Periplakin (PPL) is a main member in plakin family, which plays important role in cellular adhesion complexes supporting and cytoskeletal integrity supplying. PPL was reported to be a potential biomarker candidate for several types of cancers. However, the biological functions and underlying mechanisms of PPL in ovarian cancer (OV) remain unclear. In the present study, we used GEPIA 2, Human Protein Atlas, Oncomine, LinkedOmics, Kaplan–Meier Plotter, STRING, CytoHubba plug-in and TIMER to determine the associations among PPL expression, prognosis, and immune cell infiltration in OV. RT-qPCR and IHC analysis were conducted to validated the role of PPL in an independent OV cohort. Compared with the normal ovary tissues, the levels of PPL mRNA and protein expression were both obviously higher in OV tumors from multiple datasets (P < 0.05), and a poor survival was observed to be strongly correlated with high PPL expression (P < 0.05). Moreover, the results were further validated by RT-qPCR and IHC analysis in an independent OV cohort. A gene-clinical nomogram was constructed, including PPL mRNA expression and clinical factors in TCGA. Functional network analysis suggested that PPL participates in the important pathways like Wnt signaling pathway, MAPK signaling pathway. Ten hub genes  were identified to be positively associated with PPL. Furthermore, PPL expression was negatively correlated with infiltrating levels of CD4+ T cell, macrophages, neutrophils, and dendritic cells. In conclusion, PPL may be an unfavorable prognostic biomarker candidate in OV, which was also correlated with immune infiltrating and function in immunotherapy response.The online version contains supplementary material available at 10.1007/s12672-022-00496-z. Ovarian cancer (OV) carries a very poor prognosis as a progressive and aggressive gynecological malignancy . It rankPlakin is a large family of proteins, of which many members play important roles in cellular adhesion complexes supporting and cytoskeletal integrity supplying in different tissues . In cancIn this study, our team checked the potential prognostic value of PPL in OV for the first time based on the data from the Cancer Genome Atlas (TCGA) and multiple public databases. The functional networks related to PPL in OV patients were evaluated using GEPIA 2, Human Protein Atlas, Oncomine, LinkedOmics, Kaplan–Meier Plotter, STRING, CytoHubba plug-in and TIMER analysis. RT-qPCR and IHC analysis were further conducted to validated the role of PPL in an independent OV cohort. Moreover, we developed and validated a nomogram based on the expression of PPL and clinical factors for patients with OV to better predict the prognosis of patients. Results might provide some insights in potential targets and therapeutic strategies for OV patients.The PPL mRNA expressions from TCGA and Genotype-Tissue Expression (GTEx) projects were compared using the GEPIA 2 initially. The obvious differences were observed in Fig. high expression presented the significant shorter OS compared with the ones with PPLlow expression  was 0.867. We evaluated the OS using Kaplan–Meier method and log-rank test, and the result showed that OS of the PPLhigh expression group was significantly shorter than the PPLlow expression group  and infiltrating levels of neutrophils and macrophages cells Fig. a. Then, According to public data, the PPL expression in OV tumor was demonstrated to be significantly higher than normal ovary in this study. The higher PPL expression was, the poorer the clinical outcomes of OV patients would be, and vice versa. After that, an independent OV cohort was carried out to further validate these results. Based on PPL expression, nomogram risk score in combination with clinical characteristics was used for predicting prognosis in OV patients, which is a potential OS predicting method. What’s more, PPL expression also owned a significant correlation with immune infiltration level in OV.As a molecular bridge for cells, plakin family members could link cell–cell junctions and intracellular cytoskeleton . BesidesPPL function network was also conducted to explore the PPL co-expression and related signaling events, thus we could further reveal the underlying mechanisms of PPL in OV pathogenesis. Totally, 502 PPL expression-correlated essential genes were identified via STRING analysis, indicating that the proteins are at least partially biologically connected, as a group. Further, the ten hub genes  were identified to be positive associated with PPL. Of them, LAMC2, LAMA3, LAMB3, and LAMA5 are the members of laminin family. Dysregulated cell laminin interactions were major features of multiple cancers . A recenFurthermore, for the first time, the OV patients with higher PPL expression presenting poorer prognosis were observed from multiple cohorts and the results were validated in independent 42 OV patients. Thus, determination of PPL expression in surgical specimens of OV could aid in identifying and predicting the prognosis. What’s more, the PPL prognostic landscape was visualized in pan-cancer via public databases. Higher PPL expression also shown a correlation with poorer prognosis in uterine corpus endometrial carcinoma, pancreatic ductal adenocarcinoma, as well as bladder cancer, while the low expression of PPL was correlated to poor survival in ACC, SARC, and breast cancer, kidney renal papillary cell carcinoma, et al. PPL might play different roles in different types cancer. Besides, the heterogeneity of patients provided by TCGA dataset, particularly too many varieties on the patients’ treatment plan, might contribute the inconsistence.OV immunotherapies have been attracted increasing interest based on the results that improved clinical outcomes were associated with tumor infiltrating lymphocytes , 31. ThuFrom above all, our research studied the clinical implications of PPL expression in OV for the first time. High PPL expression was proved to be a potential unfavorable biomarker for OV. What’s more, multiple cohort studies also validated its prognostic value in pan-cancer. A potential correlation between immune cells and PPL expression implied that PPL might own the potential of crucial function in tumor immune microenvironment. However, there were several limitations in this study. First, although the study involved a bioinformatic analysis of PPL, there were no in vivo or in vitro experiments to validate the results. Therefore, future researches aiming at exploring underlying mechanism of PPL associated signaling pathways are required in further experiments. Moreover, an inadequate number of OV patients in the validate cohort enrolled in this study may be another limitation. And more precise study like prospective and multi-center study with bigger sample size should be carried out in the future to further verify these results.In this study, we explored the prognostic implications of PPL in OV patients for the first time. Our data suggested PPL might be an unfavorable independent prognostic biomarker candidate in OV. Moreover, our results suggested that PPL co-expression genes in OV might likely have far-reaching effects in pathway like Wnt signaling pathway, MAPK signaling pathway. PPL was also correlated with immune infiltrating and might play an important role in immunotherapy response.http://gepia2.cancer-pku.cn)  to Periplakin  was applied to detect PPL protein in the tissue samples. The predicted location for PPL was considered in intracellular. Specifically, the definition of strong, moderate, weak and negative staining was nuclear staining of > 75%, 25%-75%, < 25% and no nuclear staining of cells, respectively.R software v3.6.3  was applied for all statistical analysis. Pearson's chi-square test or Fisher's exact test was applied for analyzing qualitative variables. Quantitative variables analyses were performed using Wilcoxon rank-sum test (for unpaired samples). ROC was constructed using the survivalROC package in R and the optimal cut-off point was determined with the maximal sensitivity and specificity. The Kap Supplementary fig. 1. The comparison of the transcriptional level of genes from Plakin family in ovarian cancer (OV) based on TCGA and GTEx database from GEPIA2. (a) DSP (b) EVPL (c)HLA-DRB1 (d)PLEC (* P < 0.01).Supplementary file 1 (TIF 1130 KB) Supplementary fig. 2. Correlations of PPL expression with AKT1 transcriptional level in ovarian cancer (OV) patients from GEPIA2.Supplementary file 2 (TIF 859 KB) Supplementary table 1. A total of 502 PPL expression correlated essential genes were identified from Linkedomics.Supplementary file 3 (XLSX 42 KB) Supplementary table 2. The clinicopathologic data for the ovarian cancer patients from TCGA cohort.Supplementary file 4 (XLSX 382 KB)


Anterior cruciate ligament (ACL) injuries are among the most common lesions in orthopaedics practice, but still today, rates up to 24% of unsatisfactory results are reported. Unaddressed anterolateral complex (ALC) injuries have been claimed to be responsible for residual anterolateral rotatory instability (ALRI) after isolate ACL reconstruction and have demonstrated to increase graft failure. In this article, we present our technique for reconstruction of the ACL and anterolateral (ALL) ligament combining the advantages of the anatomical position and the intraosseous femoral fixation to ensure anteroposterior and anterolateral rotational stability. This video demonstrates our surgical technique for combined anatomic anterior cruciate ligament (ACL) and antero-lateral ligament (ALL) reconstruction. The knee is positioned, and examination under anaesthesia is performed confirming high-grade pivot shift. Medial and lateral landmarks are signed. A small anteromedial incision is performed, and semitendinosus and gracilis tendons are harvested using an open stripper. Care is taken to leave the distal insertion of both tendons intact. The two tendons are then whipstitched together at both extremities with high-resistance wires. Diameter is measured, and preparation of the graft is completed. On the lateral aspect of the knee, lateral femoral epicondyle, Gerdy’s tubercle and fibular head are identified and marked. A 3-cm incision is performed just posterior to the lateral epicondyle. Fascia lata is incised, and the anatomical femoral insertion of the anterolateral ligament is identified, proximal and posterior to the lateral epicondyle. With the help of an assistant, using an outside-in technique, a guide pin is positioned from the femoral insertion of the ALL to the femoral footprint of the ACL, and a tunnel of appropriate diameter is drilled. Tibial tunnel is then drilled using a standard technique. After positioning of the screw wire in the femoral socket, the graft is shuttled with two graspers through the tibial and femoral tunnels. After cyclic loading, femoral fixation is performed with the knee at 30° of flexion and neutral rotation. Lateral collateral ligament is identified and isolated, with the help of a curved forcep. The graft is then shuttled beneath the collateral ligament, from posterior to anterior. This can be done directly or with the help of a shuttling wire. A small vertical incision is performed, halfway between the Gerdy’s tubercle and the fibular head. With the help of a needle, the anatomic tibial insertion of the ALL is identified, about 10 mm below the articular surface. Fluoroscopy can be used for confirmation. The graft is passed underneath the fascia latae. Fixation of the graft in the tibial anatomical ALL insertion is performed with a low-profile staple, keeping the knee in full extension and neutral rotation. Finally, proper tension of the graft is demonstrated. If additional tension is required, the tibial portion of the ACL can be pulled using the distal whipstitch, and the tibial fixation can be added using an interference screw. Anterior cruciate ligament (ACL) injuries are among the most common lesions in orthopaedic practice, with an estimated incidence in the United States (US) ranging from 30 to 78 per 100,000 person/years.,Even if ACL reconstruction is considered by many inexperienced surgeons to be a “simple procedure”, still today, only 82% of patients are able to return to sport. When considering return to preinjury level (63%) or competitive sports (44%) percentage are even lower,,,,Even if there is still some debate on the existence and function of the anterolateral ligament (ALL), numerous authors demonstrated the ability to consistently identify this structure in nearly all analyzed specimens using specific anatomical dissection technique.,,Moreover, biomechanical studies confirmed that the anterolateral complex (ALC) acts as an important anatomical constraint against tibial internal rotation.Although different techniques have been proposed for reconstruction of ALL, consensus on which is to be considered the best is lacking, even if it is well accepted that anatomical reconstruction is to be addressed whenever possible.The patient is placed supine in a standard arthroscopic position with a lateral leg holder and a vertical post at the level of the thigh, just proximal to the knee. Leg holder is positioned in a way that allows nearly full flexion of the knee during the entire procedure. An inflatable tourniquet is placed at the base of the thigh.GR and ST are harvested using an open-ended stripper and measured, after removing muscular tissue. Total length of the graft of at least 16 cm is required to perform this procedure.The tendons are whipstitched together near the distal insertion  and at the free proximal end with a high-resistance nonabsorbable suture  .Fig 1GraAfter arthroscopic evaluation and eventual treatment of associated intraarticular lesions, femoral and tibial tunnel are drilled.A 3-cm stab incision is performed, centred on the lateral epicondyle, along the course of the fascia lata fibers. After making a small incision along the fibers of the fascia lata, the insertion of the ALL is identified just proximal and posterior to the lateral epicondyle, as previously describedThe outside-in guide for the femoral tunnel is placed so that the intra-articular part is positioned at the Isometric, Direct Fibers, Eccentric, Anatomic, Low Tension.The tibial tunnel is then drilled using the proper guide aiming for a tunnel angled ∼20° medially in the axial plane and 55° inferior in the sagittal plane. Guide pin is inserted, anatomical position is arthroscopically checked, as well as potential extension impingement; then, the tunnel is drilled at the proper diameter matching the graft dimension.The graft is grasped from the free end and pushed into the articulation from the tibial tunnel. An endoscopic grasper is then passed from outside into the femoral tunnel to grasp the proximal loop of the graft that is then pulled outside the knee. With the knee at 30° of flexion and neutral rotation, the graft is slowly pulled from the femoral side and fixed with an interference screw matching the dimension of the tunnel. At the end of the procedure, proper tensioning of the ACL portion of the graft is arthroscopically probed both at 90° and in midflexion. If additional tension is required, the tibial portion of the ACL graft can be pulled by the distal whipstitch, and tibial fixation can be added .Fig 3TenA vertical small incision is performed on the anterolateral aspect of the tibia, halfway between Gerdy’s tubercle and the fibular head, centred at a point ∼10 mm below the articular surface. The LCL is identified and, with the help of a curved dissecting forcep, the graft is passed underneath it. This is a unique feature of the present technique, aiming to obtain a structure that minimizes lateral compartment overconstrain, as reported by recent biomechanical comparison testsThe graft is then passed from the femoral tunnel to the tibial insertion, beneath the fascia latae.Tibial anatomical insertion can be checked with fluoroscopy, if required. After exposing the tibial bony surface, the graft is fixed with a low-profile staple at proper tensioning with the knee in full extension and neutral rotation .The knee is kept in a brace for the first 4 weeks, allowing 0 to 90° flexion for the first 2 weeks and then complete motion. A partial progressive weight-bearing protocol was adopted, aiming to reach full weight bearing in 2 weeks. Early rehabilitation is begun focusing on quadriceps isometric strength and activation and gaining full extension; then it progresses in a specialized centre with a standard protocol for ACL rehabilitation. Gradual return to sport is allowed starting from 5 months for nonpivoting sport and at 6 to 9 months for pivoting and contact sports. This study aimed to present our personal technique for combined reconstruction of ACL and ALL with single-stranded autologous GR and ST using a single tibial and femoral tunnel.,,,The presence of an ALL ligament was first hypothesized in 1879 by Segond, who identified the “so-called” avulsion fracture, but for many years, this anatomical structure remained largely underestimated. After consistent anatomical demonstration of the presence of this ligament,Different authors described similar techniques using a 3-folded ST and single-stranded GR for combined reconstruction of ACL and ALL.With our technique, the correct femoral anatomical position of the ALL can be consistently reproduced by pointing the external entry point of the out-in femoral guide just proximal and posterior to the lateral epicondyle. In addition, the use of the same femoral socket adopted for the ACL reconstruction avoids any risk of femoral tunnel collision and saves time, making the whole procedure faster.,,This position has demonstrated to reproduce the correct nonisometry of the ALL that is tight in extension and internal rotation at 20° and loose in flexion at 90° and 120°, as reported by different authors, and accepted by ALL Expert Group Consensus.In addition, a unique characteristic of our technique is the passage of the graft beneath the LCL, which prevents flexion overconstraint, as previously described.One of the potential criticisms to this technique is that a double-strand graft could be too thin to address the function of an ACL. We consistently found graft ranging from 7.0 mm to 7.5 mm of diameter. Although this seems apparently small to be an ACL graft, the extra-articular portion of the graft provides substantial additional anteroposterior and rotatory stability with his increased lever arm. Moreover, similar graft dimension has long been adopted for the “over-the-top” technique, with good results.In conclusion, this technique acquires some of the positive features of the “over the top” reconstruction, which has demonstrated good results at very long-term follow-up.


We found gravity level had a significant effect on 6 phases of quasi-stiffness, while speed significantly affected the quasi-stiffness in 5 phases. We concluded that the intrinsic muscle-tendon unit stiffness was the biggest determinant of quasi-stiffness. Speed had a significant effect on the R2 of all phases of quasi-stiffness. Slow walking (0.4 m/s) was the least accurately modelled walking speed. Our findings showed adaptions in gait strategy when relative power and strength of the joints were increased in low gravity, which has implications for prosthesis and exoskeleton design.Quasi-stiffness characterizes the dynamics of a joint in specific sections of stance-phase and is used in the design of wearable devices to assist walking. We sought to investigate the effect of simulated reduced gravity and walking speed on quasi-stiffness of the hip, knee, and ankle in overground walking. 12 participants walked at 0.4, 0.8, 1.2, and 1.6 m/s in 1, 0.76, 0.54, and 0.31 gravity. We defined 11 delimiting points in stance phase  and calculated the quasi-stiffness for 4 phases for both the hip and ankle, and 2 phases for the knee. The R Quantifying the quasi-stiffness of the human lower limb joints during locomotion can provide insight to gait biomechanics and be valuable for the design of robotic exoskeletons and prostheses. Quasi-stiffness, sometimes referred to as dynamic stiffness, is a linear model of the relationship between joint moment and angle during particular phases of the gait cycle ,2. The jJoint quasi-stiffness during human walking has been useful in control systems of robotic exoskeletons, prostheses, and bipedal robots which mimic the normal biomechanical behaviour of the human lower limb –12. A coStudy of joint quasi-stiffness during different gait conditions may also provide insight into human neural control ,14. JoinUsed as a gait rehabilitation therapy, simulated reduced gravity reduces the joint torques and muscle forces required to produce gait ,21. One Biomechanists use simulated reduced gravity to test hypotheses about how gravity and bodyweight affect human movement. Past studies have shown that simulated reduced gravity alters the kinematic, kinetic, and electromyographic components of gait in able-bodied humans . For exaThe purpose of this study was to understand the effect of simulated reduced gravity on ankle, knee, and hip quasi-stiffness walking overground at a range of speeds. Simulated reduced gravity will reduce joint moments during the stance phase of walking. If there is a relationship between joint moment and quasi-stiffness, it should be noticeable with a three-fold range of gravity levels. We hypothesized that joint quasi-stiffness would decrease with simulated reduced gravity as it is likely dependent on the intrinsic muscle-tendon unit stiffness which scales with muscle and tendon force ,43–45.12 able bodied individuals with no history of neuromuscular conditions participated in the study . Participant demographics are detailed in The walkway had 3 embedded force plates  to measure ground reaction forces at 1000 Hz and we used a visual motion capture system  to measure joint kinematics at 100 Hz. We palpated for and placed 22 reflective markers on the following anatomical landmarks: Ilium anterior superior, ilium posterior superior, greater trochanter of the femur, femoral lateral epicondyle, femoral medial epicondyle, fibula apex of the lateral malleolus, tibia apex of medial malleolus, the posterior of the calcaneus, heads of the 1st and 5th metatarsals, and the hallux. In addition to the anatomical markers, participants wore rigid bodies with 4 reflective markers on each shank and thigh. To control for walking speed, we used infra-red timing gates set-up before and after the force plates.Participants completed all speed conditions for a level of simulated reduced gravity, rested for 5–15 minutes, then moved onto walking at the next level of reduced gravity. For each participant, we randomized the order of gravity levels and the order of the speed conditions for each simulated gravity level. To ensure the subjects’ biomechanics adapted to simulated reduced gravity, participants practiced walking for 3 minutes at each gravity level before data were collected. Participants received no specific instructions on how to walk in reduced gravity, although we did request that they do their best to not include a flight phase in the highest walking speed and lowest gravity condition. We collected kinematic and kinetic data for 4 trials wherein the participant’s right foot struck a single force plate, and the left foot landed on a subsequent force plate or straddled the remaining two force plates.th order Butterworth filter and then cut the data to stance phase . An 18 N threshold on the vertical ground reaction force identified initial contact and toe off. To build our anthropometric model for joint angle and moment calculations in Visual3D, we used default segment definitions and estimated the hip joint location with the Coda pelvis model Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: 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 Author  Please 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 aims at investigating the effect of simulated reduced gravity (provided by a bodyweight support system) and speed on quasi-stiffness of the hip, knee and ankle during overground walking. Quantifying quasi-stiffness during simulated reduced gravity may have important implications for the design of prosthetics and exoskeletons when external bodyweight support is provided. The authors found that quasi-stiffness decrease with simulated reduced gravity and speed is a main factor in determining quasi-stiffness. They concluded that joint quasi-stiffness is determined by the inherent stiffness properties of the muscle tendon units. The manuscript is well written and the quality of the data, analysis and figures provide enough evidence to support the conclusions.Minor comment:The proposed hypothesis (“We hypothesized that joint quasi-stiffness would decrease with simulated reduced gravity as it is likely dependent on peak muscle and tendon force”) is a prediction of the results given the relationship between joint net moments and quasi-stiffness. I would suggest to reformulating the hypothesis in relation to the proposed biomechanical principles underlying the modulation of quasi-stiffness.Reviewer #2: The current paper aimed to determine how joint quasi-stiffness changes in response to different levels of simulated reduced gravity at a range of walking speeds. Quasi-stiffness is a useful tool for controlling robotic devices and has yet to be studied at different gravity levels. Twelve healthy, young participants completed the protocol, which involved overground walking over a force plate at prescribed speeds and gravity levels. The results support the authors’ hypothesis that quasi-stiffness would decrease with reductions in simulated gravity. The discussion compares and contrasts the quasi-stiffness parameters to that of previous literature and proposes explanations for the mechanism behind the changes in quasi-stiffness.While the paper is well-written, there are a handful of typos and omissions (some of which I have pointed out below). In addition, I have suggested some more impactful revisions pertaining to clarifying the methodology and focusing the intro and discussion.The most important revisions deal with ensuring that the framing of the conclusions is appropriate for the scope of this study.Intro:Throughout the intro, please include citations to back up statements of known phenomena. For example, page 9, line 23-24, multiple studies have shown a decrease in GRF due to simulated reduced gravity many of which are referenced elsewhere in this manuscript.The section on simulated reduced gravity as a gait rehabilitation therapy is lacking a clear, concise message. In the current study, gait rehabilitation appears to have motivated the use of the slowest walking speed, and in the discussion the idea of rehabilitative devices is touched on. As is, the paragraph does not appear to add meaningfully to the introduction, other than to state that body-weight support is used as a therapeutic tool. Perhaps joint quasi-stiffness has been studied in these populations and could be added to this section?Method:Page 12, lines 5-7: I think this sentence is incomplete, and may be intended to provide references for the quasi-stiffness phases, which would be helpful.Figure 1: These figures help quite a bit with visualizing and understanding the quasi-stiffness calculation and results. However, there are quite a few elements and acronyms, which make the figure difficult to understand quickly: it needs to be studied while referencing Tables 1 & 2. I recommend adding a figure that shows joint moment over time and indicates the phases over which quasi-stiffness was calculated, which will allow readers to more easily understand when the points of interest occur in the gait cyclePage 13, line 5: I assume ‘mid-point of stance’ refers to the temporal mid-point. Is this correct? Or is it based on a spatial metric? Please clarify.Page 13, line 5: planter > plantarPage 14, line12: Please specify which speeds you mean by ‘normal’.Page 14, line 22: Please add some more specifics to this section. It is not clear when the analysis is being run on a single stride, single condition, single subject, etc. For example, as written, it seems that you calculated quasi-stiffness using a least squares model for a given phase within a single stride. Then you used the resulting model to predict the joint moment from the joint angle for that same phase. It is not clear to me how that is different from the R^2 of the linear model, unless different strides or subjects were compared.Results:Figure 3: I recommend swapping the positions of the Kanpf column and Kandf column, so that the columns are more chronological from left to right. Also, the data labels for each speed level (x vs o vs triangle etc) are very clear in the legend, but not clear in the figure itself, which may make this figure hard to read for a person with color-blindness. Please make the data labels more visible. Lastly, please indicate in the figure caption which measure of error the error bars are indicating (likely standard deviation).Page 17, line 9: This appears to be the first and only use of these acronyms, so please state with words instead of acronyms.Figure 5: Similar to Figure 3, I recommend swapping the positions of Khif and Khie.Discussion:Section: Biomechanical Principles Underlying Joint Quasi-StiffnessWhile I agree with the sentiment of this section, I think it should be slightly reframed. The conclusion that ‘…quasi-stiffness is modulated by the inherent properties of the muscle-tendon units” is perhaps a stretch beyond the scope of the current study. The section could be strengthened by including discussion on how the interaction between tendon and muscle dynamics are impacted by different loads and how these changes could contribute to explaining the results of the current study. Additionally, I was unable to find/access a complete reference for citation 41, which appears to be important for this argument.Page 20, line 6 & throughout: The authors make several references to ‘normal’ walking speed. There are four speeds, and it is unclear which speed or speeds they are referring to as normal. Please be more specific when referencing results. One solution would be to use a parenthetical such as ‘…normal walking (1.2 m/s)…’ or to define what constitutes ‘normal’ at first mention.Page 21, line 14: ‘liner’ > ‘linear’Page 22, line 23: Could the change in hip kinematics be related to how the participants are interacting with the body-weight support system? According to the 2020 Maclean paper, slower speeds saw higher forward pulling forces. Perhaps it would be worthwhile to investigate trunk angle to determine how the lack of hip flexion could come about.Page 23, line 2: ‘worst’ > ‘lowest’Page 24, line 10: ‘liner’ > ‘linear’**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  31 May 2022Reviewer #1: This study aims at investigating the effect of simulated reduced gravity (provided by a bodyweight support system) and speed on quasi-stiffness of the hip, knee and ankle during overground walking. Quantifying quasi-stiffness during simulated reduced gravity may have important implications for the design of prosthetics and exoskeletons when external bodyweight support is provided. The authors found that quasi-stiffness decrease with simulated reduced gravity and speed is a main factor in determining quasi-stiffness. They concluded that joint quasi-stiffness is determined by the inherent stiffness properties of the muscle tendon units. The manuscript is well written and the quality of the data, analysis and figures provide enough evidence to support the conclusions.We thank the reviewer for their feedback and are happy to hear that the paper is well-written. Minor comment:The proposed hypothesis (“We hypothesized that joint quasi-stiffness would decrease with simulated reduced gravity as it is likely dependent on peak muscle and tendon force”) is a prediction of the results given the relationship between joint net moments and quasi-stiffness. I would suggest to reformulating the hypothesis in relation to the proposed biomechanical principles underlying the modulation of quasi-stiffness.Hypothesis has been modified to include the intrinsic muscle-tendon unit stiffness and some references to back up our hypothesis. “We hypothesized that joint quasi-stiffness would decrease with simulated reduced gravity as it is likely dependent on the intrinsic muscle-tendon unit stiffness which scales with muscle and tendon force .” Reviewer #2: The current paper aimed to determine how joint quasi-stiffness changes in response to different levels of simulated reduced gravity at a range of walking speeds. Quasi-stiffness is a useful tool for controlling robotic devices and has yet to be studied at different gravity levels. Twelve healthy, young participants completed the protocol, which involved overground walking over a force plate at prescribed speeds and gravity levels. The results support the authors’ hypothesis that quasi-stiffness would decrease with reductions in simulated gravity. The discussion compares and contrasts the quasi-stiffness parameters to that of previous literature and proposes explanations for the mechanism behind the changes in quasi-stiffness.While the paper is well-written, there are a handful of typos and omissions (some of which I have pointed out below). In addition, I have suggested some more impactful revisions pertaining to clarifying the methodology and focusing the intro and discussion.The most important revisions deal with ensuring that the framing of the conclusions is appropriate for the scope of this study.We thank the reviewer for their detailed feedback and suggestions to improve manuscript quality. We’re also happy to hear that the review found the paper well-written. We have endeavoured to incorporate your feedback to improve the manuscript.Intro:Throughout the intro, please include citations to back up statements of known phenomena. For example, page 9, line 23-24, multiple studies have shown a decrease in GRF due to simulated reduced gravity many of which are referenced elsewhere in this manuscript.We have added some references to back up statements of known phenomena. The sentences with new references in the introduction are:“Study of joint quasi-stiffness during different gait conditions may also provide insight into human neural control .”“Joint quasi-stiffness is determined by the state of all passive and active tissues that cross the joint .”“Used as a gait rehabilitation therapy, simulated reduced gravity reduces the joint torques and muscle forces required to produce gait .” “One technique to simulate reduced gravity is to apply a constant upwards force to the torso with a harness, counteracting gravity without altering body mass [22].”“Reducing the effective bodyweight also decreases joint contact forces and net muscle moments during walking, essentially making it easier to walk .”“We hypothesized that joint quasi-stiffness would decrease with simulated reduced gravity as it is likely dependent on the intrinsic muscle-tendon unit stiffness which scales with muscle and tendon force .”The references used are: 13. Wind AM, Rouse EJ. Neuromotor Regulation of Ankle Stiffness is Comparable to Regulation of Joint Position and Torque at Moderate Levels. Sci Rep. 2020;10. doi:10.1038/S41598-020-67135-X14. Rouse EJ, Hargrove LJ, Perreault EJ, Kuiken TA. Estimation of Human Ankle Impedance During the Stance Phase of Walking. IEEE Trans Neural Syst Rehabil Eng. 2014;22: 870. doi:10.1109/TNSRE.2014.230725615. Whittington B, Silder A, Heiderscheit B, Thelen DG. The contribution of passive-elastic mechanisms to lower extremity joint kinetics during human walking. Gait Posture. 2008;27: 628–634. doi:10.1016/J.GAITPOST.2007.08.00516. Silder A, Heiderscheit B, Thelen DG. Active and passive contributions to joint kinetics during walking in older adults. J Biomech. 2008;41: 1520–1527. doi:10.1016/J.JBIOMECH.2008.02.01620. MacLean MK, Ferris DP. Human muscle activity and lower limb biomechanics of overground walking at varying levels of simulated reduced gravity and gait speeds. PLoS One. 2021;16. doi:10.1371/JOURNAL.PONE.025346721. Goldberg SR, Stanhope SJ. Sensitivity of joint moments to changes in walking speed and body-weight-support are interdependent and vary across joints. J Biomech. 2013;46: 1176–1183. doi:10.1016/J.JBIOMECH.2013.01.00122. Farley CT, McMahon TA. Energetics of walking and running: insights from simulated reduced-gravity experiments. J Appl Physiol. 1992;73: 2709–2712. doi:10.1152/jappl.1992.73.6.270926. Apte S, Plooij M, Vallery H. Influence of body weight unloading on human gait characteristics: a systematic review. J Neuroeng Rehabil. 2018;15. doi:10.1186/s12984-018-0380-043. Rack PMH, Westbury DR. The short range stiffness of active mammalian muscle and its effect on mechanical properties. J Physiol. 1974;240: 331. doi:10.1113/JPHYSIOL.1974.SP01061344. Walmsley B, Proske U. Comparison of stiffness of soleus and medial gastrocnemius muscles in cats. J Neurophysiol. 1981;46: 250–259. doi:10.1152/JN.1981.46.2.25045. Krupenevich RL, Beck ON, Sawicki GS, Franz JR. Reduced Achilles Tendon Stiffness Disrupts Calf Muscle Neuromechanics in Elderly Gait. Gerontology. 2022;68: 241–251. doi:10.1159/000516910The section on simulated reduced gravity as a gait rehabilitation therapy is lacking a clear, concise message. In the current study, gait rehabilitation appears to have motivated the use of the slowest walking speed, and in the discussion the idea of rehabilitative devices is touched on. As is, the paragraph does not appear to add meaningfully to the introduction, other than to state that body-weight support is used as a therapeutic tool. Perhaps joint quasi-stiffness has been studied in these populations and could be added to this section?In this section, we introduce and discuss the concept of simulated reduced gravity. Specifically, we highlight that the technique can reduce joint torque, which links back to the previous paragraph of exploring quasi-stiffness under such conditions. We think it is important to dedicate some of the introduction to an overview of bodyweight supported walking. To the best of our knowledge, joint quasi-stiffness has not been studied in a clinical population. To improve the relevancy of the discussion of gait rehabilitation, we have explained the benefit of understanding the effect of gravity level in a healthy population as it pertains to understanding if pathological gait has an interaction with gravity level. “To the best of our knowledge, there has been no investigation of joint quasi-stiffness in clinical populations in normal or reduced gravity. To parse out the effects and interactions of gravity level, walking speed, and underlying pathological physiology on quasi-stiffness, we first need an understanding of the relationship between gravity level, walking speed, and joint quasi-stiffness in a healthy population.”Method:Page 12, lines 5-7: I think this sentence is incomplete, and may be intended to provide references for the quasi-stiffness phases, which would be helpful.This sentence was incomplete, thank you for catching that. We have completed it and added the 5 references relevant to building our quasi-stiffness models.We chose phases of quasi-stiffness delimited by points of interest (Table 2) with reference to previous studies .Figure 1: These figures help quite a bit with visualizing and understanding the quasi-stiffness calculation and results. However, there are quite a few elements and acronyms, which make the figure difficult to understand quickly: it needs to be studied while referencing Tables 1 & 2. I recommend adding a figure that shows joint moment over time and indicates the phases over which quasi-stiffness was calculated, which will allow readers to more easily understand when the points of interest occur in the gait cycleWe agree that there’s a lot of information on these figures and they required some time to be fully understood. We have endeavoured to improve clarity and split the figure into 3, with each joint moment-angle loop accompanied by the moment and angle during stance. We hope the consistency in colours and labelling between the figures, will help improve clarity. The text from each new figure is a small variation of the following:“Figure X. Joint angle, moment, and moment-angle relationships during stance phase overlaid with points of interest and phases of quasi-stiffness. The smaller subplots on the left show joint angle and moment during stance phase. The dashed vertical lines represent the timings of each point of interest, while the open circles identify the point of interest itself. The phases of quasi-stiffness are identified at the bottom of these plots, with the arrows indicating their onset and termination. The large, right figure shows the moment-angle relationship with the quasi-stiffness overlayed as dashed lines and the points of interests as “+”. Data for visualization were normalized to 60 data points and averaged across all participants walking in normal gravity at 1.6 m/s. Direction of internal joint moment and joint angle are indicated with arrows on the rightmost plot.”Page 13, line 5: I assume ‘mid-point of stance’ refers to the temporal mid-point. Is this correct? Or is it based on a spatial metric? Please clarify.You assume correctly. We have clarified the mid-point is “temporal” at multiple points in the manuscript for clarity.Page 14, line12: Please specify which speeds you mean by ‘normal’.Page 20, line 6 & throughout: The authors make several references to ‘normal’ walking speed. There are four speeds, and it is unclear which speed or speeds they are referring to as normal. Please be more specific when referencing results. One solution would be to use a parenthetical such as ‘…normal walking (1.2 m/s)…’ or to define what constitutes ‘normal’ at first mention.We agree that the speeds were somewhat confusing as they were originally were presented and have clarified walking speeds throughout the paper. Generally speaking, slow = 0.4 m/s, medium = 0.8 m/s, normal = 1.2 m/s, and fast = 1.6 m/s. Page 14, line 22: Please add some more specifics to this section. It is not clear when the analysis is being run on a single stride, single condition, single subject, etc. For example, as written, it seems that you calculated quasi-stiffness using a least squares model for a given phase within a single stride. Then you used the resulting model to predict the joint moment from the joint angle for that same phase. It is not clear to me how that is different from the R^2 of the linear model, unless different strides or subjects were compared.We appreciate ethe feedback and have added additional information for clarity. In short, for every walking trial, the linear model was used to predict moment from angle and we found the R^2 for that trial. Stiffness and R^2 values were then averaged within conditions for each participants, and then averaged across participants. “We found the 11 points of interest for each walking trial, then calculated the stiffness of each quasi-stiffness phase using a least squares linear model. For each walking trial, we applied the linear model as a function of joint angle (i.e. predicted joint moment from given joint angles). To determine the fit of the model for each phase of quasi-stiffness in each trial, we calculated the R2 value. We also calculated the duration of each phase of quasi-stiffness as a percentage of the total stance time. Finally, we averaged quasi-stiffness values, R2, and phase durations across each walking trial in a condition for every participant, and then averaged across participants.”Results:Figure 3: I recommend swapping the positions of the Kanpf column and Kandf column, so that the columns are more chronological from left to right. Also, the data labels for each speed level (x vs o vs triangle etc) are very clear in the legend, but not clear in the figure itself, which may make this figure hard to read for a person with color-blindness. Please make the data labels more visible. Lastly, please indicate in the figure caption which measure of error the error bars are indicating (likely standard deviation).We have reordered the columns to be ordered as suggested. They were originally ordered in strict chronological order, as Kandf does not follow on from Kand2, but we can see the benefit to grouping all the dorsi-flexion QS together and have implemented this in the figures and tables.We chose the colours such that they would be discernible in greyscale and for several colour-blindness types. We used an online tool (link to tool with our colours pre-loaded) to visualize the colours for different colour-blindness types and converted the figures to greyscale to check for clarity. The 0.4 and 0.8 m/s colours are the closest in greyscale, so we chose the label markers that are most visually different (cross and open circle). Unfortunately, increasing the size of the markers makes the plots too noisy and the data more difficult to read. We have included that the error bars represent standard deviation. The new figure caption reads:“Figure 5. Quasi-stiffness of the ankle in stance phase at different levels of simulated reduced gravity and speeds. Top row shows stiffness values, middle row is the quality of fit of the linear quasi-stiffness model, and bottom row is the duration of the quasi-stiffness phase as percent of stance phase. Each column of plots is a different quasi-stiffness phase for the ankle: KAnD1 is early dorsi-flexion, KAnD2 is Ankle late dorsi-flexion, KAnDF is overall Ankle dorsi-flexion, and KAnPF is Ankle plantar-flexion. The error bars represent standard deviation.”Page 17, line 9: This appears to be the first and only use of these acronyms, so please state with words instead of acronyms.Thank you for bringing our attention to this! The PKn, QKn, and RKn acronyms were accidental hold-outs of an older set of acronyms used when drafting the manuscript. These acronyms have been updated to P1Kn, P2Kn, and P3Kn respectively. The text in the manuscript reads as:“There were no instances where P1Kn, P2Kn, or P3Kn could not be identified.”Figure 5: Similar to Figure 3, I recommend swapping the positions of Khif and Khie.We have swapped the order of the appearance for this figure and relevant table. The final 2 sentences of the figure caption reads:“Each column of plots is a different quasi-stiffness phase for the hip: KHiE1 is early hip extension, KHiE2 is hip late extension, KHiE is overall hip extension, and KHiF is knee flexion. The error bars illustrate standard deviation.”Discussion:Section: Biomechanical Principles Underlying Joint Quasi-StiffnessWhile I agree with the sentiment of this section, I think it should be slightly reframed. The conclusion that ‘…quasi-stiffness is modulated by the inherent properties of the muscle-tendon units” is perhaps a stretch beyond the scope of the current study. The section could be strengthened by including discussion on how the interaction between tendon and muscle dynamics are impacted by different loads and how these changes could contribute to explaining the results of the current study. Additionally, I was unable to find/access a complete reference for citation 41, which appears to be important for this argument.We have softened this section so our discussion stays within the scope of this study. Citation 41 is still in review, so we have removed it from this manuscript. The section now reads as:“Joint quasi-stiffness tended to decrease with gravity level, which is in agreement with the supposition that quasi-stiffness is modulated by the inherent properties of the muscle-tendon units. Simulated reduced gravity reduced the load through the joints and moments about the leg joints, and reduced the muscle activation amplitudes during stance [21]. At lower muscle forces, there is less stretch of the tendon which moves the tendon stiffness into the short range stiffness known as the toe region. The toe region occurs at low levels of tendon stretch and incurs a smaller stiffness than in the subsequent linear phase of tendon length-force relationship. However, an alternative possibility is that subjects actively adjusted their muscle activation patterns to achieve close to normal joint displacements at reduced gravity. This approach would also produce a reduced joint quasi-stiffness at reduced gravity, completely independent of underlying muscle-tendon unit mechanical properties. Future research could use ultrasound imaging [45] to provide a better indication of intrinsic muscle-tendon displacements and stiffnesses during walking under simulated reduced gravity.“Our implementation of a rigid foot model may have led to an underestimation of the true ankle quasi-stiffness. We modelled the foot as a single rigid body, assuming no rotation or translation within the joints of the foot. Previous research has found that modelling the ankle and foot separately, allowing for deformation of the arch of the foot, underestimated quasi-stiffness of the ankle in hopping [50]. “Page 22, line 23: Could the change in hip kinematics be related to how the participants are interacting with the body-weight support system? According to the 2020 Maclean paper, slower speeds saw higher forward pulling forces. Perhaps it would be worthwhile to investigate trunk angle to determine how the lack of hip flexion could come about.Thank-you for your insights. We do not think the pulling forces would have impacted the lack of hip flexion. The pulling forces at all gravity levels rarely exceeded 1% of the person’s bodyweight . We suggest the lack of hip flexion is an adoption of a new walking strategy. We did not have any markers above the hips, so we cannot retroactively examine trunk angle. Page 13, line 5: planter > plantarPage 21, line 14: ‘liner’ > ‘linear’Page 24, line 10: ‘liner’ > ‘linear’Thank you for pointing out these spelling errors. We have corrected them in the revision.Page 23, line 2: ‘worst’ > ‘lowest’We agree that ‘lowest’ is a better choice than ‘worst’ and have updated the word choice in the revision. We have tracked changes in the manuscript revision, and highlighted yellow any changes we made which could not be tracked (i.e. referencing or table formatting)AttachmentResponse to Reviewers QuasiStiffness.docxSubmitted filename: Click here for additional data file. 11 Jul 2022Effects of simulated reduced gravity and walking speed on ankle, knee, and hip quasi-stiffness in overground walkingPONE-D-21-38355R1Dear Dr. MacLean,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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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**********  6. Review Comments to the Author  Please 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: (No Response)Reviewer #2: The authors have done an adequate job addressing all previous concerns, resulting in a nice manuscript describing a well performed study.**********  7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Journal of Appalachian Health is going through some changes that are reflected in the masthead and banner. We say farewell to some colleagues and welcome to the new faces.The  Journal of Appalachian Health is going through some changes that are reflected in the masthead and banner. First is the change in the managing editor. We say a sad but fond farewell to Charlotte Seidman. Her reaction to yet another retirement is chronicled by her in a separate comment in this issue of the Journal.The We are pleased and excited to have Charlotte’s replacement on board to do a smooth transition in the open managing editor position. Rachel Dixon is joining us in that role. Like with Charlotte, I have worked with Rachel over several years; she has been my student at least part of the time and has demonstrated her chops in editing and authoring books, chapters, and manuscripts. She is both a scholar herself, as well as being an editor of other’s scholarship and has done outstanding jobs in all of her roles. We welcome her contribution to the rigor and readability of the journal.Two of our editorial board members have left their current institutions for other pastures. A longtime friend and dear colleague, Linda Alexander, has decided to leave her current post as Associate Dean of Public Health at West Virginia University to occupy a new role as Chief Academic Officer at the Association of Schools and Programs of Public Health. I was involved with hiring Linda to her first appointment in public health at the University of Kentucky, so have valued her wisdom and knowledge over the years. We will miss her guidance and enthusiasm from her work at West Virginia University School of Public Health to her work with the journal.In her place, representing West Virginia University another long-time colleague and former student, Erik Carlton, has taken on the role of representing WVU on our editorial board. Like Linda, I know Erik well, having served him during his doctoral program at the University of Kentucky school of public health. The decision to put Erik in the WVU editorial board seat on the Journal was the decision of WVU, but I am delighted to have a friend and colleague, as well as a former graduate student, take on the role as a member of our editorial board. Erik is a very bright and effective academician as well as a valued colleague and friend.Erin Bouldin, who served both as a representative of Appalachia State University as a board member but also one of our Associate Editors has left Appalachia State to go to a position at the University of Utah. We wish her a fond farewell as we do the other three members of our editorial boards’ departure. Appalachia State has named Adam Hege as Erin’s replacement on the board. Adam is associate professor there and has an interest in social determinants and the health of long-haul truck drivers. He is enthusiastic about joining the board and we welcome him.These new additions to the masthead signal a renewed energy in the Journal; it is always productive to have new sets of eyes and perspectives. We welcome this turnover and the new additions to our masthead. They will bring with them new ideas and directions for the journal. We welcome these new folks and are pleased with the smooth transition that has occurred in our little corner of the publishing world. Join me and the other members of the Journal team in welcoming them to this new role. To our departing colleagues Ave Atque Vale, we thank you for your commitment and diligence in efforts to build the Journal and bring it to its level of excellence.


Oryza sativa L.) is a monocot whose reserve starch is organized into compounded structures within the amyloplast, rather than a simple starch grain (SG). The mechanism governing the assembly of the compound SG from polyhedral granules in apposition, however, remains unknown. To further characterize the proteome associated with these compounded structures, three distinct methods of starch granule preparation  were performed. Phase separation of peptides  isolated starch granule-associated proteins (SGAPs) from the distal proteome of the amyloplast and the proximal ‘amylome’ (the amyloplastic proteome), respectively. The term ‘distal proteome’ refers to SGAPs loosely tethered to the amyloplast, ones that can be rapidly proteolyzed, while proximal SGAPs are those found closer to the remnant amyloplast membrane fragments, perhaps embedded therein—ones that need isopropanol solvent to be removed from the mature organelle surface. These two rice starch-associated peptide samples were analyzed using nano-liquid chromatography–tandem mass spectrometry (Nano-HPLC-MS/MS). Known and novel proteins, as well as septum-like structure (SLS) proteins, in the mature rice SG were found. Data mining and gene ontology software were used to categorize these putative plastoskeletal components as a variety of structural elements, including actins, tubulins, tubulin-like proteins, and cementitious elements such as reticulata related-like (RER) proteins, tegument proteins, and lectins. Delineating the plastoskeletal proteome begins by understanding how each starch granule isolation procedure affects observed cytoplasmic and plastid proteins. The three methods described herein show how the technique used to isolate SGs differentially impacts the subsequent proteomic analysis and results obtained. It can thus be concluded that future investigations must make judicious decisions regarding the methodology used in extracting proteomic information from the compound starch granules being assessed, since different methods are shown to yield contrasting results herein. Data are available via ProteomeXchange with identifier PXD032314.Starch is the primary form of reserve carbohydrate storage in plants. Rice ( Zea mays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), and wheat (Triticum aestivum) are all examples of this mode of carbon sequestration [In plants, energy is stored in the form of starch, an accumulation of the glucose polymers amylose and amylopectin. Starch can either be transitory—meaning that it is synthesized in the aerial tissue during the day and is broken down during the night to sustain cellular metabolism —or storastration . In ricestration . This stThe rice amyloplast is proposed to be composed of several distinct components: an outer envelope membrane (OEM) and an inner envelope membrane (IEM), which enclose an intermembrane space (IMS); and a septum-like structure (SLS) that forms between apposing granule surfaces. The prevailing wisdom regarding compound granule agglomeration hinges on the role of the IEM. The IEM is hypothesized to form a mold in which starch molecules are deposited, forming the characteristic polyhedral granule ,5, althoThe missing pieces of information regarding the SG scaffolding or plastoskeleton may be present in the starch granule-associated protein (SGAP) proteome ,14. As sFor this study, three rice preparation methods were employed  and eachA proteomic analysis of the starch granule samples was performed. The numbers of total and uncharacterized peptides identified in each of the six starch samples by mass spectrometric analysis are shown in www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi accessed on 4 October 2019) of the NCBI Batch Conserved domain database [geneontology.org accessed on 4 October 2019) [Uncharacterized peptides were present in all three samples . No protIt was found that the use of diverse starch granule extraction methods can identify the core proteome. A common distal proteome was compiled using the mass spectrometric data obtained from all aqueous samples from all three starch preparation methods. The distinction between the distal and proximal proteomes is based on the rationale that proteins which are easily removeable from the starch granule by trypsin digest would also be more distal to the starch granule . SimilarTwenty-eight remaining proteins made up the amylome. Of these, five were glutelin isoforms, three were transcriptional/translational machinery proteins, two were seed storage proteins, and one was a membrane structural protein. The remaining 17 were related to starch biosynthesis and sucrose metabolism. They included sucrose synthase, granule-bound starch synthase, branching enzyme, ADP-glucose pyrophosphorylase, pullulanase, α-glucosidase, and α-1,4-glucan phosphorylase. Other carbohydrate-metabolism-related proteins identified included sorbitol dehydrogenase, glyceraldehyde phosphate, fructose-bisphosphate aldolase, and orthophosphate dikinase. BT1 was not found in the core proximal amylome.Both known and novel structural proteins and a novel carbohydrate-binding protein were identified in the rice SG samples . To corrA large amount of data was obtained following trypsin shaving and isopropanol treatment of the dispersion-prepared granules  and so aEach starch sample contained uncharacterized peptides, but database screening assigned functions to most of these peptides . The datActins and tubulins were present in the proteome of dispersion-prepared starch granules. Four actin proteins and ten tubulin proteins  were detected in the distal proteome of dispersion-prepared starch granules. There were two actin-depolymerizing factors (ADFs) in the same proteome: ADF-2 (Q9AY76) and ADF-3 (Q84TB6) . Plant APeptides which were uncharacterized were either run through Retrieve/ID mapping via UniProt to obtain putative function , or throNovel uncharacterized peptides were found in the aqueous fraction of dispersion-prepared starch granules treated with trypsin. Most uncharacterized peptides were found in dispersion-prepared granules. Some of these peptides could not be identified using UniProt Retrieve/ID mapping analysis but were available in the NCBI database due to the update of the rice proteome (26 March 2020). The bulk of newly identified proteins were found in dispersion-prepared granules. Some of these proteins were present in the UniProt database but were available in the NCBI database due to the update of the rice proteome (26 March 2020). Proteins that had not yet been annotated by NCBI were examined based on the presence of a conserved domain. Domain conservation is qualified by multiple sequence alignments of related proteins across multiple species that reveal the same or similar amino acid patterns . PeptideCarbohydrate-binding module (CBM) proteins were detected in the putative plastoskeleton samples. CBMs are found in a broad range of proteins that interact with carbohydrates and do not impart enzymatic activity. CBMs are still found in enzymatic proteins such as glycoside hydrolases, polysaccharide lyases, polysaccharide oxidases, glycosyltransferases, expansins, and lectins . Of all A. thaliana [Protein Q6YXZ6 has been annotated as glucan endo-1,3-beta-glucosidase 6 in the NCBI database and contains a CBMF43 or X8 carbohydrate-binding domain. These domains are 90–100 residues in length and bind β-1,3-glucans . CBMF43 thaliana .Lectins, reticulata related-like, tegument, structural, and PTST proteins were observed in the putative plastoskeleton samples. A small group of putative SLS-related proteins were identified in the proteomes of dispersion-prepared granules . The micOryza, this packaging takes the form of the compound granule, an intra-organellar anatomical feature also present in Avena.In the rice kernel, starch is the major storage carbohydrate and this metabolic reserve supplies the germinating embryo with an energy source. These molecules accumulate to high levels in the cereal endosperm and have evolved to be packaged efficiently within the cell . In OryzDispersion-based disruption of the compound granule using osmotic buffer;Microsieving;Flotation-based disruption using a cesium-chloride gradient.Proteins were extracted from rice starch granules prepared using three different methods, as follows:The dispersion preparation method obtained the highest number of unique SGAPs; conversely, flotation preparation obtained the lowest number of unique SGAPs. The top hits in each dataset were primarily seed storage and biosynthesis proteins, although the dispersion method isolated a wide variety of structural proteins in both the distal proteome and proximal amylome.Known starch granule-associated proteins (SGAPs) were detected by mass spectrometry . The staTrypsin treatments were performed on these starch samples and the water-soluble peptides liberated into the supernatant were sequenced. The remaining hydrophobic proteins were released from the starch granule surface by isopropanol solubilization. These protein extraction methods isolated primarily carbohydrate metabolism and seed storage proteins, as expected . This coAll six datasets feature the characteristic starch metabolism, biosynthesis, and storage proteins. The content is affected by the starch preparation method used, as seed proteins have varying solubilities  and, as Eleven peptides were common to the aqueous supernatants obtained from all three starch preparation methods and twenty-eight were common to the isopropanol-solubilized fraction obtained from all three methods. We found that the core proteomes are relatively sparse  considerNotably, Brittle1 (BT1)  was idenThe three methods used have shown that diverse starch granule extraction methods and proteomics analysis techniques can help to map the putative plastoskeleton of the rice SG. A hypothetical schema of the rice SG and granule has been proposed . Both inArabidopsis thaliana is involved in chloroplast formation and presents a reticulated phenotype in plant leaves [A. thaliana proteins reticulata-related 3  and reticulata-related 4  in the distal proteome of the starch granule. As with their A. thaliana homologs, these proteins feature domains of unknown function [The presence of plastid-related proteins identified by domain homology (reticulata and tegument) may also be a key towards elucidating compound starch granule organization. It was reported that the reticulata-related (rer) gene family in t leaves . RER prot leaves . This stfunction . Since tfunction , it is hfunction . The tegfunction . It is hfunction .Gleaned from these analyses, the data present many interesting avenues of future rice SGAP studies, in addition to investigating the SGAP using a plastoskeletal-focused approach: E3 ubiquitin ligases, as one example, were discovered in the distal proteome and so may play a pivotal role in the development of plastid components. There is a paucity of data regarding ubiquitination and 26S proteasome involvement in plastid development, and even less with amyloplasts. The presence of the E3 ligases begs the question of whether there is a mechanism within the amyloplast to prevent the translocation of specific proteins into the amyloplast . SimilarThis analysis used mature rice kernels, and so limits analysis on SGAPs involved in grain architecture during development (such as plastid division proteins), which will no longer be present in the mature endosperm. A time-course analysis of the SGAP proteome during rice kernel development must be performed to obtain a dynamic model. Observing the development of a simple SG, such as in maize, would provide side-by-side proteomic comparisons and could reveal novel candidates for compound SG architecture development. Whether these types of organization differ in starch mobilization rate is unknown and should also be investigated.Each method of preparation disrupts SGs into individual granules , suggestThis study revealed an extensive SGAP network, and that the outcome of proteomic analysis can differ significantly as a function of the methodology used. However, there were two major limitations that should be addressed for future SG experiments:Furthermore, this study also establishes the groundwork for the functional characterization of putative plastoskeletal candidates and other SGAPs. The development of overexpression and RNAi-knockdown plant lines can assess the impact of these candidates on amyloplast development and SG formation.Oryza sativa L. ssp. japonica cv. Nipponbare) were obtained from the USDA (Genetic Stocks—Oryza (GSOR) Collection, Stuttgart, AR, US). Kernels were de-husked manually and soaked overnight in sterile double-distilled water (18 h) at 4 °C and then de-germed prior to surface preparation.Rice kernels . Methods varied in type of physical disruption, pH, osmotic potential, and detergent use.g for 15 min at 4 °C). The supernatant was discarded, and the pellet was resuspended with 5 mL starch extraction buffer. The dispersion was subjected to vacuum sieve filtration through a 20 μm sieve. The filtrate was washed with starch extraction buffer followed by cold 95% ethanol and acetone. Centrifuging was performed between each wash . Pellets were air-dried under laminar flow for 48 h.Dispersion method : rice keg for 15 min at 4 °C) and the supernatant was discarded. Pellets were air-dried under laminar flow for 48 h.Microsieve method : rice keg for 15 min at 4 °C). The supernatant was discarded, and the pellet was resuspended in 1 mL of sterile double-distilled water overlaid with 80% w/v cesium chloride. The solution was centrifuged (4600× g for five minutes at 4 °C) and the supernatant discarded. The pellet was washed with a wash buffer , sterile double-distilled water, and acetone. Centrifuging was performed between each wash (8000× g at 10 min for 4 °C). Pellets were air-dried under laminar flow for 48 h.Flotation method : rice keg for one minute and supernatant was transferred to a fresh tube. Pellets were washed five times with a 10-fold excess of double-distilled H2O to remove residual water-soluble proteins. Following water washing, proteins remaining on the starch granule surface were extracted by adding 350 μL of 50% (v/v) isopropanol and 50 mM NaCl and gently agitated for 45 min at room temperature. The samples were centrifuged at 18,000× g for one minute, and the supernatant was collected. The peptides from both this isopropanol fraction and the previously reserved aqueous supernatant were dried in a Speed Vac . Peptide pellets were resuspended in 40 μL of double-distilled H2O, purified using ZipTips with C18 resin  to remove salt and residual starch, and dried in a Speed Vac. Peptides were resuspended in 40 μL of 0.1% formic acid.The peptides associated with the starch granule surface were collected according to a modified protocol ,17. TrypStarch samples obtained from each method were mounted on aluminum stubs and subjected to pressurized air under a vacuum hood to produce a thin layer. Stubs were sputter-coated with gold (Gatan Model 882 PECS) and analyzed by SEM .m/z 350–2000) were acquired at a resolution of 60,000. Precursor ions were filtered according to monoisotopic precursor selection, charge state (+2 to + 7), and dynamic exclusion 30 s. The automatic gain control settings were 4 × 105 for full FTMS scans and 1 × 104 for MS/MS scans. Fragmentation was performed with collision-induced dissociation (CID) in the linear ion trap. Precursors were isolated using a 2 m/z isolation window and fragmented with a normalized collision energy of 35%.Aliquots of dry rice starch powders were incubated with proteomics-grade trypsin  at a ratio of 24 μL:1 mg at 37 °C for 18 h with gentle agitation. The supernatant was collected and analyzed by LC-MS/MS using the following protocol . FurtherOryza sativa ssp. japonica protein sequences in FASTA file format from the UniProt/SwissProt database [Proteome Discoverer 2.1  was used for peptide identification . The predatabase . The Falwww.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi accessed on 4 October 2019) of the NCBI Batch Conserved domain database [www.uniprot.org/uploadlists/ accessed on 4 October 2019) [www.cazy.org accessed on 4 October 2019) [pantherdb.org accessed on 4 October 2019) [geneontology.org accessed on 4 October 2019) [Peptides with High Protein FDR ranking were selected from the mass spectrometric data for further analysis. All peptides were ranked according to SEQUEST score. Bioinformatics analysis was performed using the NCBI Web CD-Search Tool (database . Interacdatabase . UniProter 2019) , CAZy (wer 2019) , and theThe mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE  partner


Vaccine development against emerging infections is essentially important for saving people from increasing viral infections. In developing countries, Hepatitis E (HEV) is a common infection affecting millions of people worldwide. Based on In-silico analysis, different approaches have been targeted.Rationale of this study is to design an epitope-based vaccine candidates with the help of immunoinformatics that can predict promiscuous B-cell and T-cell epitopes of the most antigenic HEV-ORF2 capsid protein.This study suggests potential T-cell and B-cell epitopes of the highly antigenic HEV ORF2 capsid protein while using various In-silico tools such as NCBI-BLAST, Expassy, CLC workbench, Ellipro and Discotope.Potential antigenic and immunogenic CD8+ T-cell epitopes were predicted from the global consensus sequence of ORF2-HEV. Furthermore, twenty-two linear B-cell epitopes were predicted. Among these, “SLGAGPV” at position 587–593 and “LEFRNLTPGNTNTRVSRYSS” at position 306–325 were most antigenic with antigenicity score 1.4206 and 1.3600 respectively. Discontinuous B-cell epitopes were found by three-dimensional capsid protein structure. Epitopes predicted in this study reveal high antigenicity and promiscuity for HLA classes.Collectively, our data suggests promiscuous epitopes that can potentially acts as new candidates for the design of HEV peptide vaccine. Hepatitis E Virus (HEV) is a leading gastro-enteric viral infection that occurs worldwide. HEV shows strong hepatotropism and triggers acute infection, if gets untreated HEV poses silent yet major source of viral hepatitis in the world. HEV infection is worldwide in distribution. In developing countries, HEV is not generally considered as a serious disease and public awareness is lacking about this disease. According to a WHO survey, about 20 million cases of HEV are reported worldwide annually. Around, 60,000 individuals per year suffer from acute infections of HEV. However, the estimated mortality rate is even higher for pregnant women. During the summer, outbreak is more likely to occur. HEV mostly infects men ranging from 15–30 years of age and quite uncommon in children below the age of 10 HEV is a positive ssRNA virus with a diameter of 27–34nm. The genome is organized into three ORFs  and short non-coding regions at both 5’ and 3’ ends Nowadays, epitope-based strategies are one of the most effective strategies towards the development of vaccines. This approach was first introduced by Jacob in 1985 to develop a cholera vaccine Since HEV has the potential to get transformed into acute infections that can lead to hospital-based treatment. Therefore, there should be a proper vaccine available for this viral infection. However, HEV vaccine has been licensed in China only but not available elsewhere. Therefore, need of the hour is to design effective vaccine candidates against HEV through immunoinformatics based tools that should be validated with the wet lab analysis to make it available for everyone.Homologous sequences of HEV-ORF2 were obtained from National Center for Biotechnology Information (NCBI). The nucleotide sequences were subjected to translation using EXPASY tool. .Eighty-five protein sequences were subjected to multiple sequence alignment using CLC workbench (version 8.1) and a global consensus sequence, which is used for epitope prediction, was obtained. Furthermore, a phylogenetic tree was constructed for all the translated sequences using the CLC workbench.Using the global consensus sequence of HEV ORF2, promiscuous T-cell and B-cell epitopes were predicted through various online bioinformatics tools.Peptides binding to MHC-I and MHC-II molecules were predicted using the NetCTL1.2 server and IEDB tool respectively. For 12 supertypes such as A1, A2, A3, A24, A26, B7, B8, B27, B39, B44, B58 and B62, the NetCTL 1.2 server predicted CD8+ T-cell epitopes to be considered for MHC II binding. Peptides binding to MHC-II were predicted for HLA DRB1* allele(s): HLA DRB1*0101 and HLA DRB1*0701. The epitopes were analysed by the MHCPred 2.0 server to verify the binding affinity.Linear B-cell epitopes were predicted via the online BepiPred-2.0 and BCpred linear epitope prediction method using IEDB. VaxiJen 2.0 was used to check the antigenicity of these linear B-cell epitopes.To predict the B-cell epitopes, Ellipro and DiscoTope tools were used. ElliPro is the most reliable tool since it can model both linear and discontinuous epitopes based on the three-dimensional structures of protein. In addition, Discotope tool was used to analyze the surface's availability and score for epitope propensity. The higher score corresponds to the greater chance of the residue being involved in an epitope.In this study, HEV capsid protein crystal structure was visualized with PDB-id 3iyo. This database contains the compounds derived from the X-ray crystallography and NMR experiments. However, chimera was used to perform structural analysis of the protein.Sequence alignment of capsid protein sequence revealed 660 amino acids with highly conserved protein sequence while certain regions showed variations. Conserved regions shown with dots . ORF2 caA phylogenetic tree was constructed from eighty-five protein sequences (ORF2-HEV) across the world using CLC workbench . In thisCD8+ T-cell epitopes were predicted using NetCTL 1.2 tool. In this study we predicted total 20 epitopes which met the threshold value of 0.75 and demonstrated maximum interaction with all supertypes. The predicted T-cell epitopes were further evaluated for antigenicity and immunogenicity by the Vaxijen tool and the IEDB immunogenicity predictor. Here, 10 epitopes were below the threshold value of 0.4 on the Vaxijen server, out of 20 primarily selected epitopes, while 5 epitopes achieved an immunogenicity value of less than 0.00. Thus among 20 epitopes, 5 epitopes with positive antigenicity and immunogenicity scores have been selected . These hAntigenic Epitopes “WLSTAAEY” “IMATEASNY” “DSQPFAIPY” “ESRVVIQDY” and ‘LTTTAATRF” were selected for the identification of MHC-II alleles and their corresponding peptide or CD4+ T-cell epitope. The analysis revealed that peptides WLSTAAEYDQSTYG at position 472–486, IMATEASNYAQYRVV at position 174–188, DSQPFAIPYIHPTNP at position 47–61 and ESRVVIDQYDNQHEQ at position 435–449 showed affinity with HLA DRB1* allele(s): HLA DRB1*0101 and HLA DRB1*0701. Therefore, the strongest binding grooves for the predicted peptides may be HLA DRB1* alleles. For the effective functioning of a vaccine candidate, the epitope which binds to the MHC class II HLA DRB1* molecule is essential.B-cell epitope is a specific region of an antigen that is detected in a humoral reaction by either a specific B-cell receptor or the antibody. The epitopes of B-cell are classified into two major types: linear or continuous epitopes and conformational or discontinuous B-cell epitopes. Many of the B-cell epitopes have been shown to be of a conformational nature.The B-cell identification is the crucial step in the design of epitope-based vaccine. Therefore, an In-silico study was conducted via the web server BcPred-1.0 and BepiPred -2.0 to acquire B-cell epitopes in HEV capsid protein.By using BcPred-1.0 tool, total of 17 epitopes were predicted from the capsid protein of HEV. Among predicted epitopes, 7 epitopes “TAVAPAHDTPPVPDVDSRGA” at position 118–130, “LEFRNLTPGNTNTRVSRYSS” at position 306–325, “WEGTTKAGYPYNYNTTASD” at position 548–567, “AGHRVAISTYTTSLGAGPVS” at position 575–594, “SERLHYRNQGWRSVETSGVA” at position 248–267, “WPQTTTTPTSVDMNSITSTD” at position 212–231 and “PLQDGTNTHIMATEASNYAQ” at position 165–184 were antigenic as predicted by VaxiJen-2.0 server. Due to their elevated antigenicity score, these epitopes may be treated as potent vaccine candidates.A total of 15 linear epitopes were observed during the analysis by BepiPred-2.0. Among all these epitopes, after predicting antigenicity by VaxiJen 2.0, the antigenic epitopes were “PTNPFAPDVTAAAGAGPRVRQPARPLGSAWRDQAQRPAAASRRRPTTAGAAPLTAVAPAHDVPDVDSRGAILRRQYNLSTSPLTSSVA” at position 58–148, “PLLPLQDGTNTHIMATEASNYAQY” at position 162–185, “QTTTTPTSVDMNSITS” at position 214–229, “ASEHVIPSERLHYRNQGWRSVETSGVAEEEA” at position 241–271, “RNLTPGNTNTRVSRYSSTARHRLRRGADGT” at position 309–339, “SANGEP” at position 403–408, ”ENAQQDKGIAIPHDIDLG” at position 417–434 “QAVARSLD” at position 508–515,”LSFWEAGTTKAGYPYNYNTTAS” at position 545–566 and “SLGAGPV” at position 587-593.The threshold was set at 0.5. In the chart  the predIn the chart , predictThe three-dimensional structure of capsid protein was obtained from RCSB PDB and visualized through Chimera.This study focuses on one of the important viral infections that infect millions of people worldwide annually. This virus docks onto the intestinal epithelial linings and gets internalized into the gastro-intestinal system, leading towards severe dehydration and clinical manifestations. In the developing countries, there is lack of funding towards exploration of antiviral drugs and vaccine development. Furthermore, there is no mass communication to highlight the disease transmission and preventive measures. Therefore, we aimed towards In-silico prediction of vaccine development against HEV infection while keeping in mind the Global Sustainable Development Goals (SDGs). There are 17 interlinked SDGs, and this study comes under the Goal “Good Health and Well Being”. The endemic situation of HEV not only alarms biologists but highlights the requirement of an effective HEV vaccine. This study suggests highly promising and immunogenic epitopes that elicits B- and T- cells and acts as potential candidates for HEV vaccination. Furthermore, this study reveals ORF2 to be effective for the development of HEV vaccine. ORF2 produces capsid protein that is immunogenic in nature, and which has been used to predict epitopes that could be used as a vaccine.Previous research focused on ORF2 protein as an important immunogenic part of the HEV that has induced B- cell and T-cell since the genetic analysis of ORF2 showed over 85% similarity between genotypes 1–4 of HEV strains that infect humans In this study, by BcPred-1.0 7 linear B-cell epitopes were identified. Among these, “LEFRNLTPGNTNTRVSRYSS” was most antigenic with antigenicity score of 1.3600. However, BepiPred-2.0 B-cell epitope data showed that out of 15 linear epitopes, the epitope “SLGAGPV” was most antigenic having antigenicity score of 1.4206. The ORF2 capsid protein of HEV seemed to be an appropriate vaccine target due to its high antigenicity, as suggested by the antigenicity determination through the VaxiJen tool. We picked potent epitopes, using a variety of computer-aided tools.Conformational or discontinuous B-cell epitopes have been found using the 3D structure of the capsid protein. All the predicted discontinuous epitopes were located on the surface of HEV capsid protein, reflecting the availability of the virus that entered. Discontinuous epitopes were in the form of residues. The residues: ASP, THR and PRO showed the highest scores. The higher score leads to the greater chance of the residue being involved in an epitope. Our research has shown that HEV designed vaccine structure can activate humoral immune responses and lead to the effective activation of pathogen-specific antibodies.In the history of vaccine development, microbial species  have been used, such as live attenuated vaccines, to induce immune responses that shield the host from infections. Vaccination has therefore proved to be important for the control of multiple lethal infectious diseases. This form of vaccine, however, may contain excessive protein particles for protective immunity activation which may contribute to immunogenic responses. This has contributed to a transition to a new age of vaccine growth through reverse vaccinology. For several viruses, such as Hepatitis C virus (HCV), Human papilloma virus (HPV) and Human immunodeficiency virus (HIV), peptide vaccines have been developed consisting only of epitopes capable of inducing positive, qualifying immune responses mediated by T-cell and B-cell, and other vaccines are also being developed