To investigate the factors associated with endothelial survival after Descemet’s membrane endothelial keratoplasty (DMEK) in eyes of Asian patients with bullous keratopathy (BK).6 volume in the anterior chamber (AC) on postoperative day 1, and ECD loss rates at 12 months postoperatively.In this retrospective, consecutive interventional case series, 72 eyes of 72 patients who underwent DMEK were evaluated. Best corrected visual acuity (BCVA) and corneal endothelial cell density (ECD) were assessed at 12 months postoperatively. Multiple regression analysis was performed to assess parameters such as age, sex, axial length, preoperative visual acuity, re-bubbling, the ratio of graft to cornea area, iris damage scores, types of filling gases, air or SFP < .001). The rate of ECD loss at 12 months after DMEK was 54.4 ± 16.1%. Multiple linear regression analysis showed that a larger ratio of graft to corneal area (P = 0.0061) and higher donor ECD (P = 0.042) were the primary factors for a lower ECD loss rate at 12 months after DMEK.BCVA improved significantly at 12 months after DMEK (A relatively larger graft size compared to the host cornea and more donor ECD might help endothelial survival in patients with BK. Moreover, for such patients, the surgeon should attempt to use a relatively larger graft size when performing DMEK, particularly in Asian eyes. Corneal endothelial keratoplasty (EK), i.e., replacing only the posterior layer of the cornea, has been established as the standard treatment for corneal endothelial dysfunction. Descemet stripping automated endothelial keratoplasty (DSAEK) and Descemet membrane endothelial keratoplasty (DMEK) are the two main methods for EK and are superior to full-thickness transplantation  in terms of faster visual recovery and lower graft rejection rates . DMEK reDMEK should be an ideal treatment for endothelial dysfunction; however, a potential drawback is the relatively large decrease in endothelial cell density (ECD) postoperatively, which is similar to PKP –3. This Although endothelial survival after DMEK has been reported to be worse in patients with BK than in those with FECD ,14, thisThis retrospective, multi-center study was approved by the institutional review board of Jichi Medical University (JICHI 19–034) and adhered to the tenets of the Declaration of Helsinki. The study procedures followed all institutional guidelines and all patients provided informed consent before the procedures were performed.6) as the anterior chamber (AC) tamponade. After 2016, we switched from air to SF6 gas. DMEK was performed by three different surgeons , as previously reported , but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.  http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” . 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: NoReviewer #2: Yes**********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: YesReviewer #2: 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: This study evaluated parameters associated with 12-month endothelial cell loss in Japanese eyes treated with DMEK for bullous keratopathy.1. Introduction, line 66: please use data from the more recent 2018 EBAA report instead of the 2015 report.2. In Table 1, please report the minimum and maximum values for each parameter.3. Please recheck all the citation numbers throughout the text, because many are wrong. For example, in line 201 the citation should probably be 12 instead of 15; in line 204 the citations should probably be 18,19 instead of 16,17; and in line 205 the citation should be 19 instead of 17.4. Line 205 should note that the majority of patients in Schrittenlocher’s study were treated for FECD.5. Line 259, please clarify what is meant by “may be better at least around 65%”.6. Please add 2 scatter plots, one showing 12-month ECD vs. donor ECD and the other showing 12-month cell loss vs. the ratio of graft to cornea area. These will help the reader better assess the clinical significance of these parameters.Reviewer #2: Interesting, well written and well conducted study on the risk factors for EC loss after DMEK for bullous keratopathy in Asian eyes. Not much is known about that yet.Major comments-Discussion: how do authors explain the relatively high EC loss rates after 12 months? Axial length seems to be within comparable rates? Please give AC-Depth in Table 1.-Does AC depth correlate with EC loss?-149: How do you explain absent beneficial effect of SF6 use on detachment rates in you cohort in contrast to what is published?-259: how did you calculate the optimal ratio of 65%?-Discussion: when comparing your data with the Schrittenocher paper: please also give the absolute diameters you used in mm.-Figures: please better support your migration concept with ref. from the literature.Minor comments-Numerous spelling errors in the references need to be corrected. Some grammar and spelling errors in the text need correction.**********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,  12 May 2020Reviewer #1: This study evaluated parameters associated with 12-month endothelial cell loss in Japanese eyes treated with DMEK for bullous keratopathy.1. Introduction, line 66: please use data from the more recent 2018 EBAA report instead of the 2015 report.� Thank you for pointing this out. We have rechecked the report from EBAA and other reports, and subsequently corrected the description as follows: (Line 65–70)“As an indication, FECD and bullous keratopathy (BK) account for approximately 49% and 17% of endothelial keratoplasty cases in the United States of America, respectively [9]. In the Netherlands, FECD and BK account for approximately 85% and for 10% of DMEK cases, respectively, and the number of EK procedures has been reported to be increasing .” 2. In Table 1, please report the minimum and maximum values for each parameter.� Thank you for pointing this out. We have added the minimum and maximum values for each parameter in Table 1. Recipient characteristics Eyes N 72 Age (years), mean, ±SD, [range] 74.5 ± 8.2 [44 – 89]Male, n (%) 19 (26.4%) Preoperative BCVA (LogMAR), mean ± SD [range] 0.90 ± 0.47 [0.0458 – 2]Postoperative BCVA (LogMAR), mean ± SD [range] 0.073 ± 0.13 [-0.0792 – 0.523]Donor ECD (/mm2), mean ± SD [range] 2715 ± 231 [2020 – 3313]ECD 12 months after DMEK (/mm2), mean ± SD [range] 1246 ± 478 [363 – 2519]ECD-loss rates (%), mean ± SD [range] 54.4 ± 16.1 [15.1 – 86.1]Axial length (mm), mean ± SD [range] 23.2 ± 1.69 [21 - 31.6]Corneal diameter (mm), mean ± SD [range] 11.0 ± 0.58 [10 – 13]Graft Size (mm), mean ± SD [range] 7.87 ± 0.49 [5 - 8.5]Graft to host cornea area ratio (%), mean ± SD [range] 51.36 ± 6.65 [20.66 – 62.52] Air, n (%) 50 (69.4%) Re-bubbling  9 (12.5%) Preoperative CCT (µm), mean ±SD [range] 712 ± 94.0 [501 – 956]Iris damage scores before DMEK, mean ±SD [range] 1.44 ± 0.82 [0 – 4]3. Please recheck all the citation numbers throughout the text, because many are wrong. For example, in line 201 the citation should probably be 12 instead of 15; in line 204 the citations should probably be 18,19 instead of 16,17; and in line 205 the citation should be 19 instead of 17.� Thank you for pointing this out. We have rechecked all the citation numbers. 4. Line 205 should note that the majority of patients in Schrittenlocher’s study were treated for FECD.� Thank you for pointing this out. We agree with your comment and have now added a description.  “In contrast, Schrittenlocher et al. reported that postoperative ECD was not significantly associated with DMEK graft diameters in the range of 8–10 mm in FECD eyes.”　“Although patients with BK should have few functional endothelial cells in the peripheral cornea, most patients with FECD might have some healthy endothelium in the peripheral cornea [20]. This difference of healthy endothelium distribution between BK and FECD may be one of the reasons why the results of current study differed from those of Schrittenlocher et al.” 5. Line 259, please clarify what is meant by “may be better at least around 65%”.� Thank you for this comment. We evaluated the relation between 12-month ECD loss vs. the graft to host corneal area ratio, excluding the case of the lowest ratio of graft to the host cornea area (ratio = 20.7%). This figure is a scatter plot of 12-month ECD loss vs. the graft to host corneal area ratio showing a quadratic approximate expression (blue line) and a liner approximate expression (red line). Though there was not a significant association with a quadratic coefficient (p=0.35), this figure suggests that ECD might survive better at least around 60% to 65% of the ratio, especially in the BK eyes. Therefore, we stated that around 65% might be better. We have discussed this concept again with the co-authors, and have changed the description as follows. (line 226–232)“Although our results indicated that a relatively larger ratio of graft to host cornea area was better for ECD survival, there should be a plateau ratio, over which ECD survival would worsen. We attempted to evaluate this by drawing quadratic approximate expressions on 12-month ECD loss vs. the ratio of graft to cornea area scatter plots; however, we were unable to determine the significant ratio. Further research is needed to validate whether a larger graft is better for ECD survival or if there is an ideal graft size.”6. Please add 2 scatter plots, one showing 12-month ECD vs. donor ECD and the other showing 12-month cell loss vs. the ratio of graft to cornea area. These will help the reader better assess the clinical significance of these parameters.� Thank you for this suggestion. We have added the two scatters plots. Reviewer #2: Interesting, well written and well conducted study on the risk factors for EC loss after DMEK for bullous keratopathy in Asian eyes. Not much is known about that yet.Major comments-Discussion: how do authors explain the relatively high EC loss rates after 12 months? Axial length seems to be within comparable rates? Please give AC-Depth in Table 1.-Does AC depth correlate with EC loss?� Thank you for these comments. Our previous study* showed that ACD was strongly associated with AXL, and there was no correlation between ACD and EC loss. Although some patients had records of ACD as well as AXL, we did not record ACD in all patients. Since our data set did not include ACD, it is impossible to evaluate the correlation. It will be essential to evaluate the association in a larger number of patients in the future. https://doi.org/10.1097/ico.0000000000001698* Shimizu T, Hayashi T, Yuda K, Takahashi H, Oyakawa I, Yamazaki K, et al. Short axial length and iris damage are associated with iris posterior synechiae after Descemet membrane endothelial keratoplasty in Asian eyes. Cornea 2018; 37:1355-1359. -149: How do you explain absent beneficial effect of SF6 use on detachment rates in you cohort in contrast to what is published?� Thank you for this question. In the current study, 9 cases needed re-bubbling. Six cases were injected air, 3 were SF6. Though there was no significant difference in the current study, we think this was because the number of eyes that required re-bubbling was small. -259: how did you calculate the optimal ratio of 65%?� Thank you for this question. We evaluated the relation between 12-month ECD loss vs. the graft to host corneal area ratio, excluding the case of the lowest ratio of graft to the host cornea area (ratio = 20.7%). This figure is a scatter plot of 12-month ECD loss vs. the graft to host corneal area ratio showing a quadratic approximate expression (blue line) and a liner approximate expression (red line). Though there was not a significant association with a quadratic coefficient (p=0.35), this figure suggests that ECD might survive better at least around 60% to 65% of the ratio, especially in the BK eyes. Therefore, we stated that around 65% might be better. We have discussed this concept again with the co-authors, and have changed the description as follows: (line 226–232)“Although our results indicated that a relatively larger ratio of graft to host cornea area was better for ECD survival, there should be a plateau ratio, over which ECD survival would worsen. We attempted to evaluate this by drawing quadratic approximate expressions on 12-month ECD loss vs. the ratio of graft to cornea area scatter plots; however, we were unable to determine the significant ratio. Further research is needed to validate whether a larger graft is better for ECD survival or if there is an ideal graft size.”-Discussion: when comparing your data with the Schrittenocher paper: please also give the absolute diameters you used in mm.� Thank you for this suggestion. We always determined the graft size depending on the host cornea. As shown in our data set, most of our grafts were 7.75–8.25 mm. We have added a description regarding the absolute diameter we used. (line 93–94)“Most of the grafts were 7.75 – 8.25 mm.”-Figures: please better support your migration concept with ref. from the literature.� Thank you for this suggestion. As you mention, there are two possibilities of migration: migration from the host cornea, and from the donor cornea. We have added the references, and revised the manuscript according to your suggestion.  “Although detailed knowledge regarding the migration of corneal endothelial cell is lacking, it is considered that corneal endothelial cells close the wound gap mainly via migration and increased cell spreading [22]. The creation of a wound induces the release of chemical signals that enhance cellular motility near the wound site; cells at the wound extend towards the free surface, producing a traction force directed towards the wound . In FECD eyes with a peripheral rim of healthy endothelial cells, it has been shown that these healthy endothelial cells can be exploited to repopulate the centrally diseased endothelium either by migration or proliferation [25]. Further, a previous study suggested the possibility of treating corneal endothelial diseases by simple Descemet stripping without endothelial keratoplasty (DWEK) [26]. These studies suggest that healthy peripheral endothelium can migrate from the host cornea itself and be endothelialized. Another interesting study showed that adult endothelial cells were able to migrate in the human eye, supporting that endothelial cell in the donor graft can migrate toward the periphery of the host cornea .”Minor comments-Numerous spelling errors in the references need to be corrected. Some grammar and spelling errors in the text need correction.� Thank you for pointing this out. We have rechecked all the citation numbers and corrected the grammatical/spelling errors throughout the manuscript.Attachmentreply_to_comments_inoda.docxSubmitted filename: Click here for additional data file. 21 May 2020Factors Associated with Endothelial Cell Density Loss Post Descemet Membrane Endothelial Keratoplasty for Bullous Keratopathy in AsiaPONE-D-20-03729R1Dear Dr. Hayashi,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Hidenaga Kobashi, M.D., Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :The reviewers have completed their assessments in this revision.I would recommend to publish in Plos One.Reviewers' comments: 1 Jun 2020PONE-D-20-03729R1 Factors Associated with Endothelial Cell Density Loss Post Descemet Membrane Endothelial Keratoplasty for Bullous Keratopathy in Asia Dear Dr. Hayashi:I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Hidenaga Kobashi Academic EditorPLOS ONE


Editor-in-Chief, Journal of AnesthesiaUntil now, “the Journal of Anesthesia” and its sister journal “JA Clinical Reports” followed a policy of immediately rejecting all submissions that had posted its preprint version on the web, considering them duplicate submissions. The basis for this strict stance was to raise the bar for quality submissions, as there were endless reports of falsification in research papers by anesthesiologists in Japan.Preprints are manuscripts that have been uploaded to public servers without going through formal, rigorous peer-review. Preprints are uploaded to the Internet for free, enabling sharing of information among scientists worldwide more quickly than manuscripts published only after completing the traditional peer-review process. Rapidly advancing fields, particularly physics, mathematics, and computer science, have been using this system widely for over two decades, but it is not well established in the fields of medicine. The rationale is that, aside from the irrefutable question of a duplicate submission, as Dr. Kharasch, Editor-in-Chief of Anesthesiology, states , the infAnesthesia in the time of COVID-19,” which is openly accessible. It also created a system to simultaneously send final decisions made by the Editor-in-Chief to the authors, editors, and reviewers.With the emergence of the COVID-19 pandemic that started in Wuhan, China, journals, first in China and now around the globe, have been publishing articles about COVID-19. As specific therapies, effective treatments, and epidemical information for COVID-19 is quite important, publishers of medical journals worldwide have done what they can to post papers on the novel coronavirus as fast as possible and make them freely available. As also explained in the editorial by Dr. Kharasch, Anesthesiology introduced a system for speeding up the peer -review process to that aim. The Journal of Anesthesia adopted the strategy of promptly posting the latest information, primarily by its editors, under the title, “Nevertheless, this system cannot match the speed of preprints. Springer, the publisher of the Journal of Anesthesia, goes so far as to recommend researchers post preprints . Their sConsequently, beginning in 2021, “the Journal of Anesthesia” and its sister journal “JA Clinical Reports” will start accepting manuscripts that have posted their preprints on the web. Authors will also be able to resubmit manuscripts that the Journal of Anesthesia and JA Clinical Reports previously rejected because of their preprint version on the server. Please note that manuscripts must include information about existing preprints and that preprints cannot be cited in the manuscript, as they are presumed to have identical content.I hope that preprints will continue to be used to advance medicine for humankind and that the Journal of Anesthesia and JA Clinical Reports will continue sharing useful information in the field of anesthesiology.


Recently, health-conscious consumers have a tendency to avoid the use of modified starch in their food products because of reluctance regarding food additives or chemical processes. The present paper considers the characteristics and manufacturing methods of clean label starch, which is free from chemical modification. Clean label starch manufacturing is mainly dependent on starch blending, physical and enzymatic modification methods. Physical modifications include ultrasound, hydrothermal , pre-gelatinization , high-pressure (high hydrostatic pressure), and pulsed electric field treatments. These physical processes allow variation of starch properties, such as morphological, thermal, rheological, and pasting properties. Enzyme treatment can change the properties of starch more dramatically. Actual use of clean label starch with such altered properties has occurred in industry and is described here. This review may provide useful information on the current status and future direction of clean label starch in the field of food science. Recently, “clean label food” has emerged as a global food trend  is typically added to the product  starch blends markedly deviated from the sum of the contributions from each type of starch. These findings indicate that the non-additive effect is more obvious when a larger difference in granule size is present between the starch components of the mixture.Puncha-arnon et al.  reportedObanni and Bemiller  found thPhaseolus lunatus (lima bean) and Manihot esculenta (cassava) starch blends. The amylose contents of cassava and lima bean were 17.3% and 32.4%, respectively; a specific ratio (lima bean:cassava = 25:75) was determined to provide the best combination of characteristics because of its high viscosity (102 mPa s) without retrogradation. It was also stable in the heating–cooling cycle. These results indicated that new starches with functional properties suitable for use in various foods can be created by mixing native starches from different plant species. Juhász and Salgó  Tomasik, . Park etBlending techniques are mainly used in food industry, such as for noodles  involves the use of sound waves with a frequency higher than the threshold of human hearing (> 20 kHz)  starch. Abbas et al. , the maximum amount of curcumin could be loaded into medium-chain triacylglycerols while reducing the emulsifier concentration. Additionally, Cheng et al.  and annealing (ANN) are two common types of hydrothermal treatment that change the physicochemical properties of starch without rupturing its granular structure , slowly digestible starch (SDS), and resistant starch (RS), depending on the digestion rate    for food processing and preservation has been investigated as an alternative to traditional treatment . The extent of quinoa starch gelatinization, so thus the conversion of starch suspension to a gel, increased as pressure increased. This trend became greater as both pressure exerted and starch concentration increased. According to many other studies on the effect of high-pressure treatment on the rheological properties of various starches, including those of sorghum  without heat treatment  for short durations (< 40 μs)  method is one of these approaches  processing could change the structure of wheat starch (type A), potato starch (type B), and pea starch (type C). They used polarized light microscopy, X-ray diffractometry, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), solid-state nuclear magnetic resonance spectroscopy, small-angle X-ray scattering, and gel permeation chromatography to identify the effect of PEF on the starch. The results showed that PEF could change the structures of all three types of starch, especially potato starch. Although treatment did not change the X-ray diffraction pattern type, it changed the strength of the peaks in ATR-FTIR, as well as the molecular weight distribution  tends to reduce the peak viscosity, breakdown, and setback value of starch during pasting  in tapioca starch–water dispersion solutions . When the electric field strength was increased from 30 to 50 kV cm−1, the peak viscosity of the processed sample gradually decreased, and the difference in peak viscosity between native and processed samples showed a corresponding increase from 61 to 162 BU . However, the value did not change considerably as the intensity of the electric field increased . The RS of pea starch did not exhibit substantial change at any electric intensity   have been extensively used for clean label starch production. αGTases, which belong to the superfamily of glycoside hydrolases (GHs), cleave the α-1,4-glycosidic bond in the substrate such as amylose, amylopectin, glycogen, and maltodextrins. However, these transferases do not simply split the glycosidic bond. They transfer the removed glucan molecule from the donor to the non-reducing end of other glycosidic acceptor, thus forming a new glycosidic bond. This category includes 4-α-glucanotransferase , cyclodextrin glycosyltransferase , and branching enzyme   that have the ability to form inclusion complexes with specific guest molecules  and various sizes of large ring cyclic glucans (mainly DP 17–40 from amylose) from starch substrate, whereas CGTase significantly reduces the molecular weight of starch and mainly produces small circular products of DP 6, 7, and 8  is synthesized using branching enzyme from Bacillus stearothermophilus, and available as a food ingredient with the trade name of cyclic cluster dextrin (CCD)  of undesired components, technological advantages that include simple dosing and handling, economic advantages, and labor savings (Szente and Szejtli, In conclusion, this review has provided an overview of the currently available g clean label starch. Clean label starch, made without the use of chemicals, is not expected to completely replace conventional modified starch at the present time, due to various limitations. However, there remain many opportunities for use of clean label starch, because researchers are continuously moving the field forward with new discoveries pertaining to processes, new enzymes, and cost-effective options for consumers. Therefore, although clean label starch currently constitutes only 5% of the global modified starch market, it will increasingly replace modified starch.


Respected Editor,. This is a timely, accurate and concise editorial, addressing a topic of interest. Yes, it is true that case reports (CR) have been banned from many publications in recent years due to the reasons you have clearly alluded to. I have to support what it is in this editorial. As we need metrics, you produced actual data taken from the three most internationally important journals in our field and it is clear to the readership that CR still represents a proportion of what these journals, like the BJCVS, have also published. We have also seen this in many other publications. Some reasons may be the same; in some cases, journals may need more CR load. There may always be a reason for a CR with educational value.I read with attention your latest editorial recently published in the Brazilian Journal of Cardiovascular Surgery (BJCVS) regarding the publication of case reports. The JACC-CR focuses only on CR. The journal name is self-explanatory. After a peer-review process, accepted cases are published under the formula which contemplates publication fees. Moreover, this tendency goes on around the world.Other journals have designed a different strategy. The good example is that of the Journal of the American College of Cardiology (JACC). The JACC, like other journals, have evolved in a way that a family of journals has been created. As per the JACC, the Journal of the American College of Cardiology-Case Reports has been launched in June 2019Therefore, once again, I have to support the essence of the wise comments delivered in this recent editorial. The statement “... Case reports and case series may be the ‘lowest’ or the ‘weakest’ level of evidence, but they often remain the ‘first line of evidence’ and is where everything begins…” holds, to me, true. What we need is a very strict peer-review process that ensures that high quality CR ultimately reach every journal’s readership.


The journal retracts the article “Temporal Variations of Polycyclic Aromatic Hydrocarbons in the Seawater at Tsukumo Bay, Noto Peninsula, Japan, during 2014–2018” cited above [Adhering to our standard operating procedures, an investigation was conducted that confirmed the extent to which changes were made in PAH concentrations. Because of the significant changes to the previously reported data, it was decided that a correction would not be appropriate. Therefore, the paper has been retracted from publication.The authors agreed to this retraction.The retraction was approved by the Editor-in-Chief of the journal.


Hericium erinaceus by sodium hydroxide, β-1,3-glucanase and β-1,6-glucanase, respectively. The effects of the glucans on in vitro digestion of wheat starch were investigated by Englyst method. We found that addition of HEBGs significantly reduced the digestibility of starch, showing as decreased RDS and pGI, and increased SDS and RS content. In addition, the inhibitory effects positively correlated with the molecular weight of HEBG. The triple helix structure in HEBG plays important roles in inhibiting starch digestion. And β-1,3- glucan showed stronger inhibitor effects than those of β-1,6- glucan. This study unravels the mechanism of HEBG on inhibition of starch digestion and provides a theoretical understanding for the application of edible mushroom β-glucan to the development of low glycemic index starchy foods.β-glucan has attracted extensive attention due to its health promoting effects, such as lowering the blood sugar and lipids levels, and enhancing immunity. In this study, three different β-glucans  were obtained from  Hericium erinaceus is an edible mushroom with health beneficial effects and has attracted extensive research interests , which can be digested quickly in the small intestine, and digestion time <20 min; slowly digestible starch (SDS) refers to those can be fully digested and absorbed in the small intestine but, slowly release energy and help to maintain postprandial blood glucose stability without insulin resistance; resistant starch (RS) refers to that cannot be digested and absorbed in the human small intestine, but can be fermented by microorganisms in the large intestine to maintain the gut health. Some studies have shown that eating foods rich in SDS can effectively prevent chronic diseases such as diabetes. A number of works have confirmed the potential of H. erinaceus in the treatment of diabetes , slowly digestible starch (SDS) and resistant starch -D-glucanase  from Helix pomatia, endo-β-(1-6)-D-glucanase  from Talaromyces cellulolyticus, pepsin  from porcine stomach mucosa, invertase Grade VII  from baker's yeast (S. cerevisiae), thermostable α-amylase  from Bacillus licheniformis, they were purchased from Sigma-Aldrich Chemical Co. . Wheat starch (~98.5% starch content) was provided by Henan Kangdi Food Tech Co., Ltd, China. All the water used are deionized.Hericium erinaceus was weighted into 15 L water, mixed and heated to 100°C for 2 h, under stirring followed by cooling at room temperature. The solution was filtered through 4 layers of cloth (200 mesh). The retentate was heated again as before, concentrated to ~500 mL (dry weight of fruiting body: volume of crude extract), and centrifuged . The precipitate was collected and washed with 20% ethanol, centrifuged and repeated 3 more times to obtain white jelly-like precipitate. The solution was then dialyzed against water for 3 days (3500 Da cut-off membrane), during which the water was changed 6 times and the retentate was freeze-dried to obtain HEBG.Extraction of HEBG was conducted according to previous methods  with sliBriefly, 1 g HEBG was weighted into the 20 mL water and magnetically stirred at 60°C for 5 h. 1M sodium hydroxide solution was added, mixed, sealed and shaken intermittently for 24 h at room temperature. The solution was dialyzed and freeze dried as above, and named as HEBG-1.HEBG (100 mg) were mixed with water (50 μL) and magnetically stirred at 70°C to completely dissolve the polysaccharides. The glucan solution was cooled down to 50°C followed by addition of β-(1-3)-D glucanase or β-(1-6)-D-glucanase (3 mg). After 50 min hydrolysis, the enzymes were denatured by heating in a boiled water bath for 10 min. The hydrolysates were cooled down at room temperature and freeze-dried. The HEBG hydrolysates from β-(1-3)-D glucanase and β-(1-6)-D-glucanase was named as HEBG-2 and HEBG-3, respectively.2PO4 and 0.15 mol/LNaNO3. They were filtered through 0.22 μm filter prior to loaded into high performance size exclusion chromatography (HPSEC) system equipped with PWXL3000 and TSKPWCL4000 series gel column. The injection volume was 20 μL. The flow rate was 0.5 mL/min, the column temperature was maintained at 35°C and the laser detector light source wavelength was 623.8 nm  was dissolved in pH 7 salt solution (1 mL) containing 0.05 mol/L NaH623.8 nm .The mixtures of HEBG (5 mg) and distilled water (2 mL) were stirred at 60°C for 3 h until completely dissolved. Congo red solution  was then added and mixed thoroughly . 1M NaOHR2 = 0.9977.The standard curve drawing method of sodium periodate consumption : Sodium Periodate oxidation: HEBG-1, 2, or 3 (50 mg) and sodium periodate solution  were mixed at 4°C. 0.1 mL of samples were taken from the mixture every 24 h and diluted to 25 mL with water. The absorbance of the diluent was monitored at 223 nm until a maximum value was reached. The sodium periodate consumption of HEBG was calculated by the standard curve. Then 8 mL periodate oxidation solution and two drops of ethylene glycol was added to the original mixture to terminate the reaction. The yield of formed formic acid was titrated with 40% NaOH after adding two drops of phenolphthalein .4 holding 24 h. Then the solution dialyzed continuously for 2 days. Polysaccharide alcohol product was obtained from above solution by freeze-drying, which were analyzed by aldononitrile acetate precolumn-derivatization gas chromatography (Smith degradation was conducted according to Kocharova . The HEBtography .2. Sodium hydroxide pellet (30 mg) was then quickly added and stirred for 3 h. Methyl iodide (1 mL) was slowly added to the tube on ice bath with a flow of N2. Be operated away from light, stirred by magnetic force at room temperature for 1 h and then removed moisture by anhydrous Na2SO4 column. The methylated glucans were hydrolyzed with trifluoroacetic acid (4 mol/L), reduced with sodium boron deuteride for 3 h, and acetylated with 0.5 mL acetic anhydride at 100°C for 1 h. The formed alditol acetate was dissolved in extracted with dichloromethane and analyzed by GC-MS  equipped with a HP-5MS quartz capillary column (30 m × 250 μm × 0.25 μm). The condition was set as: column initial temperature 160°C, 2°C/min ramping rate to 240°C; injection port temperature 250°C, carrier gas was N2, with a flow rate 1 mL/min. Sample injection volume was 2 μL.Methylation analysis of the glucans were conducted according to method of Panda et al. . HEBG-1,The content of enzymatically hydrolyzed glucose was determined based on Zhuang et al.  with mint is the glucose content of wheat starch released at t min of hydrolysis, G0 is the free glucose content before digestion, TS (%) is the proportion of total dry weight (Mg) of wheat starch to total weight in each sample.C is the digestibility of wheat starch, GThe predicted glycemic index (pGI) was calculated using the equation below :90) is the percentage of total starch hydrolyzed for 90 min.which  was dissolved in 5 mL water, centrifuged, and the supernatant was diluted to a concentration of 1 μ g/mL HEBG . The diln = 3). Values with different letters in the same column differ significantly (P < 0.05) according to Duncan's multiple range test. The datas were analyzed with Origin  and SPSS was used to conduct the significant analysis between samples .All measurements were done in triplicates and results were presented as mean ± standard deviation (SD) , which can be used to assess the dispersion of the polymersization of the sample. The value close to 1 indicate high homogenous. The value increases from 1.07 in HEBG-1 to 1.54 to HEBG-3, indicating the degree of polymerizations distribute more widely using enzyme treatment of HEBG than using alkaline treatment.The HEBG is a mixture of glucans with different degrees of polymerization. The molecular weights of HEBG, HEBG-1, 2 and 3 were determined by HPSEC-MALLS-RI. The HPSEC chromatogram of HEBGs  showed aTo further elucidate its structure, degradation and methylation experiments were conducted. After mixing sodium periodate and sodium iodate in different proportions, the absorbance was measured at 223 nm, and the standard curve of periodate consumption was obtained . The calIt can be found from the Smith degradation of the HEBG-1  was shown in The product of HEBG-2 has only two peaks, 9.348 min (glycerol) and 14.352 min (erythritol), with a weight ratio of 3.18: 1. For HEBG-3, it has three peaks of 9.298 min (glycerol), 14.345 min (erythritol) and 23.727 min (glucose). No peak of glucose in Because glucanases hydrolyzes specific glycosidic linkages in polysaccharides, there is no characteristic peak in the degraded gas chromatography. The GC-MS statistics of HEBGs are summarized in The triple helix structure of polysaccharides an important for its biological activity . As can The spatial configuration of polysaccharides correlates with their biological activity. Atomic force microscope (AFM) enables to examine the structure of polysaccharides at nanoscale, which helps to build the connection with its chemical properties .Hericium erinaceus glucans indicating the structure of molecule and space upon different treatments from The typical microstructure of glucans of four kinds of It is speculated that the chains of polysaccharides are interacted via mainly hydrogen bonds. When the glycosidic linkage is cleaved by enzymatic hydrolysis, the density of hydrogen bonds between chains becomes smaller and the domain structure becomes larger. The phenomenon was consistent with the Agbenorhevis' study . The smain vitro was studied by measuring the enzymatic hydrolysis rate of wheat starch. As shown in The effect of different HEBGs on the digestion of wheat starch Hericium erinaceus β-1, 3-glucan has a greater inhibitory effect on starch digestion in the main chain structure of β-glucan of Hericium erinaceus, or it may be that the molecular length of glucan is longer than that of HEBG-2 molecular chain and has a stronger cross-linking effect with starch. This is similar to the result that oat β-glucan without branching structure can also inhibit starch digestion, which was studied by Zhang , it was directly observed that HEBG was curled and folded, accompanied by filamentous and random coil structure, but the branched chain and conformation of HEBG-2 or 3, with obvious branching characteristics and regular multi-branch juxtaposition structure. The triple helix structure in HEBG plays an important role in inhibiting starch digestion. We found the addition of Hericium erinaceus β-glucan could significantly reduce the digestibility of starch, which decreased the values of RDS and PGI, and increased the content of SDS and RS. The glucans treated by enzymatic hydrolysis also showed differences in the starch digestibility, indicating that digestibility inhibition of β-1, 3 linkage on starch digestion was stronger than that of β-1, 6 linkage. Foods added with HEBG can effectively reduce the level of GI, intake of blood sugar and prevent the occurrence of diabetes. To explore the interaction mechanism between β-glucan and starch and reveal its mechanism of inhibiting starch digestion, which provides a basis for the application of β-glucan in function food and health products. This study unravels the mechanism of HEBG on inhibition of starch digestion and provides a theoretical understanding for the application of edible mushroom β-glucan to the development of low glycemic index starchy foods.In this study, three different types of HEBGs  were prepared from The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.LY and JZ: conceptualization. SZ: methodology. DC: formal analysis. BM: data curation and writing—original draft preparation. TF: writing—review and editing. HZ and JZ: funding acquisition. All authors contributed to the article and approved the submitted version.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.


Biomimetic scales provide a convenient template to tailor the bending stiffness of the underlying slender substrate due to their mutual sliding after engagement. Scale stiffness can therefore directly impact the substrate behavior, opening a potential avenue for substrate stiffness tunability. Here, we have developed a biomimetic beam, which is covered by tunable stiffness scales. Scale tunability is achieved by specially designed plate like scales consisting of layers of low melting point alloy (LMPA) phase change materials fully enclosed inside a soft polymer. These composite scales can transition between stiff and soft states by straddling the temperatures across LMPA melting points thereby drastically altering stiffness. We experimentally analyze the bending behavior of biomimetic beams covered with tunable stiffness scales of two architectures—one with single enclosure of LMPA and one with two enclosures of different melting point LMPAs. These architectures provide a continuous stiffness change of the underlying substrate post engagement, controlled by the operating temperature. We characterize this response using three-point bending experiments at various temperature profiles. Our results demonstrate for the first time, the pronounced and reversible tunability in the bending behavior of biomimetic scale covered beam, which are strongly dependent on the scale material and architecture. Particularly, it is shown that the bending stiffness of the biomimetic scale covered beam can be actively and reversibly tuned by a factor of up to 7. The developed biomimetic beam has applications in soft robotic grippers, smart segmented armors, deployable structures and soft swimming robots. These encompass protection against localized loading such as puncture resistance14 as well as modifications to global deformation modes of more slender substrates16. Thus, scales present an exciting platform for biomimicry. When compared to many other materially engineered solutions, scales directly interface with the work environment and objects, tremendously increasing their impact on performance. Secondly, since source of multifunctionality and extreme mechanical properties are a result of both parent material and topology, it allows for a wide range of properties engineering. One of the primary effect of the biomimetic scales on soft surface is the change in the bending behavior of the overall multi-material structure. Although embedded scales will alter the overall stiffness, the exact relationships between scale properties and distributions are less obvious. Ghosh et al.15 provided a contact kinematics model predicting the bending behavior which transitions through linear-nonlinear-rigid regimes . The parameters affecting the bending deformation modes are relative stiffnesses of the scales and the substrate, scales overlap ratios, length, contact friction and some other geometrical parameters discussed in previous studies18. These nonlinear regimes are more universal than pure bending and also reported in non-uniform loads as well as torsion20. Thus clearly, changing the scales properties can lead to a change in the overall behavior of the structure. One of the most convenient parameter of change is the stiffness of the scales. This can change the deformation regimes of the scales themselves, thereby the kinematics. This complex sequence of mechanical effects have not been characterized before. In this paper we study for the first time the effect on bending rigidity, nonlinearity of the bending behavior and the effect of reversible scales deformation on the nonlinear bending behavior of the biomimetic substrate. This material change is introduced using scales with 1 layer of LMPA and 2 layers of LMPAs with different melting points are designed to demonstrate that the concept can be customized for a variety of applications discussed in the manuscript.Scales are among the most apparent and visually striking external geometrically significant features adorning animal integumentary system. Recent investigations have shown that geometry, distribution and stiffness all taken together can result in remarkable enhancement in the substrate response of both naturally occurring fishes23. These models have shown the tailorability of bending properties based on both scale geometry and materials. Such tailorability can be translated to real time tunability by altering material and geometrical characteristics of the scales. However, altering the geometry of fabricated scales is relatively difficult to achieve. In contrast, stiffness control can be achieved through a variety of well-known techniques such as thermally tunable composite wax24, vacuum-induced jamming of granular materials26, electric29/magnetic30 stimuli, smart fluids, magnetorheological (MR) or electrorheological (ER) fluids32, shape memory materials34 and rigidity-tuning conductive elastomers35. Encapsulated MR fluids within a structured elastomeric cavity provide stiffness tunability with fast switching time (millisecond range) and relatively high stiffness change ratios  have small stiffness changes (< 4×) and can be directly activated as they are conductive. Nonconductive shape memory polymers (SMPs), can have a high stiffness change ratio (> 100×) and be stimulated using an external heater. The conductive propylene-based elastomer (CPBE) is a conductive elastomer enabling direct joule heating which can be activated in 29. Phase change materials can control stiffness of the scales as they combine extreme stiffness states of smart fluids (soft in liquid phase) and shape memory materials (stiff in solid phase) providing a very large stiffness change ratio37. On the other hand, the stiffness transition speed is directly linked to the thermal conductivity, and as a result, LMPAs and SMAs are much faster in phase transition than SMPs due to 100× greater conductivity  are metals that liquefy at relatively low temperatures (47–62 °C) depending on the alloy composition weight and have elastic moduli in the order of a few GPa. These LMPAs are typically made of bismuth, cadmium, lead, tin and indium with different compositions depending on the desired melting temperature. This class of materials are thus ideal for incorporating into plate like scales to rapidly and reversibly change their stiffness. Scale covered surfaces with dynamically tunable bending stiffness have applications in a wide range of intelligent systems from soft robotics38, wearable robotics39 to devices with a safe human machine interaction40. They provide a new route to realize the next generation of smart man-made segmented armors, puncture/impact resilient and flexible structures11, deployable structures, soft robotic grippers35, soft biomimetic swimming robots41 and any other man-made systems when resilient bending, compliance and robustness are required.Theoretical underpinnings of global deformation nonlinearity have been investigated thoroughly underlying the role of scale size, distribution, orientation and stiffness on soft deformable substratesHere, for the first time, a biomimetic beam with tunable stiffness scales is developed where the stiffness of the scales could be changed via applying heat to the scales with encapsulated LMPAs. The alloy is encapsulated within each plate-like scale in two different morphologies—one with a single layer of LMPA core within the soft TangoBlackPlus polymer shell  and another where the interior of the scale is partitioned to contain two cores of the LMPAs , Fig. 42 to fabricate the two types of scales—SLS and DLS. Then, two caps are glued to both ends of the scales using Sil-Poxy silicon adhesive (Smooth-On Inc.) to encapsulate the LMPA layers in the soft shell. The fabricated scales are illustrated in Fig. m = 62 °C). The two cores of DLS had one filled with LMPA1 and another of LMPA2 . When scales are at room temperature, all LMPA layers are solid and scales are stiff. When the scales are heated up to the range of The biomimetic scale covered beams were fabricated using a multistep manufacturing process Fig. . First, To fabricate the soft substrate, a 3D-printed mold  is filled with VPS elastomer  and then cured in room temperature for 20 mins. VPS elastomer is prepared with a 1:1 weight ratio of base elastomer to catalyst agent and mixed for 1 min. This results in a soft substrate with channels defined by the features to fit the fabricated scales. In the final step, scales are bonded to the substrate using Sil-Poxy adhesive. Both SLS and DLS are fixed at an angle of 10° and embedded into the substrate by 6 mm.m = 62 °C, Young’s modulus of 9.25 GPa)43 and manual illustration of bending deformation on scale side  and plain side . Clearly scales engagement on the scale side restricts curvature when compared with the plain side . Since one of the chambers continues to be solid, the overall bending stiffness is intermediate between a beam with soft scales and fully stiff beam. As the temperature increases beyond 62 °C, both LMPA chambers completely liquefy resulting in the lowest bending stiffness value of the DLS biomimetic beam exhibiting stiffness of 0.15 N/mm.Characterization of DLS biomimetic beam under bending is illustrated in Fig. scales = 75 °C. We note that in cyclic three-point bending experiments for both soft and stiff states, a hysteresis between loading and unloading curves is observed which is due to the friction at two supports and formation of indented deformation of the scales as they transition from liquid to solid44 , then the layer 2 phase (Tm = 47 °C) changes to solid and after 78 cycles, complete solidification occurs and therefore bending behavior of the biomimetic beam did not change anymore. For SLS (Tm = 62 °C), complete stiffness was achieved much earlier, at 37 cycles, after which there was no change in bending stiffness of the biomimetic beam . Region II shows a large increase in bending stiffness of both SLS and DLS biomimetic beams, due to the onset of liquid-solid transition of LMPA1 of the scales. This phase is completed with the LMPA1 completely solidifying. This is the stiffer limit of SLS and occurs after 37 cycles. After this region, the stiffness of either SLS or DLS biomimetic beams do not change as no immediate phase change occurs. Thus region III, sets the final limit on the stiffness of the SLS biomimetic beam, indicating long term stiffness stabilization for this type of specimen. This region ends, when LMPA2 begins to solidify with falling temperature, further raising the stiffness of the second layer of the DLS. This is the onset of region IV marked by a rapid stiffness increase of the DLS. The stiffness continues to increase with each cycle as temperature continues to fall until this chamber is completely solidified and the DLS biomimetic beam attains its maximum stiffness for a given deformation. This complete solidification occurs after about 78 cycles and is characterized by complete long-term stabilization of the beam stiffness for DLS designs. In addition, the proposed biomimetic scale covered beam has been shown to exhibit consistent mechanical behavior after more than 40 activation cycles and cooling down over a span of three months, which shows the robustness of the biomimetic beam. Although accelarated cooling has not been investigated as it is beyond the scope of the paper, in practice, cooling accelaration is possible by a number of design features such as using an air gun, water spray or substrate embedded cooling mechanisms.In addition to these temperature triggered states, a wider spectrum of stiffness response exists which can be exploited for a more continuous tunability. Figure 15, including a comparison with the scales engagement (sliding effect)19. The effect of pure embedding is primarily a composite effect and measurable even before engagement. The effects of engagement are clearly visible by sharp increases in stiffness which is not the case with only inclusions19.In this paper, we have investigated for the first time, a biomimetic beam covered with tunable stiffness scales to achieve a continuous stiffness change of the underlying substrate. Scales stiffness is tuned by enclosing phase change LMPA materials into a 3D-printed soft shell. Such materials were able to reversibly transition between the extreme stiffness states through liquefying and solidifying with temperature, thereby also altering the scale stiffness. We exploited this feature to endow a large range of tunability to the fabricated scales by enclosing them into a single (SLS) and double layer (DLS) architecture. The SLS have 1 layer of phase change alloy, while DLS have two layers of phase change materials with different melting temperatures to provide multistep stiffness tunablity. These scales were then affixed on one side of a beam like soft polymeric substrate, resulting in a biomimetic scale covered beam of two different types. Bending stiffness tunability of these scale covered beams are then experimentally characterized using three-point bending tests under thermal loads to activate the phase change behavior. We find that there is a continuum of bending stiffness values  of the biomimetic beam between extreme states of embedded LMPA layers (solid or liquid). It is also shown that the stiffness of the tunable stiffness biomimetic beam can be dynamically and reversibly tuned as high as 7× as phase change of LMPA layers from liquid to solid. Scales embedding itself will lead to increase in stiffness. This effect is however, distinct and already investigated in some detail in literature45 can be potentially employed to tune the distance between scales  and develop adaptive morphing robotic grippers.It may be possible to fabricate the tunable stiffness scales of scale covered systems using alternative ways by making embedded channels of LMPAs enabling direct joule heating of individual scales. It provides localized stiffness tuning of biomimetic surfaces to dissipate local impact forces with optimized energy consumption that have applications in development of robust structures. Moreover, in the current study, SLS and DLS biomimetic beams are developed whereas it can have multi-layer of tunable stiffness materials to achieve the desired scales stiffness depending on the application. In the future, an aspiring goal would be to apply the developed biomimetic beam in a “soft robotic gripper” or “smart man-made segmented armors” to fully integrate sensing, actuations and control strategies to mimic the adaptability of biological systems. As an example, a biomimetic beam coupled with soft pneumatic actuatorsSupplementary Information 1.Supplementary Information 2.


In plants, starch is synthesized in leaves during the day-time from fixed carbon through photosynthesis and is mobilized at night to support continued respiration, sucrose export, and growth in the dark. The main crops where starch is biosynthesized and stored are corn, rice, wheat, and potatoes, and they are mainly used as food resources for humankind. There are many genes that are involved in starch biosynthesis from cytosol to storage organs in plants. ADP-glucose, UDP- glucose, and glucose-6-phosphate are synthesized catalyzed by UDP-invertase, AGPase, hexokinase, and P- hexose-isomerase in cytosol. Starch composed of amylopectin and amylose is synthesized by starch synthase, granule bound starch synthase, starch-branching enzyme, debranching enzyme, and pullulanase, which is primarily responsible for starch production in storage organs. Recently, it has been uncovered that structural genes are controlled by proteins derived from other genes such as transcription factors. To obtain more precise information on starch metabolism, the functions of genes and transcription factors need to be studied to understand their roles and functions in starch biosynthesis in plants. However, the roles of genes related to starch biosynthesis are not yet clearly understood. The papers of this special issue contain reviews and research articles on these topics and will be a useful resource for researchers involved in the quality improvement of starch storage crops. Starch is the one of three major nutrients for human life, which is the main storage carbohydrate in plants and is used for food and various other purposes. The major crops where starch is biosynthesized and stored are corn, rice, wheat, and potatoes, and are mainly used as food resources for humankind. Recently, starch has also attracted attention as a source of bioethanol and various industrial new materials. Starch is synthesized in leaves during the day-time from fixed carbon through photosynthesis and is mobilized at night to support continued respiration, sucrose export, and growth in the dark . The staStarch synthesis begins with the production of ADP glucose, a substrate for starch synthase. The production of ADP glucose in the photosynthetic chloroplasts of plant leaves occurs in the Calvin– Benson cycle by converting fructose-6 phosphate to glucose-1 phosphate (Glc-6-P) through glucose-6-phosphate (Glc-6-P). ADP-glucose pyrophosphorylase catalyzes the generation of Glc-1-P and ATP to ADP glucose and pyrophosphate (PPi) [agpS1-2 and agpL1-3 [SSs, GBSSI, BE, DBE, ISA, and PUL, use ADPglc to add glucose moieties to the amylose and amylopectin molecules. This enzyme is known to be controlled by the transcription factors, OsSERF1, OsbZIP58, and OsRPBF in rice, and ZmEREB156, ZmbZIP91, and ZmNAC36 in maize [There are many genes that are involved in starch biosynthesis from cytosol to storage organs in plants. ADP-glucose, UDP-glucose, and glucose-6-phosphate are synthesized catalyzed by UDP- invertase, AGPase, hexokinase, and P-hexoseisomerase in cytosol. Starch is synthesized by starch synthase, granule bound starch synthase, starch-branching enzyme, debranching enzyme, and pullulanase, which is primarily responsible for starch production in storage organs . Recent  agpL1-3 , catalyzin maize .However, our knowledge of the signal transduction cascade is not yet complete. In particular, there is a shortage of knowledge on the molecular identity of the sensor, intracellular signaling pathways, and integration between photosynthetic and metabolic signals. Research over the past few years has also expanded the understanding of the role of post-translational protein modification and protein–protein interactions in the regulation of starch synthesis . EvidencScientists have studied mainly the structural genes that are related to specific biosynthetic pathways and they have tried to link their functions one by one in an attempt to understand the whole biosynthesis pathway in plants. Recently, it has been uncovered that structural genes are controlled by proteins derived from other genes such as transcription factors . To obtaThe advent of genome-editing tools has enabled researchers to modify specific genomic sequences. A gain-of-function mutation  alters a gene product so that its effect is stronger (enhanced activation) or replaced by another abnormal function ,7. A los2S2) composed of two large subunits and two small subunits. The AGPase small subunit serves as a catalytic function, whereas the AGPase large subunit primarily serves to regulate the allosteric regulatory properties of AGPase [ADP-glucose is synthesized from Glc1P and ATP by ADP-glucose pyrophosphorylase (AGPase) and produced as a heterotetramer  of a protein allows rapid regulation of protein function in response to metabolic and environmental changes. Phosphorylation of proteins is known to play an important role in regulating the distribution of light energy in photosynthesis. ADP-glucose pyrophosphorylase (AGPase) was revealed to be a target of post-translational redox regulation, including the reversible formation of an intermolecular Cys bridge between Cys-82 of two small subunits of the heterotetrameric enzyme ,36. ReduIn some aspects, research points to the importance of post-translational modifications to regulate starch metabolism. That is, given that mRNA levels are not directly related to protein levels, it is necessary to study how plants regulate mRNA expression in the early stages of transcription and how proteins are post-translationally modified and used for starch biosynthesis.Mutants are useful tools for analyzing the function of a target gene; for this reason, large quantities of mutants and other research data have been produced by physical mutagenesis , chemical mutagenesis, or genetic methods . Most ofRecently, there have been more chances to improve our understanding through advanced scientific tools such as GWAS, MABc, gene pyramiding, overexpression, knock-out, RNAi, and gene editing. This Special Issue of Plants will highlight the identification, function, roles, and relationships of genes and transcription factors in starch biosynthesis, and their interactions through the processes in the starch synthesis pathway of plants.


The aim of this study was to evaluate magnetic resonance elastography (MRE) as a response parameter in patients who received transarterial chemoembolization (TACE) for the treatment of colorectal liver metastases.Forty-two patients  with repeated TACE therapy of colorectal liver metastases underwent on average 2 repetitive magnetic resonance imaging (MRI) and MRE exams in 4- to 6-week intervals using a 1.5-T scanner. MRE-based liver stiffness measurements were performed in normal liver parenchyma and in metastatic lesions. Moreover, the size of the liver metastases was assessed during treatment and compared with the results of the MRE analysis.p < 0.001). However, only a weak correlation was found between the lesion size and stiffness . MRE analysis revealed an increase in stiffness of the colorectal liver metastases from 4.4 to 7.1 kPa after three cycles of TACE (p < 0.001). Also, the mean size of the metastases decreased from 17.0 to 11.3 cm2 (p < 0.001). Finally, the entire liver stiffness increased from 2.9 to 3.1 kPa over the three cycles of TACE therapy.Liver metastases showed a significantly higher degree of stiffness compared with the normal liver parenchyma (In conclusion, MRE showed a significant change in stiffness and size of liver metastases. Therefore, MRE may provide an added value for an evaluation of treatment response in patients with colorectal liver metastases undergoing TACE.• MRE showed an increase in stiffness of the colorectal liver metastases during TACE therapy.• Liver metastases showed a significantly higher degree of stiffness compared with the normal liver parenchyma.• However, only a weak correlation was found between the lesion size and stiffness. Colorectal cancer is one of the most prevalent forms of cancer in the world , and theIf neither surgery nor local-ablative methods show response, transarterial chemoembolization (TACE) is an option for local tumor control. This therapy has shown promising results in patients with unresectable colorectal liver metastases . TACE isThe aim of our study was to assess MRE as a response parameter in patients with TACE of colorectal liver metastases.This retrospective study was approved by the institutional ethical committee of our university hospital. Data was collected between April 2017 and December 2017. All patients had met our inclusion criteria as follows: (a) age between 18 and 90 years old, (b) histopathological and/or radiological evidence of colorectal liver metastases, (c) current therapy with TACE, (d) no contraindications for MRI scanning. Exclusion criteria were as follows: (a) liver metastases of different origin, (b) inflammation in or around the liver, (c) abbreviations from the standard imaging protocol.Forty-two patients  who had met the general inclusion and exclusion criteria were analyzed in the present study. The patients underwent regular treatment with TACE  and MRI with additional MRE. The MRI was performed either on the same day as the TACE therapy or in the following 2 days after the intervention, so that a range of 0–2 days in between MRE and TACE can be given. As treatment effects will vary over time and inconsistencies in timing between MRE snapshots and TACE could bias the results, patients with longer ranges were not included. The majority of MRI and MRE examinations (93%) were performed immediately after TACE on the same day. All study patients underwent TACE in 4–6 weeks intervals. Twelve patients had evidence of metastases in the left liver lobe, while the other 30 showed metastases in the right lobe. In the case of multiple metastatic lesions in a single patient, only the largest lesion was evaluated. On average, 3.3  TACE interventions were performed before the first MRE imaging.2 body surface), gemcitabine (500 mg/m2 body surface), and cisplatin (30 mg/m2 body surface) in all patients. After administration of the chemotherapeutic substances, a selective injection of lipiodol was performed. The lipiodol capitation was not correlated with the efficacy of the TACE because of the frequently inhomogeneous appearance. All TACE therapies were performed using a robot-supported angiography system , and the median time of the whole procedure was 35 min including the installation of the coil and positioning checks [After the puncture of a femoral artery, a pigtail catheter was inserted via the Seldinger technique . Thereafg checks .Unenhanced and contrast-enhanced MRI was performed directly before or after the intervention using gadobutrol . T1- and T2-weighted MRI and MRE scans were acquired in transverse and sagittal orientation with 5 mm slice thickness using a 1.5-T system . Commercially available system was used for MRE measurements (Resoundant) which consisted of an active driver located outside the scan room connected to passive actuators in the scan room. The time of vibrations was between 15 s for five slices of EPI (WIP measurement) and 23 s for a single slice of GRE. The median time for the MRE measurement including patient and hardware preparation was 18 min. The applied sequence protocol consisted of the listed parameters in Table n = 16) were only used for the collection of the general elastography data. Patients with two (n = 11) or more (n = 15) MRE series were evaluated to assess therapy response , total liver stiffness (kPa) , total liver MAP T1 (ms) and MAP T2 (ms), left and right lobe separated extent, stiffness and MAP T1/T2 measurement, metastasis extent, stiffness and MAP T1/T2 . This approach allows for a high-resolution segregation of intrahepatic structures and exact measurements of intrahepatic elastography, differentiating metastases and healthy tissue. Three different groups of patients with MRE imaging were investigated, as not every patient had the same number of performed MRE series due to the retrospective design of the study. Patients with one MRE examination n = 6 were onnse Fig. . Evaluat/T2 Fig. . Additiop value < 0.05 indicated statistical significance. Spearman’s test was used to assess correlation.Minimum and maximum values with standard deviations were calculated for continuous variables, whereas medians with ranges were calculated for categorical variables. The Kolmogorov-Smirnov test was applied to test for normal distribution. The Friedman test was used to determine whether differences between the measurements were significant. Statistical software  was used and a 2 in the first MRI to 12.5 ± 8.0 cm2 in the second MRI and to 11.5 ± 10.0 cm2 in the third MRI (p = 0.03). Simultaneously, a significant change in stiffness was detected in the liver metastases increasing from 4.8 ± 1.4 kPa in the first MRI to 7.0 ± 2.6 kPa in the third MRI (p < 0.001). In addition, the metastases (5.9 ± 2.2 kPa) were significantly stiffer than the normal liver parenchyma (3.2 ± 1.0 kPa) (p < 0.001) .However, only a weak correlation was found between size and stiffness measurements (p = 0.04) compared with the right lobe  during the three cycles of TACE therapy. The left lobe parenchyma showed a significantly lower stiffness (3.3 ± 1.3 kPa) compared with the metastases (5.4 ± 1.8 kPa)  during therapy . Measurements in the healthy liver tissue showed an increase in stiffness of only + 0.87% with an average of 2.2 ± 0.6 kPa during therapy (p > 0.05) (Table The stiffness of the liver as a whole slightly increased from 3.0 ± 0.9 kPa in the first cycle to 3.3 ± 1.2 kPa in the last cycle (5) Table .Table 4Sp > 0.05) and the MAP T2 of the whole liver increased by only 0.003% . The MAP T1 and MAP T2 measurements of the metastases showed similar low changes than the rest of the liver. The MAP T2 in the metastases showed a significantly decrease   who had undergone yttrium-90 radioembolization, TACE, or radiofrequency ablation (RFA) . In theiMoreover, few studies evaluated the effects chemotherapy on liver stiffness in animal experiments. Pepin et al compared the stiffness of treated and untreated tumors in a mouse model . After sThese studies all demonstrate that MRE can be used for early response evaluation of tumor cells under therapy with chemotherapeutic agents. The studies showed in general a decrease of stiffness which is contrary to our results. This might be attributable to the different tumor origin and therapy in our study. Moreover, the accumulation of lipiodol in the metastases could have modified the stiffness of the lesions and may explain the different results compared with those in the literature.Our results showed a higher degree of stiffness in metastases than in healthy liver areas measured. However, we found only a weak correlation between stiffness and size measurements of liver metastases. To our knowledge, no previous studies have analyzed the stiffness of colorectal liver metastases using MRI and MRE. Moreover, the difference between the left lobe and right liver lobe may be explained by the method, as the vibration source is located on the right anterior chest wall with intercostal approach. In this area, the compression might be less effective than in the left lobe which can be compressed by the elastic band that maintains the system. However, almost no drug-related effects were found in the healthy liver tissue.MRE allows for highly accurate analysis of specific liver structures. Although ultrasound elastography has a higher plane resolution than MR, the depth is very limited . The acqThere were certain limitations in our study. First, the patient cohort was relatively small, and we did not include a control group. Second, patients with different tumor stages were included in this study. The impact of TACE varies according to different tumor stages, as well as the measured stiffness. Third, patients with MRE were treated during different phases of TACE therapy. Some patients included in the current study received their first cycle while others had more than 20 therapies completed. Fourth, there were different periods of time between the start of therapy and image acquisition that may affect our data. As our study was in a retrospective setting and many patients received their therapy for a longer period of time, it would be interesting to evaluate MRE-based parameters in a prospective study with more patients. Furthermore, some of the MRE data were extrapolated that might also create bias . In geneIn conclusion, the MRI and MRE are useful tools to evaluate tumor response after TACE therapy in patients with colorectal liver metastases. Our results showed a significant increase in stiffness of the metastases and a simultaneous decrease in size of the metastases. Therefore, MRE imaging may provide additional value to evaluation of tumor response after TACE therapy. In addition, MRE measurements may be used to calculate the remaining healthy liver proportion and function.


Tumor-derived extracellular vesicle (TEV) protein biomarkers facilitate cancer diagnosis and prognostic evaluations. However, the lack of reliable and convenient quantitative methods for evaluating TEV proteins prevents their clinical application.Methods: Here, based on dual amplification of hybridization chain reaction (HCR) and CRISPR-Cas12a, we developed the apta-HCR-CRISPR assay for direct high-sensitivity detection of TEV proteins. The TEV protein-targeted aptamer was amplified by HCR to produce a long-repeated sequence comprising multiple CRISPR RNA (crRNA) targetable barcodes, and the signals were further amplified by CRISPR-Cas12a collateral cleavage activities, resulting in a fluorescence signal.Results: The established strategy was verified by detecting the TEV protein markers nucleolin and programmed death ligand 1 (PD-L1). Both achieved limit of detection (LOD) values as low as 102 particles/µL, which is at least 104-fold more sensitive than aptamer-ELISA and 102-fold more sensitive than apta-HCR-ELISA. We directly applied our assay to a clinical analysis of circulating TEVs from 50 µL of serum, revealing potential applications of nucleolin+ TEVs for nasopharyngeal carcinoma cancer (NPC) diagnosis and PD-L1+ TEVs for therapeutic monitoring.Conclusion: The platform was simple and easy to operate, and this approach should be useful for the highly sensitive and versatile quantification of TEV proteins in clinical samples. Extracellular vesicles (EVs) are a heterogeneous class of membrane-bound phospholipid vesicles that are shed by cells into a variety of bodily fluids The hybridization chain reaction (HCR) is a simple but robust and efficient isothermal amplifycation process in vitro selection methods Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a has recently been demonstrated to cleave the nonspecific single-stranded DNA (ssDNA) reporter induced by Cas-CRISPR RNA (crRNA) target recognition-and-cleavage events + TEV detection. Given that circulating EV programmed death ligand 1 (PD-L1) has been reported to be an ideal predictive biomarker for monitoring clinical responses to anti-PD-1/PD-L1 therapy + TEV detection.Aptamers are a low-cost alternative to antibodies but exhibit comparable excellent affinities and specificities towards target proteins + and PD-L1+ TEVs. Based on the concept of apta-HCR-CRISPR, we developed our sensitive, simple, clinically feasible and cost-effective platform as a potential broad biosensing strategy for the detection of EV proteins.Here, we combined aptamers that specifically bind to protein targets on EVs to transduce the target recognition event signal to DNA effectors, HCR for DNA amplification and the production of multi-repeated CRISPR-targetable DNA units, and CRISPR/Cas12a collateral endonuclease activity for amplification, real-time DNA detection and readable signal outputs to develop an alternative CRISPR Cas12a strategy, termed the apta-HCR-CRISPR assay, to detect nucleolin2 and HEPES were obtained from Sigma-Aldrich . Other chemical reagents were procured from Guangzhou Chemical Reagents Factory . All reagents used in this study were of analytical grade and were used directly without any purification. All solutions were prepared using RNAase-free water.The oligonucleotides used were synthesized and purified by polyacrylamide gel electrophoresis (PAGE) or HPLC by Sangon Biotechnology Co., Ltd.  or Ruibiotech Co. . All of the DNA sequences and modifications are listed in 2HPO4, 1 mM NaCl and 5 mM EDTA, pH 7.0) and incubated at 37 °C for 1 h. Agarose gels (2%) and PAGE gels were run to confirm the formation of HCR products. After Goldview  staining, the gels were transferred to the Tanon-2500  for gel imaging.DNA sequences of the HCR system were designed according to the literature and modified to include an aptamer region in vitro as previously reported ex: 535 nm; λem: 572 nm).For a standard CRISPR-Cas12a method, crRNAs were transcribed For heterogeneous HCR-CRISPR, H0 at a specified concentration was added to the 0.72 mg/mL streptavidin MBs and incubated at 37 °C for 0.5 h followed by three washes. Then, the beads were resuspended with 50 μL of 2 μM H1 and 50 μL of 2 μM H2 and incubated for 1 h at 37 °C. After three washes, we carried out the standard CRISPR-Cas12a method.2. EVs secreted from SUNE2 cells were isolated by the ultracentrifugation method as described in our previous study The human nasopharyngeal carcinoma cancer (NPC) cell line SUNE2  was cultured in RPMI-1640 medium  containing 10% heat-inactivated FBS  at 37 °C with 5% COThe isolated EVs were observed using a JEM-1200EX transmission electron microscope (TEM) , and the concentration and size were verified by nanoparticle tracking analysis (NTA) performed with a NanoSight NS300 . The characteristic proteins of the EVs were analyzed by Western blotting as previously reported 6 particles/µL) were added to the anti-CD63, anti-CD81 and anti-CD9 MBs and incubated with 100 μL of 500 nM biotin-labeled aptamer at 37 °C for 1 h. After washing three times, 100 μL of diluted streptavidin-HRP  was added per well, followed by incubation for 20 min at room temperature. The MBs were washed three times with wash solution (0.01% Tween 20 and 5 mM MgCl2 in PBS) between each binding incubation. Finally, the 100-μL substrate (tetramethylbenzidine) to HRP was added, followed by incubation at 37 °C for 10-15 min in the dark. The reaction was stopped with 50 μL of 2 N H2SO4, and the OD was measured immediately at a wavelength of 450/630 nm.Next, 100-µL serial dilutions of SUNE2-derived EVs  were captured and recognized by 500 nM H0 using the same protocol as for the apta-ELISA. Then, the beads were resuspended with 50 μL of 2 μM H1 and 50 μL of 2 μM H2 and incubated for 1 h at 37 °C; after washing, the HCR products contained repetitive sequences bound to the beads, serving as targets for subsequent CRISPR-Cas12a recognition. Then, the standard CRISPR-Cas12a method was performed.First, 100-μL serial dilutions of the EVs  were also processed with a similar protocol to that described above.+ EV changes after immunotherapy initiation, ten advanced cancer patients with serum collected within 7 days before (day -7) and after 4 courses of nivolumab or pembrolizumab therapy were enrolled and analyzed.For the NPC detection experiment, pretreated serum from 20 pathologically confirmed NPC patients was obtained from Sun Yat-sen University Cancer Center (SYSUCC), and 20 volunteers who underwent routine physical examinations with negative results were also selected from the health examination department at our center. To evaluate PD-L1The serum from the enrolled subjects was centrifuged at 3600 rpm for 8 min and then stored at -80 °C until use. The clinical characteristics of the patients with NPC and the healthy controls are shown in This study was reviewed and approved by the Institutional Review Board and Ethics Committee of SYSUCC (GZR2018-197). Written informed consent for the use of clinical parameters and collected samples for further studies at the time of patient admission was obtained as a general standard procedure; the samples were anonymous and de-identified before use.+ EVs for immunotherapy surveillance, a paired t-test was applied. The diagnostic efficacy of nucleolin+ EVs was evaluated using the area under the receiver operating characteristic (ROC) curve (AUC). All tests were two tailed, and a p-value less than 0.05 was considered statistically significant. Three replicates were performed to improve the statistics.Statistical analyses were performed using GraphPad Prism version 8.0 software and Microsoft Excel. A two-tailed Student's t-test was used to analyze differences between the two groups. To evaluate the value of PD-L1Our apta-HCR-CRISPR assay comprised three processes Figure : aptamerFrancisella novicida (Fn) exhibits a fast cleavage effect on dsDNA Prior to conducting the apta-HCR-CRISPR, we set up HCR-CRISPR using nucleolin and PD-L1 as examples. The feasibility of HCR was validated by both agarose and polyacrylamide gel electrophoresis (PAGE) Figure A. As CasThe composition and length of complementarity between the crRNA and DNA largely modulate Cas12a nuclease activation Given that HCR produces nicked dsDNA structures, to explore whether the nicks affect Cas12a-crRNA2 duplex cleavage events, we introduced ligase to close the nicks A. A stanNext, target specificity was explored using H1, H2, and H0. We observed that the fluorescence intensity (FI) increased with the reaction time for Cas12a-crRNA cleavage in both the HCR products and the H2 ssDNA group but not in the H1 ssDNA and H0 groups  and considered H2 to be the cognate target. The fluorescence signals in duplicate consecutive H2 dsDNA were almost twice those in individual H2 dsDNA at three concentrations Figure . We alsoCas12a cleavage could be improved by buffer, crRNA, Cas12a concentration, and reaction temperature optimization . With thPrior to the detection of EV-derived proteins using the proposed apta-HCR-CRISPR platform, nasopharyngeal carcinoma cancer (NPC) EVs secreted from SUNE2 cells were harvested. The enriched EVs were confirmed to be cup-shaped vesicles with diameters of approximately 100 nm and were positive for the CD63, CD9, TSG101 and CD81 markers , indicat6 particles/µL) spiked in PBS were used as samples, and PBS served as a blank. After subtracting the PBS background OD, we found that the OD values of apta-ELISA were barely detectable until the EV concentration reached 106 particles/µL for both nucleolin+ and PD-L1+ TEVs  with 5-fold serial dilutions spiked in PBS (64 - 106 particles/µL) were used as samples . The LOD of apta-HCR-CRISPR for nucleolin+ TEVs was 102 particles/µL based on 3σb , which was approximately four orders of magnitude lower than that of the apta-ELISA and 100-fold lower than that of the apta-HCR-ELISA  and the FI (Y) ranging from 64 to 106 particles/µL was observed .Therefore, we detected EV-derived proteins by combining the apta-ELISA and the highly efficient signal amplification HCR-CRISPR/Cas12a platform Figure E. SUNE2 s Figure F, and anA Figure C. A lineicles/µL D, S9E. T+, with the help of CRISPR-Cas12a, the HCR time could be reduced to 1 h with a low LOD of 102 particles/µL.We believe that our approach is potentially suitable for analyzing TEVs in clinical applications given the low cost, easy operation, high sensitivity and wide linear range. A comparison of our proposed platform and recently reported methods for EV-derived protein detection is shown in + and PD-L1+ EVs (2 particles/µL (4 particles/ul) and apta-ELISA (106 particles/ul). Therefore, the proposed assay can work well for complex biological samples and has great potential for use in clinical serum samples  at three different concentrations, and comparable results between the signal of the EVs spiked in FBS and those in PBS were observed Figure . This as-L1+ EVs . The resicles/µL , surpass+ TEVs in 20 healthy people, 10 early-stage (stages I and II) NPC patients, and 10 advanced NPC patients were measured with the apta-HCR-CRISPR assay. No significant differences in sex or age were found between the three groups. As shown in Figure + EVs varied across individual participants. Both immunofluorescence , and the advanced NPC group exhibited higher levels compared to the early-stage NPC group , suggesting that nucleolin+ TEVs may be potential biomarkers for NPC diagnosis. Receiver operating characteristic (ROC) curve analysis and the area under the curve (AUC) were used to assess the diagnostic effect. A superior diagnostic effect  undergoing anti-PD-1 therapy. After 4 courses of anti-PD-1 treatment, the level of circulating EV PD-L1 decreased significantly in the clinical responders . These results are consistent with those of other studies showing that dynamic changes in PD-L1 levels are ideal predictive biomarkers for immune checkpoint blockade therapy responses We also examined serum PD-L12 particles/µL directly from complex sample environments. The multiple HCR barcodes recognized by crRNA/Cas12a provide an easily designed and highly adaptable aptamer-based protein detection method. Considering its high sensitivity, cost efficiency and ease of operation, this structurally versatile and stable method has considerable potential to be exploited as a routine bioassay for efficient clinical diagnosis or therapy surveillance. Our experiment with a small sample preliminarily confirmed that serum nucleolin+ TEVs are potential biomarkers for NPC detection and that changes in PD-L1+ TEVs levels are potential predictive biomarkers for immune checkpoint blockade therapy responses.In conclusion, we successfully constructed a novel apta-HCR-CRISPR assay to detect TEV surface proteins. Our method can detect TEV 10Supplementary figures and tables.Click here for additional data file.


Here, we evaluated the ability of PCPS immunoproteasome cleavage model to discriminate CD8+ T cell epitopes.We previously introduced PCPS (Proteasome Cleavage Prediction Server), a web-based tool to predict proteasome cleavage sites using + T cell epitopes and their source proteins. We then analyzed cleavage predictions by PCPS immunoproteasome cleavage model on this dataset and compared them with those provided by a related method implemented by NetChop web server. PCPS was clearly superior to NetChop in term of sensitivity (0.89 vs. 0.79) but somewhat inferior with regard to specificity (0.55 vs. 0.60). Judging by the Mathew’s Correlation Coefficient, PCPS predictions were overall superior to those provided by NetChop (0.46 vs. 0.39). We next analyzed the power of C-terminal cleavage predictions provided by the same PCPS model to discriminate CD8+ T cell epitopes, finding that they could be discriminated from random peptides with an accuracy of 0.74. Following these results, we tuned the PCPS web server to predict CD8+ T cell epitopes and predicted the entire SARS-CoV-2 epitope space.We first assembled an epitope dataset consisting of 844 unique virus-specific CD8+ T cell epitope features. iPCPS is available for free public use at https://imed.med.ucm.es/Tools/pcps/.We report an improved version of PCPS named iPCPS for predicting proteasome cleavage sites and peptides with CD8 Moreover, it has been shown that the C-terminus of peptides presented by MHC I molecules results from proteasome cleavage [Proteasomes are multicatalytic protease complexes that play a central role in cellular protein homeostasis by degrading damaged and misfolded proteins –3. Withicleavage .+ T cell epitopes result from immunoproteasome cleavage [The essential role of the proteasomes in class I antigen presentation has been elucidated in mammals, where two main forms of the proteasome exist, the constitutive proteasome and the immunoproteasome . Most cecleavage .+ T cell epitopes, researchers have developed different approaches to predict proteasome cleavage sites [n-grams to model and predict immunoproteasome cleavage sites [n-grams models for free public use at https://imed.med.ucm.es/Tools/pcps/.Given that proteasomes/immunoproteasomes determine the repertoire of CD8ge sites . Since tge sites –18. The ge sites . Moreove+ T cell epitope discrimination when combined with peptide-MHC I binding predictions [+ T cell epitopes. Using a dataset of 844 virus-specific CD8+ T cell epitopes, we discriminated epitopes from random peptides with an accuracy of 0.74 or more, regardless of the presenting MHC I molecules. Following these results, we have enhanced PCPS to a new version named iPCPS, enabling CD8+ T cell epitope prediction and we applied the tool to identify the entire Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) epitome.Proteasomal cleavage site predictions serve to enhance CD8dictions , 19, 20.+ T cells in the context of MHC I molecules results from cleavage by the immunoproteasome [n-grams models for such a task that were implemented in PCPS online tool. In cross-validation, PCPS immunoproteasome n-grams reached an MCC of 0.47. Here, we evaluated cleavage predictions by the default PCPS immunoproteasome model using a larger independent dataset consisting of 257 proteins encompassing 844 9-mer virus-specific CD8+ T cell epitopes . Moreover, judging by Mathews Correlation Coefficient, PCPS performance was overall better than that of NetChop . It is worth noting that we used the original PCPS n-gram models while NetChop models have been retrained in increasingly larger datasets (current version is 3.1), indicating that we already have enough data to capture proteasome cleavage sites.According to the results, summarized in Table + T cells only recognize peptides presented by MHC I molecules and prediction of peptide-MHC I binding is therefore the main basis for anticipating CD8+ T cell epitopes [+ T cell epitope discrimination [+ T cell epitopes. To that end, we tested the ability of the PCPS immunoproteasome model to distinguish our set of 844 CD8+ T cell epitopes by cleavage at their C-terminus in their source proteins with regard to random peptides generated from the same proteins. The results obtained at different cleavage site score thresholds are summarized in Fig. + T cell epitopes.CD8epitopes . Proteasmination . In partmination . Here we+ T cell responses  with regard to the human leukocyte antigen class I (HLA I) molecules known to restrict the CD8ymorphic  and the + T cell epitopes covering all HLA I molecules, which is of particular relevance when no peptides are available for developing HLA I-binding models. Following these results, we have enhanced PCPS to enable CD8+ T cell epitope predictions. In the next section we illustrate the usage of the improved version of PCPS, which we renamed as iPCPS.In sum, our results clearly show that the prediction of C-terminal cleavage sites in peptides by PCPS immunoproteasome model alone can serve to predict CD8n-grams. This option is still available in iPCPS  with different settings and compared them with those predicted by Grifoni et al. [+ T cell epitopes drop down to 1682  [https://www.uniprot.org/) after the accession numbers (ACN) provided by IEDB.We obtained virus-specific CD8e (IEDB) . We limin-grams models that were trained on fragments obtained upon peptides eluded from human MHC I molecules (proteasome models) and upon naturally restricted CD8+ T cell epitopes (immunoproteasome models). In this work, we selected the default immunoproteasome model, which was specifically trained on 8 residue peptide fragments, each including a cleavage site (P1 and P1′ residues). Peptide fragments included the 4 last residues of CD8+ T cell epitopes followed by 4 proximal residues that flank the C-terminus of the epitopes in the relevant source proteins. We compared PCPS cleavage site predictions with those provided by NetChop (https://www.cbs.dtu.dk/services/NetChop/) [We predicted proteasome cleavage sites using PCPS . PCPS imetChop/) . In NetCetChop/) .+ T cell epitopes following the approach described elsewhere [Th) of 0.5. This approach assumes that cleavage after the epitope C-terminus has to be favored with regard to other cleavage sites within the epitope . Briefly, by evaluating cleavage scores (Cs) on each epitope residue, cleavage sites defined by epitopes were classified as follows:Cs is ≥ Th.True positive (TP): The C-terminus Cs is ≥ Th and ≥ than that of the C-terminus.False Positive (FP): At least one internal Cs are < Th or < than that of the C-terminus.True Negative (TN): All internal Cs is < Th.False Negative (FN): The C-terminus We evaluated the performance of PCPS and NetChop immunoproteasome models on sequences containing CD8lsewhere , 25 and The performance of the predictions was then measured in terms of Sensitivity (SE), Specificity (SP), Accuracy (ACC) and Matthews Correlation Coefficient (MCC) using Eqs. + T cell epitopes was analyzed by determining their ability to discriminate viral CD8+ T cell epitopes from random 9-mer peptides. Random peptides were considered as non-epitopes and were selected randomly from the same protein sources than the CD8+ T cell epitopes at a 1:1 ratio. Cleavage sites predictions were carried over the protein sources containing both epitopes and randomly selected peptides. TPs, TNs, FPs and FNs were obtained at different cleavage thresholds  by examining the cleavage scores after the C-terminal residue of both CD8+ T cell epitopes (positive instances) and random peptides (negative instances). CD8+ T cell epitopes with a cleavage score above threshold were TPs and with cleavage scores below threshold were FNs. Conversely, random 9-mer peptides with a cleavage score below threshold were TNs and those with cleavage scores above threshold were FPs. The performance of the predictions was subsequently assessed by computing SE, SP and ACC, using Eqs. The capacity of C-terminal cleavage predictions provided by PCPS immunoproteasome model to anticipate CD8Additional file 1. Dataset with virus-specific CD8+ T cell epitopes.Additional file 2. SARS-CoV-2-specific CD8+ T cell epitopes predicted using iPCPS with different settings.