Authors predominantly applied MBI’s default settings and chose “mechanistic/non-clinical” rather than “clinical” MBI even when testing clinical interventions . Using these data, we can estimate the Type I error rates for the typical MBI study. Authors frequently made dichotomous claims about effects based on the MBI criterion of a “likely” effect and sometimes based on the MBI criterion of a “possible” effect. When the sample size is n = 8 to 15 per group, these inferences have Type I error rates of 12%-22% and 22%-45%, respectively. High Type I error rates were compounded by multiple testing: Authors reported results from a median of 30 tests related to outcomes; and few studies specified a primary outcome (14%). We conclude that MBI has promoted small studies, promulgated a “black box” approach to statistics, and led to numerous papers where the conclusions are not supported by the data. Amidst debates over the role of p-values and significance testing in science, MBI also provides an important natural experiment: we find no evidence that moving researchers away from p-values or null hypothesis significance testing makes them less prone to dichotomization or over-interpretation of findings.Magnitude-based inference (MBI) is a controversial statistical method that has been used in hundreds of papers in sports science despite criticism from statisticians. To better understand how this method has been applied in practice, we systematically reviewed 232 papers that used MBI. We extracted data on study design, sample size, and choice of MBI settings and parameters. Median sample size was 10 per group  for multi-group studies and 14 (IQR: 10–24) for single-group studies; few studies reported  Magnitude-based inference (MBI) is a controversial statistical method that has been used in hundreds of papers in the sports science and medicine literature. MBI has laudable goals. The method arose out of a desire to address over-reliance on tests of statistical significance, inattention to estimation, and distortion from unpublished null results . MBI wasHowever, the development and dissemination of MBI have been unusual for a statistical method. MBI has never been published in a journal following peer review by statisticians, and equations with formal notation have never been provided. In fact, since its introduction into the peer-reviewed literature in 2006 , the metst, 2019), thus necessitating further scrutiny of the method. We note that in late 2019, magnitude-based inference was renamed to magnitude-based decisions (MBD) . A wild-card symbol (*) was used with “sport” to capture variations on that word. Searches included August 2nd 2018 to the earliest available date.The resulting 491 results were combined with 84 results identified in a previous search , see the® survey hosted at the University of Utah. A re-creation of the data extraction tool is shown in a priori power calculation, information related to bias, and the choice of MBI settings and parameters (which are described in more detail below).All authors assisted in the development of a data extraction tool that went through 4 rounds of revision before being converted into a REDCapArticles were randomly assigned to random pairs of five of the authors  and each author entered the relevant data into REDCap. When data extraction was complete, the full dataset was downloaded and agreement between authors was calculated. Percentage agreement was moderate to high across items, median = 77%, IQR = . A third reviewer (KRL) then conducted consensus coding for all entries on all items in which there was disagreement. Remaining points of disagreement or areas of ambiguity where discussed with a fourth reviewer (KLS). Both the third and fourth reviewer were blind to the identities of the initial raters. See Selection, performance, and detection bias were assessed for individual studies. Details of this assessment are presented in MBI is implemented in downloadable Excel spreadsheets available at the website sportsci.org . Though δh and δb, respectively—define this trivial range. The default setting for the trivial range is −δh = −0.20 to δb = +0.20 standard deviations [MBI practitioners define a trivial range of effects that they would consider clinically irrelevant. The thresholds for harm and benefit—which we will denote −viations .0: true effect ≤ δb), MBI would conclude that there is a 25% chance that the intervention is not beneficial and thus a 75% chance that the intervention is beneficial. MBI practitioners choose parameters called the maximum risk of harm and minimum chance of benefit—which we will denote ηh1 and ηb1. An effect is deemed “unclear” when the harm and benefit probabilities exceed ηh1 and ηb1, respectively. Otherwise, an effect is deemed “clear.”MBI returns probabilities that an effect is negative (or harmful), trivial, or positive . These probabilities are based on interpreting p-values from two one-sided hypothesis tests as if they were Bayesian posterior probabilities ,8. For e0: true effect ≤ δb) at a significance level of .25 but not .05. A “possibly positive” result is equivalent to rejecting H0: true effect ≤ δb at a significance level of .75 but not .25.When an effect is deemed clear, MBI returns a categorical inference such as “possibly positive,” “likely positive,” or “very likely positive.” These categories are determined by the MBI probabilities: ≤0.5% is most unlikely; 0.5–5% is very unlikely; 5–25% is unlikely; 25–75% is possible; 75–95% is likely; 95–99.5% is very likely; ≥99.5% is most likely or almost certainly ,18,21. Fηb1 and ηh1, whereas non-clinical MBI treats both directions equivalently, such that ηb1 = ηh1, and (2) clinical and non-clinical MBI have different default values—0.5% and 5%, respectively—for ηh1 (the “maximum risk of harm”). The choice of when to use clinical MBI versus non-clinical MBI is not clearly defined and a source of confusion in MBI [MBI is divided into two types of inferences—“clinical” and “non-clinical/mechanistic”; correspondingly, the MBI spreadsheets return two sets of results, one for clinical MBI and one for non-clinical MBI. There are two main differences: (1) clinical MBI allows different values for n in MBI . As we wηh1 = ηb1 = 5%, an effect is deemed “unclear” if the harm and benefit probabilities both exceed 5%; this is equivalent to the 90% confidence interval for an effect overlapping both the positive  and negative (or harmful) ranges  is much more stringent than for non-clinical MBI (5%), which means that it is harder to find “clear” positive effects with clinical MBI than with non-clinical MBI . When an observed effect is in the positive direction, it will be declared “unclear” in clinical MBI but “clear” in non-clinical MBI if the 99% confidence interval overlaps the negative range but the 90% confidence interval does not. We speculate that this is why most users report the non-clinical MBI results.) ranges . The def0: true effect ≤ δb) at a significance level of .05; a “likely positive” result is achieved when one rejects the null hypothesis of not positive at a significance level of .25 (but not .05); and a “possibly positive” result is achieved when one rejects the null hypothesis of not positive at a significance level of .75 (but not .25). Thus, one might expect that the Type I error rates for these inferences would equal these significance levels. However, the calculation of Type I errors is complicated because otherwise beneficial/positive results may sometimes be deemed “unclear”, which can lower the Type I error rate. Whether results are clear or unclear is affected by the standard error  and the choice of MBI parameters. Therefore, unlike standard hypothesis testing, the Type I error rate for MBI depends on the sample size, study design, outcome variance, and the choice of MBI parameters. Additionally, the Type I error rates depend on the size of the true effect , and must account for errors in both directions .MBI inferences are based on one-sided hypothesis tests with significance levels of .005, .05, .25, and .75. For example, a “very likely positive” result is achieved when one rejects the null hypothesis of not beneficial/positive  or the percentage of studies where p < .05 (for standard hypothesis testing). Simulations were conducted in SAS 9.4. See We simulated the Type I error rates for MBI for both a between-group comparison and a within-group comparison assuming a range of sample sizes. For the between-group comparison, we generated 200,000 simulated trials with We also calculated the Type I error rates mathematically, using previously derived mathematical equations for the Type I error rate . These eηh1 = 5%; thresholds of harm/benefit of 0.2 standard deviations; and a true effect size of 0, to facilitate comparison with standard hypothesis testing. We assumed variances of either 1.0  or 0.364 . We then varied these parameters.We calculated Type I error rates for a range of scenarios. All reported values were confirmed by both simulation and math. For the base-case, we assumed Our systematic review identified 232 studies that used MBI. The topics covered in these articles spanned the range of sport and exercise science and were published in highly cited journals in these fields, see a priori power analysis. In cases where authors reported sample size calculations based on the MBI calculators, they often reported very low sample size requirements, such as 5, 6, or 7 total participants [The studies covered a range of experimental designs, including randomized controlled trials, crossover trials, and observational studies, see icipants –24. We aicipants –29. For icipants  wrote ofFocusing solely on analyses related to the main hypotheses (excluding baseline and other ancillary comparisons), the median number of dependent variables was 7 and the median number of statistical tests run was 30. Despite this multiplicity of outcomes, few studies (14%) defined one or more variables as “primary.”ηh1 = 5%. Authors explicitly noted the use of clinical MBI (3.4%), non-clinical MBI (16%) or both (1.3%) in a minority of papers. Given descriptions in the texts, we identified five additional papers where we believe clinical MBI was used, for a total of 16 papers (7%) that used clinical MBI. We believe that non-clinical MBI was used in almost all other cases . In these cases, authors wrote a version of the following statements: “When the positive and negative values were both >5%, the inference was classified as unclear” or, equivalently, “If the 90% confidence interval overlapped the thresholds for the smallest worthwhile positive and negative effects, effects were classified as unclear”; this means they used ηh1 = 5% and treated both directions equivalently, consistent with non-clinical MBI. We cannot rule out that authors used clinical MBI with both ηh1 = 5% and ηb1 = 5%, but this is less likely given that these are not the usual default choices for clinical MBI.The vast majority of studies used non-clinical MBI with the same values: trivial thresholds between -0.20 and +0.20 SD with likely at least small beneficial effects (effect sizes≥.20) on absolute and relative VO2max , and total and moderate PA  in the intervention compared to the usual care group. We found no likely beneficial improvements in any other outcome” (emphasis added). One can infer from their description and ensuing positive recommendation  that they used “likely” as their minimum evidence threshold, though this was never explicitly stated.MBI provides categories of evidence, such as “possible”, “likely”, and “very likely” beneficial. We found that researchers employed MBI’s evidence thresholds much like significance thresholds: They highlighted results that met the bars of “possible”, “likely”, or higher in tables and figures with symbols to distinguish the achieved thresholds much like the use of symbols for p<0.05 or p<0.01; for examples, see: –32. Theyconservative approach to inference whereby substantial effects were only declared clear when the probability likelihood for the effect was ≥75% ” (emphasis added).We found that most authors used a minimum evidence threshold of “possible” (38%) or “likely” (43%) . We founAlmost half (47%) of studies performed null hypothesis testing alongside MBI. Most authors reported using the Excel spreadsheets to run their MBI analyses, but a few authors reported running frequentist statistical models in other programs, such as SAS, but then interpreted the resulting frequentist confidence intervals in MBI terms. We found no studies out of 232 that described their statistical approach as Bayesian.ηb1 = ηh1 = 5%. In fact, clinical MBI with ηb1 = ηh1 = 5% (rather than ηb1 = 25% and ηh1 = 0.5%) was given as the default choice in at least one version of the MBI spreadsheets that we found online in August of 2019 —often in the 0.2 to 0.3 range—were declared “likely” or higher by MBI [For multi-group studies, the interquartile range of sample size was 8 to 15 per group. At these sample sizes, the Type I error rate for the “possible” threshold is 22% to 45% and for the “likely” threshold is 12% to 22% . Consistr by MBI –52. See In some examples, high p-values were reported alongside the MBI results. When p-values were reported alongside MBI results, we found that MBI descriptors were frequently prioritized above p-values in the interpretation of results. Indeed, the results were often used to justify the superiority of MBI over conventional approaches. Examples include:“Although parametric analysis was unable to demonstrate significant differences, magnitude-based inferences indicated that the change in 1-RM squat showed a likely benefit from PA on increasing lower body strength and a very likely benefit for increasing lean body mass (LBM).”“Because a repeated measures ANOVA could not detect any significant difference in the variables between the fastest and slowest trials (P = .136 –>.999), magnitude-based inference approach was adopted to analyse the differences at each step.”“There were no statistically significant differences between the non-runners and runners groups in the CRP values for any of the coupling pairs analysed, however, the magnitude-based inferences enabled more precise tracking of differences between groups and some of the differences were substantially clear.”“There were no statistical differences in NB between groups (P = 0.23); however, magnitude-based inferential statistics revealed likely small  and moderate (0.80 ± 0.91) increases in NB for PULSE and INT compared to BOLUS and possible small increase (0.42 ± 1.00) for INT vs. PULSE. Conclusion: We conclude that the pattern of ingested protein, and not only the total daily amount, can impact whole-body protein metabolism.”In these instances, and others, the authors emphasized the more favorable MBI results in their conclusions, ignoring that the observed effects could easily have arisen due to sampling variability.o = 0 as: P(T21>(.46/(.5/1.72)))+P(T21<-(.46/(.5/1.72))) = .13. Notably, this paper reported that the intervention “almost certainly” improves countermovement jumps with a 100% probability of benefit [In many other examples, authors did not report p-values, but we back-calculated high p-values from confidence intervals . For exa benefit .ηh1 from 5.0% to 0.5% lowers the Type I error rates for these small sample sizes, but as previously noted, researchers rarely chose this value .Until now, our discussion has focused on a single test with default MBI parameters. MBI’s high Type I error rates are further compounded by multiple testing . Though a priori power calculations (15%) or specified a primary outcome (14%). Multiple testing was common with a median of 7 dependent variables and 30 tests related to the main hypotheses of interest; this exacerbates the Type I error issue.Through our systematic review, we have documented that studies using MBI are typically small in size and often make strong claims based on weak evidence. MBI authors routinely made claims based on achieving MBI inferences of “likely” and sometimes “possible” effects. These inferences carry high Type I error rates—12%-22% and 22%-45%, respectively, for the way that MBI is typically applied in the literature. MBI users predominantly applied MBI’s default settings, used a maximum risk of harm of 5%, and chose “non-clinical” rather than “clinical” MBI even when testing interventions in humans. This is likely related to “non-clinical” MBI providing stronger inferences for a given set of data as well as to a lack of precise guidelines about when to use non-clinical versus clinical MBI. Few studies reported a priori sample size calculations, and even made statements suggesting that MBI circumvents the need for an adequate sample size [The median sample size was 10 per group for multi-group studies and 14 total for single-group studies. As a benchmark, it is worth noting that a sample size of 10 per group has a mere 40% power to detect an effect size of 0.8, which is commonly described as a large effect (given α = 5%). Inadequate sample sizes and poor sample size planning are certainly not unique to MBI. However, we believe that the way that MBI has been advertised to the sports science community has encouraged the use of small studies. A central justification for MBI has been its ability to generate more “publication-worthy” results from under-powered studies. For example, proponents of the method state that MBI, “provides an avenue for publishing previously unpublishable effects from small samples” . Thus, iple size –29.Overinterpretation of statistical results is also not unique to MBI, but features of the method and its portrayal may exacerbate the problem. MBI has been described as a Bayesian procedure  and “defnecessarily carry a high Type I error rate, because MBI’s error rates depend on the choice of MBI parameters and settings as well as study design and sample size. However, we found that MBI is predominantly applied in a way that results in high Type I error. For example, use of a maximum risk of harm of 0.5% keeps Type I error rates low for samples of 8 to 15 per groups, but only 8% of users chose this value. Despite conducting clinical research in human populations, authors primarily used non-clinical MBI rather than clinical MBI, likely because it has a default maximum risk of harm of 5% and thus makes it easier to find “clear” beneficial/positive effects.MBI’s proponents have repeatedly denied that MBI has elevated Type I error rates –17. We hThere has been disagreement about what constitutes a Type I error in MBI. The developers of MBI sometimes refer to “clinical Type I error,” which they define as claiming benefit when the true effect is harmful; however, this ignores the larger source of Type I error, which is claiming benefit when the true effect is trivial . The deva priori. In the MBI literature, we found no cases where MBI practitioners acknowledged higher Type I error rates, let alone justified them.We acknowledge that the appropriate tradeoff between Type I and Type II error may be different in sports science than in other biomedical fields, such as cancer research. Thus, there may be cases where a Type I error rate higher than 0.05 may be justified. However, we emphasize that: (1) 0.05 is itself considered a weak level of evidence  (2) 0.05a priori. Of course, MBI is not unique in encouraging a black box approach to statistics. Many statistical programs allow users to implement statistical methods that they do not understand. However, MBI’s lack of rigorous review and documentation may exacerbate the problem [We found that the MBI spreadsheets encourage a black box approach to statistics . For exa problem .The MBI spreadsheets are also not designed to handle many common statistical situations: repeated-measures or clustered data, interactions, count data, and adjustment for more than a single covariate . The inaWe acknowledge that Type I error rates for null hypothesis significance testing can also be greatly inflated due to “p-hacking,” the practice of manipulating data and analyses to produce p-values under 0.05 . Though Proponents of MBI argue that small studies are unavoidable in sports science and thus that MBI is needed to ensure a path to publication for otherwise “unpublishable” null results . We disaProponents of MBI have additionally argued that MBI is necessary because it provides the only alternative to null hypothesis significance testing, for example writing: “Generations of statisticians before us have also criticized the null-hypothesis test, but magnitude-based inference appears to be the first practical alternative that properly takes into account the uncertainty arising from sampling variation” . This arThe conclusions of past studies that have used MBI should be treated with skepticism. However, these papers do still contain valid information—such as means, standard deviations, and confidence intervals—that can be used to re-evaluate the studies’ conclusions. For example, one study reporting that an intervention “almost certainly” improved countermovement jumps also reported a 90% confidence interval for this improvement of -0.10 to 0.91 standard deviations . It is co: effect size ≤-δh); and subtracting this value from 1 gives the p-value associated with the null hypothesis of no harm (Ho: effect size ≥-δh). Similarly, the benefit/positive probability is the p-value associated with the null hypothesis of benefit (Ho: effect size ≥δb); and subtracting this value from 1 gives the p-value associated with the null hypothesis of no benefit (Ho: effect size ≤δb). One may use these p-values to perform equivalence testing and/or minimal effects testing [Furthermore, some MBI papers report the exact harm and benefit probabilities given by the MBI spreadsheets, which are useful because they reveal the p-values associated with specific one-sided hypothesis tests. The harm/negative probability is the p-value associated with the null hypothesis of harm .In cases where papers do not report the exact MBI harm and benefit probabilities, it is possible to back-calculate the relevant p-values from confidence intervals. We include SAS and R code for doing this, specifically for between-group comparisons, in Basic and Applied Social Psychology after the journal banned p-values found that many authors over-interpreted their results far beyond what the data supported [As a case-study, MBI has larger lessons for the statistical community. Some academics have suggested that moving researchers away from p-values and/or significance testing will improve applied statistical practice. MBI provides a natural experiment for testing this hypothesis. We found that MBI papers were no less prone to dichotomization or over-interpretation of findings; in fact, use of MBI generally led to greater exaggeration of results than standard significance testing. Other empirical investigations have drawn similar conclusions: A review of 31 papers published in upported . Misuse We have conducted a systematic review of the MBI literature and undertaken mathematical analyses of MBI methods. We found that MBI has done direct harm to the sports science and medicine literature by causing authors to draw overly optimistic conclusions from their data. MBI has also promoted small studies, promulgated a simplistic “black box” approach to statistics, and reinforced the harmful view that the purpose of research is to get publishable results for the researcher. Sports scientists should stop using MBI. Past empirical studies that have used MBI may well include useful descriptive statistics, but their conclusions should be treated with skepticism. As a case-study, MBI has larger lessons for the statistical community: Our findings suggest that moving researchers away from significance testing and p-values does not improve applied statistical practice.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendixwww.sportsci.org in August 2019.Implemented in Microsoft Excel. Obtained from (XLS)Click here for additional data file.S1 File(DOC)Click here for additional data file.S2 File(CSV)Click here for additional data file.


Ganoderma lucidum (Fr.) Krast, commonly known as “Lingzhi” in Chinese, is a medicinal mushroom that is rich in biologically active substances. Polysaccharides and triterpenoids are the two major components responsible for the bioactivity of this fungus. In the present study, the ultrasonic-assisted co-extraction (UACE) of polysaccharides and triterpenoids from G. lucidum was optimized using response surface methodology with a desirability function, with the equal importance for the two components. Following single factor experiments, the optimal conditions were determine as ultrasonic power of 210 W, extraction temperature of 80C, ratio of liquid to solid of 50 mL/g, and 100 min extraction time, using aqueous ethanol  as the extracting solvent. Under the optimal conditions, the extraction yields of polysaccharides and triterpenoids reached 0.63% and 0.38%, respectively. On the basis of the scavenging capacity of 2,2-diphenyl-1-picrylhydrazyl and evaluation of reducing power, the antioxidant capacities of the polysaccharides obtained by optimal UACE process were higher than those of polysaccharides extracted using traditional hot water extraction, whereas the triterpenoid-rich extracts showed antioxidant activities similar to those obtained using the ethanol maceration method. The present study is the first report on the simultaneous extraction of polysaccharides and triterpenoids from G. lucidum. The developed UACE process could be useful in preparation of a polysaccharide- and triterpenoid-rich ingredient that holds great promise for application in the Ganoderma industry. Ganoderma lucidum (Fr.) Krast belonging to the family of Polyporaceae, commonly known as “Lingzhi” in Chinese and “Reishi” in Japanese, is one of the most highly prized medicinal mushrooms. Its fruiting bodies have been used as a folklore remedy to treat various illnesses and promote longevity for thousands of years in many oriental countries like China and Korea Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: YesReviewer #3: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: No**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The manuscript describes the antioxidant capacity of polysaccharides and triterpenoids obtained from Ganoderma lucidum, using ultrasonic-assisted co-extraction (UACE) and response surface methodology (RSM). The paper is interesting but need major revision.� The MS title is not appropriate and need revision.� The abstract is generalized. Incorporate your significant data into the abstract.� In introduction, the extraction procedures are focused rather than the importance of Ganoderma constituents and their ability to cure various ailments.� In materials and method section, references are missing in subsections. Add pertinent references to the methods adopted.� In results section, the authors have added methods too, which needs to be shifted to the relevant section. For instance line 265-268.� The discussion is very shallow and needs major input.� The linguistic quality of the MS is very poor (for instance line 237). Edit the MS drastically for grammatical mistakes and typos.� I would suggest the authors to make a graphical model incorporating all information presented in the paper.� The authors may add the recent relevant reference, Ryua DH, JY Cho, NB Sadiq, JC Kim, BY Lee, M Hamayun et al. (2020) Optimization of antioxidant, anti-diabetic, and anti-inflammatory activities and ganoderic acid content of differentially dried Ganoderma lucidum using response surface methodology. Food Chemistry, 335: 127645Reviewer #2: The simultaneous recovery of polysaccharides and triterpenoids were reported by the authors using ultrasound-assisted extraction. The authors first performed one factor at a time experiment in order to determine the domain of the experimental design and optimised the influence of four significant variables . The introduction of the manuscript is well prepared and follows the theme of the article. A good methodology was established, the authors provide a clear rationale for each experiment conducted and the results are discussed thoroughly. Discussions are very well reported, and the authors focused on comparing their finding with previous reports. The figures are self-explanatory, clear, and easy to understand. However, I provided a few suggestions and recommend the acceptance of this paper in PLOSONE.1. Please rephrase line 30-33 in the abstract for more clarity2. Line 45: Include the classificator of Ganoderma lucidum mushroom for the first time (Karst.)3. Line 37-38: "the triterpenoids showed antioxidant activities similar to those isolated using the ethanol maceration" This sentence is not correct because the authors performed only extraction. Hence, it should be corrected as "the triterpenoid rich extract showed antioxidant activities similar to those obtained using ethanol maceration.4. Rephrase line 765. Line 208-210: The quantification of triterpenes should be better explained. Dis the authors used a standard (Ursolic acid for example). If yes, then the results should be expressed in the equivalent of the standard6. In its current state, the manuscript has some grammatical errors and instances of badly constructed sentences. Please check the manuscript and refine the language carefully.Reviewer #3: This study describes a methodology for simultaneously extracting polysaccharides and triterpenes.However, the goals of bioactivity differ greatly between polysaccharides and triterpenes. For example, there are many types of triterpenes, and each triterpene has a unique bioactivity. Isn't each activity weakened by simultaneous extraction?In this study, the author describes the usefulness of simultaneous extraction, but I think it is not enough to confirm with DPPH alone.If you have a reason, please state why you expressed the bioactivity only with the DPPH result. In some cases, I suggest removing the DPPH results and changing to a paper with only extraction methods.P3L62: Please add a description of GA-MT and GA-TP5 Materials and Methods: Particle size after sample grinding should be specifiedP6L119-L147: The description of the extraction method is redundant. Please correct it to a concise description.Figures: I can't consider the data because the figure doesn't have a legend.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  25 Nov 2020Responses to the reviewer’s commentsManuscript number: PONE-D-20-24854Title: Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacitiesDear Editors,We appreciate your comments and suggestions on our manuscript submitted previously. The manuscript has been carefully revised according to the suggestions and recommendation by the reviewers, and now all problems have been addressed in revised manuscript. Revised portion are marked in red in the paper. Point-by-point responses to these comments are showed below in this letter.Sincerely yours,Shi-Zhong Zheng, Wei-Rui Zhang, Sheng-Rong LiuReviewer 1#: Some doubts, suggestions and recommendationComment 1: The manuscript describes the antioxidant capacity of polysaccharides and triterpenoids obtained from Ganoderma lucidum, using ultrasonic-assisted co-extraction (UACE) and response surface methodology (RSM). The paper is interesting but need major revision.Response: Thanks. We have carefully and extensively revised the manuscript.Comment 2: The manuscript title is not appropriate and need revision.Response: Accept. It has been revised.Comment 3: The abstract is generalized. Incorporate your significant data into the abstract.Response: Thanks. The abstract has been carefully revised as suggested, and the quality was greatly improved. See in the revision.Comment 4: In introduction, the extraction procedures are focused rather than the importance of Ganoderma constituents and their ability to cure various ailments.Response: Thanks. Some abundant description has been deleted, and the focus has been paid to the extraction techniques and optimization. See in the revision.Comment 5: In materials and method section, references are missing in subsections. Add pertinent references to the methods adopted.Response: Accept. Several related references have been cited in the revised manuscript. Comment 6: In results section, the authors have added methods too, which needs to be shifted to the relevant section. For instance line 265-268.Response: Accept. The extraction conditions were deleted in Results section, and described in Materials and methods section. See in the revision.Comment 7: The discussion is very shallow and needs major input.Response: Thanks. We have carefully revised this section, and more in-depth explanation has been provided. See in the revision.Comment 8: The linguistic quality of the MS is very poor (for instance line 237). Edit the manuscript drastically for grammatical mistakes and typos.Response: Thanks. We have carefully checked the whole manuscript.Comment 9: I would suggest the authors to make a graphical model incorporating all information presented in the paper.Response: Accept. A graphical model was made and provided. Comment 10: The authors may add the recent relevant reference, Ryua DH, JY Cho, NB Sadiq, JC Kim, BY Lee, M Hamayun et al. (2020) Optimization of antioxidant, anti-diabetic, and anti-inflammatory activities and ganoderic acid content of differentially dried Ganoderma lucidum using response surface methodology. Food Chemistry, 335: 127645Response: Accept. It has been cited in the manuscript. See in the revision.Reviewer 2#: Some doubts, suggestions and recommendationComment 1: Please rephrase line 30-33 in the abstract for more clarity.Response: Accept. We has revised it for clarity.Comment 2: Line 45: Include the classificatory of Ganoderma lucidum mushroom for the first time (Karst.)Response: Accept. It has been described. See in the revision.Comment 3: Line 37-38: “the triterpenoids showed antioxidant activities similar to those isolated using the ethanol maceration" This sentence is not correct because the authors performed only extraction. Hence, it should be corrected as” the triterpenoid-rich extract showed antioxidant activities similar to those obtained using ethanol maceration.Response: Thanks. It has been revised as suggested.Comment 4: Rephrase line 76: “For extraction of triterpenoids of G. lucidum, maceration using organic extracting solvents such as ethanol is often used”.Response: Accept. It has been rephrased. See in the revision.Comment 5: Line 208-210: The quantification of triterpenes should be better explained. Did the authors use a standard (ursolic acid for example). If yes, then the results should be expressed in the equivalent of the standard.Response: Accept. We used D-glucose as the standard for polysaccharides measurement, and ursolic acid as the standard for the measurement of triterpenoids. The results were expressed in the equivalent of the standard. There information have been described in the manuscript.Comment 6: In its current state, the manuscript has some grammatical errors and instances of badly constructed sentences. Please check the manuscript and refine the language carefully.Response: Thanks. We have carefully checked the manuscript.Reviewer 3#: Some doubts, suggestions and recommendationComment 1: This study describes a methodology for simultaneously extracting polysaccharides and triterpenes. However, the goals of bioactivity differ greatly between polysaccharides and triterpenes. For example, there are many types of triterpenes, and each triterpene has a unique bioactivity. Isn’t each activity weakened by simultaneous extraction?Response: Thanks. We are very sorry that the separation of individual triterpenoids was not carried out, and the activity of each triterpene was not investigated in this study.Comment 2: In this study, the author describes the usefulness of simultaneous extraction, but I think it is not enough to confirm with DPPH alone. If you have a reason, please state why you expressed the bioactivity only with the DPPH result.Response: Thanks. For a systematic comparison, we have performed additional experiments on the reducing power of the extracts, and the results have been provided in the manuscript. See in the revision.Comment 3: In some cases, I suggest removing the DPPH results and changing to a paper with only extraction methods.Response: Thanks. We provided additional results on the antioxidant assay, and the results on the antioxidant assay were present in the manuscript. See in the revision.Comment 4: Page 3, Line 62: Please add a description of GA-MT and GA-TResponse: Accept. It has been added.Comment 5: Page 5 Materials and Methods: Particle size after sample grinding should be specified.Response: Accept. The particle size was specified in the revision.Comment 6: Page 6, L119-L147: The description of the extraction method is redundant. Please correct it to a concise description.Response: Accept. We have shortened it. See in the revision.Comment 7: Figures: I can’t consider the data because the figure doesn’t have a legend.Response: Accept. Legends have been provided. See in the revision.List of the changes in revisionTitle1. Line 1-3 of page 1, “Desirability function approach for optimization of ultrasound-assisted extraction of polysaccharides and triterpenoids from Ganoderma lucidum and evaluation of their in vitro antioxidant capacities” was revised as “Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacities”.Abstract1. Line 24-25 of page 2, “Ganoderma lucidum is a medicinal mushroom that is rich in biologically active substances” was revised as “Ganoderma lucidum (Fr.) Krast, commonly known as “Lingzhi” in Chinese, is a medicinal mushroom that is rich in biologically active substances”.2. Line 25 of page 2, “and widely used as a traditional Chinese herbal medicine in Asian countries” was deleted.3. Line 26-27 of page 2, “Polysaccharides and triterpenoids are the two major bioactive ingredients of this mushroom” was changed as “Polysaccharides and triterpenoids are the two major components responsible for the bioactivity of this fungus”.4. Line 28 of page 2, “of” was revised as “from”.5. Line 29 of page 2, “with the equal importance for the two components” was inserted.6. Line 29 of page 2, “single factor tests” was revised as “Following single factor experiments”.7. Line 30-31 of page 2, “by RSM with the associated D value as the response variable” was deleted.8. Line 33 of page 2, “With” was changed as “Under”.9. Line 35 of page 2, “and evaluation of reducing power” was inserted.10. Line 36 of page 2, “crude” was inserted.11. Line 37 of page 2, “crude” was inserted.12. Line 37-38 of page 2, “while the triterpenoids showed” was revised as “whereas the triterpenoid-rich extracts”.13. Line38-39 of page 2, “using the ethanol maceration” was changed as “using the ethanol maceration method”.14. Line 39 of page 2, “The present study is the first report on the simultaneous extraction of polysaccharides and triterpenoids from G. lucidum” was inserted.Keywords1. Line 42-43 of page 2, “ultrasonic extraction” was inserted.Introduction1. Line 45 of page 3, “Ganoderma lucidum is the most famous medicinal mushroom in the world” was revised as “Ganoderma lucidum (Fr.) Krast belonging to the family of Polyporaceae, commonly known as “Lingzhi” in Chinese and “Reishi” in Japanese, is one of the most famous medicinal mushrooms”.2. Line 47 of page 3, “in oriental countries” was revised as “in many oriental countries”.3. Line 48-51 of page 3, “Nowadays, this mushroom is widely used to prevent and treat many challenging human diseases, such as hepatitis, hyperglycemia, high blood pressure, and cancers, and as a nutraceutical to improve health, and promote spiritual growth for human in many countries” was deleted.4. Line 51-53 of page 3, “Owing to its medicinal properties and perceived health-promoting effects, there has been a growing worldwide interest in utilizing G. lucidum in the biomedical industry in recent years” was revised as “In recent years, there has been a growing worldwide interest in utilizing G. lucidum in the biomedical industry due to its pharmacological properties”.5. Line 53 of page 3, “many G. lucidum products” was revised as “a variety of G. lucidum products”.6. Line 54 of page 3, “in many parts of the world” was inserted.7. Line 55 of page 3, “G. lucidum contains” was revised as “G. lucidum in the form of fruiting bodies, mycelium, and spores contains”.8. Line 57-59 of page 3, “the two key bioactive components namely polysaccharides and triterpenoids are receiving special attention from researchers in the field of biomedicine” was revised as “the polysaccharides and triterpenoids are receiving special attention from researchers in the field of biomedicine”.9. Line 59 of page 3, “Several kinds of polysaccharides” was revised as “Several polysaccharides”.10. Line 60 of page 3, “are recognized” was revised as “generally recognized”.11. Line 60 of page 3, “such as antitumor, antioxidant and immunomodulatory effects” was inserted.12. Line 61-63 of page 3, “Regarding various chemically diverse triterpenoids, ganoderic acids such as GA-Me and GA-T have been proven to have potent pharmacological activity” was revised as “The triterpenoids from this fungus exhibit structural diversity, and many of them such as ganoderic acids Me and T (a kind of highly oxygenated lanostane-type triterpenoids) have been proven to have potent antitumor activity”.13. Line 63-65 of page 3, “To date, a wide variety of important bioactivities, including antitumor, antioxidant, antimicrobial, and immunomodulatory effects, have been shown for both the polysaccharides and triterpenoids” was deleted.14. Line 65-66 of page 3, “much attention” was revised as “much effort”.15. Line 69-71 of page 4, “Hot water extraction (HWE) is a conventional method, and widely used in extraction of polysaccharides from mushrooms due to the fact that no special equipments are required in the extraction” was revised as “Polysaccharides are generally prepared by traditional hot water extraction due to its easy operation and low instrument input”.16. Line 73 of page 4, “a variety of new techniques” was revised as “a variety of novel techniques”.17. Line 75-76 of page 4, “for a higher yield and efficacy” was deleted.18. Line 76-77 of page 4, “For extraction of triterpenoids of G. lucidum, maceration using organic extracting solvents such as ethanol is often used” was revised as “For the extraction of triterpenoids, maceration as a conventional method is adopted widely using organic solvents as an extraction solvent”.19. Line 77-78 of page 4, “and microwave irradiation was shown to be effective for the promotion of extraction of triterpenoids from G. lucidum” was revised as “A novel technique of microwave irradiation was shown to be effective for the extraction of triterpenoids from this fungus”.20. Line 80-82 of page 4, “To improve the extraction efficacy, the simultaneous extraction of multiple biologically active components from plant materials has become increasingly popular in recent years” was changed as “In recent years, the simultaneous extraction of multiple biologically active components from plants has become increasingly popular in order to improve extraction efficacy and reduce the number of extraction steps”.21. Line 82 of page 4, “there have been no available reports on the simultaneous extraction of multiple objective constituents from mushrooms to date” was inserted.22. Line 82 of page 4, “most studies” was revised as “most of previous studies”.23. Line 84-85 of page 4, “The application of these available processes may result in the loss of either polysaccharides or triterpenoids during the extraction” was revised as “Hence, the significant loss of either polysaccharides or triterpenoids occurred during the extraction, and this would lead to a waste of resources”.24. Line 86 of page 4, “a process for” was deleted.25. Line 88-89 of page 4, “ultrasound technology was examined for the simultaneous extraction of polysaccharides and triterpenoids” was revised as “ultrasound technology was examined for the co-extraction of polysaccharides and triterpenoids”.26. Line 89 of page 4, “and an optimization study is essential” was inserted.27. Line 90-91 of page 4, “Response surface methodology (RSM) is a collection of mathematical and statistical techniques widely used for optimizing complex processes” was revised as “Response surface methodology (RSM), a collection of mathematical and statistical techniques, is widely used for optimizing complex processes”.28. Line 93-94 of page 4-5, “RSM has been used extensively in optimizing the extraction processes of bioactive components” was changed as “RSM has been employed for optimizing the extraction of G. lucidum polysaccharides and ganoderic acids and triterpenoids from the medicinal fungus Sanghuangporus sanghuang”.29. Line 94 of page 5, “On the other hand” was deleted.30. Line 96 of page 5, “with different relative importances” was inserted.31. Line 101-103 of page 5, “In an attempt to better utilize G. lucidum resources, the simultaneous extraction of polysaccharides and triterpenoids was examined using ultrasound technology in this work” was revised as “In the present study, the ultrasonic-assisted co-extraction (UACE) of polysaccharides and triterpenoids was evaluated using ultrasound technology as an attempt to better utilize G. lucidum”.32. Line 103-104 of page 5, “The parameters of ultrasonic-assisted co-extraction (UACE) of polysaccharides and triterpenoids were optimized using RSM integrated with a desirability function” was changed as “The extraction parameters were optimized using RSM with a desirability function by setting the equal importance for the two components”.33. Line 104 of page 5, “Furthermore” was inserted.34. Line 107-108 of page 5, “using conventional approaches” was revised as “using conventional extraction approaches”.35. Line 110-112 of page 5, “The present study is the first report on the simultaneous extraction of polysaccharides and triterpenoids from the medicinal mushroom G. lucidum” was deleted.Materials and methods1. Line 115 of page 5, “The dried G. lucidum fruiting bodies were” was revised as “The dried G. lucidum was”.2. Line 117 of page 5, “ferric chloride, and trichloroacetic acid” was inserted.3. Line 119 of page 6, “Conventional hot water extraction of polysaccharides” was changed as “hot water extraction of polysaccharides”.4. Line 120 of page 6, “The G. lucidum sample was processed into a fine powder in a grinder” was revised as “The dried G. lucidum was grounded in a grinder to fine powder (40 mesh)”.5. Line 121 of page 6, “treated” was revised as “pretreated”.6. Line 123 of page 6, “in the matrix” was deleted.7. Line 123-125 of page 6, “The obtained powder (1 g) was placed into a 150 mL flask containing 40 mL deionized water, and extracted at 95�C in a water bath for 2 h” was revised as “The treated powder (1 g) was mixed with 40 mL of deionized water in a 150 mL flask, and extracted for 2 h at 95�C”.8. Line 125 of page 6, “The extraction procedure was repeated once” was inserted.9. Line 125 of page 6, “The whole extract” was revised as “The extract”.10. Line 126 of page 6, “The supernatant was collected, and the residue was extracted again” was deleted.11. Line 127-128 of page 6, “The supernatants were combined, and used for the estimation of polysaccharides or for further purification before the assay of antioxidant activity” was revised as “The combined supernatants were used for the estimation of crude polysaccharides or for further purification by ethanol precipitation when used in the assay of antioxidant activity”.12. Line 129 of page 6, “Conventional method for extraction of triterpenoids” was revised as “Ethanol maceration for extraction of triterpenoids”.13. Line 131-132 of page 6, “The mouth of the flask was sealed with aluminum foil to avoid solvent evaporation during the extraction” was revised as “The flask was sealed with aluminum foil during the extraction”.14. Line 132-134 of page 6, “The residue was collected after centrifugation at 8000 ×g for 10 min. The supernatant was harvested, and the residue was extracted again” was revised as “The extraction procedure was repeated once. The extraction solution was centrifuged at 8000 ×g for 10 min”.15. Line 136-137 of page 6, “Ultrasonic-assisted co-extraction of polysaccharides and triterpenoids” was revised as “UACE of polysaccharides and triterpenoids”.16. Line 138-139 of page 6, “An ultrasonic bath having a 3 L usable capacity was used” was revised as “The UACE was performed in an ultrasonic cleaning bath with a 3 L usable capacity”.17. Line 139-140 of page 6, “and ultrasonic power can be set between 35 and 310 W” was deleted. 18. Line 140-141 of page 6, “The G. lucidum fine powder (1 g) was extracted with an ethanol-water solution in a 150 mL flask” was revised as “Each of 1 g of the fine powder was extracted with an aqueous ethanol in a 150 mL flask, and the other conditions were described elsewhere”.19. Line 141-142 of page 6-7, “The mouth of the flask was sealed with aluminum foil during the extraction” was deleted.20. Line 143-144 of page 7, “and the resulting supernatant was used for measurements of polysaccharides and triterpenoids” was revised as “The polysaccharides and triterpenoids in the resulting supernatant were determined”.21. Line 144-145 of page 7, “The number of extractions varied with experiments” was deleted.22. Line 168 of page 8, “Single factor test” was revised as “Single-factor experiments”.23. Line 169-170 of page 8, “Prior to RSM optimization, the preliminary range of process variables for the extraction was determined using one-factor-at-a-time approach with the desirability function” was revised as “The single-factor experiment was carried out to determine the preliminary range of the extraction variables”.24. Line 170-174 of page 8, “The G. lucidum fine powder (1 g) was extracted in 150 mL flasks with varying temperatures (40 to 90�C), ethanol concentrations (20% to 80%), ultrasonic power (70 to 245 W), ratios of liquid to solid (10 to 60 mL/g), extraction times (30 to 180 min), and number of extractions (1 to 4)” was revised as “The G. lucidum fine powder (1 g) was extracted in 150 mL flasks with aqueous ethanol. The optimum extraction temperature was first determined by varying temperature from 40 to 90�C under other conditions: 40% ethanol, ultrasonic power 140 W, liquid/solid ratio 40 mL/g, extraction time 60 min, and number of extractions 1. Then, the optimum values of ethanol concentration (20%−80%), ultrasonic power (70−245 W), ratios of liquid to solid (10−60 mL/g), extraction time (30−180 min), and number of extractions (1−4) were determined sequently using determined optimal values in the extraction”.25. Line 174 of page 8, “Each experiment was conducted in triplicate” was inserted.26. Line 175 of page 8, “and the D values were calculated” was inserted.27. Line 176 of page 8, “Box-Behnken design (BBD)” was changed as “Box-Behnken design (BBD) of RSM”.28. Line 177 of page 8, “On the basis of the results of the single-factor experiments” was revised as “After the single factor experiments”.29. Line 184-185 of page 8, “To simplify the research, one extraction number was used in all experiments” was revised as “To simplify the study, only one extraction number was tested for all experiments in this design”.30. Line 185 of page 8, “Each trial” was revised as “Each experiment”.31. Line 187 of page 9, “To determine the relationship” was revised as “To correlate the relationship”.32. Line 201 of page 9, “Assay of polysaccharides and triterpenoids” was revised as “Quantitation of polysaccharides and triterpenoids”.33. Line 206 of page 9, “at 8000 ×g for 10 min” was deleted.34. Line 208-209 of page 10, “The vanillin-glacial acetic acid method was used for analysis of the triterpenoid amount in each sample” was revised as “The vanillin-glacial acetic acid method was used for analysis of the triterpenoid with ursolic acid as the standard”.35. Line 210 of page 10, “and expressed as the equivalent amount of ursolic acid” was inserted.36. Line 211 of page 10, “using D-glucose as the standard” was inserted.37. Line 224 of page 10, “and A is the absorbance for the solution” was revised as “and A is the absorbance for the DPPH solution”.38. Line 226 of page 10, “Reducing power assay” was inserted.39. Line 226 of page 10, “The reducing capacity of a natural component can employ as a significant indicator of its potential antioxidant activity” was inserted.40. Line 226 of page 10, “A higher absorbance of the reaction mixture at 700 nm shows a higher reducing power” was inserted.41. Line 226 of page 10, “The reducing power of the polysaccharides and triterpenoids was determined following the method described by Ahmadi et al [35], with minor modifications” was inserted.42. Line 226 of page 10, “Briefly, 2 mL sample at varying concentrations (0.1−0.5 mg/mL) was mixed with 2 mL potassium phosphate buffer , then incubated at 50�C for 20 min. The reaction was terminated by adding 2 mL trichloroacetic acid solution . Then, the reaction solution (2 mL) was mixed with 0.4 mL ferric chloride  and distilled water (2 mL), and the absorbance was measured at 700 nm against a blank after 10 min” was inserted.Results1. Line 237 of page 11, “by HWE” was revised as “conventional HWE (two numbers of extraction)”.2. Line 237-238 of page 11, “and 0.59% was obtained for the triterpenoids using the ethanol maceration” was revised as “For triterpenoids extraction using the conventional maceration method, an extraction yield of 0.59% was obtained using two extraction times”.3. Line 240 of page 11, “on the yields of polysaccharides and triterpenoids” was added.4. Line 241-243 of page 11, “Extraction temperature was tested between 40 and 90�C, while other variables were constant ” was deleted.5. Line 243 of page 11, “As shown in Fig 1A” was revised as “As presented in Fig 1A”.6. Line 244 of page 11, “within the tested range from 40 to 90�C” was inserted.7. Line 244-246 of page 11, “The highest polysaccharide yield (0.41%) was achieved at the highest extraction temperature (90�C)” was revised as “The highest polysaccharide yield was 0.41% at 90�C”.8. Line 246-248 of page 11, “The triterpenoid yield increased from 0.11% to 0.15% as extraction temperature changed from 40 to 80�C. When extraction temperature increased beyond 80�C, the triterpenoid yield decreased” was revised as “The triterpenoid yield increased from 0.11% to 0.15% as extraction temperature changed from 40 to 80�C, and then decreased above 80�C.”.9. Line 248 of page 11, “As to the combined performance” was inserted.10. Line 250 of page 11, “Above this, it started to decline” was revised as “Above this, it started to decline”.11. Line 252 of page 12, “on the yields of polysaccharides and triterpenoids” was added.12. Line 253-255 of page 12, “Ethanol concentrations ranging between 20%−80% were tested. Other conditions were as follows: temperature, 80�C; ultrasonic power, 140 W; ratio of liquid/solid, 40 mL/g; extraction time, 60 min; and number of extractions, 1” was deleted.13. Line 255-257 of page 12, “Polysaccharide yield decreased in a rapid manner with increasing ethanol concentration , with the maximum yield of 0.46% observed at the lowest studied concentration (20%)” was revised as “Fig 1B shows that the polysaccharide yield decreased in a rapid manner with increasing ethanol concentration in the scope of test, with the maximum yield of 0.46% observed at the lowest concentration of 20%”.14. Line 257-258 of page 12, “the highest triterpenoid yield (0.34%)” was revised as “the highest triterpenoid yield of 0.34%”.15. Line 260-261 of page 12, “The optimal level of ethanol was therefore around 50%” was revised as “The optimal ethanol concentration was therefore around 50%”.16. Line 263 of page 12, “50% ethanol was used and not included in a further optimization” was revised as “the ethanol concentration of 50% was selected, and not included in BBD for further optimization”.17. Line 264 of page 12, “on the yields of polysaccharides and triterpenoids” was inserted.18. Line 265-268 of page 12, “The effects of ultrasonic power varying between 70 and 245 W were investigated, while other operational parameters were: extraction temperature, 80�C; ethanol concentration, 50%; ratio of liquid to solid, 40 mL/g; extraction time, 60 min; and number of extractions, 1” was deleted.19. Line 268 of page 12, “As observed in Fig 1C” was revised as “As observed in Fig 1C”.20. Line 271 of page 12, “applied” was inserted.21. Line 273-274 of page 12, “An ultrasonic power of 175 W was therefore appropriate in the subsequent optimization” was changed as “An ultrasonic power of 175 W was therefore desirable in the extraction”.22. Line 275 of page 12, “on the yields of polysaccharides and triterpenoids” was inserted.23. Line 276-278 of page 13, “Extraction was conducted at different liquid/solid ratios from 10 to 60 mg/L under the following conditions: extraction temperature, 80�C; ethanol concentration, 50%; ultrasonic power, 175 W; extraction time, 60 min; and number of extractions, 1” was deleted.24. Line 278-279 of page 13, “As illustrated in Fig 1D” was revised as “As presented in Fig 1D”.25. Line 280 of page 13, “increased from” was revised as “rose from”.26. Line286 of page 13, “As to the combinAttachmentResponses to Reviewers.docSubmitted filename: Click here for additional data file. 1 Dec 2020PONE-D-20-24854R1Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacitiesPLOS ONEDear Dr. Liu,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.==============================Authors have addressed all the major concerns raised in the reviewers' reports. However, one sentence in the conclusion section fails to present the verity of the study: "The antioxidant activity of the polysaccharides was enhanced by ultrasound."(L480), which does not hold the true since it means that you have somehow extracted polysaccharides, measured their antiox activity, let out the ultrasound through them and, measured their antiox activity once again and, finally, compared the two values. Please elaborate this.Additionally, more attention should be paid to the Conclusion section .==============================plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Jan 15 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsIf applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Branislav T. Šiler, Ph.D.Academic EditorPLOS ONE[Note: HTML markup is below. Please do not edit.]https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  13 Dec 2020Responses to the reviewer’s commentsManuscript number: PONE-D-20-24854Title: Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacitiesDear Editors,We appreciate your comments and suggestions on our manuscript submitted previously. The manuscript has been carefully revised according to the suggestions and recommendation by the reviewers, and now all problems have been addressed in revised manuscript. Revised portion are marked in red in the paper. Point-by-point responses to these comments are showed below in this letter.Sincerely yours,Shi-Zhong Zheng, Wei-Rui Zhang, Sheng-Rong LiuReviewer 1#: Some doubts, suggestions and recommendationComment 1: Authors have addressed all the major concerns raised in the reviewers’ reports. However, one sentence in the conclusion section fails to present the verity of the study: “The antioxidant activity of the polysaccharides was enhanced by ultrasound”(L480), which does not hold the true since it means that you have somehow extracted polysaccharides, measured their antioxidant activity, let out the ultrasound through them and, measured their antioxidant activity once again and, finally, compared the two values. Please elaborate this.Response: Accept. We have carefully revised this sentence. See in the revision.Comment 2: Additionally, more attention should be paid to the Conclusion section.Response: Accept. We have carefully revised this section, and we believe that the quality was greatly improved. See in the revision.Comment 3: If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.Response: Thanks. We have updated statement on the financial disclosure in our cover letter.List of the changes in revisionAbstract1. Line 34 of page 2, “crude” was deleted.2. Line 35 of page 2, “crude” was deleted.Introduction1. Line 46 of page 3, “The most famous” was revised as “the most highly prized”.Results1. Line 322 of page 15, “the model was statistically very significant” was revised as “the model was statistically significant”.2. Line 396 of page 19, “triterpenoids” was revised as “triterpenoid-rich extracts”.Discussion1. Line 435 of page 20, “in a convenient extraction process” was revised as “in a simple extraction process”.2. Line 448 of page 21, “have been reported” was revised as “have been reported widely”.3. Line 458 of page 21, “was shown” was revised as “was also shown”.4. Line 461 of page 21, “were” was revised as “may be”.Conclusion1. Line 476 of page 22, “the simultaneous extraction” was revised as “the simultaneous and efficient extraction”.2. Line 477 of page 22, “from G. lucidum” was revised as “from the medicinal fungus G. lucidum”.3. Line 477 of page 22, “with ultrasonic technology” was revised as “with ultrasonic extraction technology”.4. Line 480 of page 22, “On the basis of the scavenging capacity of DPPH and evaluation of reducing power” was inserted.5. Line 480 of page 22, “The antioxidant activity of the polysaccharides was enhanced by ultrasound” was revised as “the antioxidant activity of the extracted polysaccharides by ultrasonic action in optimal UACE process was significantly enhanced”.6. Line 480 of page 22, “while the triterpenoid-rich extracts demonstrated unchanged activity” was inserted.AttachmentResponses to Reviewers.docSubmitted filename: Click here for additional data file. 16 Dec 2020Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacitiesPONE-D-20-24854R2Dear Dr. Liu,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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Šiler, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 18 Dec 2020PONE-D-20-24854R2 Ganoderma lucidum and evaluation of their in vitro antioxidant capacities Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Dear Dr. Liu:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Branislav T. Šiler Academic EditorPLOS ONE


On PDF page 1, in the abstract, in the sentence “Four high-risk international CNPA clones  dominated, but the distribution of these clones changed significantly after the intervention was implemented,” “ST375” within the parentheses should be “ST357.” This change does not change the conclusions of the paper.Volume 10, no. 6, e02384-19, 2019,


Results: At 11 and 13 years, 6.8% and 8.2% of children respectively consumed SSB daily. Neither SSB frequency at 11 nor at 13 years was associated with subsequent BMI z-score or overweight/obesity up to 18 years, or with waist circumference, waist-to-hip ratio, or body fat percentage at 16–19 years adjusted for age, sex, socioeconomic position, health status, physical activity and other food consumption, although bias to the null from under-reporting cannot be eliminated. Conclusion: Although we cannot definitively exclude a small association of SSB frequency with adiposity, lack of association of SSB frequency with adiposity in a non-Western setting with low SSB consumption suggests that the role of SSB in adiposity appears to be minor.Background: Observationally, sugar-sweetened beverage (SSB) consumption is associated with adiposity in Western children but could be confounded. We examined the association of SSB frequency with adiposity in the non-Western setting of Hong Kong. Methods: We examined the associations of SSB consumption frequency at 11 and 13 years assessed by using a food frequency questionnaire with subsequent body mass index (BMI) z-score and overweight/obesity up to 18 years using generalized estimating equations, and with waist circumference, waist-to-hip ratio, and body fat percentage at 16–19 years using linear regression in a population-representative Chinese birth cohort “Children of 1997” ( Mean body mass index (BMI) and the prevalence of obesity in children and adolescents increased dramatically from 1975 to 2016 ,4. Meta-Given the inconclusive evidence concerning the effect of SSB on adiposity, assessing the association in settings with different confounding patterns can be helpful . Unlike n = 8327) that included 88.0% of all births in Hong Kong from April 1, 1997 to May 31, 1997, as described in detail elsewhere [The “Children of 1997” birth cohort is a population-representative Chinese birth cohort , “not at all in the last week” , “1–3 times a week” , “4–6 times a week” , “once a day” , “2–3 times a day” , and “4 or more times a day” . Reponses were combined into the following categories: “less than weekly”, “1–3 times per week”, “4–6 times per week” and “daily” because of the relatively small numbers in some categories. The food frequency questionnaire in Survey I showed some validity regarding the consumption of milk and non-milk dairy products [SSB consumption at 11 years was assessed by asking parents in Survey I “On average, how often did your child drink canned pop/soda/soft drinks in the last week?” with responses “never ever drinks”   to compaTo handle missing data, we combined multiple imputation (MI) and inverse probability weighting (IPW). We used multiple imputation for missing confounders  based on a flexible additive regression model with predictive mean matching incorporating data on sex, birth order, maternal age at birth, mother’s birthplace, father’s birthplace, highest parental education, highest parental occupation, household income per head, main caregiver, general health status, physical activity, fruit, vegetable, and meat consumption and BMI z-score at 11 years, SSB consumption, and outcomes . We imputed missing data 20 times using the “Hmisc” R package. We calculated the probability of observations being present using logistic regression models to identify potential predictors of missingness, from which inverse probability weights of response were obtained for each observation. We analyzed the 20 imputed datasets using these weights and summarized the results into single estimated beta-coefficients or odds ratios (OR) with 95% confidence intervals (CI) adjusted for missing data uncertainty . We repeAll statistical analyses were performed using R version 3.6.1 .2 and BMI z-score was 0.21 ± 1.27. Of the 8327 individuals originally recruited to the “Children of 1997” birth cohort, as of December 2018, 28 had permanently withdrawn. Of the remaining 8299, 7934 were potentially contactable for Survey I in 2008–2009, whilst 75 migrated without trace, 12 deceased, and 278 were untraceable (probably migrated or dead). Of these 7934, 3678 responded to Survey I, of whom 98.6% provided information on SSB consumption at about 11 years (mean age at assessment 11.5 ± 0.3 years) . The proIn this large, population-representative birth cohort of Hong Kong Chinese children, SSB frequency at 11 or 13 years was not associated with subsequent BMI z-score or overweight (including obesity) up to 18 years, nor with WC, WHR or BFP at 16–19 years.To our knowledge, this is the first study to examine the association of SSB consumption with adiposity using a prospective cohort of Chinese children. Cross-sectional studies in Chinese children ,28 have SSB is the largest source of added sugars in US children , althoug2 or greater) for 1–3 times or more per week as compared to less than weekly, thus we cannot exclude a small effect of SSB on adiposity [Despite being conducted in a setting where diet has been reported quite accurately in terms of total calories , and condiposity ,30, or aSSB consumption is seen as an important target of intervention to combat obesity in Western countries. However, added sugars and SSB intake have declined while obesity has risen steeply over the same time period in Australia, which to some extent challenges the assumption that reduced consumption of SSB can, in itself, help reverse the upward trend of obesity . SSB conIn Hong Kong Chinese children with low SSB consumption and less clear socioeconomic patterning of SSB consumption and obesity, SSB consumption frequency was unrelated to adiposity. Although we cannot rule out the possibility of small effects of SSB, especially for overweight and obese children, our study highlights the role of evidence from different social contexts in examining empirically driven hypotheses from long-term economically developed populations.


Synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome is a rare disease without standard treatments. Tripterygium wilfordii hook f (TwHF) is a traditional Chinese herb with anti-inflammatory effect, and 1.0 mg/(kg·d) dose of Tripterygium glycosides has been reported to significantly improve the disease activity of a SAPHO patient in a case report. However, the optimal dose of TwHF is still unclear. Here, we report the first case of SAPHO patient who achieved rapid remission in clinical symptoms after receiving 1.5 mg/(kg·d) dose of Tripterygium glycosides treatment.A 67-year-old woman noted palmoplantar pustulosis and pain in the anterior chest wall and waist. Bone scintigraphy demonstrated the typical tracer accumulation feature and magnetic resonance images showed bone marrow edema in lumbosacral vertebra.The diagnosis was made by dermatological and osteoarticular manifestations and classical signs in bone scintigraphy in accordance with the diagnostic criteria proposed in 2012.Tripterygium glycosides was given with a primary dose of 1.5 mg/(kg·d) for 1 month and then reduced at a rate of 10 mg every 2 weeks until 1.0 mg/(kg·d) for a long-term maintenance.Fast-induced remission on clinical manifestations was achieved and magnetic resonance imaging abnormality was improved significantly. Additionally, no apparent side effects were observed.1.5 mg/(kg·d) dose of Tripterygium glycosides seems to have fast-induced remission than 1.0 mg/(kg·d) with reliable safety. Besides, Tripterygium glycosides may also have a pharmacological effect of inhibiting osteolysis and enhancing bone strength. The increase of proinflammatory cytokines such as IL-6 and IL-8, the imbalance of the ratio between Th17 and regulatory T cells, and the decrease of TGF-β1 contribute to the pathogenesis of SAPHO syndrome.–5 Treatments for SAPHO syndrome include nonsteroidal anti-inflammatory drugs (NSAIDs), antibiotics, corticosteroids, bisphosphonates, immunosuppressants, conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), biological agents like TNF-α inhibitors. However, there is no standard treatment for the disease.Synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome is a rare disease, characterized by osteoarticular manifestations and skin lesions.,7 Tripterygium glycosides tablet is the most commonly used TwHF drug in the clinic. The regular therapeutic dose of Tripterygium glycosides is 20 mg 3 times per day, which was selected based on the dose of 1.0 mg/(kg·d). Our team has reported a case of remarkable remission of SAPHO syndrome in response to 1.0 mg/(kg·d) dose of TwHF. However, the recommended dose of Tripterygium glycosides is 1.0 to 1.5 mg/(kg·d); therefore, whether a higher therapeutic dose is still safe and can lead to more significant and comprehensive improvement of SAPHO is currently unclear. Here, we report the first case of SAPHO patients who achieved significant remission without apparent side effects in a short period of time after receiving 1.5 mg/(kg·d) dose of Tripterygium glycosides treatment.Tripterygium wilfordii hook f (TwHF) is a traditional Chinese herb with anti-inflammatory effect, which has been used in the treatment of rheumatoid arthritis, ankylosing spondylitis, and other immunologic diseases.2Written informed consent was obtained from the patient for publication of this case report, and the study was approved by the Ethics Committee of Peking Union Medical College Hospital and the Chinese Academy of Medical Sciences.A 67-year-old woman noted palmoplantar pustulosis (PPP) in November 2017. Thereafter, the skin lesions suffered repeated remission and occurrence, but aggravated gradually. Four months later, the patient presented with pain in the anterior chest wall and waist. Although the bone pain could be relieved after receiving NSAIDs, the symptoms relapsed with pain and movement restriction in the lumbar region. The patient was admitted to our hospital in November 2018, and there was no family history of similar symptoms reported.99mTc-MDP showed increased radionuclide uptake in the sternum, left first anterior rib, and lumbar vertebrae, demonstrating a characteristic “bull's head” sign of SAPHO syndrome  6.87 mg/L and erythrocyte sedimentation rate (ESR) 36 mm/h. Blood test, liver, and kidney functions were within the normal range. Her rheumatoid factor, antinuclear antibody, and human leukocyte antigen-B27 were all negative. Whole body bone scintigraphy via ome Fig. A. Magnetome Fig. B. Based The patient weighed nearly 60 kg and was treated with oral Tripterygium glycoside tablets 30 mg 3 times a day based on a dose of 1.5 mg/(kg·d). After 1 month, the dose was reduced by 10 mg every 2 weeks and finally reduced to 1.0 mg/(kg·d) for long-term maintenance.After the treatment, the bone pain and disease activity of the patient were significantly relieved, showed by the decrease of Visual Analog Scale, Bath Ankylosing Spondylitis Activity Index, Bath Ankylosing Spondylitis Functional Index, and Ankylosing Spondylitis Disease Activity Score Fig. B. Her PPDuring the whole follow-up, the patient strictly complied with the drug treatment regimen. There were no abnormalities in routine blood tests and laboratory examinations of hepatic and renal function, and no significant adverse events such as gastrointestinal disorders, fatigue, headache, or dizziness were observed. As the patient is a menopausal woman, reproductive toxicity was not concerned.3This is the first case report on 1.5 mg/(kg·d) dose of Tripterygium glycosides in the treatment of SAPHO syndrome. The changes in clinical symptoms, disease activity score, and serological inflammatory factors showed that the disease activity significantly decreased in a short period of time after treatment and could be maintained at a low level during the follow-up. Additionally, no apparent side effects such as gastrointestinal disorders, fatigue, headache, or dizziness were observed.Compared to the previously reported case of 1.0 mg/(kg·d) TwHF treatment, the present case could add 3 new findings. First, PPP as a major skin manifestation of SAPHO patients could also be significantly improved by Tripterygium glycosides, which is meaningful for the life quality and overall prognosis of SAPHO patients. Second, 1.5 mg/(kg·d) dose of Tripterygium glycosides seems to have faster-induced remission than 1.0 mg/(kg·d). Third, in addition to the mechanism of anti-inflammation, Tripterygium glycosides may also exert beneficial effects through the regulation of bone metabolism. PPP is a chronic recurrent skin disease with eruption of sterile pustules on palms and soles. Patients with PPP usually present with pain or itching, and there is a significant correlation between disease severity and quality of life. The improvement of skin lesions is meaningful for SAPHO patients to get a better quality of life and overall prognosis.94.6% of SAPHO patients suffer from cutaneous lesions, mainly manifested as PPP, followed by severe acne.,14 and according to our own clinical experience and observation, 1.5 mg/(kg·d) dose of Tripterygium glycosides might have faster-induced remission than 1.0 mg/(kg·d). The previously reported case receiving 1.0 mg/(kg·d) treatment achieved maximum remission after 3 months, and maintained at a low-level inflammatory state with the concomitant use of MTX. In the present case, the patient achieved comparative maximum remission only after 1 month, and such improvement could be maintained during the follow-up without apparent adverse effects. However, given the heterogeneity of SAPHO patients, the difference in efficacy and toxicity between these 2 doses of Tripterygium glycosides needs to be confirmed by further prospective research. Considering that high-dose exposure may also lead to greater hepatotoxicity, nephrotoxicity, and reproductive toxicity, 1.5 mg/(kg·d) dose of Tripterygium glycosides should be prescribed to patients with normal hepatic and renal function and no desire to have children. To avoid irreversible damages, the hepatic and renal function should be tested regularly during the treatment period, and for premenopausal female patients, their menstrual condition and, if necessary, hormone levels should also be monitored.Previous studies have found that the anti-inflammatory efficacy of TwHF is dose-dependent, In this case, 1.5 mg/(kg·d) dose of Tripterygium glycosides not only play an anti-inflammatory role in improving osteoarticular pain and skin lesions, but also showed benefits in bone metabolism. Osteocalcin and β-CTX are common biochemical markers for bone metabolism. Osteocalcin is a vitamin K-dependent calcium-binding protein, which is mainly synthesized by osteoblasts and reflects the activity of osteoblasts and bone turnover. β-CTX is a decomposition product of type I collagen, which is used to measure the activity of osteoclasts and bone resorption. A retrospective analysis showed that the main mechanism of bone destruction in SAPHO syndrome was the enhancement of bone resorption, and the level of β-CTX could reflect the disease activity of SAPHO syndrome. After the 1.5 mg/(kg·d) Tripterygium glycosides treatment, the increase of serum osteocalcin accompanied by the decrease of serum β-CTX, suggested that Tripterygium glycosides may also have a pharmacological effect of inhibiting osteolysis and enhancing bone strength, which is consistent with the study of Huang et al.The inflammatory response induced by infection or others is closely related to bone metabolism. The inflammation characterized by the T cells activation and cytokines release can lead to the imbalance of ratio between Receptor activator nuclear factor κ B ligand and osteoprotegerin, and such abnormal bone metabolism will eventually result in bone destruction.Compared with other treatments, Tripterygium glycosides has the advantage of lower cost and more comprehensive efficacy; therefore, it should be widely recommended for the treatment of SAPHO syndrome. In addition, the combination of Tripterygium glycosides with other csDMARDs or biological agents might achieve a better therapeutic effect.4This case suggests that through the mechanism of anti-inflammation and bone metabolism regulation, 1.5 mg/(kg·d) dose of Tripterygium glycosides had reliable safety and could lead to significant and rapid remission on both osteoarticular and cutaneous lesions. Therefore, it is necessary to design further study to evaluate the therapeutic effect and safety of different doses of TwHF.Conceptualization: Liang Gong, Lun Wang, Yihan Cao, Chen Li.Data curation: Liang Gong, Lun Wang, Yihan Cao, Chen Li.Funding acquisition: Chen Li.Methodology: Liang Gong, Lun Wang, Yihan Cao, Chen Li.Project administration: Liang Gong, Lun Wang, Yihan Cao, Chen Li.Supervision: Chen Li.Visualization: Liang Gong, Lun Wang, Yihan Cao.Writing – original draft: Liang Gong, Lun Wang, Yihan Cao, Chen Li.Writing – review & editing: Liang Gong, Lun Wang, Yihan Cao, Chen Li.


Malus rootstock, scion, and their interaction in Cd accumulation and tolerance, four scion/rootstock combinations consisting of the apple cultivars “Hanfu” (HF) and “Fuji” (FJ) grafted onto M. baccata (Mb) or M. micromalus “qingzhoulinqin” (Mm) rootstocks differing in relative Cd tolerance were exposed either to 0 µM or 50 µM CdCl2 for 18 d. Cd accumulation and tolerance in grafted Malus plants varied within rootstock, scion, and rootstock–scion interaction. Cd-induced decreases in photosynthesis, photosynthetic pigment level, and biomass were lower for HF grafted onto Mb than those for HF grafted onto Mm. Reductions in growth and photosynthetic rate were always the lowest for HF/Mb. Cd concentration, bioconcentration factor (BCF), and translocation factor (fT) were always comparatively higher in HF and FJ grafted onto rootstock Mm than in HF and FJ grafted on Mb, respectively. When HF and FJ were grafted onto the same rootstock, the root Cd concentrations were always higher in HF than FJ, whereas the shoot Cd concentrations displayed the opposite trend. The shoot Cd concentrations and fT were lower for HF/Mb than the other scion/rootstock combinations. Rootstock, scion, and rootstock–scion interaction also affected subcellular Cd distribution. Immobilization of Cd in the root cell walls may be a primary Cd mobility and toxicity reduction strategy in Malus. The rootstock and scion also had statistically significant influences on ROS level and antioxidant activity. Cd induced more severe oxidative stress in HF and FJ grafted onto Mm than it did in HF and FJ grafted onto Mb. Compared with FJ, HF had lower foliar O2-, root H2O2, and root and leaf MDA levels, but higher ROS-scavenging capacity. The rootstock, scion, and rootstock–scion interaction affected the mRNA transcript levels of several genes involved in Cd uptake, transport, and detoxification including HA7, FRO2-like, NRAMP1, NRAMP3, HMA4, MT2, NAS1, and ABCC1. Hence, the responses of grafted Malus plants to Cd toxicity vary with rootstock, scion, and rootstock–scion interaction.To understand the roles of  Cd is an health . From agSolanum torvum rootstocks decreased foliar Cd accumulation (Cucumis sativus L.) scions onto “Power” (C. maxima × C. moschata) rootstocks reduced Cd absorption and transport  reductase proteins and natural resistance-associated macrophage protein 1 (NRAMP1) are located in the PM and control Cd penetration into root cell cytosols  and M. domestica “Fuji” (FJ) scions grafted onto either M. baccata (Mb) or M. micromalus “qingzhoulinqin” (Mm) rootstocks and exposed them either to 0 µM or 50 µM CdCl2 for 18 d. We hypothesized that (i) the rootstock, the scion, and their interaction influence Cd accumulation and translocation, and (ii) the rootstock–scion interaction regulates physiological acclimation and the key genes involved in Cd uptake and transport, thereby alleviating Cd toxicity. To test these hypotheses, we evaluated and compared growth characteristics, Cd accumulation and translocation, subcellular Cd distribution, oxidants, antioxidants, and the key genes regulating Cd uptake, translocation, and detoxification.In a previous study, we compared Cd accumulation and tolerance among various apple rootstocks. fication . To elucM. baccata (Mb) or M. micromalus “qingzhoulinqin” rootstocks and used in this study. Hereafter, “Hanfu” and “Fuji” grafted onto M. baccata are referred to as HF/Mb and FJ/Mb while “Hanfu” and “Fuji” grafted onto M. micromalus “qingzhoulinqin” are referred to as HF/Mm and FJ/Mm, respectively. Seeds of the M. baccata and M. micromalus “qingzhoulinqin” rootstocks were stratified in sand at 0–4°C for 60 d. The seedlings were transferred in early April 2016 to nursery plates containing substrate and cultivated for 40 d in a greenhouse under natural light and temperature conditions . Seedlings with similar growth from two rootstock species were selected and transplanted in plastic pots (20 cm × 20 cm ×18 cm) containing sand. One seedling was planted per pot. Each plant was slowly chemigated every 2 d with 50 mL Hoagland solution. In April 2017, virus-free “Hanfu” and “Fuji” budwood was grafted onto Mb and Mm rootstocks that were uniform in diameter (4–5 mm). All plants were cultivated in a greenhouse under the aforementioned environmental conditions. Twelve weeks after budding, healthy and uniform grafted plants (height 35 cm) were selected and transplanted to aerated Hoagland nutrient solution (pH 6.0) that was renewed every 3 d. After 2 weeks, 36 plants similar in height and growth performance were selected from each graft combination, divided into two groups of 18 plants, and cultivated for 18 d in Hoagland solution containing either 0 µM or 50 µM CdCl2. Six plants per group were randomly selected for histochemical root and stem analyses. The remaining 12 plants per group were harvested after gas exchange measurements.The apple cultivars “Hanfu” (HF) and “Fuji” (FJ) were grafted onto 2 assimilation rate (A), stomatal conductance (sg) and transpiration rate (E) were measured on sunny days between 9:00 and 11:00 h with the CIRAS-2 photosynthesis system  as described previously  of each plant from four apple scion/rootstock combinations. The COeviously .via separation of root, rootstock stem, scion stem and leaf tissues. The roots were carefully rinsed in 20 mM EDTA disodium solution for 5 min to remove Cd2+ from the root surface, and then washed with deionized water. All of the harvested samples were wrapped with tinfoil after the fresh weight of samples were recorded and frozen immediately in liquid nitrogen. The frozen samples were milled to ﬁne powder in liquid nitrogen with a ball mill  and stored at −80°C. To perform biochemical and gene expression analysis, equal amounts of powder from different tissues of four plants that received the same treatment were pooled to form a biological sample . The fresh powder (ca. 50 mg) obtained from each tissue per plant was dried at 60°C for 72 h to determine the fresh-to-dry mass ratio, which was used to calculate the dry mass of each tissue.After gas exchange measurement, each plant was harvested 2O. Subsequently, the samples were exposed to a staining solution  for 90 s. After a brief rinse in deionized H2O, the well-stained samples with Cd-dithizone precipitates that displayed as red coloration were immediately photographed under an Eclipse E200 light microscope  using a CCD camera  connected to a computer.Cd distribution was investigated in the root and scion stem tissues of apple trees using the histochemical staining method as described by To measure contents of foliar pigments, fine powder of fresh leaves (ca. 50 mg) was extracted in 5 ml of 80% acetone for 24 h in darkness until the color disappeared completely. The contents of chlorophyll in the extracts were determined using a spectrophotometer  as suggested by 3 and 1 ml concentrated HClO4 at 170°C as described previously (−1) in roots, rootstock stems, stem scions and leaves to that in the solution (μg g−1) (fT) was defined as Cd concentration (μg g−1) in the aerial tissues of a plant divided by that in the roots and multiplied by 100% (The fine powder (ca. 100 mg) samples obtained from the root, rootstock stem, scion stem and leaf tissues were digested in a mixture containing 7 ml concentrated HNOeviously . The Cd (μg g−1) . The tra by 100% .3 and 1 ml concentrated HClO4) at 170°C. Cd concentration in different fractions was determined by graphite furnace atomic absorption spectrophotometry .Root and leaf samples were separated into four subcellular fractions  using the gradient centrifugation technique according to 2.− and H2O2 in the roots and leaves of plants were measured using a spectrophotometer at 530 and 410 nm, respectively, as suggested by The concentrations of OThe concentrations of MDA in the roots and leaves were determined spectrophotometrically at 450, 532 and 600 nm, according to the previous study .The concentration of free proline and soluble phenolics was determined spectrophotometrically as reported previously . AscorbaThe soluble proteins in the root and leaf tissues were extracted and quantified according to −ΔΔCt method (M. baccata (Mb) rootstock not exposed to Cd. The corresponding fold changes in the transcripts were calculated for other scion/rootstock combinations subjected to 0 µM or 50 µM CdCl2. A gene expression heatmap was generated for the log base 2 average expression fold values using the heatmap.2  command in the “gplots” package in R (http://www.r-project.org/).Total RNA extraction and purification and RT-PCR were performed according to the methods of t method . The exp2 (Cd), rootstock (R), and scion (S) as the three factors to test all parameters for significant changes. A posteriori means comparisons were performed by Tukey’s method when statistically significant interactions were detected. All the results were presented as means ± standard error (SE) of three biological replicates. Differences between means were considered significant at P < 0.05 according to the three-way ANOVA F-test.All data were processed in Statgraphics . All data were tested for normality before the statistical analyses. Three-way ANOVA was applied using CdCl2 assimilation rates and sg were markedly higher in HF than FJ, regardless of the rootstock  followed by the soluble fraction (F IV), the organelle-rich fraction (F II), and the membrane-containing fraction (F III) of the roots and leaves of the various apple graft combinations Table 2.2•−, H2O2 and MDA concentrations were significantly increased in all four combinations exposed to Cd, relative to their untreated controls,  reductase proteins and NRAMP1 also regulate Cd uptake  concentration, and shoot biomass than HF and FJ grafted onto Mm  but the lowest Cd distribution ratios in their organelle-rich fraction (F II). As a result, HF/Mb had the lowest Cd accumulation and translocation capacity and, by extension, the highest Cd tolerance and the strongest ability to prevent Cd from interfering with the organelles.In plants, subcellular Cd distribution is vital to Cd accumulation, migration, and detoxification . Cell wathe Fuji . Here, t2•−, H2O2, and MDA in the roots and leaves of all apple scion/rootstock combinations exposed to Cd indicated the presence of Cd phytotoxicity. However, the changes in ROS and MDA concentration in response to the Cd treatment differed among the four scion/rootstock combinations. There was less pronounced induction of O2•− and MDA in the roots and leaves and H2O2 in the roots of the apple cultivar scions grafted onto Mb than there was in those grafted onto Mm. Therefore, Mb more effectively alleviated Cd toxicity than Mm. The tomato cultivar “Ikram” grafted onto “Maxifort” had lower ROS and MDA levels than self-grafted “Ikram”  than it did in the roots of grafted cucumber Jinyu No. 1  when Figleaf Gourd was the rootstock. Overall, both rootstock and scion control the tissue ROS and MDA concentrations in plants subjected to Cd stress. Thus, the rootstock–scion interaction might regulate the antioxidant system.Cd is non-essential and toxic to most organisms . The obs “Ikram” . The sci2•− and MDA concentrations observed in the same scion/rootstock combination. These results suggest that all apple scion genotypes have relatively higher capacities to scavenge Cd-induced ROS when they are grafted onto Cd-tolerant rootstocks such as Mb rather than Cd-sensitive ones such as Mm. Similar findings were reported for Citrus  reductase, and NRAMPs regulate the uptake of divalent cations such as Cd2+ in plants (HA7 and FRO2-like encode PM-bound H+-ATPase and Fe(III) reductase, respectively. HA7, FRO2-like, and NRAMP1 downregulation in M. baccata roots lowered net Cd2+ influx in this species more than it did in other apple rootstocks , the Liaoning Revitalization Talents Program (Grant No. XLYC1907105), the Scientific Research Foundation of Liaoning (Grant No. LSNQN201916), the National Key Research and Development Program of China (Grant No. 2016YFD0201115), the Program for Liaoning Excellent Talents in University (Grant No. LJQ2015098), the China Agriculture Research System (Grant No. CARS-27), and the Scientiﬁc Research Foundation of Talent Introduction of Shenyang Agricultural University (Grant No. 20153007).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.