This study aims to clarify whether normobaric oxygen therapy improves the survival of auricular composite grafts in rats.2 composite grafts were harvested from bilateral ear regions including whole auricles. The harvested grafts were transferred caudally and sutured there. The 10 rats were randomly divided into two groups and kept for 21 days in two different circumstances. The first group (Control group: five rats carrying 10 grafts) was kept in room air (20% oxygen) throughout the 21 days, and the second group―named NBO (normobaric oxygen) group (five rats carrying 10 grafts)―was kept in normobaric 60% oxygen for 3 days and then in room air for 18 days. All the 10 rats were sacrificed on the 21st day. Surviving areas of the grafts and the height of the surviving auricular cartilage were examined for statistical comparison of the two groups. Furthermore, the conditions of chondrogenesis occurring around the perichondrium were compared between the two groups.For 10 male SD rats, 1.5 cmSurviving areas did not present statistically significant differences between the two groups. The height of surviving cartilage was significantly greater for the NBO group (2610 ± 170 SD µm) than that for the Control group (1720 ± 190 SD µm). Chondrogenesis occurred at positions more distant from the recipient bed in the NBO group than that in the Control group.Normobaric oxygen therapy increases the thickness of surviving cartilage in auricular composite grafting in rats, thus suggesting that NBO therapy may also be effective in composite grafting for humans. Free composite grafting is a surgical procedure in which different kinds of combined tissues are transplanted without vascular anastomosis. Plastic surgeons often use auricular composite grafting—consisting of cartilage, subcutaneous fat, and skin―in their clinical practices. The skin of the ear is similar to the skin of the face in texture and color. Furthermore, the inclusion of cartilage allows surgeons to form the graft into desired three-dimensional shapes to fit defects by using cartilage as a frameworkOn the other hand, graft survival is not necessarily reliable in auricular composite grafting. It takes 2–3 days for angiogenesis from the recipient bed into the graft to occur. Furthermore, the supply of oxygen to the graft depends solely on diffusion from the recipient bed to the graft. Hence, parts of the graft distant from the recipient bed cannot receive sufficient oxygen to survive, thus developing necrosis. Thus, oxygen supply regulates the size of the graft that can be safely transplanted.Therefore, while performing the reconstruction of defects above certain sizes with auricular composite grafting, efforts should be made to maximize the part of the graft capable of survival. Specific examples of clinical trials to improve outcomes by increasing oxygen supply include hyperbaric oxygen (HBO) therapy for diabetic feet10In the present study, we focus on normobaric oxygen (NBO) therapy. NBO therapy—putting patients under high-density oxygen at normal pressure—is conducted to improve metabolic effects by increased oxygen supply. NBO therapy is easier to apply clinically than HBO therapy.12When patients are put under NBO circumstances, oxygen supply to auricular grafts is expected to increase. Subsequently, a greater part of the graft is expected to survive. The present study aims to verify this hypothesis by experiments with rat models.The present study was conducted in accordance with the Declaration of Helsinki, under the approval and supervision of the Institutional Review Board of Kagawa University .Ten male SD rats weighing 300–350 g were included in the study. The rats were anesthetized by intra-abdominal injection of medetomidine hydrochloride (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol tartrate (5 mg/kg). Combined tissues consisting of a square-shaped section of skin and the whole ear were harvested. The length of the square's side was 15 mm. The grafts were removed from the head at the layer of the superficial fascia. Defects were made at bilateral scapular angles of each rat to match the shape and size of the harvested auricular grafts. The harvested grafts were transferred to cover the defects and sutured there with 4-0 Nylon suture .Figure 2Normobaric oxygen) group  group . All ratOn the 21st day after surgery, the rats were anesthetized, and photographs of the transplanted composite grafts were taken with a digital camera . The areas of surviving parts of the grafts were evaluated with graphics software  by counting the number of pixels included in the parts. Survival/necrosis of a part was judged by observing whether the tissues in contact with the recipient bed survived. Even if the overlying tissues of a certain part developed necrosis, the part was included in the surviving area when the underlying tissues―directly in contact with the recipient bed―survived .Figure 4The grafts were stained with Alcian blue and then sliced for histological observation. The as-produced slices were observed with an optical microscope image analysis software system . For each graft, the maximum distance of the surviving cartilage from the recipient bed was measured. This distance was defined as the height of surviving cartilage .Figure 5After staining the harvested grafts with anti-type 2 collagen antibody  and Anti-Aggrecan Antibody , cartilage cells that had generated around the perichondrium were observed and evaluated regarding the three items given below.The maximum distance of the point at which chondrogenesis occurred was measured .Figure 6The area of generated cells was measured.The locations of chondrogenesis were observed in terms of position between the regenerated chondrocyte and the grafted cartilage. To be more specific, whether regenerated cartilage cells existed above (the side away from the recipient bed) or below (the side facing the graft bed) was observed.t test by using analysis software . P values equal to or smaller than 0.05 were considered statistically significant.The above-stated items were compared between the Control (10 grafts in five rats) and NBO (10 grafts in five rats) groups. Statistical analyses were performed with Student's The photos of the grafts on the 21st day are shown in The height of surviving cartilage was significantly greater for the NBO group (2610 ± 170 SD µm) than that for the Control group (1720 ± 190 SD µm) .Chondrogenesis was observed around the perichondrium in six samples of the Control group and seven samples of the NBO group. Statistical comparison of these samples revealed the following findings.Chondrogenesis distance from the recipient bed did not present statistically significant differences between the Control group (1670 ± 276.0 SD µm) and the NBO group (1940 ± 172 SD µm) .2) and the NBO group (94100 ± 32400 SD µm2)  .In six of the 10 samples of the Control group, chondrogenesis occurred only on the recipient bed side. In contrast, in seven of the 10 samples of NBO group, chondrogenesis occurred on the contralateral side as well as the recipient bed side of the cartilage .Figure 8Auricular composite grafting is a useful surgical technique for the reconstruction of various defects of the face. However, the survival of the graft is unstable because the supply of oxygen to the graft depends on effusion from the recipient bed in the early stage after transplantation. Though the regions adjacent to the recipient bed can survive safely, those distant from the recipient bed can develop necrosis because of early-stage hypoxia. To achieve optimal results, efforts should be made to maximize the surviving part of the graft. We hypothesized that increasing the supply of oxygen to the graft with NBO therapy may extend the surviving part of the graft, and this was performed in the present study to verify this hypothesis.In clinical practice, HBO therapy is occasionally used to increase oxygen supply to tissues.In the present study, 60% oxygen was provided for the NBO group for 3 days. This period was defined considering a time-related shift in the mechanism providing oxygen to the graft. After angiogenesis occurs from the recipient beds into transferred grafts, the grafts can receive oxygen from the blood coming through the newly generated vessels. However, before angiogenesis, oxygen supply to the grafts is dependent on the influx from the recipient beds by diffusion. Angiogenesis starts 48 hours after transplantation. Hence, to assure oxygen supply to the graft in the early stage, high-density oxygen needs to be provided at least for 48 hours. In terms of treatment cost, on the other hand, the period of high-density oxygen provision should be minimized. Balancing these effect-versus-cost factors, the rats were placed in 60% oxygen for 3 days in this study. Better survival of the graft might have been achieved if 60% oxygen was provided for more than 3 days, which can be elucidated in future studies.The findings of the present study are summarized in terms of the following three issues.First, surviving areas of the grafts showed no statistical differences between the Control and NBO groups. Araki et al.―by studying the effects of NBO therapy on the survival and contracture ratios of composite grafts—reported that NBO therapy increased the survival ratio and decreased the contracture ratio.Second, the height of surviving cartilage increased by 1.5 times in the NBO group compared to that in the Control group. According to the previous study by Araki, the partial oxygen pressure of inguinal fat and subcutaneous tissue increased by 195% and 178%, respectively, under 60% oxygen in comparison to that under room air.Third, chondrogenesis occurred on both sides of the graft  in the NBO group, whereas it occurred only on the recipient bed side in the Control group. We hypothetically explain this phenomenon as follows.,In the zone close to the recipient bed, grafted tissues can receive sufficient oxygen supply and survive safely. Conversely, grafted tissues die because of the lack of oxygen in the zone distant from the recipient bed. These zones are temporarily defined as the safe zone and hypoxic zone, respectively. In the zone between these two zones, grafted tissues barely survive because of receiving a minimal amount of oxygen necessary to maintain metabolism. This third zone is defined as the border zone. Gómez-Leduc stated that the stress of ischemia induces the differentiation of mesenchymal stem cells into chondrocytes.Among the three main findings of the present study, the second finding—the height of surviving cartilage increased by NBO therapy—would appeal to reconstructive surgeons most because of its potential to innovate auricular composite grafting.,,,Auricular composite grafting is frequently used to reconstruct total or partial thickness defects of the eyelid caused by trauma or tumor removal.Naturally, the safety of NBO therapy―the problem of potential oxygen toxicity in particular―needs to be carefully considered before using NBO therapy for the given purpose. Our investigation revealed an old report stating that dizziness, tachycardia, and deterioration of vital signs appeared after application of 90% oxygen for 65 hours.p value of 0.17―a larger sample size could present a statistically significant difference. Hence, we hope that the present experiment is reproduced with a greater sample size by other researchers.The present study is limited by its relatively small sample size, because survival was evaluated with only 10 composite grafts. If the experiment is repeated with a greater sample size, more clinically meaningful results might be obtained. For instance, although there is no statistically significant difference in the area of chondrogenesis between the Control and NBO groups in the present study―with a Nevertheless, the effect of NBO therapy for auricular composite grafting in rats is demonstrated by the present study. We believe that this achievement will allow us to extend postoperative NBO therapy to auricular composite grafting for humans. Under the approval of the institutional review board, we will plan clinical trials in humans in the near future.None.


JFMK is a member of the Committee on Publication Ethics (COPE). To verify the originality of content submitted to our journals, we still use iThenticate to check submissions against previous publications. MDPI works with Publons to provide reviewers with credit for their work and MDPI Scitations Alert to provide our authors information on new publications in their research field. Kinesiology is the study of human movement or physical activity related to many sub-disciplinary areas, such as athletic training, biomechanics, exercise physiology, psychology, leisure, measurement, motor behavior, public health, and sociology.The Journal of Functional Morphology and Kinesiology , which was firstly released in March 2016 [We are proud to let you know that thanks to your continuous support, Journal of Functional Morphology and Kinesiology has continued to grow in the fields of morphology, kinesiology, movement, biomechanics, sport medicine, and musculoskeletal disorders. It is my pleasure to confirm the progress recorded in 2017 and 2018 .Indeed, the number of published manuscripts has jumped from 89 in the 2017 volume to 154 in the 2018 volume, with 16 scientific contributions for each issue, and we rejected in total 34 contributions to maintain the high standards of our journal. The Journal of Functional Morphology and Kinesiology receives more manuscripts than it is able to publish and the decision as to which papers are accepted or rejected is a difficult one. The decision is based on several factors, including originality, experimental design, scientific quality, data interpretation, clarity, and English quality, to maintain the high standards of our journal.In 2018, the sections of JFMK have been updated to eleven parts: Functional Anatomy; Kinesiology and Biomechanics; Sport Medicine and Nutrition; Motor Control and Rehabilitation; Athletic Training and Human Performance; Gait and Posture; Mechanobiology; Movement and Neurodegenerative Diseases; Musculoskeletal Disorders; Physical Exercise for Health Promotion; and Strength and Conditioning.In 2018, different special issues were activated thanks to the huge support of our editors. They include the following: “Resistance Training for Performance and Health”, edited by Prof. Dr. Antonio Paoli ; “ArrhytIn 2018, ten distinguished scientists joined the Editorial Board: Prof. Dr. John E. Kovaleski ; Prof. Dr. Paul Arciero ; Prof. Conrad P. Earnest ; Prof. Laura Capranica ; Assist. Prof. Dr. Helmi Chaabene ; Dr. Abdul Rashid Aziz ; Dr. Michael O. Harris-Love ; Dr. James Fisher ; Dr. Roland van den Tillaar ; and Dr. Cristina Cortis , for a total of 72 editorial board members, eight advisory board members, and the editor-in-chief.2018 ORS (Orthopedic Research Society) Annual Meeting, 10–13 March 2018, at the Hyatt Regency New Orleans, USA, ;The 23nd Annual Congress of the ECSS, Dublin, Ireland; 4–7 July 2018 ;ISBS 2018, Auckland, New Zealand; 10–14 September 2018 (banner advertisement in newsletter);International Conference on Kinesiology and Exercise Sciences, Athens, Greece; 30–31 July 2018 (keynote speaker);6th International Congress on Sport Sciences Research and Technology Support—icSPORTS 2018, Seville, Spain; 20–21 September 2018 ;72° Italian National Congress of SIAI , Parma ; 20–22 September 2018 ;X° Italian National Congress of SISMES , Messina ; 05–07 October 2018 .Journal of Functional Morphology and Kinesiology confirmed its status as an international journal by being present as a media partner, an exhibitor, or a sponsor at national and international meetings, among which were the following:For the first time this year we recognized the 2018 JFMK Travel Award. This award (600 CHF) is granted to Dr. Gavin Lenton, a postdoctoral research fellow under the supervision of Prof. David Lloyd from Griffith University. Dr. Lenton’s research focuses on musculoskeletal biomechanics. He will present his work at the 2018 World Congress of Biomechanics, which will be held on 8 July 2018, in Dublin, Ireland. It was a very difficult decision with such high-quality applications for the award, and we would like to thank all the applicants in various fields of study for their participation and all of the Award Committee members for their evaluation of the abundant excellent applications. We congratulate Dr. Gavin Lenton on his accomplishment. I take this opportunity to thank again the Evaluation Committee.In 2019, we shall continue our efforts to improve the journal through further growth and increased visibility. Follow up the planned papers from editorial board members;Contact international conferences recommended by the editor-in-chief or by editorial board members and try to establish media partnerships with them to make JFMK increasingly well-known among scholars;Communicate with editorial board members regularly and ask for their help and suggestions for journal development;Post high-quality papers through social media  and increase online readership;Reduce the processing time of each manuscript;Try to have publications indexed by the Emerging Sources Citation Index (Web of Science) and by EMBASE (Elsevier);Try to be included in the SCImago Journal Rank in the kinesiology-related section;Accomplish, for our authors, the best JFMK paper award and the JFMK travel grant award;Garner, for the sake of journal promotion, support from sponsors for our editors to participate in, and disseminate our journal to, international conferences.In order to achieve this target and lay a strong foundation for publications in 2019 and application for indexing, we have made the following plans:We hope that you share our enthusiasm for this new journal and we look forward to working with you to make JFMK a leader in its field. Your contributions are vital for the success of this new journal. We look forward to receiving your contributions  and proposals for special issues are always welcome.It is my pleasure to end this editorial by wishing you a healthy and prosperous new year. This is also the opportunity for me to warmly thank, for their confidence, the following: our authors, readers, and reviewers, as well as our editorial advisors, eminent scientists in these fields that with their experience and important suggestions, guide us in this great enterprise; our excellent editorial board members whose depth of experience covers a very broad spectrum on different disciplines related to morphology and kinesiology arenas; the managing editor Olivia Yu for her huge support, the publishing manager Martyn Rittman, the assistant managing editor Molly Lu, and assistant editor Sydney Tang, who day after day, thanks to their valuable contributions, ensure the growth of this journal; and, finally, all members of our teams in Basel, Barcelona, Beijing, Belgrade, and Wuhan, as well as our sponsors.


Journal of Biological Research—Thessaloniki in 2019—your generosity is much appreciated and we hope your association with the journal continues in the future.We would like to thank all those listed below for taking the time to review for the Editor-in-Chief: Theodore J AbatzopoulosAharon OrenAlessandro ChiarucciAlexandra StaikouAna Claudia Oliveira CarreiraAnargyros MoulasAndigoni MalousiAndriana C KalioraCamilla Vieira EstevesChristopher M. RoundyDavid G. HarrisonDimitris KardassisDimitris KontoyiannisEirini KanataEleftheria GalatouEleni DouniEleni NikolakakiElissavet GeorgiouEmanuela ChiarellaEudoxia HatzivassiliouFlorian GoedeckeGeorge MosialosGrigorios GeorgolopoulosIlias KappasIoannis A. GiantsisIoannis VizirianakisJuliette K. TinkerKarl DuffyKim B. SeroogyKimon MoschandreouKonstantina PsathaKonstantinos XanthopoulosKostas FeidantsisKostas KougioumoutzisMakoto NodaMaria G RoubelakisMaria LambropoulouMaria StarodubtsevaMaria TourakiMichal RahatMinas YiangouMohammad Reza JabalameliNadia ShabanNiel Van WykNikoleta KaraiskouPanayotis KaloyannidisSalih PasaSinos GiokasSonia LópezSpyridon GkelisSpyros TsiftsisSybille D. ReichardtVirginia Díez-ObreroWanning WangXian-Si ZengΜ. Doulberis


When standing, intrinsic ankle stiffness is smaller when measured using large perturbations, when sway size is large, and when background torque is low. However, there is a large variation in individual intrinsic ankle stiffness. Here we determine if individual variation has consequences for postural control. We examined the relationship between ankle stiffness, ankle torque and body sway across different individuals. Ankle stiffness was estimated in 19 standing participants by measuring torque responses to small, brief perturbations. Perturbation sizes of 0.2 & 0.9 degrees (both lasting 140 ms) measured short- and long-range stiffness respectively, while participants either stood quietly on a fixed platform or were imperceptibly tilted to reduce stability . The spontaneous body sway component  and background ankle torque were averaged from sections immediately before perturbations. The results show that, first, intrinsic ankle stiffness is positively associated with ankle torque, and that this relationship is stronger for long-range stiffness. Second, intrinsic ankle stiffness is negatively associated with body sway, but, in contrast to the relationship with torque, this relationship is stronger for short-range stiffness. We conclude that high short-range intrinsic ankle stiffness is associated with reduced spontaneous sway, although the causal relationship between these two parameters is unknown. These results suggest that, in normal quiet standing where sway is very small, the most important determinant of intrinsic ankle stiffness may be stillness. In less stable conditions, intrinsic ankle stiffness may be more dependent on ankle torque. Although posture is flexible and dependent on the coordination of all joints and muscles of the body, it can appear that the single point of the estimated whole body centre-of-mass (COM) is controlled as a means to achieve standing balance –5. PreviIn standing, the ankle joint holds particular interest due to its strategic position linking the long upright body with the horizontal feet, the only body surface normally able to exert gravitational counteractive torques against the ground. Standing demands active neural control of ankle torque because the intrinsic stiffness of the ankle joints is too low for even minimal stability , 16, 17.Intrinsic ankle stiffness has been measured by applying small, brief rotations to the ankle joint and analyzing the resultant torque change. By limiting the time window of the analysis, neural responses are excluded. Previous studies using this approach have revealed several clear tendencies. First, intrinsic stiffness depends on the size of the perturbation used to measure it–it increases as the measuring perturbation becomes smaller –21. Secobetween individuals have not been considered in detail. However a large range of intrinsic ankle stiffness in standing individuals has been reported  [As the main concern in previous studies was to identify the general properties of intrinsic stiffness, differences  torque) , 17–20. -1; mean±SD).The study was approved by the local human ethics committee at the University of Birmingham (ERN_15–0674). All participants gave a written informed consent. It took place at the School of Sport, Exercise and Rehabilitation Sciences, University of Birmingham, UK. Nineteen healthy adults, aged between 24 and 37 years were recruited for this non-invasive experiment Fig 1A). ® webbing at shoulder, waist and calf levels. We assumed that, as the board was rigid, its position would give a good estimate of the overall body rotation by minimizing any hip or knee motion. The recording of sagittal body rotation was taken from a laser-reflex sensor  pointing directly at the board (inverse tangent of linear data from laser-reflex sensor divided by laser height above the ankles), subtracted from foot rotation recorded by the precision Hall effect potentiometer located on the platform axis.We were focused on the properties of the ankles, so to reduce the participant’s use of hip and knee strategies a light wooden board  was strapped to the participant’s back with TeryleneFor the measurement of intrinsic ankle stiffness, small, brief raised-cosine type perturbations (140 ms duration) were applied to the footplates. The interval between them , and their direction (toes-up or toes-down) and amplitude (0.2 and 0.9 deg), were all randomized. Since we found no difference between toes-up and toes-down estimates of stiffness, these were combined.Intrinsic stiffness is the resistance to stretch when the ankle is rotated. It becomes greater as the size of the measuring perturbation is reduced so cannot be described by a single value –22, 24. Each trial period involved 64 perturbations (32 of 0.2 deg and 32 of 0.9 deg). The principle of calculating stiffness by decomposing torque responses to a perturbation is well established –20, 23. Intrinsic stiffness refers to the natural elastic resistance of the ankle joint to ankle rotation, assuming an unchanging level of muscle activity. It is a passive mechanical property of the muscles and tendons acting at the ankle joint. Passive does not imply that the musculature is relaxed, but that the level of activity is not altered by the nervous system. Conversely, the active mechanism is the alteration of the calf muscle activity by the nervous system.We examined the relationship between mean intrinsic stiffness, mean sway size and mean torque when the participant stood quietly in a normal attitude on a fixed platform. In separate trials we repeated these investigations when the platform was slowly and slightly sinusoidally tilted (either 0.2 deg or 0.4 deg) about the medio-lateral ankle axis.et al. [We expected that this would induce synchronous sway at the low frequency of the platform and decrease stability. Gurfinkel et al.  showed tTherefore, we studied the interrelationship between mean intrinsic ankle stiffness, mean sway size and mean torque in three conditions below:Normal: locked footplates;Sine 0.2: footplates were continuously oscillated by a 0.1 Hz sine waveform of 0.2 deg peak-to-peak amplitude;Sine 0.4: footplates were continuously oscillated by a 0.1 Hz sine waveform of 0.4 deg peak-to-peak amplitude.Two trials were conducted for each of the three conditions, totaling 6 trials. Another two trials without perturbations were recorded to normalize data between participants . Altogether, 8 trials of approximately 150 s were recorded from each participant. All participants were tested in one session of approximately 2 hours.θ = ankle angle (deg), -1), B = viscosity (Nm s deg-1) and I = moment of inertia of the foot (kg m2) are the variables estimated from the fitting equation. A 40 Hz low-pass Butterworth filter was applied to the very fast stiffness-measuring data to remove high frequency noise while preserving the integrity of the signal amplitude. The result was the mean intrinsic stiffness of the participant during the trial.The estimation of intrinsic stiffness (K) is described elsewhere . This caThere is minimal movement of body parts during normal standing. The range of ankle rotation is normally small enough that gravitational torque exerted by the body COM is very nearly linearly related to the COM rotation around the ankle joint –3, 31. Tversus body sway was influenced by baseline ankle torque. We averaged left ankle torque sections of 20 ms prior to each perturbation, after filtering with a 10 Hz low-pass Butterworth filter. This baseline ankle torque was multiplied by 2 to account for two ankles and divided by mgh to normalize it for height and body mass, then multiplied by 100 to obtain % estimates. As ankle torque in standing depends on the size and mass of the subject and on her/his forward angle of lean, normalized ankle torque is a value which is, in effect, the mean angle of forward lean of a participant during the trial.In addition to being dependent on the size of the perturbation used to measure it, intrinsic stiffness has been shown to be positively dependent on ankle torque , 22, 23.Sway consists of an apparently random fluctuation in body angle and angular velocity during standing. The mean time between stationary instants (points of zero velocity) shown in the literature is around 1 s . As the Statistical analysis was performed with RStudio: Integrated Development for R . Descriptive statistics (mean and standard deviation) were generated to describe the profile of the sample. Coefficient of variation (CV) was used to determine relative variability between participants in these different conditions. Two-way repeated measures ANOVA was used for each variable to verify if the conditions of increasing platform tilt  and increased perturbation amplitude (0.2 and 0.9 deg) induced significant changes in average intrinsic stiffness, postural sway and baseline torque. Pearson’s correlation coefficient (r) was used to verify the relationship between these 3 variables to answer the main question of this study, i.e. is sway related to stiffness and torque. Following suggestion by Cohen , we consIntrinsic ankle stiffness was measured using both small (0.2 deg) and large (0.9 deg) perturbations, during fixed platform and tilting conditions . There w-1, and for 0.2 deg perturbations, 5.0±0.4 deg s-1. It is a feature of biological tissues such as tendon and muscle that they exhibit non-Newtonian viscosity which is higher at high stretching velocities [Estimated viscosity was larger for larger perturbations, F = 107.9, p <.001, but remained similar across conditions, F = 0.01, p = .988. The interaction of these two factors was significant, F = 8.5, p <.010. Increase in viscosity for larger perturbations was expected because 0.9 deg perturbations were much faster than 0.2 deg perturbations, as the raised cosine-type perturbation always had a 140 ms duration. Peak velocity for 0.9 deg perturbations was on average 20.3±0.8 deg slocities .Baseline torque did not differ much between conditions , decreas-1 and 0.48±0.13 deg s-1, for 0.2 and 0.9 deg perturbation amplitudes, respectively) to sine 0.2 (0.50±0.13 deg s-1 and 0.51±0.12 deg s-1), and sine 0.4 conditions (0.54±0.14 deg s-1 and 0.54±0.14 deg s-1)  . The maiPrevious research has shown that people can have very different intrinsic ankle stiffness –20, 22. -1   . Most paine 0.4) . ParticiOur participants were all young healthy adults . To our satisfaction, even with this limitation, we found a large range of intrinsic ankle stiffness of 94% mgh difference between maximum (134% mgh) and minimum (40% mgh) values for short-range stiffness and 65% mgh difference (range 91–26% mgh) for long-range stiffness . When apWe fitted linear regression models between intrinsic stiffness and spontaneous sway velocity . These rTo investigate the relationship between intrinsic stiffness and baseline torque  we also et al. [The level of significance chosen in our results (0.05%) is naturally arbitrary, so an economical interpretation of Figs et al.  have shoet al.  have recet al.  showed tThe data we present Figs –4 descriThese results show that individuals with small spontaneous sway velocity tend to have high short-range intrinsic stiffness. Torque has a smaller and less significant effect on short-range intrinsic stiffness. A possible explanation is that the short-range stiffness of the ankle is limited by compliance of connective tissues. Numerous in vitro and in vivo experiments have shown the existence of high short-range stiffness in skeletal muscle tissue. That is, as a pull is applied to a muscle tendon unit, the muscle length changes very little and the series component extends. Somewhat counter-intuitively, at relatively small tensions, the muscle is much stiffer than the tissues which transmit the forces to the load. A muscle tendon unit cannot be stiffer than its most compliant part so the intrinsic stiffness will reflect the stiffness of the connective tissue. This explains why increasing torque (which will stiffen the muscle by increasing the number of cross-bridges) cannot greatly increase short-range ankle stiffness. Even if the muscle becomes completely rigid the stiffness cannot rise above the limiting value of the series component.  . This coIn accordance with previous studies we show that there is a wide range of intrinsic ankle stiffness in different people and that some people spontaneously sway more than others. We show that in normal, minimally disturbed, standing there is an inverse correlation between the short-range intrinsic ankle stiffness of our participants and their spontaneous sway velocity. When intrinsic stiffness is measured with larger perturbations or while the participants are destabilized by tilting its magnitude is decreased and the correlation with stasis destroyed. Participants with higher baseline torque tend to have higher intrinsic stiffness.


Scientific Reports 10.1038/s41598-018-36551-5, published online 22 January 2019Correction to: The original version of this Article contained an error in the Abstract.“Cell velocity in the highly metastatic population shows a relative insensitivity to matrix stiffness”.now reads:“Cell velocity in the highly metastatic population shows a relative stability at higher matrix stiffness”.Additionally, this Article contained an error in Figure 2, where panel (c) used incorrect units.Furthermore, this Article contained errors in the Results section.“At lower stiffness of 10 kPa and 40 kPa, the more metastatic B16 F10 had a significantly higher dry mass growth rate of 1092 pg/hr and 1157 pg/hr, respectively, as compared to the less metastatic B16 F0, which had a dry mass growth rate of 641 pg/hr and 930 pg/hr, respectively (p = 0.00086). At the higher stiffness of 100 kPa, B16 F0 had similar dry mass growth to the lower stiffnesses at 880 pg/hr , whereas B16 F10 decreased to 740 pg/hr (p = 0.27).”now reads:“At lower stiffness of 10 kPa and 40 kPa, the more metastatic B16 F10 had a significantly higher dry mass growth rate of 109.2 pg/hr and 115.7 pg/hr, respectively, as compared to the less metastatic B16 F0, which had a dry mass growth rate of 64.1 pg/hr and 93.0 pg/hr, respectively (p = 0.00086). At the higher stiffness of 100 kPa, B16 F0 had similar dry mass growth to the lower stiffnesses at 88.0 pg/hr , whereas B16 F10 decreased to 74.0 pg/hr (p = 0.27).”“On average, the more metastatic B16 F10 cells had similar VDW of 2.43 µm/hr, 2.28 µm/hr, and 2.06 µm/hr at 10 kPa, 40 kPa, and 100 kPa, respectively, indicating that stiffness has little influence on B16 F10 migration velocity . In contrast, stiffness plays a pronounced role in the migration profile of the less metastatic B16 F0. While B16 F0 had a VWD of 1.74 µm/hr and 1.56 µm/hr at the lower stiffnesses of 10 kPa and 40 kPa, VDW increased to 2.01 µm/hr at 100 kPa.”now reads:“On average, the more metastatic B16 F10 cells had VDWs of 5.2 μm/hr, 4.0 μm/hr, and 3.5 μm/hr at 10 kPa, 40 kPa, and 100 kPa, respectively, indicating that lower stiffness has some influence on B16 F10 migration velocity . As well, stiffness plays a role in the migration profile of the less metastatic B16 F0. While B16 F0 had a VWD of 2.5 μm/hr and 3 μm/hr at the lower stiffnesses of 10 kPa and 40 kPa, VDW increased to 3.5 μm/hr at 100 kPa.”Finally, this Article contained an error in the Conclusion section.“In addition, high metastatic potential corresponds with higher migration profiles, as determined by the velocity width distribution, which was relatively insensitive to changes in stiffness.”now reads:“In addition, high metastatic potential corresponds with higher migration profiles, as determined by the velocity width distribution, which did not increase with increasing stiffness.”These errors have now been corrected in the PDF and HTML versions of the Article.


Asian/Pacific Island Nursing Journal would like to thank the following people who served as peer-reviewers for the journal from January 2020 through December 2020. Without your support the journal could not exist.The Editor-in-Chief of Kamomilani Anduha WongRichard ArakakiUmanthesani BalakrishnanRuel BillonesKathryn BraunMirella BrooksHa Do ByonRose ConstantinoChad CrossAlona Dalusung-AngostaKathryn DaubFelicitas dela CruzGabriel GarciaArtemis GonzalesNaoko HikitaEun-Ok ImKamalnand SinghIra Kantrowizt GordonMerle Kataoka-YahiroCarina KatigbakMasashi KawanoMay KealohaSera Kim SunadaPauline LasqueteHeeyoung LeeWen-Wen LiNada LukkahataiMiguel Luzviminda BanezAngelina NguyenConnie Nguyen-TruongDonna Marie MalakikoMary Frances OnehaMisty PachecoAndrew ReyesKarol RichardsonReiko SakashitaLeorey SaliganTakuichi SatoHiroaki SatohReimund SeraficaChiraporn TachaudomdachHatsumi TanigiuchiLillian Tom-OrmeJenny TsaiJohnny YaoJenny Zhong


The assessment of spinal stiffness by manual palpation in clinical settings has demonstrated both poor accuracy and reliability. More recently, mechanical methods for assessment of spinal stiffness have demonstrated superior accuracy and reliability. However, mechanical methods of spinal stiffness assessment can be expensive, time consuming and/or unsuited to clinical practice. While a new device has been designed to address these issues (VerteTrack), its benchtop performance remains unknown.To measure the bench-top performance of VerteTrack.A series of laboratory-based experiments were conducted in February 2018 to investigate the accuracy (precision and bias) of load and displacement measurements obtained by VerteTrack and then were compared against an appropriate reference standard. Measurements of both multiple-level continuous assessment , and single-level assessment  were performed on a viscoelastic foam medium  and the resulting stiffness calculated.p < .001), and no systematic measurement bias for measured versus reference displacement . The magnitude of stiffness obtained during multiple-level continuous assessment was on average 0.25 N/mm (2.79%) less than that for single-level assessment .VerteTrack demonstrated high precision at all loads and displacements. There was minimal systematic measurement bias identified for applied versus reference load  under bench-top conditions. The difference in stiffness found between multiple versus single spinal levels should be considered in the research context, but is unlikely to be clinically relevant. The results of this study demonstrate that VerteTrack may be suitable for both single and multi-level spinal stiffness measurements in-vivo. Manual therapists’ commonly use manual spinal stiffness assessment (MSSA) to guide diagnosis and treatment decisions for patients with non-specific spinal pain , 2. In M3, k 0.95–100) and good to excellent between-day reliability  in the clinical setting [VerteTrack is a new device developed to address the limitations of existing mechanical devices that quantifies bulk measurement of spinal stiffness . A novel setting , however setting , 25. TheThis was a laboratory-based accuracy study conducted in February 2018 . Experimwww.stepperonline.com, China) . These loads were selected as they represent loads that have previously been used in VerteTrack studies [The VerteTrack frame  suspends an aluminium gantry that supports movement of a rolling indenter head (RIH) in three axes: X-axis , Y-axis  and Z-axis  via stepper motors  Fig. . A strin studies , 23, 26  studies , 20. Pla studies .Fig. 3AThe VerteTrack can perform two modes of indentation testing: single-level and multiple-level continuous indentation. Single-level indentation assesses a single spinal level and requires the operator to position the RIH directly above the target tissue. Loads are then applied incrementally to the spine in a posterior to anterior direction with the resulting deformation of the spinal tissues recorded (Z-axis displacement). Multiple-level continuous indentation requires the operator to first identify the spinal trajectory that the RIH will travel within the horizontal (X-Y) plane. This is achieved by manually aligning each spinous process  with the RIH using the embedded laser pointer. The laser points are memorised by the device and then replayed to move the RIH continuously along the same pre-defined trajectory for each successive load. The resolution of the RIH is identical to the resolution of stepper motors (0.007 mm).2). Displacement precision  of the VerteTrack was also estimated using coefficient of variation over 10 repetitions at each of 6 discrete levels of the RIH on a custom-engineered wooden wedge to simulate tracking of a spinal sagittal curve  of the VerteTrack was estimated by the coefficient of variation (CV = SD / load mean) over 10 repetitions for each load. The RIH was measured using recently calibrated digital scales   was measured ten times. Displacement bias was also estimated using the same method employed to determine load bias. Mean displacement bias was determined over 10 repetitions at each of 6 discrete levels as reported by the VerteTrack, compared to an external digital calliper   was estimated by comparing each load delivered through the VerteTrack against the same load externally. Mean load bias was estimated by calculating the differences between reference loads and loads measured by the VerteTrack, and the 95% confidence interval of the difference . Referenmm) Fig. , panel bA method-comparison experiment was conducted to evaluate the performance of VerteTrack for measurement of stiffness during multiple-level continuous and single-level (reference) modes of operation. Terminal stiffness values (i.e. the ratio of the maximum load to the maximum displacement)  were usemultiple) and single-level indentation (Stiffnesssingle) at three discrete locations  on the medium. Incremental loads (plates) were added to the RIH in a predefined sequence . Between each trial, 90 s elapsed to allow for any residual deformation to resolve. Between each cycle , an additional 5 min elapsed to allow any residual deformation to resolve after the maximum load was applied to the medium. A total of ten cycles were performed.Precision of the VerteTrack during measurement of stiffness on the test medium was estimated by the coefficient of variation (CV = SD / stiffness mean) over 300 trials for both single-level and multiple-level continuous indentation. Stiffness was measured during multiple-level continuous indentation  was reported for load and displacement. LinCCC tests both agreement and linearity [Each trial for Stiffnessinearity . The strinearity . Alpha wVerteTrack) were obtained beginning with no load 16.557 N  then increasing by the addition of single masses. The coefficient of variation (CV) ranged from 0.03 to 0.27% depending upon the applied load (Table Six measures relating to RIH loading (LoadVerteTrack) beginning at a baseline value of 60.03 mm  then increasing to 12.08 mm . The CV ranged from 0 to 0.32% depending upon the level of the wedge (Table Six discrete RIH displacements were then measured (Displacementref) ranged from 27.757 N  to 71.461 N . There was a statistically significant (p < .001) systematic mean bias for the VerteTrack load (LoadVerteTrack), compared to the reference load (Loadref) of − 0.123 N   as measured by the digital calliper ranged from 12.03 mm (95%CI: 11.98 to 12.08 mm) to 60.08 mm (95%CI: 60.02 to 60.13 mm). There was no statistically significant (p = .001) systematic bias for the VerteTrack displacement (DisplacementVerteTrack) as compared to the reference displacement (Displacementref)  , and moderate to high loads as 49.583 N, 60.687 N and 71.461 N (3–5 plates). The terminal stiffness values for low load conditions ranged from 6.09 to 8.81 N/mm, and moderate-high load conditions yielded stiffness values ranging from 5.70 to 6.38 N/mm. Under low load conditions, the terminal stiffness grand mean was 7.43 N/mm. As for moderate to high load conditions, the terminal stiffness grand mean was 6.03 N/mm  at three locations on the AIREX balance beam, CV was calculated for each load (Table single ranged from 2.0 to 2.3% and Stiffnessmultiple ranged from 1.4 to 3.2%.To determine the precision of stiffness measured by the VerteTrack (Stiffnessad Table . The CV multiple) and single-level indentation (Stiffnesssingle) were compared at three discrete locations  on the AIREX balance beam. There was a negative systematic bias for Stiffnessmultiple, compared to Stiffnesssingle of − 0.25 N/mm  , which is essential to establish internal validity of the VerteTrack. Both single-level and multiple-level continuous indentation modes demonstrated high levels of precision and agreement, despite a small negative systematic bias for multiple-level continuous compared to single-level indentation . It is unclear if this difference is clinically relevant as there is currently no published data to support a minimal clinically important difference (MCID) for the assessment of spinal stiffness, nor standards for different indentation modes , 11. MorThe plot of raw single-level and multiple-level continuous stiffness values demonstrated that higher stiffness values were obtained under low loads compared with moderate-high loads where stiffness remained at approximately 6 N/mm Fig. . This isThis study was performed on a viscoelastic foam medium, without the presence of physiological properties known to influence spinal stiffness  , 11. AlsIt is unclear whether the use of mechanical spinal stiffness measurement devices in a clinical setting would aid in the patient’s diagnosis, prognosis, treatment, or clinical outcomes. There are inconsistencies in the literature regarding the relationship between pain, disability and spinal stiffness, however emerging research into the sub-grouping of patients into responders and non-responders to spinal manipulative therapy have yielded promising results . MechaniThe Vertetrack demonstrated good bench-top performance through high precision, linearity, and low systematic bias compared to reference values. When combined with recently published clinical reliability data, the VerteTrack demonstrates high levels of internal validity. The novel multiple-level continuous indentation mode offers potential for increased time efficiency in future clinical trials without compromise in stiffness measurement or patient comfort.Additional file 1: Figure S1. Panel a) The Bland-Altman plot demonstrates a statistically significant bias (p < .001) for loads delivered by the VerteTrack compared to the calibration sample . Open circles (50 data points) represent the magnitude of bias (N) = Loadref - LoadVerteTrack. Panel b) The Bland-Altman plot demonstrates no statistically significant bias (p = .001) for displacement as measured by the VerteTrack compared to a digital calliper . Open circles (60 data points) represent the magnitude of bias (mm) = Displacementref - DisplacementVerteTrack.Panel c) The Bland-Altman plot demonstrates a statistically significant (p < .001) negative bias for multiple-level continuous vs. single-level stiffness, of 0.25 N/mm . Open circles (150 data points) represent the magnitude of bias (N) = Stiffnessmultiple - Stiffnesssingle. Legend: RIH – Rolling indenter head, d0 – displacement 0, d1 – displacement 1, d2 – displacement 2, d3 – displacement 3, d4 – displacement 4. Figure S2. Panel a) Lin’s Concordance Correlation Coefficient for VerteTrack load vs. the reference sample to demonstrate almost perfect agreement . Open circles (50 data points) represent co-ordinates  at loads . Panel b) Lin’s Concordance Correlation Coefficient for VerteTrack displacement vs. the digital calliper demonstrated an almost perfect agreement . Open circles (60 data points) represent co-ordinates  for each wedge level (d0–d5). Legend: RIH – Rolling indenter head, d0 – displacement 0, d1 – displacement 1, d2 – displacement 2, d3 – displacement 3, d4 – displacement 4.


The surface roughness and water contact angle of the zirconia surfaces were measured. The tetragonal-to-monoclinic (t–m) phase transformation and residual stresses (sin2ψ method) were studied by X-ray diffraction. The resin-bonded zirconia specimens were subjected to shear bond strength (SBS) tests before and after thermocycling  (n = 10). As the particle size and pressure increased, the roughness gradually and significantly increased (p = 0.023). However, there were no significant differences in roughness-corrected contact angle among all the sandblasted groups (p > 0.05). As the particle size and pressure increased, the m-phase/(t-phase + m-phase) ratios and compressive residual stresses gradually increased. After thermocycling, there were no significant differences in SBS among the sandblasted zirconia groups (p > 0.05). In conclusion, increased surface roughness and residual stress do not directly affect the resin bonding durability.The influence of residual stress induced by sandblasting the zirconia ceramic surface on the resin bonding to the ceramic is still unclear. The effect of four different sandblasting conditions  on the bonding of adhesive resin cement (Panavia F 2.0) to zirconia (Cercon In dentistry, the introduction of computer-aided design/computer-aided manufacture (CAD/CAM) systems significantly increased the use of all-ceramic restorations . ZirconiUnlike silica-based ceramics, zirconia is a silica-free polycrystalline ceramic, which makes it resistant to traditional hydrofluoric acid etching and silane coupling agent treatments . The bonSandblasting involves impacting the substrate surface with hard particles at high velocities, thereby eroding the material and leaving a roughened surface with high wettability . In the Residual stress can affect the bond strength of the coating layer on a substrate . Yang an® ht, DeguDent, Hanau, Germany) were prepared by sintering following the manufacturer’s recommendations and polished up to a 1 μm diamond suspension. Excluding for the control (polished surface) group, sandblasting was carried out on the zirconia surfaces with two different sizes of alumina particles  of 50 and 110 μm [A total of 210 rectangle-shaped (10 × 10 × 2 mm) zirconia plates (Cercond 110 μm  and two n = 1). In addition, the chemical composition of the surfaces was verified with energy dispersive X-ray spectroscopy .The zirconia surfaces were examined after gold coating with scanning electron microscopy  at a magnification of 1000× and an accelerating voltage of 10 kV (Ra) and average peak-to-valley height (Rz) of the zirconia surfaces were measured with a profilometer . The stylus speed was 0.1 mm/s, the cutoff was 0.8 mm, and the range was 600 μm. The values for each zirconia specimen were recorded as the average of three readings (n = 5).The average roughness (n = 5). The CAs were subjected to roughness correction according to Wenzel’s equation, which states the relation between the measured CA (θm) and Young’s CA (θY) [θm = r cosθY, in which r denotes the ratio between the rough and the smooth surface areas of the specimen and can be calculated from the equation: r = 1 + Sdr/100, where Sdr refers to the developed interfacial area ratio. The Sdr values of the zirconia surfaces were measured using a phase shift interferometer in the goniometer.CAs of water droplet (5 μL volume) on the zirconia surfaces were measured with a CA goniometer  ( CA (θY) : cosθm =n = 1). Spectra were obtained for 2θ between 25° and 100°. Cu Kα with wavelength λ = 1.5406 Å was employed as the radiation source.The t–m phase transformation of the polished and sandblasted zirconia surfaces was investigated by XRD   reflection of the zirconia specimens. The diffraction angle 2θ = 62.5° was chosen and a linear elastic distortion of the crystal lattice plane was assumed. The stress producing the strain was calculated from the strain versus sin2ψ plots by considering the gradient of the line and the elastic properties of the zirconia, using the following equation [φσ is the single stress acting in a chosen direction, E is the modulus of elasticity in MPa, v the Poisson’s ratio, (hkl) the Miller indices describing a family of crystalline planes, d0 the unstressed lattice spacing, and φ the angle between a fixed direction in the plane of the specimen and the projection in that plane of the normal of the diffracting plane. The lattice spacing φψd is a linear function of sin2ψ.XRD was also employed to measure the residual strains induced by polishing and sandblasting for known orientations relative to the zirconia surfaces. The sinpecimens , in whicn = 30 per sandblasting condition) were subdivided into three groups depending on the thermocycling conditions (n = 10). The adhesion procedure was carried out using the notched SBS test [® 20i, Ivoclar Vivadent, Schaan, Liechtenstein). The bonded specimen was then removed from the button mold. Only one resin cylinder was prepared on each zirconia specimen [The polished (control) or sandblasted  zirconia specimens  until failure occurred (crosshead speed = 1.0 mm/min) . Bond stThe surface roughness, CA, and SBS data did not meet the assumption of equal variances (Leven’s test). Thus, the Kruskal–Wallis test, followed by the Mann–Whitney test with significance level adjustment using the Benjamini and Hochberg method, was used for the statistical analysis of the data, using SPSS 17.0 for Windows  at α = 0.05.Ra and Rz) and water CAs (before and after roughness correction) for the polished and sandblasted zirconia specimens are summarized in Ra and Rz values. As the alumina particle size and pressure increased, both the Ra and Rz values gradually and significantly increased (p = 0.023). The measured CAs significantly decreased after sandblasting (p = 0.042) and gradually decreased by increasing the abrasive particle size and pressure. The 110M-4B group showed significantly lower CA than the 50M-2B and 50M-4B groups (p = 0.029). After roughness correction, however, there were no significant differences in CA among all the sandblasted groups (p > 0.05).The surface roughness values . At 0 cycles, the 50M-4B and 110M-4B group values were significantly higher those of the 50M-2B and 110M-2B group (p < 0.05). At both 10,000 and 30,000 cycles, however, there were no significant differences in SBS among the sandblasted zirconia groups (p > 0.05). The polished zirconia surfaces showed only adhesive failure, irrespective of the thermocycling condition. Before thermocycling, the distribution of the failure modes for the sandblasted surfaces was 20–30% adhesive failures and 70–80% mixed failures. No cohesive failure within the resin cement was observed. After thermocycling, all sandblasted zirconia specimens exhibited exclusively adhesive failure after debonding.The purpose of the present study was to investigate the effect of four different sandblasting conditions (two blasting particle sizes and two pressures) on the resin bonding to zirconia ceramic in terms of residual stress. Based on the findings, the first null hypothesis, that the different sandblasting conditions would not result in different residual stresses of the zirconia surfaces, was rejected . The resIt is known that the sandblasting of zirconia conditions the surface to increase the roughness, as well as to clean and activate the surface . HoweverSdr values of the surfaces [The surface roughness values of the zirconia specimens were dependent on the sandblasting pressure as well as the alumina particle size . Likewissurfaces . After rThe XRD analysis showed that the m-phase newly formed on the sandblasted zirconia surfaces increased with an increase in particle size and blasting pressure . The shaThe combination of mechanical and chemical procedures is essential to the efficacy of the resin bonding procedure to zirconia . BisphenSandblasting of zirconia increases the surface roughness and thus also the area for mechanical interlocking, resulting in acceptable micrometer scale roughness . At the Strong micromechanical interlocking can only be achieved when a resin luting material effectively penetrates and flows into the microretentions created on the sandblasted zirconia surface . HoweverIn this study, the effect of four different sandblasting conditions (two blasting particle sizes and two pressures) on the resin bonding to zirconia was investigated in terms of residual stress. The residual stresses created on the zirconia surfaces directly depended on the aggressiveness of sandblasting. However, such increased surface roughness and residual stress did not affect the bonding durability of the adhesive resin cement with relatively high viscosity. Although sandblasting is favorable for improved resin bonding to zirconia, it can affect the mechanical properties of the ceramic due to accelerated t–m phase transformation, which may put stress on the surfaces and cause fatigue in the structure that could lead to premature and catastrophic failure . Therefo


In vitro digestibility test showed that RS content of the rice cultivars and HCN was in the range of 85.5 to 99.5%. The RS extracted from the rice cultivars and HCN showed significant difference in the apparent amylose content (AAC), ranging between 31.83 to 40.68% for rice and 45.79% for HCN. Water absorption capacity (WAC), swelling and solubility index of RS ranged from 112–133.9%, 5.28–7.25 g/g and 0.033–0.044 g/g, respectively. The rice RS granules were polyhedral and irregular shaped with granular length in the range of 4.8–5.9 µm. The HCN RS granule morphology showed smooth surfaced, round, elliptical, irregular and oval shapes with average granular length of 21 µm. Pasting behavior also varied significantly between rice RS and HCN RS with later showing the lower values of pasting properties. Thermal properties  and ΔH gel also varied considerably between the rice RS and HCN RS, wherein the highest values for peak gelatinization temperature and gelatinization enthalpy were seen for CH-1039. X-ray diffraction pattern of rice RS and HCN RS showed the characteristic A type of pattern in consonance with cereal starches.In this study resistant starch (RS) was extracted from five Himalayan rice cultivars and Indian Horse chestnut (HCN) using porcine pancreatin enzyme following which it was subsequently characterized for its physicochemical, structural and functional properties.  For nutritional purpose, starch is mainly classified into rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) depending upon its rate of digestion2. RS is one of the naturally occurring functional carbohydrates accounting to about more than 30% of the fiber content in foods3. RS is defined as the sum of starch and its degradation products which escapes the normal route of digestion but undergoes fermentation by the intestinal microflora, thereby results in the formation of short chain fatty acids (SCFA) such as acetic acid, butyric acid and propionic acid that are thought to protect against large bowel cancer besides possessing varied health benefits to the colon by reducing pH and increasing regularity4. The functional properties of RS has been acknowledged for the control of lifestyle syndromes like obesity, diabetes and subsequently, for reducing the risk of cardiovascular diseases5. RS is further sub classified into four major types based on its source and processing conditions2: RS1, RS2, RS3 and RS4. RS1 is physically inaccessible or entrapped starch and is mainly confined to the tough coatings of whole unprocessed cereal grains and legumes. RS2 is inaccessible to enzymatic action due to its crystalline nature such as high amylose maize starch (HAM). Both RS1 and RS2 belong to the category of native RS. RS3 is formed when starch rich foods are cooked and cooled during food processing applications by which the starch undergoes gelatinization and subsequent retrogradation thereby rendering the starch less soluble. RS4 refers to the category of starches that are chemically modified by various techniques to resist the normal score of digestion process such as starch esters, cross linked starches etc2. Rice (oryzea sativa) serves as an important and prime dietary source to over half of the world’s population. Rice with its high starch content is of particular interest for extraction of RS as it will implement in the dietary prevention of obesity, diabetes and cardiovascular diseases, similarly Indian horse chestnut (Aesculus indica) with about 38.3% starch content6 also serves as an important source for extraction of RS.The growing importance for functional foods has led to an intense awareness among individuals for healthy foods. Starch being one of the most important food components serves as an efficient and prime dietary source of energy contributing 60–70% of the total energyThough several researchers have developed the RS from rice and HCN starches by using physical, chemical and enzymatic modifications, no systematic study has been done so far on the extraction of natural RS from rice and HCN. Therefore, the objective of this study was to extract and characterize the natural RS from the five Himalayan rice cultivars and HCN.Five rice cultivars viz. SK-46, SK-338, SK-406, CH-1039 and CH-1007 were procured from SKUAST-K. The seeds of Indian Horse Chestnut (HCN) were harvested from the trees of Botanical garden, Department of Botany, University of Kashmir, Hazratbal, Srinagar, India, 190006. Seeds were dehulled and stored at refrigeration temperature until further use. Glucose oxidase/peroxidase reagent (GOPOD) was purchased from Megazyme International, Wicklow, Ireland. Pancreatin, amyloglucosidase and α-amylase were obtained from Sigma-Aldrich, St. Louis, USA. All other reagents used in the study were of analytical grade.7.Starch was extracted from five rice cultivars and HCN by the alkaline steeping methodBriefly 4 g starch (dry weight basis) and 160 mL of enzyme mixture were added, mixture was vortexed and incubated in a shaking water bath 37 °C for 16 h (200 strokes/min) to hydrolyze the digestible starch . At the end of incubation period, the suspension was mixed with absolute ethanol (160 mL) and vortexed to deactivate the enzymes. RS was recovered as a pellet by centrifugation . Pellet was washed twice with 50% ethanol to remove the digested starch. The RS was dried in an oven at 40 °C.8.The extracted RS was analyzed for moisture (925.10), protein (920.87), fat (920.85) and ash (923.03) according to the methods of AOAC, 19909.Apparent amylose content (AAC) of RS samples was determined by the colorimetric method10. Briefly 4 mL of α-amylase (10 mg/mL) containing amyloglucosidase (3 U/mL) was added to 100 mg of each RS sample, mixture was vortexed followed by incubation at 37 °C in a shaking water bath (200 strokes/min) for 16 h to hydrolyze digestible starch. Then absolute ethanol (4 mL) was added to deactivate the enzymes, followed by centrifugation at 1,500 × g for 10 min. The pellet obtained was washed twice with 50% ethanol to remove the digested starch. The sediment was dissolved in KOH  by vigorous stirring for 20 min. The solution was neutralized with sodium acetate buffer . Then 0.1 mL of amyloglucosidase (3300 U/mL) was added followed by incubation for 30 min at 50 °C. The samples were centrifuged at 3000×g for 10 min. GOPOD (3 mL) was added to aliquots (0.1 mL) of the supernatant and incubated at 50 °C for 20 min. Absorbance was measured at 510 nm using a spectrophotometer. RS was calculated as the amount of glucose × 0.9.RS content was determined by the approved AACC method 32–40 et al.8.Swelling & solubility index was determined according to Ashwar et al.8.Water absorption capacity (WAC) of RS was determined according to Ashwar Pasting properties of RS samples were measured using a Rapid Visco Analyzer . RS was dispersed in water . The dispersion was equilibrated at 50 °C for 1 min, followed by heating to 95 °C at the rate of 12.2 °C/min, held at 95 °C for 2.5 min, cooled to 50 °C at the rate of 11.8 °C/min and again held at 50 °C for 2 min. A rotating paddle (160 rpm) was used throughout the entire analysis, except for rapid stirring (960 rpm) for the first 10 s to disperse the sample.The thermal properties of RS samples were studied with a Differential Scanning Calorimeter . 3.5 mg of RS sample and 8 µL of deionized water were weighed in a platinum pan. The sample was heated from 20 to 200 °C at 10 °C/min. An empty platinum pan was used as a reference.The RS samples were placed on an adhesive tape attached to an aluminum specimen stub. After coating with gold-palladium, the samples were scanned at 5 kV in a scanning electron microscope .−1 at room temperature.Spectra of RS samples were recorded using ATR-FTIR system  in the range of 4000–400 cmX-ray diffraction pattern of the RS samples was recorded using X-ray diffractometer  with a wavelength of 0.154 nm. The diffractometer was operated at 40 kV and 35 mA. The diffractograms were obtained at 25 °C over a 2 Ø range of 4–40° with a step size of 0.02 and sampling interval of 10 s. The relative crystallinity (RC) of RS samples was calculated by the equation:Data reported are the averages of triplicate observations. Analysis of variance (ANOVA) with a significance level of 5% was applied and Duncan’s test was used to determine the differences between means with the help of commercial statistical package (SPSS 16.0).Moisture content of the RS samples ranged from 4.20% (SK-406) to 6.73% CH-1007) (Table 007 (Tabl11 and the AAC of native HCN was found to be 30.1512. The huge increase in AAC of RS samples might be due to the action of amyloglucosidase and amylase enzymes on the α-(1–6) linkages of amylopectin molecules during the extraction of RS13. Similar inferences were reported in literature14. However higher amylose content (46.78–50.18%) was reported in resistant starch of elephant foot yam15. Differences in the amylose content of different rice starches have been reported to be influenced by the botanical source of starch, climatic and agronomic conditions, cultivar, starch isolation procedures and analytical methods17. The activity of the enzymes involved in starch biosynthesis may also be responsible for the variation in amylose content among the various starches18.The AAC of the rice RS samples varied significantly (p ≤ 0.05) with the values ranging from 31.83% (SK-338) to 40.68% (CH-1007), however the RS extracted from HCN showed the highest value of AAC (45.79%) Table . The AAC19. Besides AAC, various other factors were suggested to affect RS content like molecular association between starch components, degree of crystallinity, and amylose-lipid complexes20.The RS contents (%) of five rice cultivars also varied significantly (p ≤ 0.05) with the values ranging from 85.54% (SK-338) to 92.75% (SK-46), however HCN showed the highest value for RS content (99.5%). Higher RS contents of CH-1007, SK-46 and HCN were due to their higher amylose contents. Several reports also showed a positive correlation between the amylose and RS contents20. The gels formed from these crystalline regions are weak and have greater tendency towards retrogradation because of the covalent bonds15. The swelling power of rice RS ranged from 5.28 g/g (SK-46) to 7.25 g/g (SK-338) whereas solubility index ranged from 0.033 g/g (SK-46 and CH-1007) to 0.040 g/g (CH-1039)  which could be probably due to its highest amylose content, as swelling is primarily a property of amylopectin24 plus some water absorbing pentosans that may have remained during the extraction process. The swelling and solubility index was found to be influenced by various factors like variation between different cultivars, topographical conditions, starch isolation methods, degree of debranching, length of chains, conformation of the molecules and the ratio of amylose and amylopectin molecules27.The swelling power indicates the capacity of the starch granules to uptake water at elevated temperature. Starch granules with greater crystalline areas along with strong covalent interactions in the crystalline regions usually show little swelling power in cold water than subsequent heating21 and intensive association of hydroxyl groups in between starch chains resulting in the formation of covalent and hydrogen bonds28. However HCN showed the highest value of WAC (133.9%) among all the RS samples. The highest WAC value of HCN may be due to its highest amylose content that results in higher water uptake and loose compaction of amylose and amylopectin29. The WAC of starch is greatly dependent on the source, content of amylose/amylopectin, extraction procedure and thermal stability31.Water absorption capacity represents the ability of the starch molecules to associate with limited water addition. Among the five rice RS samples, SK-338 showed the highest value of WAC (114.3%) whereas SK-46 showed the lowest value 112%)  lowest value of pasting temperature (70.07 °C). Trough viscosity of rice RS ranged from 636 cP (SK-338) to 888.5 cP (SK-406), however trough viscosity was considerably lower for HCN (180.5 cP). Final viscosity of rice RS ranged from 1412 cP (SK-46) to 2021.7 cP (CH-1007), however HCN RS showed lowest final viscosity (239) cP. The final viscosity is the measure of the ability of the starch to form a viscous paste and is because of the re-association of amylose and amylopectin molecules39. Lowest breakdown viscosity was seen for SK-46 (169.0 cP) and highest for CH-1007 (482.5 cP), similarly setback viscosity was seen to range from 636 cP (SK-338) to 1351 cP (CH-1007). Due to low amylose content in SK-46 and SK-338, breakdown and setback viscosities were substantially low; similarly the highest setback in CH-1007 was attributed to its highest amylose content. The disintegration of swollen starch granules results in BD (breakdown), while the variations of BD among rice starches may be attributed to the differences in rigidity of swollen granules. SB shows the extent of recovery of starch viscosity during cooling of the heated starch pastes, and is usually related to the AAC of starch41. The pasting properties of the rice and Horse chestnut resistant starches were lower than their corresponding native starches42 that could be because of the enzymatic hydrolysis of starch which includes increased formation of short linear chain molecules and increased RS content which could be substantiated to the decrease in the pasting viscosity along with the reduced ability of forming gels43.Pasting properties indicates the behavior of starch granules that ultimately determines the functionality and cooking quality of starch0, Tp and Tc) and enthalpy of gelatinization (ΔHgel) varied significantly (p ≤ 0.05) between the rice and Horse chestnut RS. In general CH-1039 and SK-406 showed the highest peak gelatinization temperature while as the lowest value was shown by SK-338, probably because of its lowest amylose content. Previous reports have also related the gelatinization temperature of starch to its amylose content45. The highest peak gelatinization temperature of CH-1039 recorded by the DSC was also consistent with its high pasting temperature obtained from the RVA data. CH-1039 shows highest value for ΔHgel (370.87 J/g). This increase in the enthalpy of gelatinization has been attributed to the compact physical structure because of lower amylose and the higher quantity of amylopectin as it forms a dense, highly ordered structure46 hence more energy is needed to disrupt intermolecular hydrogen bonds linking adjacent chains or helices within starch structure47. The differences in T0, Tp and Tc among the rice resistant starches may be attributed to differences in AAC, granular architecture, molecular weight distribution and structure of amylopectin molecules48. The T0, Tp and Tc of HCN were 40.89, 75.23 and 121.06 °C. The high gelatinization temperature of HCN might be because of its higher AAC, wherein amylose restricted the hydration of amorphous regions, hindered the gelatinization of crystalline areas and raised the gelatinization temperature.Thermal properties of rice and Horse chestnut RS samples are presented in Table 42. The morphology of starch granules depends on the biochemistry of chloroplast or amyloplast, as well as the physiology of plant48. Some reports claimed that the variation in size and shape of starch granules of different plant sources is attributed to biological origin49, however several other studies suggested that starch biosynthesis results in natural variability in amylose and amylopectin molecules that might be responsible for diversity of starch granules50. Other factors responsible for starch granule diversity are climatic conditions, agronomic factors and processing51.Scanning Electron Microscopy (SEM) images of the rice and HCN resistant starches are shown in Fig. −1 attributed to hydrogen bonded O-H stretching vibration; the sharp band ranging from 2924–2927 cm−1 attributed to C-H bond stretching associated with the ring methane hydrogen atoms; the band ranging from 1559–1650 cm−1 ascribed to H-O-H bending vibration; the band from 1339–1363 cm−1 attributed to the bent modes of O-C-H, C-C-H, and C-O-H, the bands at 1075 cm−1 and 1147–1148 cm−1 had both been assigned as the coupling of C-O, C-C and O-H bonds and the band at 1175–1140 cm−1 has been assigned to the C-O-C glycosidic bond52. The C-O-H bending modes observed at 1047 and 1022 cm−1 have been used to express the crystalline and amorphous structures of starch, respectively54. The FTIR analysis of HCN RS showed close results with the reported values for native horse chestnut starch12. There was also no remarkable band difference among the RS extracted from five rice cultivars suggesting that the basic structure of RS samples is similar which is being reflected in their similar absorption bands.The FTIR spectrum of the rice and horse chestnut RS samples is shown in Fig. 55. The XRD pattern of horsechestnut RS also showed the same characteristic peaks at 15° and a feeble peak at 23° which is in conformity with the results reported in literature56. The relative crystallinity of the rice RS varied from 16.75 (SK 406) to 17.72% (SK 46) however the lowest relative crystallinity was shown in horsechestnut (13.90%). The relative crystallinity of all rice and HCN RS were lower than the native rice and HCN56. This might be because of their higher amylose contents as amylose disrupts the crystalline packing of amylopectin57.The X-ray diffraction patterns of rice and HCN RS samples are presented in Fig. In vitro digestibility test showed that RS content ranged significantly between the rice cultivars and HCN, ranging between 85.5 to 92.7% for rice and 99.5% for HCN. The RS extracted from the rice cultivars and HCN showed significant difference in the apparent amylose content (AAC), ranging between 31.83 to 40.68% for rice and 45.79% for HCN. Water absorption capacity (WAC), swelling and solubility index of RS ranged from 112–133.9%, 5.28–7.25 g/g and 0.033–0.044 g/g, respectively. The rice RS granules were polyhedral and irregular shaped with granular length in the range of 4.8–5.9 µm. The HCN RS granule morphology showed smooth surfaced round, elliptical, irregular and oval shapes with average granular length of 21 µm. Pasting properties also varied significantly between rice RS and HCN RS with the later showing lesser values. Thermal properties  and ΔH gel also varied considerably between the rice RS and HCN RS, wherein the highest values for peak gelatinization temperature and gelatinization enthalpy were seen for CH-1039. X-ray diffraction of rice and HCN RS showed the A type of crystallinity pattern. Physicochemical and structural parameters of the rice and HCN RS successively revealed the potential of the extracted RS to be used for various functional purposes. HCN might serve as an efficient source for RS extraction as presently it goes waste. It was also observed from the above study that HCN has high amylose content and low swelling power than rice RS hence best suited to maintain the texture of foods.In this study RS was extracted from the five Himalayan rice cultivars and HCN using porcine pancreatin enzyme following which it was subsequently characterized for its physicochemical and structural properties.


Multiple HBV-derived T cell epitopes have been reported, which can be useful in a therapeutic vaccination strategy. However, these epitopes are largely restricted to HLA-A*02, which is not dominantly expressed in populations with high HBV prevalence. Thus, current epitopes are falling short in the development of a global immunotherapeutic approach. Therefore, we aimed to identify novel epitopes for 6 HLA supertypes most prevalent in the infected population. Moreover, established epitopes might not all be equally effective as they can be subject to different levels of immune escape. It is therefore important to identify targets that are crucial in viral replication and conserved in the majority of the infected population. Here, we applied a stringent selection procedure to compose a combined overview of existing and novel HBV-derived T cell epitopes most promising for viral eradication. This set of T cell epitopes now lays the basis for the development of globally effective HBV antigen-specific immunotherapies. in vitro assay to validate their HLA-binding capacity. Using this method, a total of 13 HLA binders derived from HBx and 33 binders from Pol were identified across HLA types. Subsequently, we demonstrated interferon gamma (IFN-γ) production in response to 5 of the novel HBx-derived binders and 17 of the novel Pol-derived binders. In addition, we validated several infrequently described epitopes. Collectively, these results specify a set of highly potent T cell epitopes that represent a valuable resource for future HBV immunotherapy design.Immunotherapy represents an attractive option for the treatment of chronic hepatitis B virus (HBV) infection. The HBV proteins polymerase (Pol) and HBx are of special interest for antigen-specific immunotherapy because they are essential for viral replication and have been associated with viral control (Pol) or are still expressed upon viral DNA integration (HBx). Here, we scored all currently described HBx- and Pol-derived epitope sequences for viral indispensability and conservation across all HBV genotypes. This yielded 7 HBx-derived and 26 Pol-derived reported epitopes with functional association and high conservation. We subsequently predicted novel HLA-binding peptides for 6 HLA supertypes prevalent in HBV-infected patients. Potential epitopes expected to be the least prone to immune escape were subjected to a state-of-the-art IMPORTANCE Multiple HBV-derived T cell epitopes have been reported, which can be useful in a therapeutic vaccination strategy. However, these epitopes are largely restricted to HLA-A*02, which is not dominantly expressed in populations with high HBV prevalence. Thus, current epitopes are falling short in the development of a global immunotherapeutic approach. Therefore, we aimed to identify novel epitopes for 6 HLA supertypes most prevalent in the infected population. Moreover, established epitopes might not all be equally effective as they can be subject to different levels of immune escape. It is therefore important to identify targets that are crucial in viral replication and conserved in the majority of the infected population. Here, we applied a stringent selection procedure to compose a combined overview of existing and novel HBV-derived T cell epitopes most promising for viral eradication. This set of T cell epitopes now lays the basis for the development of globally effective HBV antigen-specific immunotherapies. Chronic hepatitis B virus (CHB) infection affects roughly 250 million people worldwide  and is aImmunotherapy had already emerged in the 1990s as a promising option to treat CHB. T cell responses are considered essential for viral clearance but are scarce or exhausted in CHB patients –7. Still–8–in vivo. Others have explored strategies to predict HLA-I epitopes from Pol but focused exclusively on a single HLA type or assessed only a limited number of HBV sequences  or core antigen (HBcAg). However, the proteins X (HBx) and polymerase (Pol) also pose interesting targets, as both are vital for viral persistence –27 and i–30–equences –40. TakeIn addition to a limited epitope repertoire, there is another hurdle in the development of HBV-directed immunotherapy. Established epitopes might not all be equally effective, as they can be subjected to different levels of viral mutagenesis and subsequent immune escape. Indeed, previous reports clearly demonstrate that HBV is subject to immune pressure and that mutation of epitope sequences leads to immune evasion –43 or ev–46–Here, we have taken an effort to tackle the above-mentioned issues by integrating viral indispensability, genomic variation, HLA binding, and immunogenicity to identify the best HBx- and Pol-derived T cell epitopes for immunotherapy across 6 of the most prevalent HLA supertypes within the HBV-infected population. The results of this study pave the way for the development of globally effective HBV antigen-specific immunotherapies.n = 14) and Pol (n = 50)  , left, w , left, (n = 50)  Fig. 1,,n = 14 a = 50) 4648, 52–69We argued that epitopes in which the least conserved amino acid was still present in 80% of all sequences tested would be targetable in the majority of the population. Combining this criterion with the preference for functional association, we found 7 HBx-derived and 26 Pol-derived sequences reported as epitopes across HLA types to be preferential targets for global immunotherapy . We first predicted binders spanning 8 to 14 amino acids for supertype-representative HLA types using the established in silico prediction tool NetMHCpan to make a frequency distribution of predicted binders (P = 0.57 by a Mann-Whitney test). Predicted binders spanning 9 to 11 amino acids were subsequently aligned to our maps outlining conservation and function .The prediction yielded totals of 251 potential novel HLA binders for HBx and 1,655 for Pol , right, ed assay . For praed assay . For HLAin vitro binding assay. Peptides were classified as HLA binders when their binding capacity was higher than 25% of that of a known high-affinity peptide (Table S4). HLA-A*11:01 and HLA-A*03:01 were both tested as members of the HLA-A*03 supertype since many HBV-infected patients are Asian and HLA-A*11:01 is more prevalent in this population than the supertype representative HLA-A*03:01, which is more prevalent in Caucasians  from blood donors who had previously resolved an HBV infection were expanded in the presence of peptide pools, followed by single-peptide restimulation and an interferon gamma (IFN-γ) enzyme-linked immunosorbent assay (ELISA). As expected, IFN-γ production was detected in response to the well-established epitopes c18-27 and p549-557 . FurtherThe aim of this study was to rationally address two major hurdles in developing a generic antigen-based immunotherapy for HBV: (i) the lack of prioritization of epitopes in further studies toward clinical implementation and (ii) the shortage of non-HLA*02-restricted epitopes.To address the first issue, we ranked all currently described HBx- and Pol-derived epitopes according to conservation and association with viral indispensability. Conservation patterns were similar to those previously reported, with the most conspicuous observation being that the spacer domain of Pol is extremely variable , 42, 73.Four Pol-derived epitopes were more frequently described in acute or resolved infection than in chronic infection. Of these, p756-764 and p573-581 scored the highest in conservation. p756-764 contains at least 1 amino acid important for viral persistence, but no functional relevance has been described for p573-581. Since the DNA sequence coding for p573-581 completely overlaps that of HBsAg, we interrogated the literature for the functional relevance of amino acids in this overlapping part of HBsAg. However, no functional association was reported 7375. Altho76–Although we rationalized to prioritize conserved peptide sequences, we reckon that regions containing prevalent sequence variation could still be interesting if immunogenicity is preserved. Novel T cell responses may arise due to cross-reaction between the variant and the original sequence , 81. Thi+ epitopes, while HBx seemed more subject to immune-pressure-induced deletion of epitopes  vaccines. SLPs can be designed to harbor both HLA-I and HLA-II epitopes and are processed more efficiently by dendritic cells than whole proteins types covering the vast majority of the HBV-infected population that target the virus where it is most vulnerable. Collectively, the results of this study provide a valuable resource to guide future development of HBV-specific immunotherapies.n = 8,127) and Pol . Positions where a gap (indicated by “−”) was most frequent were deleted, after which the dominating amino acid at each position was determined. Percentages of sequences containing the dominant amino acid were calculated as the conservation score. Combining all dominant amino acids for Pol led to the consensus sequence MPLSYQHFRKLLLLDDEAGPLEEELPRLADEGLNRRVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFPDIHLQEDIINRCQQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKPYYPEHVVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGRLVFQTSKRHGDESFCSQSSGILSRSPVGPCIQSQLKQSRLGLQPQQGSLARRQQGRSGSIRARVHPTTRRSFGVEPSGSGHIDNSASSSSSCLHQSAVRKAAYSHLSTSKRQSSSGHAVELHNIPPSSARSQSEGPVFSCWWLQFRNSKPCSDYCLSHIVNLLEDWGPCTEHGEHHIRIPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGNTRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPHLLVGSSGLSRYVARLSSNSRIINNQHGTMQNLHDSCSRNLYVSLLLLYKTFGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSYMDDVVLGAKSVQHLESLYTAVTNFLLSLGIHLNPNKTKRWGYSLNFMGYVIGSWGTLPQEHIVQKIKQCFRKLPVNRPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQAKQAFTFSPTYKAFLCKQYLNLYPVARQRPGLCQVFADATPTGWGLAIGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKLIGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDPSRGRLGLYRPLLRLPFRPTTGRTSLYAVSPSVPSHLPDRVHFASPLHVAWRPP.A frequency table was downloaded from HBVdb V42.0 , 51 baseThe resulting consensus sequence for HBx was determined to be MAARLCCQLDPARDVLCLRPVGAESRGRPLSGPLGTLPSPSPSAVPADHGAHLSLRGLPVCAFSSAGPCALRFTSARRMETTVNAHQVLPKVLHKRTLGLSAMSTTDLEAYFKDCVFKDWEELGEEIRLKVFVLGGCRHKLVCSPAPCNFFTSA.in vitro binding assay as described below.These sequences were loaded into NetMHCpan3.0  to prediin vitro binding assay as described previously (Synthetic peptides (Peptide 2.0 Inc.) of selected potential HLA binders were used in an eviously . In briein vitro HLA binding assay were assessed for immunogenicity. Briefly, PBMCs were isolated by Ficoll  density centrifugation from buffy coats of 9 donors who had previously resolved HBV infection. Buffy coats were provided by the local blood bank with corresponding 2-digit HLA types. Four-digit HLA typing was performed for 7 out of 9 donors using the global screening array (GSA)  (Peptides with >25% binding in the tterdam) . All donP value was <0.05. For the few epitopes with a response in at least 25% of HCC patients, patient groups could not be compared due to the lack of data for CHB and acute hepatitis patients.The Hepitopes initiative previously performed an extensive literature search of the Medline and Embase databases to collect all HBV-derived HLA-I epitopes from 112 papers , 49. All55–