways which are required for viral replication and inflammatory responses (The EV71 virion is composed of 60 capsid copies, each of which is made up of four capsid proteins: VP1, VP2, VP3 and VP4 . Among tesponses . Thus, Pesponses .Fig. 7A in vitro anti-EV71 effects of PC were mirrored in EV71-infected newborn mice. Herein, we found that intramuscular therapy of EV71-infected mice with PC markedly improved their survival and increased the body weights of EV71 infected mice (The ted mice . PC treain vitro and in vivo with low toxicity. PC can block EV71 infection via targeting VP1 protein to interfere with the interaction between VP1 and its receptor SCARB2. Further studies of the inhibition of PC against other EV71 strains in animal models and clinical research will be required to advance it for drug development. Nevertheless, PC merits further studies as a novel antiviral agent for prophylaxis and therapy of EV71 infection.In summary, PC possessed anti-EV71 activities both Conceptualization, W.W., C.H., Funding acquisition, W.W, C.H., Investigation, J.Q.S., X.Y.M., Data analysis, J.Q.S., L.S.S., Y.Z., Writing, J.Q.S., W.W., Review and editing, C.H., W.W.Jiqin Sun: Investigation, Methodology, Formal analysis, Writing – original draft. Xiaoyao Ma: Investigation, Validation, Data curation. Lishan Sun: Methodology, Formal analysis. Yang Zhang: Formal analysis. Cui Hao: Conceptualization, Writing – review & editing, Funding acquisition. Wei Wang: Conceptualization, Supervision, Writing – review & editing, Funding acquisition.The authors declare no conflict of interests.


Ketogulonicigenium vulgare and Bacillus megaterium for the production of the vitamin C precursor, 2-keto-L-gulonic acid (2-KLG), has been widely used for large-scale industrial production. To further investigate the cell–cell communication in microbial consortia, a microbial consortium consisting of Ketogulonicigenium vulgare and Bacillus pumilus was constructed and the differences in protein expression at different fermentation time points (18 h and 40 h) were analyzed by iTRAQ-based proteomics. The results indicated that B. pumilus was subjected to acid shocks in the coculture fermentation system and responded to it. In addition, the quorum sensing system existed in the coculture fermentation system, and B. pumilus could secrete quorum-quenching lactonase (YtnP) to inhibit the signaling pathway of K. vulgare. This study offers valuable guidance for further studies of synthetic microbial consortia.Microbial consortia play a key role in human health, bioenergy, and food manufacturing due to their strong stability, robustness and versatility. One of the microbial consortia consisting of  With thKetogulonicigenium vulgare and Bacillus spp. have been widely used in a two-step vitamin C fermentation process and have achieved great economic benefits  from L-sorbose via L-sorbosone but with an extremely low yield. Bacillus strains , as helper strains, are cocultured to significantly stimulate the growth of K. vulgare and 2-KLG yields  to reduce costs and increase product quality. Microbial consortia of benefits . K. vulgG yields . The relK. vulgare and the helper strain have been well explained with the development and application of omics technology   . Most ofenomics) . The helr strain . In addiuctivity . AlthougK. vulgare produced signals in the environment and that the oxidization products, including 2-KLG, could inhibit the growth of the helper strain. To test these hypotheses, iTRAQ-based proteomics analysis of an artificial consortium of K. vulgare and B. pumilus was performed, and the results used to derive novel information for further understanding of the synthetic consortium in Vc microbial fermentation and hopefully shed light on other complex microbial consortia.In this paper, our aim was to investigate the cell–cell communication and the reasons for inhibiting the growth of the helper strain in the coculture fermentation system. We therefore hypothesized that 2.2.1.Ketogulonicigenium vulgare 25B-1 (K. vulgare 25B-1) and Bacillus pumilus SY-A9 (B. pumilus SY-A9) were kindly provided by Northeast Pharmaceutical Group Co. Ltd. . These strains were stored at −80°C in glycerol.2PO4, 0.4 g/L MgSO4, 1 g/L CaCO3, 20 g/L agar powder, pH 6.7. The seed medium was the same as the isolation medium without the addition of agar powder. Fermentation medium: 80 g/L L-sorbose (autoclaved separately), 12 g/L urea, 10 g/L corn-steep liquor, 0.2 g/L MgSO4, 1 g/L KH2PO4, 5 g/L CaCO3, pH 6.7. These media were autoclaved at 121°C for 15 min before use.Three media were used. Isolation medium: 20 g/L L-sorbose (autoclaved separately), 10 g/L peptone, 1 g/L urea, 5 g/L corn-steep liquor, 3 g/L yeast extract, 3 g/L beef extract, 1 g/L KH2.2.K. vulgare were collected from the isolation medium plate (K. vulgare had been incubated for 48 h at 28°C) and suspended in 2 mL of sterile water. Second, one colony of B. pumilus with a diameter of 1 mm from the isolation medium plate (B. pumilus had been incubated for 24 h at 28°C) was mixed with 2 mL of sterile water. Then, 1 mL of B. pumilus solution and 2 mL of K. vulgare solution was mixed and the mixture was inoculated into a 20-mL seed culture medium in a 250-mL flask. After shaking (170 rpm) for 16 h at 28°C, the seeds were obtained, and K. vulgare colony number reached 1 × 109 CFU/mL and B. pumilus colony number reached 1 × 106 CFU/mL. Subsequently, 2 mL of seed was inoculated into a 20-mL fermentation medium in a 250-mL flask and the flask was cultivated on a shaker with 170 rpm at 28°C.First, approximately 1,000 colonies of 2.3.g for 20 min to obtain 0.5 g of wet bacteria for protein extraction. Each sample was mixed with 300 μl of breaking buffer , 1 mM ethylenediaminetetraacetic acid (EDTA) and 5% glycerol) and complete protease inhibitor cocktail  on ice, followed by centrifugation at 4°C and 15,000 × g for 15 min. The supernatant containing intracellular proteins was further quantified for protein concentration using the Bradford assay. 200 μg protein was mixed with 5 mL of 1 M dithiothreitol (DTT) at 37°C for 1 h and alkylated with 20 μL of 1 M indole acetic acid (IAA) at room temperature for 1 h in the dark. Then, trypsin digestion (protein:trypsin ratio of 20:1) was performed for 24 h at 37°C. Using an iTRAQ Reagent-8Plex Multiplex Kit (AB SCIEX), peptides from the four samples were labeled with the iTRAQ tags as follows: K. vulgare + B. pumilus18 (tags 113 and 114) and K. vulgare + B. pumilus40 (tags 115 and 116), respectively.The fermentation broths at 18 h and 40 h were centrifuged at 13,000 × 2O, pH 10.0, consisting of 20 mM NH4Ac) and eluted at a flow rate of 0.2 mL/min with the following gradient: 5 to 35% mobile phase B (90% acetonitrile (ACN), pH 10.0, consisting of 20 mM NH4Ac) for 30 min, 35 to 90% mobile phase B for 5 min, 90% mobile phase B for 10 min. The elution peak was monitored at 214 nm and one component per minute was collected and then lyophilized.The peptides were further fractionated using a C18 column  with a high-performance liquid chromatography (HPLC) system  at a flow rate of 0.2 mL/min. The peptides were suspended in mobile phase A (100% H2.4.The lyophilized peptides were resuspended in 5 μl of 0.5% formic acid (FA), transferred to a C18 trap column , and then separated using an Easy nLC 1,000 system with a C18 column  at a flow rate of 200 nl/min. The mobile phases were solvent A (0.1% FA) and solvent B (0.1% FA in ACN). The elution gradient was as follows: 0–1 min, 1–5% solvent B; 1–6 min, 5–10% solvent B; 6–85 min, 10–30% solvent B; 85–87 min, 30–40% solvent B; 87–90 min, 40–90% solvent B; 90–100 min, 90% solvent B; 100–101 min, 90–1% solvent B; 101–120 min, 1% solvent B. The end of the nanoliter liquid-phase separation was connected directly to the Q-Exactive mass spectrometer  with a set of the following parameters: polarity, positive ion mode; MS scan range, 300–1,800 m/z; MS/MS scan resolution, 17,500; normalized collision energy, 30.2.5.p-values <0.05, fold changes ≥1.2 or ≤ 0.83, were identified as differentially expressed proteins (DEPs) between the different B. pumilus strains, whereas p-values <0.05, fold changes ≥1.1 or ≤ 0.91 were identified as DEPs between the different K. vulgare strains. In other words, in B. pumilus, the fold changes of the upregulated DEPs were more than 1.2, while those of the downregulated DEPs were less than 0.83. In K. vulgare, the fold changes of the upregulated DEPs were more than 1.1, while those of the downregulated DEPs were less than 0.91. For the convenience of expression, B. pumilus18 (B18) vs. B. pumilus40 (B40) and K. vulgare18 (K18) vs. K. vulgare40 (K40) were used to compare the proteome of the former with that of the latter to screen DEPs.The acquired raw mass spectrometry data were processed and identified using the Sequest server and Proteome Discoverer (PD)  software. The spectra extracted using the PD software were searched against the Uniprot-Ketogulonicigenium+vulgare&Uniprot-Organism%3Abacillus+ subtilis database using the Sequest server with the following identification parameters: max missed cleavages, 2; fixed modifications, carbamidomethyl (C), iTRAQ8plex (N-term), iTRAQ8plex (K), iTRAQ8plex (Y); variable modifications, oxidation (M), deamidated ; peptide mass tolerance, 10 ppm; fragment mass tolerance, 0.02 Da; enzyme, trypsin. Proteins were quantified based on the PD software. In this paper, proteins with two unique peptides, 2.6.Hierarchical cluster analysis of DEPs was performed using R package. Clusters of orthologous genes (COG) annotations of DEPs were carried out using NCBI Blast.2.7.−ΔΔCT method . The synthesis of the first-strand cDNA was performed by an Evo M-MLV RT Permix for quantitative PCR (qPCR)  after measuring the RNA quality and quantity by a BioDrop Spectrophotometer . Specific primers of selected DEPs were designed by NCBI Primer-BLAST , and 16ST method , followe3.3.1.B. pumilus SY-A9 and 5,475 peptides and 1,134 proteins of K. vulgare 25B-1 were obtained via iTRAQ labeling and 2D LC-MS/MS analysis in this study. Compared with the control group (samples at 40 h fermentation broth), 82 DEPs (38 upregulated and 44 downregulated) of B. pumilus SY-A9 and 96 DEPs (47 upregulated and 49 downregulated) of K. vulgare 25B-1 were identified in the 18 h fermentation broth group , [E] amino acid transport and metabolism , [J] translation, ribosomal structure and biogenesis , [S] function unknown , and [K] transcription . The detailed functional annotation of the up- and downregulated DEPs of B. pumilus SY-A9 was shown in Among all the DEPs of K. vulgare 25B-1, three were annotated with multiple functions, and 14 were not annotated. The three main functional categories were as follows: [J] translation, ribosomal structure and biogenesis , [E] amino acid transport and metabolism , and [S] function unknown . The detailed functional annotation of the up- and downregulated DEPs of K. vulgare 25B-1 was shown in Among all the DEPs of 3.3.p < 0.05) were obtained based on the DEPs of B. pumilus (B18 vs. B40) (p < 0.05) were acquired based on the DEPs of K. vulgare (K18 vs. K40) (Seven significantly enriched KEGG pathways (vs. B40) , includivs. K40) , includi3.4.B. pumilus SY-A9 was higher than that of the upregulated proteins, and the core proteins were mostly related to energy production and conversion.Protein–protein interaction (PPI) network analysis  showed tK. vulgare 25B-1 was higher than that of the upregulated proteins, and the core proteins were mostly related to translation, ribosomal structure and biogenesis.PPI network analysis  showed t3.5.B. pumilus and K. vulgare, respectively, which showed similar trends in terms of changes in their transcript levels and protein expression levels.To test the correspondence between transcript levels and protein expression levels, the transcript levels of 9 DEPs were measured by qPCR, and the result are shown in 4.B. pumilus, iTRAQ-based proteomic analysis was used in a coculture fermentation system, and a total of 178 DEPs were obtained in the B18 vs. B40 and K18 vs. K40.To explore the reasons for inhibiting the growth of B. pumilus, including M5P1Y9, G4EPA3, A0A0C5CAF1, Q3EJU2, M4KMP7, W7R788, L8ASG9, A0A0C5CB21, A0A0K6MRG1, A0A0C5CMU8, Q9KEE6, and A0A132BIK5, were predicted to be involved in the response to acid stress. Here, the acid resistance mechanisms of B. pumilus including the general stress response, pH homeostasis maintenance, metabolic rearrangements and alkali production, and the secondary oxidative stress response are discussed. In Gram-positive bacteria, the alternative sigma factor σB, as the key sigma factor, plays a vital role in regulating the expression of approximately 150 genes in response to significant changes in the surroundings and appear to control, at least in part, the general stress response  complex , general stress protein 39 , glucose 1-dehydrogenase , and uncharacterized oxidoreductase . The negative regulator of genetic competence ClpC/MecB  is reported to be required for various processes, including the stress response, sporulation and competence in B. subtilis  involved in electron transport in the respiratory chain was downregulated, and the nucleotide-binding protein  that displays ATPase activity was also downregulated. The downregulation of these two DEPs suggested that B. pumilus could use both ATP synthesis and hydrolysis to regulate and thereby maintain intracellular pH homeostasis.Under acidic conditions, bacteria activate enzymes such as Feostasis . In aeroolecules . B. pumi+ to produce NH4+ to raise the internal pH , which controls the respiratory pathway by regulating transcription in response to changes in the cellular NADH/NAD+ radox state, was downregulated  and virulence. The LysR-type transcriptional regulator  was upregulated and regulated a diverse set of genes involved in virulence, metabolism, QS and motility , efflux transporter, RND family, MFP subunit , and oligopeptide/dipeptide (Opp/Dpp) ABC transporter, ATPase subunit  were downregulated. In Gram-negative bacteria, the multidrug efflux mechanism is mediated by transport proteins that are resistant to toxic compounds, and these transporters are associated with intrinsic and acquired antimicrobial resistance  . When th factors . In vitaK. vulgare contains an N-acyl-L-homoserine lactone synthetase-like protein (KVU_2075) and LuxR (KVU_2076), indicating that K. vulgare could use the AHLs-LuxR QS system to regulate its own cell density (KVU_2075 (encoded N-acyl-L-homoserine lactone synthetase-like protein), KVU_2076 , and ytnP (encoded probable quorum-quenching lactonase) were measured in monoculture and coculture fermentation systems at 6, 18, 40, and 72 h, respectively  can be found in the article/SL contributed to conception and design of the study. QZ performed the statistical analysis and wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported by the National Science Foundation of China (Grant number: 31370077) and Liaoning Province Science Research Fund (Grant number: LSNJC201915). Shenyang Agricultural University Postgraduate Innovation Cultivation Project (Grant number: 2022YCXB11).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cupressus sempervirens extract. Hesperetin represents the greatest concentration , while other compounds, such as pyro catechol, rutin, gallic acid, chlorogenic acid, naringenin, and quercetin, were recognized in concentrations of 2922.53 µg/mL, 1313.26 µg/mL, 1107.26 µg/mL, 389.09 µg/mL, 156.53 µg/mL, and 97.56 µg/mL, respectively. The well diffusion method documented the antibacterial/antifungal activity of C. sempervirens extract against E. faecalis, E. coli, C. albicans, S. typhi, S.aureus, and M. circinelloid with 35, 33, 32, 25, 23, and 21 mm inhibition zones, respectively, more than the standard antibiotic/antifungal agent. Low values ranging from 7.80 to 15.62 µg/mL of MIC and MBC were recorded for E. faecalis, E. coli, and C. albicans. From the 1- diphenyl-2-picryl hydrazyl (DPPH) assay, promising antioxidant activity was recorded for C. sempervirens extract with IC50 of an 8.97 µg/mL. Moreover, ferric reducing antioxidant power (FRAP) and total antioxidant capacity assays (TAC) confirmed the antioxidant activity of the extract, which was expressed as the ascorbic acid equivalent (AAE) of 366.9 ± 0.2 µg/mg and 102 ± 0.2 µg/mg of extracts, respectively. α-amylase and α-glucosidase inhibition % were determined to express the antidiabetic activity of the extract in vitro, with promising IC50 value (27.01 µg/mL) for α-amylase compared to that of acarbose (50.93 µg/mL), while IC50 value of the extract for α-glucosidase was 19.21µg/mL compared to that of acarbose 4.13 µg/mL. Prothrombin time (PT) and activated partial thromboplastin time (APTT) revealed the role of C. sempervirens extract as an anticoagulant agent if compared with the activity of heparin. Binding interactions of hesperetin and gallic acid were examined via the Molecular Operating Environment (MOE) Dock software against E. faecalis (PDB ID: 3CLQ), C. albicans (PDB ID: 7RJC), α-amylase (PDB ID: 4W93), and α-glucosidase (PDB ID: 3TOP). The obtained results shed light on how molecular modeling methods might inhibit the tested compounds, which have the potential to be useful in the treatment of target proteins.In the last decade, the urgent need to explore medicinal plants or drug development has increased enormously around the world to overcome numerous health problems. In the present investigation, HPLC indicated the existence of 18 phenolic and flavonoid compounds in the  Cupressus sempervirens is an evergreen tree, one of the traditional aromatic and medicinal plants that contain a wide range of bioactive metabolites. It belongs to the Cupressaceae family, and it is a species of cypress native to the eastern Mediterranean region. Phytochemical screening of C. sempervirens indicated the existence of several classes of chemical compounds, such as phenols, flavonoids, alkaloids, and terpenoids , and 50 mL of 10% trichloroacetic acid were added to each labeled Eppendorf tube. For 10 min, the mixture was centrifuged at 3000 rpm. Following centrifugation, 33.3 mL of ferric chloride  and 166.66 mL of the supernatant from each sample were put to 96-well plates. Microtiter plate reader  readings were collected at 630 nm. Ascorbic acid (1 mg/mL) was utilized as a positive control and DMSO as a negative control. The ascorbic acid equivalent (AAE) mg/mg of extract was used to express the results.The potassium ferricyanide and trichloroacetic acid technique  was utilp-nitrophenyl-α-D-glucopyranoside (p-NPG) as enzyme substrate was added to initiate the reaction after 20 min, and the mixture was then kept for 30 min. With the addition of 50 μL of Na2CO3 (0.1 N), the reaction was stopped, followed by measuring the absorbance at 405 nm using a spectrophotometer (Biosystm 310 Plus). α-glucosidase inhibition was calculated via the following Formula (2):The used extract was tested for α-glucosidase inhibitory activity using a slightly modified version of the procedure described by Pistia-Brueggeman and Hollingsworth . In totaOne unit of the α-glucosidase was defined as the quantity of α-glucosidase necessitated for the creation of one μmol of p-nitrophenol from the p-NPG per minute.2HPO4 (0.02 M)/NaH2PO4 (0.02 M) and 0.006 M of NaCl adjusted at pH 6.9 as a buffer to obtain ranging concentrations from 1.9 up to 1000 μg/mL. The reaction mixture contained of 200 μL of 2 units/mL of α-amylase with 200 μL of the tested extract, followed by incubation at 30 °C for 10 min. Then, 200 μL of suspended starch in water (1%) was added to the reaction mixture and kept for 3 min. DNSA reagent (200 μL) was added to the reaction mixture (require to stop the reaction) with boiling in a water bath at 90 °C for 10 min. After cooling of the reaction mixture (at 25 °C), it was diluted with 5 mL of distilled H2O. Followed by measuring the absorbance at 540 nm using spectrophotometer (Biosystm 310 Plus). The 100% activity of α-amylase as a control 100% was formulated by alternating the tested extract by 200 μL of buffer, while the blank reaction was formulated utilizing the tested extract without the α-amylase solution. α-amylase inhibition was calculated via the following Formula (3):The used extract was tested for α-amylase inhibitory activity using a slightly modified version of the procedure described by Wickramaratne  using 3,50, μg/mL) was calculated employing regression equation achieved via plotting concentration ranged from 1.95–1000 μg/mL and developed % inhibition.The concentration extract necessitated to inhibit 50% of the activity of α-glucosidase/α-amylase  (unite of time with seconds) and prothrombin time (PT) (time in seconds). Nine/one parts of human blood (from corresponding author of the current paper)/sodium citrate (3.2%) (v/v) were mixed, and then centrifuged for 10 min at 5000 rpm, followed by supernatant collection containing plasma. Different doses  of C. sempervirens extract were dissolved in 0.9% aqueous solution of NaCl. The reaction mixture contained plasma and C. sempervirens extract, and then kept at 37 °C for 3 min. The reagent of APTT (0.10 mL) (previously pre-kept at 37 °C for 3 min) was added to the reaction mixture and then kept at 37 °C for 5 min. Later, 0.10 mL of 0.025 mol/L CaCl2 (pre-kept at 37 °C for 3 min) was added to the reaction mixture, followed by recording clotting time. For PT test, the plasma was mixed with C. sempervirens extract, and then kept at 37 °C for 3 min. The reagent of PT (0.20 mL) (pre-kept at 37 °C for 3 min) was added to the reaction mixture, followed by recording clotting time. The cited method was re-performed with heparin as appositive control and with plasma without C. sempervirens extracts as control [The anticoagulant activity of  control .E. faecalis (PDB ID: 3CLQ), C. albicans (PDB ID: 7RJC), α-amylase (PDB ID: 4W93), and α-glucosidase (PDB ID: 3TOP). Each protein used downloaded from the Protein Data Bank . At first, the protein files were prepped using built-in “Quickprep” function. Then, water molecules and bound ligands were manually deleted from the downloaded proteins. The file of the prepared protein active site was loaded; the docking site was specified as dummy atoms, the placement methodology as triangle matcher, and the scoring methodology as London dG. The refinement methodology was adjusted as a rigid receptor and the scoring methodology as GBVI/WSA dG for selecting the best 5 poses from 100 different poses for each tested molecule. The library of compounds was imported and prepped into MOE database file and dock calculations were run automatically. At the end of the docking processes, the obtained poses were carefully studied, and the ones having the best ligand–protein interactions with the most acceptable RMSD values were selected.Molecular operating environment (MOE) software on Dell Core i7 processor 1.9 GH, 16 GB memory with Windows 10, 64-bit operating system was used to conduct molecular docking investigations on hesperetin and gallic acid binding properties in The achieved antimicrobial, antioxidant, anticoagulant, and antidiabetic results were obtained in triplicate, so the results were calculated as mean values of ±standard deviation (±SD).C. sempervirens extract with the estimation antimicrobial, antidiabetic, antioxidant, and anticoagulant activities. C. sempervirens extract was richened with several phenolic and flavonoid compounds, such as hesperetin , chlorogenic acid (389.09 µg/mL), naringenin (156.53 µg/mL), and quercetin (97.56 µg/mL), which play a vital role in the biosystems. Pathogenic bacteria as well as fungi were inhibited with C. sempervirens extract but with different levels of inhibition depending on the type of microbe ranging from 21 to 35 mm inhibition zone. The values of the MIC and MBC of the extract were low for some microorganisms such as E. faecalis, E. coli, and C. albicans, ranging from 7.80 to 15.62 µg/mL. Antioxidant potential via DPPH, FRAP, and TAC was referred to the antioxidant potential of C. sempervirens extract. Moreover, antidiabetic activity in vitro via measuring α-amylase (with IC50 of 27.01 µg/mL) and α-glucosidase (with IC50 of 19.21µg/mL) inhibition; moreover, anticoagulant through PT and APPT assays were linked to C. sempervirens extract. The molecular design was performed to investigate the binding mode of the proposed compounds with E. faecalis (PDB ID: 3CLQ), C. albicans (PDB ID: 7RJC), amylase (PDB ID: 4W93), and glucosidase (PDB ID: 3TOP). The data obtained from the docking studies were fitted with that obtained from the biological screening. All the tested compounds showed significant inhibitory activities against selected proteins. The obtained results indicated that the active compounds could be useful as a template for future design, optimization, adaptation, and investigation in order to generate more potent and selective inhibitors with higher analogs.Several investigations were planned in the present study, including analytical characterization of phenolic and flavonoid constituents in


Journal of Radiation Research, Volume 64, Issue 1, January 2023, Pages 44–52, https://doi.org/10.1093/jrr/rrac066This is a correction to: Inga Solgård Juvkam, Olga Zlygosteva, Delmon Arous, Hilde Kanli Galtung, Eirik Malinen, Tine Merete Søland, Nina Jeppesen Edin, A preclinical model to investigate normal tissue damage following fractionated radiotherapy to the head and neck, In this work, we used a lead collimator to limit the radiation field to certain parts of the mouse head, as described in the original manuscript. The original dosimetry was performed without the lead shield, as we were concerned that the resulting collimation would lead to partial volume effects, and thus systematic errors, in the ion chamber measurements. However, the impact of the lead plate on the dose to the irradiated area was underestimated. This was evident from additional dosimetry of the irradiation setup with and without the plate performed after the original work was published. This revised dosimetry is given below.™ EBT3 dosimeter films at a source-surface distance representing sagittal skin entrance of the mouse. Calibration was done using an unshielded beam. EBT3 films (lot No. 02122001) were irradiated and processed according to the recommended protocols specified for radiochromic film dosimetry in the report of AAPM Task Group 235 [3].The absorbed dose rate to water of the X-ray system was measured to be 0.63 ± 0.02 Gy/min for an unshielded beam using a FC65-G Farmer type ionization chamber  together with a MAX-4000 electrometer  according to IAEA report TRS-277 [1-2]. To measure the dose in the collimated case, we employed GafchromicThe results showed that the actual dose with the use of the collimator lead plate was 12% lower than the dose reported in the original manuscript. Therefore, all the doses in the manuscript should be corrected accordingly: 3 Gy to 2.6 Gy, 4.4 Gy to 3.9 Gy, 5 Gy to 4.4 Gy, 5.75 Gy to 5.1 Gy, 6.5 Gy to 5.7 Gy, 7.5 Gy to 6.6 Gy, 8.5 Gy to 7.5 Gy.The authors apologize for any inconvenience this might have caused.[1]: IAEA, Report No. ISBN 92-0-115 087-3, 1987.[2]: IAEA, Report No. ISSN 1011-4289, 1996.Grams M P, Lewis D F, Soares C G, Van Battum L J, Das I J, Trichter S, Kissick M W and Massillon-Jl G 2020 Report of AAPM task group 235 radiochromic film dosimetry: an update to TG-55 Med. Phys  475 986-6025.[3]: Niroomand-Rad A, Chiu-Tsao S-T, NB: the error in dosimetry is emended in this correction notice alone. The errors have not been emended throughout the paper online.


The magnetic CoxFe3−xO4 phases with x = 0.0, 0.5, and 1.0 were obtained by thermal decomposition in triethylene glycol media. The magnetoelectric nanocomposites were synthesized by the decomposition of barium titanate precursors in the presence of a magnetic phase under solvothermal conditions and subsequent annealing at 700 °C. X-ray diffraction revealed the presence of both spinel and perovskite phases after annealing with average crystallite sizes in the range of 9.0–14.5 nm. Transmission electron microscopy data showed two-phase composite nanostructures consisting of ferrites and barium titanate. The presence of interfacial connections between magnetic and ferroelectric phases was confirmed by high-resolution transmission electron microscopy. Magnetization data showed expected ferrimagnetic behavior and σs decrease after the nanocomposite formation. Magnetoelectric coefficient measurements after the annealing showed non-linear change with a maximum of 89 mV/cm*Oe with x = 0.5, 74 mV/cm*Oe with x = 0, and a minimum of 50 mV/cm*Oe with x = 0.0 core composition, that corresponds with the coercive force of the nanocomposites: 240 Oe, 89 Oe and 36 Oe, respectively. The obtained nanocomposites show low toxicity in the whole studied concentration range of 25–400 μg/mL on CT-26 cancer cells. The synthesized nanocomposites show low cytotoxicity and high magnetoelectric effects, therefore they can find wide applications in biomedicine.Nowadays, magnetoelectric nanomaterials are on their way to finding wide applications in biomedicine for various cancer and neurological disease treatment, which is mainly restricted by their relatively high toxicity and complex synthesis. This study for the first time reports novel magnetoelectric nanocomposites of Co Two-phase ME materials, known as ME composites, attract more attention due to a significantly higher ME effect compared to single-phase materials because magnetic and electric orders must coexist in an asymmetric structure in single-phase materials. The ME effect itself is a result of coupling between the magnetostrictive effect, which is the property to change shape during the process of magnetization in the magnetic phase; and the ferroelectric effect, which is the property to polarize under mechanical deformation in the ferroelectric phase [During the last decade, magnetoelectric (ME) materials have attracted significant interest for application in various branches of science and technology, such as magnetic-field sensors, spintronics, energy harvesters, and biomedicine ,2,3,4,5.3  and YMnO3 [3 . The sec3 [3 . Usuallyic phase . TherefoIn connection with the biomedical applications of ME composites, a novel concept, based on the idea of using both magnetic and electric fields to control drug delivery and release processes, was presented in the publications ,11,12,132O4 core and BaTiO3 shell [2O4-BaTiO3, consist of rather toxic elements: cobalt and barium. While iron [The core-shell structure is expected to have a better arrangement of magnetic and ferroelectric phases because the higher interface contact leads to a higher ME effect . Thus, eO3 shell ,18,19,20O3 shell ,17,20 anO3 shell ,12,21. MO3 shell  and magnO3 shell . Unfortuile iron ,25,26 anile iron ,28 materile iron ,30 and bile iron  is restr2+, the value is 1.8–3 × 105 J/m3 at 300 K [2+ [2+ compared to stoichiometric compounds [In all the cases mentioned above, cobalt ferrite has been used as a magnetic core due to its high magnetostriction, which correlates with the magnetocrystalline anisotropy of the magnetic atoms in the lattice. For Coat 300 K , which i00 K [2+ . The aniompounds ,35. More3 nanoparticles as well [3 phase and surface connections between phases were obtained by annealing. Moreover, MENC with three different types of CoxFe3−xO4 magnetic core with x = 0.0; 0.5; 1.0 were synthesized to study the effect of magnetic phase on the ME effect. However, the methods of ME composite synthesis for biomedical applications mentioned above are largely based on citrate synthesis; magnetic nanoparticles are usually synthesized by coprecipitation of titanium and barium citrates with subsequent evaporation of the solvent. The final mixture of citrate complexes and magnetic phase is annealed, resulting in the formation of a core-shell structure . Unfortu as well ,39,40. T3; Sigma-Aldrich, St. Louis, MO, USA), cobalt (II) acetylacetonate (Co(acac)2; Sigma-Aldrich, St. Louis, MO, USA), triethylene glycol, 99% , titanium (IV) isopropoxide , barium hydroxide octahydrate (II) (Ba(OH)2·8H2O; Sigma-Aldrich, St. Louis, MO, USA), 1-octadecene, ≥95% , oleic acid, ≥99% , ethanol, ≥95% , MTS , NaOH, 99.5% , polyvinylpyrrolidone, 10 kDa , Pluronic F127 , and deionized water.Iron (III) acetylacetonate (Fe(acac)xFe3−xO4, where x = 0.0 (x = 0.0-0); 0.5 (x = 0.5-0); 1.0 (x = 1.0-0), were synthesized by thermal decomposition of x × 4 mmol Co(acac)2 and (3 − x) × 4 mmol Fe(acac)3 in 120 mL TEG medium at 265 °C for 1 h under argon blanketing and cooled to room temperature.The magnetic phase was synthesized according to a slightly modified version of the protocol . FerritexFe3−xO4 were synthesized via dropwise injection of x × 12 mmol Co(acac)2 and (3 − x) × 12 mmol Fe(acac)3 in 240 mL of TEG into 60 mL of seeds solution at 225 °C under argon blanketing for 1.5 h. Then, the reaction medium was kept at 225 °C for an extra 30 min and cooled down to room temperature. Finally, three types of MNP with Fe3O4 (x = 0.0-1), Co0.5Fe2.5O4 (x = 0.5-1) and CoFe2O4 (x = 1.0-1) structure were obtained.The final magnetic nanoparticles (MNP) of CoxFe3−xO4-BaTiO3 composites with x = 0.0 (x = 0.0-BT); 0.5 (x = 0.5-BT); 1.0 (x = 1.0-BT) were synthesized using a solvothermal approach based on the published protocol with significant modifications [2·8H2O in 12 mL of TEG. Solvothermal synthesis was conducted at 200 °C in a 75 mL glass autoclave for 4 h in an oil bath followed by cooling to room temperature. The obtained sol was diluted three times with ethyl acetate and the sample was collected by magnetic decantation for annealing. Coications . Sixty mThe samples were annealed at 700 °C for 5 h. The annealed samples were dispersed in a 1-octadecene solution with oleic acid (mass ratio sample: oleic acid: octadecene—1:1:8) in a specialized 80-milliliter steel container using 10 balls (d = 10 mm) and 50 balls (d = 5 mm) of tungsten carbide. The mixture was ground in a planetary ball mill  in the air at 400 RPM for 12 h with a 5 min pause for every hour. The grounded mixture was washed with isopropanol twice, and the magnetic phase was collected by magnetic decantation and redispersed in toluene. The grounded samples were hydrophilized through magnetic stirring of 2 mg/mL samples solution in toluene with 2 mM Pluronic F127 solution in water according to the previously published protocol . FinallyThe nanoparticles’ morphology and microstructure were imaged via a JEOL JEM-1400 transmission electron microscope  with an accelerating voltage of 120 kV and a JEOL JEM-2100 transmission electron microscope  with an accelerating voltage of 200 kV. Structural phase analysis was carried out on a Rigaku Ultima IV diffractometer  using CoKα radiation and a graphite monochromator. Static magnetic properties were measured in magnetic fields ranging from −18 to 18 kOe at 300 K on a Quantum Design Physical Property Measurement System . Elemental analysis and concentration measurements were conducted using a 4200 MP–AES machine . Elemental composition was also studied via SEM/EDX analysis using a scanning electron microscope JSM-6480LV . The hydrodynamic size of the samples was measured by dispersing 50 μL of the as-synthesized colloid sols in 2 mL ethanol or water using a Zetasizer Nano ZS analyzer .b is the thickness of the sample, and The ME properties were estimated using a dynamic method described elsewhere ,43. For 2) in T-25 cultural flasks . Upon reaching a high confluence, the cells were subcultured at a ratio of 1:10 according to the standard trypsinization method.Murine syngeneic cell-lines of colon-rectum (CT-26) were purchased from ATCC (CRL-2639™). The cell line was cultured in RPMI 1640 media  supplemented with antibiotics , GlutaMax Supplement , and 10% fetal bovine serum . The cells were cultured under standard conditions  were added to the cells. After another 24 h incubation, the cells were washed with PBS, and a fresh growth medium with MTS reagent was added to each well. Non-treated cells by the samples of x = 0.0-BT, x = 0.5-BT, and x = 1.0-BT were used as a positive control. The cells were incubated in MTS reagent for 4 h at 37 °C and 5% CO2 in a humid atmosphere. The assay was conducted in three replicates. Optical density was measured using a Multiscan GO plate reader , λ = 490 nm.Cytotoxicity tests for the samples of x = 0.0-BT, x = 0.5-BT, and x = 1.0-BT in CT-26 cells were performed via CellTiter 96 AQueous One Solution Cell Proliferation Assay  according to the manufacturer’s protocol. CT-26 cells were seeded at 12 × 10s is the optical density of the sample wells, Ab is the optical density of the blank wells, and Ac is the optical density of the positive control wells.Cell viability was calculated as:xFe3−xO4 seeds: x = 0.0-0; x = 0.5-0; x = 1.0-0, were synthesized in a TEG medium by thermal decomposition of Co(acac)2 and Fe(acac)3. The average size, as determined by transmission electron microscopy (TEM), was 6.6 ± 1.4 nm, 6.4 ± 1.1 nm, and 5.6 ± 1.0 2 were injected dropwise into the seed media. The Co to Fe stoichiometry was kept the same as it was for the initial seeds. Finally, the series of CoxFe3−xO4 nanoparticles x = 0.0-1, x = 0.5-1, and x = 1.0-1, with average sizes of 15.2 ± 1.9 nm, 13.9 ± 1.6 nm, and 17.1 ± 2.2 nm ± 2.2 nm A–C, resp± 2.2 nm D. The ob2O4  (s) and remanence magnetization are significantly high for such size of nanoparticles: 70.9 ± 0.7 A•m2/kg, and 5.14 ± 0.05 A•m2/kg for the x = 0.0-1; 70.5 ± 0.7 A•m2/kg, and 7.66 ± 0.08 A•m2/kg for the x = 0.5-1; 66.3 ± 0.7 A•m2/kg, and 6.65 ± 0.08 A•m2/kg for the x = 1.0-1. The σs decrease with the increase of Co content, which is reasonable, because the magnetic moment of Co is less than Fe.Then, the synthesized series of MNP x = 0.0-1, x = 0.5-1, and x = 1.0-1 were studied by X-ray diffraction (XRD) and vibrating sample magnetometry (VSM) measurement. According to structure studies, all samples have a spinel structure (Space group Fd3m) specific for magnetite (PDF Card No.: 01-074-1909) and cobalt ferrite (PDF Card No.: 01-078-4451) as well as other ferrites with the structure: Me(II)Fe(III)2O4 . The cry= 1.0-1) . This te0.42Fe2.58O4 and Co0.93Fe2.07O4, respectively. This may be due to the reduction of Fe(III) to Fe(II) by the glycol media, which competes with Co(II) during MNP growth [The elemental analysis of the ferrite MNP was conducted using the MS AES method. The x = 0.5-1 and x = 1.0-1 samples have a lower amount of Co compared to the loaded ratio of Co to Fe precursors . The actP growth ,49. xFe3−xO4-BaTiO3 series.All the synthesized samples x = 0.0-1, x = 0.5-1, and x = 1.0-1 were further used for the synthesis of the Co2+ ions:As part of this research, a synthesis of final ME composites was carried out according to a modified solvothermal procedure for obtaining a crystalline shell of barium titanate . This texFe3−xO4-BaTiO3 in variable conditions, where x = 0.0, was carried out. Therefore, for all the further syntheses x = 0.0-1 samples were used. Based on the study mentioned, a synthesis of the series CoThe first series of samples were obtained by the solvothermal synthesis in polyols with the addition of 0%, 5%, and 15% of water by dissolving barium hydroxide in a TEG and the addition of a subsequent amount of water . All syntheses were carried out using a PVP stabilizer. PVP is a common polymer with low toxicity, which is actively used to stabilize nanoparticles including ferrites. It consists of monomeric units of N-vinylpyrrolidone, which is a lactam (cyclic amide) with lone electron pairs on the nitrogen and oxygen atoms, which, on the one hand, are capable of coordinating with the surface of the magnetic core, and, on the other hand, play the role of a matrix for subsequent formation of the barium titanate shell. The use of this polymer made it possible to significantly increase the stability of the obtained nanocomposites at the stage of solvothermal synthesis. The samples turned out to be relatively polydisperse, but a tendency was revealed by DLS and TEM that the higher the amount of water, the higher the hydrodynamic sizes of nanoparticles turn out to be A,D. AccoThe obtained sample (x = 0.0-BT5%) with 5% extra water volume added was further studied by XRD and Energy-dispersive X-ray spectroscopy (EDX) elemental analysis . The samThe initial mass ratio of the magnetic phase to the ferroelectric phase was 1 to 2.5. It was chosen because the magnetic phase should be surrounded by barium titanate to gain a higher ME effect, otherwise the presence of interfacial magnetic connections leads to an increase in the electric conductivity of the whole material (ferrites are semiconductors) and a decrease in ME effect. On the other hand, the larger amount of ferroelectric phase leads to less interfacial connections between magnetic and ferroelectric phases and therefore the lower ME effect. Thus, maintaining the optimal ratio of phases is necessary. For this reason, extra samples (x = 0.0-BT 1–4) with 1 to 4 magnetic to ferroelectric phases were synthesized and studied by TEM, XRD, and DLS analyses. According to TEM, the samples were crystalline, and a lack of an amorphous phase was observed A. The naThe MENC series of x = 0.0-BTO, x = 0.5-BTO, x = 1.0-BTO was synthesized according to the protocol given in the section Materials and Methods. According to the TEM studies the magnetic cores of the samples were covered with amorphous barium titanate precursors shell A,C,D. ThThe samples after the annealing were studied by XRD and using magnetic measurements. The XRD study revealed that the composites are highly crystalline and consist of barium titanate (PDF Card No.: 01-075-1606), ferrite phase, and barium carbonate (PDF Card No.: 01-078-4343) phases , Table 1s and σr due to the presence of the non-magnetic phase: barium titanate , that helps to avoid side evaporation during ball milling and/or excludes ignition, which is possible in the case of more commonly used flammable liquids. Oleic acid has a very high boiling point as well, but moreover, it can adsorb onto nanocomposite surfaces and plays the role of surface stabilizer, seeing as it is used for ferrite nanoparticle stabilization. To find the optimal time of ball milling the dynamics of size and polydispersity index (PdI) vs. time were studied using the DLS method  was recorded in the Co0.5Fe2.5O4-BaTiO3 sample, which combines the higher crystallite size and medium cobalt content. All investigated samples demonstrated high ME properties in a diapason of 50–89 mV/cm·Oe. The cytotoxic studies on the cancer cell line CT-26 revealed that all samples are non-toxic in a concentration range of 25–400 μg/mL. The obtained MENC shows low cytotoxicity and high magnetoelectric effect and can find a wide application in neural stimulation, cell regeneration, hyperthermia, drug delivery, and other biomedical applications.The aim of this research was to study the influence of magnetic core composition on the magnetic properties, the magnetoelectric effect, and the cytotoxicity of magnetoelectric nanocomposites composed of Co


Furthermore, COS could reduce ROS formation, NF-κB overactivation, phosphorylation of Erk1/2 and Akt and Nrf2/HO-1 in LPS-exposed cells. These results indicate that COS manifests anti-inflammatory activity and antioxidant action via NF-κB, Erk1/2, Akt and Nrf2/HO-1 signaling with an increasing relevance for inflammatory disorders.Chitooligosaccharide (COS), found in both insects and marine sources, has several bioactivities, such as anti-inflammation and antioxidant activities. However, the mechanism of shrimp shell COS on retardation of inflammatory and antioxidant effects is limited. Therefore, the aim of this study is to examine the mechanism of the aforementioned activities of COS in LPS-activated RAW264.7 macrophage cells. COS significantly improved cell viability in LPS-activated cells. COS at the level of 500 µg/mL could reduce the TNF-α, NO and IL-6 generations in LPS-activated cells ( Inflammation is the bodily reaction in preventing tissue injury governed by several results, such as bacteria and chemical agents . Lipopol2O2 method had high antioxidant and antimicrobial activities, especially when 1% H2O2 was used. Varieties of COS production showed that commercial COS had some ability to inhibit IL-6 and TNF-α productions of LPS-activated RAW264.7 macrophage and possessed high anti-inflammatory activity in BALB/c mice [Chitooligosaccharide (COS) is the hydrolyzed result of chitosan, which is easily soluble in water . CommonlB/c mice ,23. AlthB/c mice ,25. Thus®, Thermo Fisher Scientific, Inc. . Chitosan (MW: ~2.1 × 103 kDa and degree of deacetylation: 82%) was given by Marine Bio Resources Co., Ltd., Samutsakhon, Thailand. All the other chemicals and reagents were analytical grade and obtained from commercial sources.RAW264.7 cells were obtained from ATCC . Griess reagent, ELISA kit and MTT were procured from Sigma-Aldrich . Fetal bovine serum and RPMI 1640 were supplied by Gibco2O2) [2O2 was mixed by CS solution and heated at 60 °C for 2 h. The mixtures were cooled in iced water and pH adjusted to 7. The undissolved matter was removed by centrifugation and the supernatant was pooled. The COS solution was lyophilized, and the COS powders were kept at −40 °C until used. The preparation process of COS is shown in MW were determined using a MALDI-TOF-MS and gel permeation chromatography techniques, respectively [COS was processed using the oxidative hydrolysis method with the aid of hydrogen peroxide (H2O2) ; 1% chitectively . DP of C4 cells/well) were seeded in a 96-well plate. Thereafter, LPS at 0.001–10 µg/mL or COS at 25–1000 µg/mL were added to the cells. Subsequently, cell viability was estimated using an MTT assay [RAW264.7 cells  for 24 h, followed by adding 1 µg/mL LPS for another 24 h. After adding LPS, cytotoxicity was examined . ApoptotCOS at 500 µg/mL was added to cells and kept in a cell incubator for 24 h . After i4 cells/well) were pre-incubated with COS and subsequently added LPS for 24 h. The cells were probed with dye for 1 h, as detailed by Chotphruethipong et al. [The intracellular ROS level was tested by staining with DCFH-DA dye. Cells  were seeded into a 35 mm dish for 24 h and subsequently 500 μg/mL COS was added for 24 h with or without an antagonist for 1 h, and treated with LPS (1 µg/mL) for 30 min. The cells were lysed and the protein content was measured [w/v) or skimmed milk  was used to block the membranes before incubating with primary antibodies against caspase-3, cleaved caspase-3, Akt, p-Akt, Erk1/2 and p-Erk1/2 for overnight. The secondary antibody grafted with HRP was incubated for 2 h. The protein signals were detected with chemiluminescence substrate. Loading control used was β-actin.Cells (1 × 10measured . The proFour independent experiments were examined. The data were reported as the average ± standard deviation (SD). The significant difference between groups was done by one-way ANOVA with Tukey’s post hoc test.p > 0.05), whereas a high level of COS (1000 µg/mL) resulted in reduced cell viability (p < 0.05). Since cell viability was similar between COS-treated cells at 25–500 µg/mL and control, these levels were chosen for further study. When LPS at different concentrations (0.001–10 µg/mL) was used to assess cell viability (p < 0.05). Conversely, LPS-treated cells at 10 µg/mL (75.08%) had lower cell viability than LPS-treated cell at 1 µg/mL (84.73%). Therefore, LPS at the level of 1 µg/mL was selected for the subsequent trials. To verify whether COS could diminish LPS-induced cytotoxicity, cells were pre-incubated with COS at 25–500 µg/mL for 24 h before incubation with LPS at 1 µg/mL for 24 h. As depicted in p < 0.05). Nevertheless, pretreatment with COS at high concentrations (100–500 µg/mL) could inhibit LPS-induced cytotoxicity (p < 0.05). The highest inhibitory effect was found for cells treated with COS at 500 µg/mL. Thus, COS at the concentration of 500 µg/mL was an appropriate level for further investigations.To investigate the protective effect of COS, LPS was applied to trigger cytotoxicity in RAW264.7 cells. The impact of COS on cell viability is presented in iability b, high lp < 0.05)  c. The ex < 0.05) d. These κB [p < 0.05). With pretreating COS into LPS-induced cells, a decrease in the NO level was observed. Also, the NO level of LPS-induced cells pretreated with COS was similar to control (p > 0.05), demonstrating that COS had excellent anti-inflammatory activity. When considering TNF-α (κB expression than LPS-treated cells (p < 0.05)  d.p < 0.05) while COS or NAC (ROS scavenger) alone did not result in any significant alteration in ROS production. In contrast, pretreatment with COS and NAC dramatically decreased the intracellular ROS formation in the LPS-treated group. Results suggested that COS could be used for protecting LPS-induced ROS generation in cells.A previous study demonstrated that LPS enhanced ROS production . Thus, tp < 0.05) (p < 0.05). These data indicate that COS can protect LPS-induced RAW264.7 cells. Taken together, the results reveal that COS has potential in the prevention of inflammation and ROS production as formed by the LPS involved with the Nrf-2 and HO-1 signaling pathway.Previous study has reported that Nrf-2 and HO-1 play an essential role in regulating ROS-stimulated cell injury in inflammatory cells . To stud < 0.05) a–d. Decrp < 0.05), while cells pretreated with COS before exposure to LPS were decreased (p < 0.05) (p < 0.05), reflecting that COS had an ameliorating effect on LPS-activated inflammation associated with downregulating Erk1/2 and Akt.A recent study reported that LPS activated Erk and Akt signaling pathway during inflammation . To inve < 0.05) . To veriκB [LPS is a potent stimulator for macrophages to release pro-inflammatory cytokines such as IL-6 and TNF-α, NO and NF-κB . AccumulκB ,27. IncrκB ,34. COS κB . NeverthκB , who repWhen the protective effect COS on the apoptotic pathway of RAW264.7 cells was studied, COS could protect from damage to cells as influenced by LPS activation. Anil  revealedκB also indicated the ability of bioactive compounds to prevent inflammation [κB activation signaling pathway generates IL-6 and TNF-α proinflammatory cytokines [κB was perceived in COS-pretreated cells before adding LPS. This finding reflected that COS had an inflammatory activity towards LPS-activated cells.When RAW264.7 cells were acclimatized to assess the anti-inflammatory activity of COS, LPS at the selected level initiates inflammation in RAW264.7 cells by activating mediators of the inflammatory response by an increase in IL-6, NO and TNF-α protein expressions. COS could arrest LPS-generated inflammation by decreasing the expression of NO and TNF-α and IL-6 proteins. These results are similar to a previous finding that COS could prevent LPS-activated inflammation . Macrophammation . The NF-ytokines ,47,48. AκB and pro-inflammatory cytokines, namely TNF-α, IL-6 [κB, TNF-α and IL-6 levels in this study appeared to increase in response to ROS treatment, whereas the decreased levels of NF-κB, TNF-α and IL-6 activated by LPS treatment were found in the presence of COS. In addition, our data revealed that antioxidant agent NAC suppressed NF-κB levels. Several lines of evidence indicate that Nrf2 is a crucial molecular mechanism of oxidative stress and inflammation [Additionally, the effectiveness of COS on ROS inhibition of LPS-activated RAW264.7 cells was investigated. In general, ROS generation plays a crucial role in various aspects; however, excessive level of ROS can induce the pathogenesis of many diseases . It was -α, IL-6 . Consistammation . Nrf2 fuκB and MAPK signaling pathways take part in inflammation [κB and Nrf2/HO-1 pathways, in which COS might be used for a promising functional compound for the ministration of inflammatory disease.When RAW264.7 cells were used to test the influence of COS on controlling of Erk1/2 and Akt signaling pathways, COS exerts anti-inflammation associated with Erk1/2 and Akt signaling pathways and contributes to releasing TNF-α, NO and IL-6 inflammatory mediators in RAW264.7 cells activated by LPS. Also, COS could alleviate LPS-activated oxidative stress through Nrf2/HO-1 signaling. Thus, COS had a potent effect on inhibiting oxidative stress and inflammation in macrophage cells induced by LPS. Several studies demonstrated that the Akt, NF-ammation ,51. Our ammation . Thus, CκB, IL-6 and TNF-α proteins in LPS-induced cells. Moreover, it could inhibit ROS generation in cells via the Nrf2/HO-1 pathway. Also, COS decreased the expression of p-Erk1/2/Erk1/2 and p-Akt/Akt proteins in LPS-treated cells related to cell survival. Therefore, COS can be a functional ingredient with bioactivities, especially antioxidant and anti-inflammatory activities.COS from Pacific white shrimp shell chitosan had no cytotoxicity on RAW264.7 cells when levels lower than 1000 µg/mL were used. The use of COS at the level of 500 µg/mL could protect against the death of LPS-induced cells better than other levels. COS at 500 µg/mL had the ability to fight inflammation by inhibiting the expression of NO, NF-


Spirodela polyrhiza subjected to different boric acid concentrations. Plants were grown for 7 and 10 days in ½ Schenck-Hildebrandt media supplemented with 0 to 56 mg B.L-1, the non-structural and structural carbohydrates, and related genes were evaluated. The results showed that B altered the morphology and carbohydrate composition of this species during plant development. The optimum B concentration (1.8 mg B.L-1) led to the highest relative growth and biomass accumulation, reduced starch, and high pectin and apiose contents, together with increased expression of UDP-apiose/UDP-xylose synthase (AXS) and 1,4-α-galacturonosyltransferase (GAUT). The toxic state (28 and 56 mg B.L-1) increased the hexose contents in the cell wall with a concomitant reduction of pectins, apiose, and growth. The pectin content of S. polyrhiza was strongly associated with its growth capacity and regulation of B content within the cells, which have AXS as an important regulator. These findings suggest that duckweeds are suitable for B remediation, and their biomass can be used for bioenergy production.Pectic polysaccharides containing apiose, xylose, and uronic acids are excellent candidates for boron fixation. Duckweeds are the fastest-growing angiosperms that can absorb diverse metals and contaminants from water and have high pectin content in their cell walls. Therefore, these plants can be considered excellent boron (B) accumulators. This work aimed to investigate the relationship between B assimilation capacity with apiose content in the cell wall of  B is stDuckweeds have a distinct cell wall composition with high levels of pectin , a complex polysaccharide class built up from galacturonic acid chains with substitutions and ramifications of different sugars . These p−1)   and rela−1) . The sev−1) . Besides−1) . This me−1) . Other c−1) . There a−1) . Evidenc−1) . The evahydrates . Furtherhydrates . These s0 mg L−1 , but no 22.1Spirodela polyrhiza 9509  was obtained as in vitro culture from the Rutgers Duckweed Stock Cooperative (RDSC) collection and was cultivated under axenic conditions in 250-ml borosilicate erlenmeyers with 100 ml of in ½ Schenk-Hildebrandt (SH) medium (Sigma-Aldrich®) containing 2 M (NH₄)₃PO₄, 0.08M H3BO3, 1.28 M CaCl2, 0.42 mM CoCl2•6H2O, 1.25 mM Cu.SO4*5H2O, 0.05 M Na2-EDTA, 0.05 FeSO4•7H2O, 1.62 M MgSO4, 0.06 mM MnSO4*H2O, 1.28 mM H2MoO3•2H2O, 6 mM KI, 22.7 M KNO3, and 3.5 mM ZnSO4•7H2O (pH 6.5), and supplemented with 0.5% sucrose. Then, the standard ½ SH was modified by adding different concentrations  of boric acid  to evaluate the effect of this micronutrient on duckweed growth. The boron concentrations correspond to 0.007, 0,014, 0.028, 0.056, 0.112, 0.450, and 0.900 M of boron, and the DI water used in media preparation has no boron content. The regular growth is performed with 0.4 mg L−1 B, used as a control S. polyrhiza were grown for 7 and 10 days at 25°C with a photoperiod of 16 h of light (photosynthetic active radiation intensity of 100 µmol m−2 s−1). At each harvest (7 and 10 days), the fresh biomass was weighed, frozen in liquid nitrogen, and ground to a fine powder with a mortar and pestle. Samples were stored at −80°C and freeze-dried for molecular and biochemical analyses.2.2−1 of B single element, Inorganic Ventures).B remaining in the culture media and B content in the duckweed biomass were quantified after the disposal in the borosilicate erlenmeyers; therefore, if there was any leak, it was incorporated in the mensuration. At each harvest (after 7 and 10 days of growth), the media pH was measured with a pH meter (Metler Toledo Seven Compact), and 5 ml of culture media was frozen at −20°C to evaluate B concentration by inductively coupled plasma-optical emission spectrometry . To determine B content in biomass, 50 mg of fresh biomass was digested using nitric acid . To each2.3S. polyrhiza were initially inoculated into the culture medium, corresponding to one biological replicate. Simultaneously, five replicates were harvested . RGR was calculated by Equation 1, simplified into Equations 2 and 3, where x represents the data of fresh biomass and t represents elapsed time in days .The plants’ relative growth rate (RGR) was calculated according to the International Steering Committee on Duckweed Research and Applications (ISCDRA). Growth measurements followed the procedure described by 2.4® Savant SC 250 EXP), and resuspended in 1 ml of deionized water with 1 ml of chloroform to remove pigments. Sucrose, fructose, glucose, and raffinose were analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) in a Dionex® system  using a CarboPac PA1 column and eluted with 150 µM sodium hydroxide in an isocratic run of 27 min (Soluble sugars were extracted three times from 10 mg of powdered dry biomass with 1.5 ml of 80% ethanol (v/v) at 80°C for 20 min each. The supernatants containing the soluble sugars were recovered by centrifugation at 14,000 rpm for 10 min, vacuum concentrated (ThermoScientificf 27 min .−1 of α- amylase (E.C. 3.2.1.1) of Bacillus licheniformis (Megazyme®) in 10 mM MOPS buffer (pH 6.5) at 75°C for 1 h. Afterward, 30 U ml−1 of amyloglucosidase (E.C. 3.2.1.3) of Aspergillus niger (Megazyme®) in 100 mM sodium acetate (pH 4.5) was added, and the mixture was incubated at 50°C for 1 h. To the recovered supernatants, a mixture containing glucose oxidase , peroxidase (700 U ml−1), 4-aminoantipirin (290 µmol L−1), and 50 mM of phenol at pH 7.5 was added to determine the released glucose by a colorimetric assay. The reactions were further incubated for 15 min at 30°C, and the absorbance was measured at 490 nm.The alcohol-insoluble residue (AIR) was dried overnight at 45°C before starch digestion . Starch 2.5®). Apiose, arabinose, fucose, galactose, glucose, mannose, rhamnose, and xylose were analyzed by HPAEC-PAD  on a CarboPac SA10 column (Dionex-Thermo®). Sugars were eluted isocratically with 99.2% of water and 0.8% sodium hydroxide (v/v) (1 ml min−1) and detected using a post-column base containing 500 mM NaOH (0.5 ml min−1).Aliquots of 2 mg of de-starched AIR were hydrolyzed with 1 ml of 2 M trifluoroacetic acid for 1 h at 100°C to obtain non-cellulosic monosaccharides. Then, the samples were vacuum dried and resuspended in 1 ml of MilliQ water. Cellulosic hydrolysis was performed with 72% sulfuric acid at 45°C for 30 min, diluted to 4%, and incubated at 100°C for 1.5 h. Samples were filtered through 0.22-µm filters (Millipore2.6Uronic acids were determined as described by 2.7®) followed by DNase treatment according to the manufacturer’s instructions. The RNA quantification and purity were assured using a Nanodrop ND-100 spectrophotometer (Thermo-Fischer Scientific®), and samples with 260/280 ratios between 1.8 and 2.2 were considered sufficiently pure. The samples’ integrity was also checked by electrophoresis on a 1% agarose gel and stained with SYBR Safe DNA gel stain (Thermo-Fischer Scientific®). Approximately 1 µg of each RNA sample was reverse transcribed with random hexamers by SuperScript III Reverse Transcriptase (Thermo-Fischer Scientific®). The samples were tested for the absence of genomic DNA with UPD-apiose/UDP-xylose synthase (AXS) primers  as described by RNA was extracted using the ReliaPrepTM RNA Tissue Miniprep System  ab initio gene prediction was performed with the Augustus prediction tool (version 3.3.2) on an A. thaliana gene model (http://augustus.gobics.de/) and the functional annotation was verified by BLASTp, InterProCan, and Gene Ontology as described by A. thaliana were used as queries to S. polyrhiza 9509 and the recovered S. polyrhiza 9509 genes were compared with the Reference Sequence (RefSeq) database of NCBI by BlastX , InterPro database, and HMMER scan (https://www.ebi.ac.uk/Tools/hmmer/) for protein family association and functional domain validations. Primers were designed with Primer-BlastR (NCBI) (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) according to MIQE guidelines. The primer sequences are shown in S. polyrhiza 9509 was measured by qRT-PCR analysis using a QuantiStudio 6 Flex Real-Time PCR system . The PCR reactions were performed with 1.4 µl of cDNA (1:10), 7 µl of 2X SYBR Green Master Mix  800 nM primer set, and the following cycling conditions: 95°C for 10 min, 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. A melting curve analysis confirmed the amplification of a single product. The cycle quantification (Cq) values and the efficiency of each primer were determined using LinRegPCR software (UDP-apiose/UDP-xylose synthase (AXS), UDP-glucuronate decarboxylase (UXS), rhamnose biosynthesis (RHM), and α-galacturonosyltransferase (GAUT) was quantified compared to the average expression, normalized by the highest Cq value, and the Cq values of the mentioned targets were normalized by the geometric average of the reference gene combinations [Elongation factor 1-α (EF1) and F-box family protein (FBOX)]. The specific transcripts were selected based on the highest expression levels, as reported by The relative abundance of target transcripts in software . The rel2.9p< 0.05). In addition, a comparison between 7 and 10 days was made with Student’s t-test (p< 0.05) for each concentration. The analyses were carried out with R software version 3.6.1.Five replicates were used for the relative growth rate, biomass accumulation, boron dosage in media, pH evaluation, soluble sugars, starch, uronic acids, monosaccharide quantification, and gene transcript analyses. Owing to plant biomass availability, three replicates were used for B dosage in plants. Data from each harvest day (7 and 10 days) were evaluated by one-way ANOVA followed by Tukey’s test (33.1S. polyrhiza. Higher B levels (between 28 and 56 mg B L−1) led to chlorosis, dark leaf pigmentation, and reduction in growth (−1 (control) had the highest growth rate and biomass accumulation at 7 days , no stipe abscission (connectors from mother frond and daughter fronds) occurred  from 7 to 56 mg B L−1, while at 10 days, the pH was somewhere neutral from 0 to 7 mg B L−1, getting acidic (pH 5.4 and 4.4) at the toxic concentrations (28 and 56 mg B L−1)  and 10 days (0.08–1.69 µg mg−1) with a tendency to increase over time (S. polyrhiza under a toxic (28 and 56 mg B L−1) state accumulates 4.6 and 15 times more B than the control (0.4 mg B L−1) at 7 and 10 days, respectively  to 55.4% (131.1 µg mg−1 DW), while 28 mg B L−1 B reduced pectic content (uronic acids) by 26.3% (62.2 µg mg−1 DW) when compared to 0.4 mg B L−1 (84.3 µg mg−1 DW) (GAUT - Spipo12G0021200), whose relative expression was higher at 0.4 mg B L−1  and 17% (10 days) under high B (>7 mg B L−1) (−1), the apiose levels were the highest identified for both 7 (151.4 µg mg−1 DW) and 10 (167.6 µg mg−1 DW) days (−1 have an elevated biomass accumulation and UDP-apiose/UDP-xylose synthase (AXS - Spipo0G0011100) expression  and toxicity (28 mg B L−1). At the same time, glucose revealed an inverse pattern in 7 and 10 days, with accumulation in 10 days in higher B concentrations , while the walls of eudicots have higher pectin content and a greater need for B (20–30 B µg g−1 DW). Pteridophytes, lycophytes, and bryophytes have 21, 15.1, and 12.4 µg B g−1 DW, respectively  (Mimulus guttanus) and fuzzy water clover (Marsilea drummondii) can accumulate up to 1,000 mg B kg−1 DW   .S. polyrhiza may be related to B uptake, which at a toxic point (>28 mg B L−1) resulted in growth reduction and a decrease in pectin content by 26%. In contrast, the opposite was observed at 0.4 to 7 mg B L−1 (−1 (−1) , which is essential for plant growth and development  . Further(−1 (−1) . The glu(−1) , S. polyrhiza reduced the apiose content by 50.8% and 17.2% at 7 and 10 days, respectively, when compared to the control (0.4 mg B L−1). This reduction is hypothesized as a mechanism to reduce the B content stored in the plant tissues and block B uptake. As apiose has a trade-off with starch accumulation and growth, the starch was investigated. The starch accumulation was prominent in 7 days, especially for 28 and 56 mg B L−1  changed under B deprivation and toxicity in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.DP, AG, and MB planned and designed the experiment. DP, AG, and JP performed the experiments. JU and MC performed the boron quantification. DP and AG analyzed the data. DP, AG, and MB wrote the original draft, reviewed it, and edited it. All authors contributed to the article and approved the submitted version.