Open Access is an upcoming paradigm to communicate scientific knowledge. The Trans-O-MIM Project works on strategies, models, and evaluation metrics for the goal-oriented, stepwise, sustainable, and fair transformation of established subscription-based scientific journals into open-access-based journals. This research intends to present an evaluation metric and the associated identified appropriate parameters for such transformations. To develop the evaluation metric, it has been implemented in the context of a business management method for planning, steering and controlling action and corporate strategies. The central element was a 3-step procedure for developing the metric. In stage 1 necessary preconditions for a transformation were considered. Stage 2 is the actual elaboration of the evaluation metric by means of a scenario analysis and stage 3 comprises the exemplary testing at the journal Methods of Information in Medicine. The three methodological steps have primarily resulted in 5 scenarios with 9 different final states from the scenario analysis. Thus, the metric is now composed of these 5 scenarios, which can be used to evaluate the success or failure of a transformation. A list of 65 suitable parameters to measure changes in scenario were compiled. So, it is possible to evaluate the transformation and to find the current final state. Parameters like submissions, publications, and time as well as the scenario states could be applied to the transformation process of the Methods of Information in Medicine journal. The proposed evaluation metric can be used to evaluate the transformation processes of subscription-based journals into open-access-based journals.The online version of this article (10.1007/s10916-020-01651-1) contains supplementary material, which is available to authorized users. Open Access (OA) is an important and established form of knowledge and science communication . The revTo investigate the possibility of switching under fair conditions from the existing science communication model to science communication with OA, the project ‘Strategies, models and evaluation metrics for the goal-oriented, stepwise, sustainable and fair transformation of established subscription-based scientific journals into open-access-based journals with Methods of Information in Medicine as example’ (Trans-O-MIM) – funded by the German Research Foundation (DFG) – was carried out. Haux et al. describe the details and general conditions of this project in their 2016 paper .This project aims to transform an existing Subscription-based Journal (SJ) into an Open Access-based Journal (OAJ) under fair conditions . Haux etThe current status of the evaluation of OAJ refers to testing and evaluating Journals and the articles they contain . An evalThis paper reports on this metric. The tool of choice for the metric is a scenario analysis, which is used in the context of futurology e.g. as a method for the observation and evaluation of processes in economics , 8. The For this purpose, the parameters, influencing and disruptive factors that influence the transformation process are determined. These factors are then used to create scenarios to describe the possible final states after a transformation. In this way, assessments can be made to predict the result of the transformation process. With regard to the objectives, the concrete area of scenario analysis lies in the monitoring and evaluation of the transformation from MIM to Methods Open. The tandem model is used as a basis, whereby the journal is divided into a subscription part and an OA part. Both parts are compared and evaluated. Thus, data are collected and evaluated on parameters that are decisive for the success or failure of the transformation. The result is compared with the defined scenarios and the trend in the transformation is determined based on the current figures. Thus, it is examined whether conversion from a subscription journal to an OAJ is possible using the tandem model.The scenario analysis consists of three development phases: the preparation phase, the scenario development phase, and the monitoring or evaluation phase. The methods and results of these three phases are described in the following sections. The preparation phase for the transformation is an important and necessary step, especially on the part of the publisher, as it allows the possible framework conditions and tasks, but also the first environmental and influencing factors that are decisive for the scenario analysis to be determined. In addition, these tasks are important for the publisher, so that it can be assessed whether a transformation is at all possible for the publisher. The second phase is based on the results and insights from phase one and now enables the exact determination of the decisive parameters, influencing and disruptive factors for the entire transformation process as well as the scenarios resulting from the possible developments of these parameters. The third phase then uses the example of MIM to show an implementation of the transformation with accompanying evaluation and assessment of the current state of the journal through the defined scenarios and the current figures for the decisive parameters for success and failure of the transformation.Due to the lengthy process of transforming a SJ into an OAJ, a publication service provider is confronted with various questions and necessary preparation work. During the Trans-O-MIM project, we were able to identify the following five essential and necessary preliminary tasks for publication service providers (PSP).journal-specific definition of OA, it must be decided how and where future articles are to be published. For example, whether articles are to be published on the PSP’s website or full-text indexing within a database such as PubMed Central. Deciding on the appropriate license is also an important factor.For the authors’ interest in an OAJ and willingness to publish. A good instrument for investigating is a survey and continuous communication with stakeholders, either through social media or being present at congresses and conferences.An important point in the preparation phase is to establish the financing methods must be considered. These could, for example, take the form of advertising revenue, subsidies from professional associations, or an “author pays” model in the form of the so-called Article Publication Charges (APC) [Since subscription fees are not charged for an OAJ, alternative es (APC) . To be aconvert the editorial processes in the run-up to an OA transformation should not be underestimated. Editing and publication of OA articles require the creation of a suitable workflow. The article layout and the integration of clean and complete metadata must be defined. Also, the processes related to invoicing, such as the rapid dispatch of invoices and cross-departmental information on incoming payments, must be worked out. Functioning communication is also important for final publication of the articles. For this purpose, the editorial office, accounting department, and the electronic media department must be integrated into the communication process to be able to place finished and paid articles online.The effort required to marketing strategy is also part of the preparation phase of a transformation. Here the aim is to inform all stakeholders early and continuously about the changes in the journal. Thus, professional societies should be actively informed and involved in the project. Of particular importance are the authors. They must be informed about the innovations and convinced of the advantages. To ensure sustainability, the marketing strategy should be long-term and planned at least one year in advance.The preparation of a To evaluate the transformation, a scenario analysis is recommended for the preparation phase, in which the possible final scenarios of a journal transformation are developed. It is then possible to use these scenarios to make an assessment in the monitoring phase. Scenario analysis is a method of strategic control in which alternative environmental scenarios are created. A scenario represents a possible future situation, from which different conclusions and development possibilities can be derived .0. Depending on the development of the influencing factors and the environment, different scenarios can be described for different points in time in the future [Best-case scenario – describes the most positive case for the transformation to be performed.Worst-case scenario – describes the worst case for the execution of the transformation.Trend scenario – should occur if the trend of the underlying data and parameters remains the same .In every scenario development, the starting point for the development of a scenario is the current status, i.e. the actual situation at time te future . The scee future , 12. It e future . To limie future , 12.BestThus, the definition of scenario analysis as “a systematic methodology for developing scenarios”  or “a syThe system boundaries, basic conditions, participants, target groups, parameters, and environmental influences were developed in cooperation with Schattauer GmbH (Schattauer). This meant the processes of the publishing house for publishing science journals could be analyzed. By interviewing experts during project meetings, we received relevant information on the respective partial aspects of the scenario analysis and were able to derive the described scenarios on this basis. The inclusion and analysis of the scientific journal MIM was decisive in this context.In our opinion, the transformation of a SJ should meet the requirements of being goal-oriented, stepwise, sustainable, and fair . These rThe cost coverage should be availableThe business model should cover production costsFinancial resources must be made availableParallel to these requirements is another category of basic conditions for scenario analysis. These are financial conditions. In particular, the initial investment costs and running costs play a central role. The basic conditions that must be taken into account for the running costs are:One-off internal costs for the transformationFor investment costs, the basic condition to be taken into account is:Individuals in the group of participants describe the roles they must participate in during the transformation process and the decisions they must make. The following participants are identified as being relevant groups: executive management, accounting and finance department, editorial offices, marketing department, press department, electronic media department, production department, advertising department, and publishers.The target group describes the groups that are addressed by the transformation process. The groups’ publication organizations , authorship, readership, and advertising clients are identified as target groups for the transformation.For creation of scenarios, identification of parameters influencing journal and transformation process is an essential step. These parameters are an essential part of the scenarios, and their specific developments results in the current valid trend scenario. Definition of characteristics of the parameters is necessary for statements and measurability of the respective scenarios. The parameters are divided into primary parameters and secondary parameters . The remaining data set has been divided for evaluation of the primary and secondary parameters. For evaluation of the primary parameters, a direct comparison was made between the subscription part and the OA part of MIM. This includes all publications and submissions of the two journal sections as well as the resulting acceptance rates since the introduction of the OA part in January 2017. For evaluation of the secondary parameters, a comparison was made between the two journal sections since January 2017, as well as a comparison of the time factors before and after the transformation with data from January 2014 onwards. For the before-and-after comparison, the cut-off date of 1 January 2017 (introduction of the OA part of the MIM) was established. A compilation of all the figures can be found in Online Resource 1.For the evaluation of the transformation process, the primary parameters from the category publication parameters and the time-related secondary parameters from the category process quality parameters should be considered. The selection of the parameters was based on the criteria to which were subjected within the framework of the Trans-O-MIM project and on the requirements of the publishers Schattauer und Georg Thieme Verlag KG (Thieme) for the transformation of MIM. Predominantly these were the easy or even public availability of the used data as well as the simple realization of possible calculations and evaluations. The remaining parameters are just as relevant for the evaluation of such a transformation, but they are better applicable in the context of publisher-internal evaluations. A data set based on information and data from Manuscript Central™ , the MIMbMed.gov  was compThe presented data report the current status as a partial result of the scenario analysis after two and a half years after transformation.The numbers of this dataset show that submissions in the subscription part of the journal decreased in the last two and a half years. Whereas figures for the OA part show as almost constant over the entire period. The concrete figures for the individual months from January 2017 to September 2019 are shown in Fig. A comparison of the figures for publications in MIM also shows a consistently low trend for publications in the OA part and a strongly fluctuating trend in figures for the subscription part. The total numbers for the period January 2017 to September 2019 are shown in Fig. Tables Based on these numbers, we have calculated the quarterly figures for submissions and publications. From these figures, we were able to determine the acceptance rate for the quarters Q1/2017 to Q3/2019. The total figures can be seen in Table For analysis of the transformation, we also looked at the secondary parameter time factor: analyzing the time between important steps of the publication process. To do this, we looked at a larger time period to compare the submissions and publications for both journal parts. The time period for analyzing the time factor starts in January 2014 and ends in September 2019. The distribution of all data are shown in Fig. First, we looked at the time factors before and after transformation. The box plot in Fig. A comparison of the two journal sections of MIM shows that the publication process for a manuscript in the subscription section takes an average of about 10 weeks more than for a manuscript in the OA section see Fig. . This loAfter considering the individual parameters in comparison to each other as well as before and after the transformation, an overall evaluation of the new state can now be derived for the MIM on the basis of the resulted numbers and in accordance with the rules of a scenario analysis. Based on the current numbers, the transformation of MIM should be assigned to a worst-case scenario. In particular, the numbers in Tables This paper presented a methodology for evaluating the transformation of a SJ into an OAJ. The scenario analysis was described in general terms and with the specific facts for the use case of a journal transformation. Thus, there is now a list of suitable parameters and the description of five scenarios to monitor and evaluate a transformation from a SJ to an OAJ. This methodical approach was successfully tested with the journal MIM, and the current status and scenario for MIM were presented.The application of the metric to evaluate the transformation of a journal from a SJ to an OAJ should be possible for a journal which is comparable to the MIM. The metric was developed to work for this use case for different journals. Especially the primary parameters should be applicable to any scientific journal. The level of detail of the parameters is left to the publishing house or editor of the journal. In addition, the metric is designed to be flexible enough to allow for an easy extension, for example, the possible final scenarios. It is also easy to add to or rearrange the parameters to evaluate the scenarios. Thus, the metric is adaptable to most of the contingencies known in scientific journals. This makes it also possible to use the metric in case changes are necessary.The result that the MIM is on the way to a worst-case scenario is to be seen as positive after the current runtime of the transformation, because the runtime observations show that further measures such as intensive advertising measures or the registration in different open-access journal directories are necessary to successfully complete the transformation. In particular, the information about the current development is very valuable for the editor and the publisher to control the whole process.During the Trans-O-MIM project and the preparation of this paper, various limitations arose, which limit the results and thus, the informative value of the facts presented.Deviations in metrics in relation to other scientific journals are possible since other publishers may have different processes and thus different influences. However, extensive literature studies on the current status of Open Access across different disciplines have not provided any further indications of possible deviations from our metrics . TherefoThe metric was tested only on the example of MIM since no other SJ is known to have carried out a transformation into an OAJ using the tandem model. With the example of MIM it could be shown that the metric is an objective tool. Insofar it is possible to accompany and evaluate the transformation of a SJ into an OAJ and to take measures according to the resulting situations and numbers, which make it possible to achieve the desired target scenario.There are various restrictions on the parameters, which are based on how much effort is required to collect the relevant data. This results in an imbalance between the effort required to collect data and the significance of the evaluation of the transformation. Among the primary parameters, cost parameters must be considered under this aspect. The costs for a journal are complicated to record since they are not available as individual costs for a journal and always refer to a mixed calculation based on all costs incurred. It is almost impossible to separate individual elements from this calculation, since determining the proportional costs is extremely time-consuming. In addition, costs are very company-specific and are often subject to secrecy. For the secondary parameters, it has been shown that the effort required to collect data for the parameter groups quality of publications, author satisfaction, demand comparison between the user groups, and topics between both journal parts is so high that the question of the actual benefit for an evaluation of the transformation arises. The evaluation can already be very well accompanied and evaluated based on collection and analysis of the primary parameters. Insofar a survey of the secondary parameters is not overly necessary, and it should be seen as additional information to make it possible to assess the scientific character and acceptance of the journal in the scientific community over several years. These parameters can thus contribute, for example, to sharpening the target group or investigating measures taken about scientific influence.Our results for the MIM are only interim results related to the duration of the transformation and the investigation and monitoring with help from a scenario analysis. From these intermediate results, it can be seen that after the initial positive development, the transformation is now moving in the direction of an undesired final state. Being aware of this effect is positive because the editor and publisher can now take measures to counteract its development.The analyzed data set includes only the original papers and the Focus Theme original papers. All other contributions such as editorials, discussions or memorandums at the invitation of the editor were not included in the analysis. We have therefore excluded 79 papers from the data set (January 2014 to September 2019). The main reason for this decision was that these papers do not undergo the classic review process and thus lack the quality control of papers by independent experts. In addition, many of these articles could not provide the necessary data for the planned evaluations.A serious limitation that occurred during the project is the sale of Schattauer to Thieme. First of all, we have to mention that, fortunately, the Trans-O-MIM project received the same full support from Thieme as it has received from Schattauer before. This change was not foreseeable in the run-up to the transformation but had consequences for the transformation process. Work processes that were defined and developed in advance could, of course, not be reconciled with the new structures. For example, it was no longer possible to address the income and cost parameters of the journal within the framework of evaluation, since the collection of income and expenditure for the MIM journal was, for obvious reasons, not comparable and feasible between Schattauer and Thieme.The methodology of scenario analysis is suitable for evaluating the transformation from a SJ to an OAJ. The results of the thesis show that it is possible to use the parameter submissions, publications, acceptance rate, and duration to evaluate the transformation of a journal. The use of the other parameters should be decided depending on the cost-benefit ratio for the data collection. These parameters are well suited for assessing the scientific reach and the presence of the journal among the target groups. The interim status for the evaluation of the transformation of the MIM journal could be shown, and the developed scenarios are applied here. Corresponding to the situations described by the scenarios, measures should be taken within the framework of such a transformation to achieve the desired target scenario.ESM 1(XLSX 74 kb)


Arenaviridae, is an ambisense RNA virus that causes severe hemorrhagic fever with a high fatality rate in humans in West and Central Africa. Currently, no FDA approved drugs or vaccines are available for the treatment of LASV fever. The LASV glycoprotein complex (GP) is a promising target for vaccine or drug development. It is situated on the virion envelope and plays key roles in LASV growth, cell tropism, host range, and pathogenicity. In an effort to discover new LASV vaccines, we employ several sequence-based computational prediction tools to identify LASV GP major histocompatibility complex (MHC) class I and II T-cell epitopes. In addition, many sequence- and structure-based computational prediction tools were used to identify LASV GP B-cell epitopes. The predicted T- and B-cell epitopes were further filtered based on the consensus approach that resulted in the identification of thirty new epitopes that have not been previously tested experimentally. Epitope-allele complexes were obtained for selected strongly binding alleles to the MHC-I T-cell epitopes using molecular docking and the complexes were relaxed with molecular dynamics simulations to investigate the interaction and dynamics of the epitope-allele complexes. These predictions provide guidance to the experimental investigations and validation of the epitopes with the potential for stimulating T-cell responses and B-cell antibodies against LASV and allow the design and development of LASV vaccines.Lassa virus (LASV), a member of the  Arenaviridae1, is an ambisense RNA virus that causes a severe hemorrhagic Lassa fever in humans. LASV is endemic, particularly in the West African countries of Sierra Leone, The Republic of Guinea, Nigeria, and Liberia3. The transmission of LASV to humans occurs through the urine or feces of infected Mastomys rats and the virus spreads human-to-human through direct contact with the blood, urine, feces, or other bodily secretions of an infected person. LASV can be fatal and no approved effective therapeutics are currently available. The development of therapeutics such as antibodies and vaccines for the treatment of LASV is therefore of significant urgency6.Lassa virus (LASV), a member of the 7. GP1 is the receptor-binding subunit, and GP2 is the membrane-spanning fusion subunit10. The virion envelope protein spikes are composed of three heterotrimers, with each heterotrimer containing signal peptide, GP1, and GP212, shown in Fig. 13. GP is considered to be a key factor for LASV growth, cell tropism, host range and pathogenicity, and as it is the only protein situated on the LASV virion surface, GP becomes a primary target for vaccine design4.Of the four proteins that are encoded by the two RNA segments of the LASV genome, the glycoprotein (GP) is the only protein on the viral surface. GP results from the cleavage of a 75 kDa precursor polypeptide, GPC by signal peptidase and then further glycosylated and processed into GP1 and GP214. Although the antibody predominantly binds to GP2, GP1 is required to maintain the proper prefusion conformation of GP2 for antibody binding4.The crystal structure of the trimeric LASV GP in complex with the 37.7 H neutralizing antibody from a human survivor  tools. Subsequently, we identified strongly binding alleles to the MHC-I T-cell epitopes and modeled the allele structures and performed docking to understand the interaction between alleles and epitopes. We further investigated the stability and dynamics of the epitope-allele complexes using molecular dynamics simulations. Analyses of root-mean square deviations, hydrogen bond, interaction energy, and solvent accessibility showed that epitope-allele complexes are stable, indicating that the epitopes strongly bind to the alleles. The identified B and T-cell epitopes of LASV GP in the study can be useful for the development of effective vaccines against Lassa hemorrhagic fever.Identification of epitopes is an essential step for understanding disease etiology, immunotherapy, immunodiagnostics, and the discovery and development of epitope based-vaccines. An epitope-based vaccine has fewer side effects compared to conventional vaccines. Experimental identification of a promiscuous epitope involves many expensive and time-consuming steps, including the production of antibodies to map antigenic regions on a target protein, animal models, and determination of the crystal structure of antigen-antibody complexes using X-Ray crystallography. Computational identification of epitopes is often employed as a powerful and fast approach to facilitate the identification of potential epitope candidates that can decrease the number of validation experiments and time19. Subsequently, multiple sequence alignments were performed between the sequences using Clustal Omega20 to select a conserved LASV GP for sequence-based epitope prediction. The corresponding X-Ray crystal structure of the Mouse/Sierra Leone/Josiah/1976 LASV GP was obtained from the Protein Data Bank (PDB ID: 5VK2)21 for structure-based B-cell epitope prediction. The missing residues were modeled using the Charmm-Gui24.The sequence of GP for different LASV strains was obtained from the NIAID Virus Pathogen Database and Analysis Resource (ViPR)25, BCPREDS26 and BcePred27 servers separately. These servers predict epitopes based on physico-chemical properties of amino acids, and these servers accept the primary sequence of LASV GP as an input.Sequence-based B-cell epitope prediction was performed with the use of BepiPred2.028, Epitopia29 and DiscoTope30. These servers predict epitopes regions based on the geometrical and solvent surface-accessibility of a protein structure, and these servers accept the 3D structure of a protein as input. The consensus epitopes from both sequence and structure-based predictions were selected as potential epitopes for further analysis.Structure-based B-cell epitope prediction for the LASV GP (PDB ID: 5VK2) was carried out using three different programs separately: ElliPro31, CTLPred32 and NetCTL1.233. To predict their alleles, the consensus epitopes among these three prediction methods were analyzed using IEDB34. The epitopes that strongly bind to the alleles (lowest IC50) were selected for further analysis.Sequence-based MHC-I T-cell epitope predictions for LASV GP were carried out by using three different servers, ProPred-I35, NetMHCII2.336 and EpiTOP3.037. The antigenicity score of the selected epitopes was predicted by VaxiJen 2.038.Sequence-based MHC-II T-cell epitope predictions for LASV GP were performed with the use of three different servers: ProPred39], HLA-A*02:03 (A2) [PDB ID: 3OX839], and HLA-B*35:01 (A3) [PDB ID: 2CIK40] were obtained from the PDB. The experimental structure for the HLA-A*32:01 (A4) allele is not available, and thus, the sequence of this allele was obtained from the UniProt database (UniProtKB ID: P01892), and subsequently its structure was modeled using Swiss-Model43. The selected consensus MHC-1 epitopes were extracted from the crystal structure of LASV GP (PDB: 5VK2). The epitopes and the alleles were prepared for docking using Autodock Tool version 1.5.644. Autodock Vina 1.1.245 was used for peptide docking with a grid space that covered the entire allele. The best peptide-allele complexes were selected for further investigation based upon visual inspection of peptide-allele interactions and the Autodock Vina criteria. The stability and dynamics of the selected peptide-allele complexes were further studied using molecular dynamics simulations.The structure of HLA-A*02:06 (A1)  as a highly conserved strain, and we thus selected this strain for the sequence-based MHC-I and MHC-II T-cell epitope predictions and for both structure and sequence-based B-cell epitope predictions. In addition, a search of this strain with the experimentally determined structure available in the PDB displayed the 3.2 Å resolution crystal structure of the prefusion GP trimer of LASV in complex with the human neutralizing antibody 37.7 H. [PDB ID: 5VK2] as shown in Fig. -I Table . Althoug-I Table  which is50), antigenicity, and allele. Among these four consensus epitopes, the nanomer E1 epitope FATCGLVGL shows the lowest average IC50 value of 34 nM against the A1 allele as predicted by the IEDB, and it has also a reasonable antigenicity score of 1.65. This was followed by the E3 epitope FSRPSPIGY, which has an average IC50 value of 88 nM against the A3 allele, and also has a better antigenicity score of 2.50 compared to the FATCGLVGL epitope. Interestingly, the E4 epitope RRGTFTWTL is predicted by all three methods though its IC50 and antigenicity scores are not as good as the other epitopes 37 to predict the MHC-II T-cell epitopes. The epitopes that were predicted by at least two methods are listed in Table 57.MHC-II T-cell epitope prediction with the LASV GP sequence was performed using three different methods separately: ProPred, NetMHCII 2.3, and EpiTOP 3.0, and the results are shown in Supplementary Table 25, BCPREDS26, and BcePred27. The BepiPred predicts the epitopes based on a random forest algorithm trained on epitopes annotated from antibody-antigen structures. BCPREDS predicts epitopes by using SVM combined with a different kernel method, including string kernels, radial basis kernels, and subsequence kernels. The BcePred locates B-cell epitopes using four physicochemical properties like hydrophilicity, polarity, exposed surface and beta-turns27. The epitope E30 containing 10 residues was predicted by all three of these sequence methods  with the modeled missing residues was used. ElliPro predicts linear and conformational epitopes by incorporating the antigenicity, solvent accessibility, and flexibility of protein structureset al.14 have recently reported the cloning of many human monoclonal antibodies derived from memory B cells of Lassa fever survivors in West Africa. These antibodies specifically bind to both GP1 and GP2 epitopes of LASV. The comparison of our predicted B-cell epitopes with those epitopes shows that there are five consensus epitopes  as the best template for constructing models. The sequence identity between A4 and the template was 92%. The best model was then selected based on multiple validation methods, including GMQE  and QMEAN. The GMQE and QMEAN values45. However, we validated the capability of the docking protocol before docking the epitopes by redocking the epitopes into the allele crystal structure (PDB ID:3OX8) to see whether the crystal bound conformation of the peptide could be reproduced or not. The docked allele-epitope complex showed the same residue-epitope interactions observed in the epitope bound crystal structure, indicating that the Autodock Vina docking protocol was capable of reproducing the experimentally observed binding mode of the epitope. We applied Autodock Vina to each of the four MHC-I allele-epitope complexes. Autodock Vina found that the highest ranked docking structure had the following binding affinities: −5.5 kcal/mol for A1::E1 −5.0 kcal/mol for A2::E2, −6.8 kcal/mol for A3::E3, and −6.0 for A4::E4. These epitopes-alleles docking complexes are shown in Fig. Docking of the four consensus MHC-I epitopes Table  was perfet al.61 on MHC-I epitopes.In order to investigate the dynamics and stability of the four MHC-I allele-epitope complexes, we performed 200 ns all-atom, explicit solvent MD simulations. To quantitatively understand the stability of the allele-epitope complex, we calculated the root mean square deviations (RMSD) of the backbone atoms of the allele-epitope complexes as a function of simulation time as shown in Fig. Since the interactions between protein and epitope peptide are mostly influenced by non-covalent interactions, we computed the number of hydrogen bonds and the interaction energy between the allele and epitope as a function of the MD simulation time. The hydrogen bond was calculated between the protein interface atoms with a distance cut-off of 3.5 Å and angle cut-off of 30o between the donor and acceptor heavy atoms. As shown in Fig. Figure 34. The IEDB database contains the epitopes that are annotated based on scientific literature. The IEDB showed that the E1, E4, E18, E22, E27, E29 epitopes, which bind to solvent exposed regions on the protein  segment was found to be especially stable. This study demonstrates that the adopted consensus epitope prediction strategy is valuable for in-silico investigations of known epitopes and the identification of new epitopes. Experimental validation of these epitopes may lead to the design and development of effective LASV vaccines.LASV hemorrhagic fever is endemic in West Africa, and no approved effective therapeutics are currently available. Therefore, there is an urgent need for the discovery and development of potential antiviral therapeutics. The LASV GP spike has emerged as a promising selective target for the development of novel vaccines as it plays an essential role in the virus-host interaction. Several in-silico studiesSupplementary Information.


Substituting starch with digestible fiber (dF) can improve digestive health of rabbits and reduce costs. Therefore, it is necessary to develop a criterion for dF and starch supply. Effects of the dietary dF-to-starch ratio on pellet quality, growth and cecal microbiota of Angora rabbits were evaluated.Five isoenergetic and isoproteic diets with increasing dF/starch ratios  were formulated. A total of 120 Angora rabbits with an average live weight of 2.19 kg were randomly divided into five groups with four replicates. At the end of 40 day feeding trial, cecal digesta were collected to analyse microbiota.Ruminococcus in the 0.71 dF/starch treatment was higher than that in the 0.59 dF/starch treatment (p<0.05)The results showed that the dF/starch ratio had linear effects on pellet variables (p< 0.01). When the dF/starch ratio was 1.44, the pellets had the lowest powder and highest durability. The dF/starch ratio had unfavorable linear effects on growth variables (p<0.001). When analyzed by quadratic regression, the optimal dF/starch ratios for average weight gain and feed/gain were 0.59 and 0.74, respectively. There were differences in wool yield, fiber length and fiber diameter caused by the dF/starch ratio (p<0.05), and the dF/starch ratios that ranged from 0.66 to 1.06 were appropriate for good results. The cecal microbiota operational taxonomic unit (OTU) number index in the 1.05 dF/starch treatment was higher than that in the 0.66 and 0.71 dF/starch treatments. The higher dF/starch ratio resulted in a higher cecal microbiota OTU number index (p<0.05). The proportion of The most suitable dF/starch ratio for feed pellet quality is 1.44, and for rabbit growth the optimal range of ratios is from 0.59 to 0.74. With combination of the wool growth, output cost, and cecal microbiota, we suggest that a dietary dF/starch ratio ranging from 0.74 to 1.06 is optimal. Angora rabbit produces 1.0 to 1.4 kg/yr of pure fine animal fiber. This represents about 30% of its live weight. However, there is a considerable paucity of information on Angora rabbit nutrition compared with published work on the production of meat from rabbits or wool from sheep. The special feed for Angora rabbit is also extremely rare on the market. Therefore, the nutritional research of Angora rabbit requires particular attention.vice versa on digestive parameters was described by Gidenne and Perez [The carbohydrate fraction (fiber and starch) is a major component of commercial rabbit diets. Fiber and starch both affect the stability of the microbial ecosystem and gut health. Dietary fiber accounts for 40% to 50% of the total diet. Its importance is related to the influence on feed consumption, rate of digestive passage and role as a substrate for cecal microbiota . Sufficind Perez , and Gidnd Perez . Substitnd Perez . It has nd Perez . Becausend Perez . Therefond Perez . Howeverad libitum, a transition from standard to experimental diet was followed by a 40 d feeding trial. Natural lighting and automatic ventilation were used in the rabbit house; the temperature ranged from 12°C to 24°C, and the relative humidity ranged from 40% to 50% throughout the trial. Determination of digestive energy by the endogenous indicator method was conducted in the last week of the feeding trial.The experiment was conducted in accordance with the Institutional Animal Care and Use Committee at Shenyang Agricultural University . It was carried out at the Jinhua Angora Rabbit Industrial Park , during the period from October 2016 to December 2017. A total of 120 growing female  Wan strain Angora rabbits (83 d old and 2.19±0.07 kg) were selected and randomly allocated in galvanized metal wire cages with automatic drinkers with nipples, to five treatments each with 4 replicates containing six rabbits. After an adaptation period of 7 d during which the rabbits were fed, The diets corresponded to a linear decrease in starch level (28% to 16%) and to a nearly linear increase in dF (16% to 23%); dF/starch ratios were 0.59, 0.66, 0.71, 1.05, and 1.44. The diets were approximately isoenergetic and isonitrogenous. The dietary gross energy was analyzed by a full-automatic calorimeter . Crude protein was analyzed by a semi-automated Kjeldahl technique. Neutral detergent fiber (NDF), ADF, and acid detergent lignin used detergent method of Van-Soest by an automatic fiber analyzer . Starch was measured according to national food safety standard (GB 5009.9-2016 Determination of starch in foods).We used a 150 mm long vernier caliper, with a precision of 0.02 mm, to determine particle size.We filled the feed pellets into a 1,000 mL cylinder, leveled the particles off on the top edge of the cylinder, weighed this sample, and calculated the weight to volume ratio.We used a sclerometer  to determine pellet hardness.Powder ratio determination was based on the national standard (GB/T 16765-1997). We sieved the sample with a 2-mm metal sieve, weighed the particles passing through the screen, and calculated the percentage of the weight of screened particles to the total weight of the sample.We used the national standard (GB/T 5169-91) and a pellet durability index (PDI) instrument  to score the PDI.The number of replications for particle size, bulk density, hardness, powder ratio, and PDI determination of feed pellet were 30, 5, 15, 3, and 3 respectively.At the start and end of the experiment, rabbits were weighed in the morning. The feed consumed was recorded daily. Weight gain, feed/gain (F/G), and wool yield were measured after the feeding trial.All of the hair shed by each rabbit was individually weighed.Fiber samples were collected from a 10×10 cm patch area in the rib area of the left side of each rabbit, and their lengths were measured using a ruler. A total of 200 fibers from each rabbit were measured.Diameters of fiber samples were measured using a fiber fineness meter .At 123 d of age (at the shearing period), 20 rabbits (4 per group) in apparently healthy condition were euthanized by sudden cervical dislocation. For each rabbit, the cecal digesta was collected in tube and stored at −80°C until processed.Genomic DNA from the cecal digesta (0.2 g) was extracted using the cetyltrimethylammonium bromide (CTAB) method. The extracted DNA (2 μL) was then subjected to electrophoresis on a 7 g/L agarose gel (W/V) to estimate the amount and integrity of the DNA products. The DNA solution was preserved at −20°C until further analysis.Amplification of the variable V3-V4 region of bacterial 16S rDNA was performed using the general primer sequences 340F and 805R. The details of these primers are as follows: upstream primer 340F: 5′-CCTACGGGNBGCASCAG -3′; downstream primer 805R: 5′-GACTACNVGGGTATCTAATCC -3′. Polymerase chain reaction (PCR) was performed using model PTC220  and PCR amplification kits  in reaction mixtures containing 5 μL 10× PCR buffer; 1 μL dNTP (2.5 mM); 0.2 μL rTaq (5 U/μL); 2 μL 340F (20 mM); 2 μL 805R (20 mM); 1 μL template DNA, and made up to a final volume of 50 μL using double distilled water. The thermal cycle used the following conditions: initial denaturation at 95°C for 3 min, followed by 30 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 60 s, and final elongation at 72°C for 7 min.Samples were mixed according to the concentration of the PCR products. After complete mixing, the PCR products were purified using 0.5×tris-boric acid buffer of 1.5% agarose gel electrophoresis. The targets were then recovered using a MinElute Gel Extraction Kit .The complementary DNA Library was constructed using a NEB Next Ultra DNA Library Prep Kit  for Illumina. The constructed library was tested by Qubit quantitative and library and, after qualification, MiSeq 2500 was used for sequencing.http://ccb.jhu.edu/software/FLASH/. Paired-end reads were assigned to each sample according to the unique barcodes and follow up analysis with the processed sequence; operational taxonomic units (OTUs) cluster and Species annotation. Sequence analysis was performed by QIIME 1.8 software package [Paired-end reads assemblies: We used FastQC software to evaluate the number and quality of sequencing. Paired-end reads from the original DNA fragments were merged using FLASH v1.2.7  package . We filtData were analyzed using one-way analysis of variance with SPSS 21.0 . Contrast was established using linear and quadratic polynomial methods to determine the effects of increasing the dF/starch ratio. The data are presented as means, standard error of the mean, and p-values. Multiple comparisons were made using least significant difference method. Differences were considered significant at p<0.05.1, p<0.001) and hardness  were observed. Based on the regression equation , when the dF/starch ratio was 0.67, the pellet bulk density was the highest, and when the dF/starch was 1.53, the hardness was the greatest. When the dF/starch ratio was 1.44, the feed had the best pellet quality (the lowest powder ratio and the highest PDI).Effects of the dietary dF/starch ratio on pellet quality are presented in Effects of different dietary dF/starch ratios on growth and wool yield in Angora rabbits are shown in The wool yield in the 0.66 dF/starch treatment was the highest (140.6 g), and this was higher than that in the 1.44 dF/starch treatment by 9.37% (p<0.05), but there was no significant difference compared with the other groups (p>0.05). The fiber diameter in the 1.05 dF/starch treatment was the finest, and this was finer than that in the 0.59 dF/starch treatment (p<0.05).The shortest fiber length (5.63 cm) occurred in the 0.66 dF/starch treatment and this was shorter than that in the 1.05 dF/starch treatment by 17.81% (p<0.05). A significant favorable quadratic effect on fiber diameter was seen (p<0.05). Based on the regression equation , when thThe microbial 16S rDNA V3-V4 region of 20 cecum contents in 5 treatments were sequenced based on Illumina Miseq sequencing technology, in which 18 samples met the requirements. OTUs were classified according to the similarity of 97%; the effective sequence range of each sample was 239,496 to 963,776 and the range of OTUs was 4,403 to 10,959.Alpha diversity is the analysis of species diversity within a single sample. We annotated the microbial species found in this study. A total of 14 phyla, 24 classes, 26 orders, 25 families, and 17 genera were obtained from the 18 samples. The effect of different dF/starch ratios on the proportion of microbiota phylum is shown in The cecum microbiota was similar at the phylum level, among the different dietary dF/starch ratio treatments (p> 0.05). The dominant bacteria phyla were Firmicutes and Bacteroidetes, followed by Verrucomicrobia and Proteobacteria. The relative microbiota abundance of Bacteroidetes, Proteobacteria, Actinobacteria, Acidobacteria, Chloroflexi, Gemmatimonadetes, Planctomycetes, as well as Nitrospirae, and Crenarchaeot in the 0.59 dF/starch treatment was the highest among all other groups. The microbiota abundance of Firmicutes in the 0.66 dF/starch treatment was the highest among all other treatments. The relative microbiota abundance of Verrucomicrobia in the 0.71 dF/starch treatment was the highest among all treatments. The 1.44 dF/starch treatment had the largest proportion of Tenericutes, TM7 and Euryarchaeota.Ruminococcus (4.58%), Bacteroides (4.88%), Akkermansia (3.21%), Faecalibacterium (2.43%), and Oscillospira (1.22%). The abundance ratio of bacteria at the genus level was not affected by the dF/starch ratio (p>0.05) except for Ruminococcus. The proportion of Ruminococcus in the 0.71 dF/starch treatment was higher than that in the 0.59 dF/starch treatment (p<0.05).There were 3 genera  found only in the 0.59 dF/starch treatment.At the genus level, 14 shared bacteria taxa were identified . The perTo reflect OTU positioning among the samples, two dimensional clustering of samples and OTUs was conducted, resulting in a graphical heatmap . In the Ruminococcaceae (26.13%), S247 (10.42%), Lachnospiraceae (10.39%), Bacteroidaceae (4.63%), Verrucomicrobiaceae (3.16%), and Rikenellaceae (2.90%). Other families .At the family level, unknown bacteria accounted for 33.46% of the total, followed by The pellet quality of rabbits is mainly a function of the particle size, pellet durability and hardness. The diameter and length of the pellets affect the feeding, digestion and processing cost of rabbits. The length of pellets is preferentially between 8 to 10 mm . If pellPellet durability and hardness are major quality charac teristics of rabbit pellets because rabbits do not eat the fines between the pellets. When provided diets with a mixture of pellets of different hardness, rabbits tend to eat a greater percentage of the hard particles. However, very hard pellets with low elasticity are fragile and can produce a large amount of fines . MorisseThe digestive energy present in rabbit diet is mainly from starch and dF but excessive dF will reduce rabbit growth. Perez et al  reportedWe found that a diet combining a low dF level with rela tively high starch (dF/starch: 0.59 to 0.74) enhanced rabbit growth. These results are consistent with the results of Tazzoli et al  who obseFor regular diets, without lipid addition, growing rabbits regulate their feed intake based on the digestible energy level of the diet . If the In this paper, the highest wool yield was obtained in the 0.66 dF/starch treatment, but there was no significant difference compared with the other groups except for the 1.44 dF/starch ratio treatment. Some impairment in the performances of Angora rabbits was observed in those fed the highest dF/starch ratio (1.44). This might be explained by higher fermentation losses in the cecum . The estFiber degradation is ultimately determined by microbial activity, digesta retention time in the cecum and fiber chemical composition and structure . Jehl anWe found that the cecal microbiota remained relatively stable at the phylum level. The dominant bacteria phyla (%) were Firmicutes (59.14), Bacteroidetes (21.38), Verrucomicrobia (3.47), and Proteobacteria (3.25). This result was consistent with results of Xu et al  who repoRuminococcus spp. is the main Fibrolytic bacteria found in the rumen of cattle [Ruminococcus in the rabbit cecum in all treatments exceeded 1%, followed by Bacteroides and Akkermansia. These data could explain why both high dF and high starch diets can promote the growth of Fibrolytic bacteria. However, when the dF/starch ratio was 0.71, the abundance ratio of Ruminococcus was the highest (6.24%). So, the 0.71 dF/starch diet seems to be more beneficial to the decomposition of fiber and this result was consistent with the growth rate. We speculate that appropriate dF/starch ratio diet can provides both long and digestible fiber, which allows an adequate transit time of the digesta and a balanced growth of the cecal microbiota. There was a high percentage (77.18%) of unidentified bacteria genera in this study, indicating that there are many unidentifiable species of intestinal microbiota in Angora rabbits. More attention should be given to the characteristics and functions of unknown bacteria for a better understanding of the intestinal microbiota.f cattle . We demoIn conclusion, the most suitable dF/starch ratio for feed pellet quality is 1.44 and for rabbit growth the optimal range of ratios is from 0.59 to 0.74. In addition, for wool growth, the optimal range of ratios is from 0.66 to 1.06. With the combination of the output cost and cecal microbiota, we suggest that a dietary dF/starch ratio for Angora rabbits ranging from 0.74 to 1.06 is optimal.


Subtalar joint (STJ) dysfunction can contribute to movement disturbances. Vibration energy with color Doppler imaging (VECDI) may be useful for detecting STJ stiffness changes.(1) Support proof-of-concept that VECDI could detect STJ stiffness differences; (2) Establish STJ stiffness range in asymptomatic volunteers; (3) Examine relationships between STJ stiffness and foot mobility; and (4) Assess VECDI precision and reliability for examining STJ stiffness.in vivo. Three reliability measurements were collected per variable; Rater-1 collected on all subjects and rater-2 on the first ten subjects. Subjects were classified into three STJ stiffness groups.After establishing cadaveric testing model proof-of-concept, STJ stiffness , ankle complex passive range-of-motion (PROM) and midfoot-width-difference (MFWDiff) data were collected in 28 asymptomatic subjects In vivo VECDI measurements demonstrated good intra-rater (0.76–0.84) versus poor inter-rater (-3.11) reliability. Significant positive correlations were found between STJ stiffness and both dorsum (r = .440) and posterior (r = .390) PROM. MFWDiff exhibited poor relationships with stiffness (r = .103) and either dorsum (r = .256) or posterior (r = .301) PROM. STJ stiffness ranged from 2.33 to 7.50 ΔTUs, categorizing subjects’ STJ stiffness as increased (n = 6), normal (n = 15), or decreased (n = 7). Significant ANOVA main effects for classification were found based on ΔTU (p< .001), dorsum PROM (p = .017), and posterior PROM (p = .036). Post-hoc tests revealed significant: (1) ΔTU differences between all stiffness groups (p < .001); (2) dorsum PROM differences between the increased versus normal (p = .044) and decreased (p = .017) stiffness groups; and (3) posterior PROM differences between the increased versus decreased stiffness groups (p = .044). A good relationship was found between STJ stiffness and dorsum PROM in the increased stiffness group (r = .853) versus poor, nonsignificant relationships in the normal (r = -.042) or decreased stiffness (r = -.014) groups.Cadaveric VECDI measurement intra-rater reliability was 0.80. A significantly lower STJ ΔTU (p = .002) and ankle complex PROM (p < .001) was observed during the screw fixation versus normal condition. A fair correlation (r = 0.660) was observed between cadaveric ΔTU and ankle complex PROM. PROM may not clinically explain all aspects of joint mobility. Joint VECDI stiffness assessment should be considered as a complimentary measurement technique. Throughout the gait cycle, the subtalar joint (STJ) acts as a shock absorber and assists in propulsion . This joStudies assessing the relationship between STJ dysfunction and foot mobility are limited in number and scope. Calcaneal fractures have been found to display altered joint kinematics ,9 where Few approaches to STJ mobility assessment have been reported. Loaded and unloaded plain-film stress radiographic imaging can speculate STJ dysfunction, where larger foot posture differences correspond with clinical instability . Diagnosin vivo skin-mounted infra-red markers overlying bony structures ) using an electrogoniometer (ELGON); as well as MFWDiff. We hypothesized that a good to excellent correlation would be observed between STJ stiffness (i.e. ΔTU) and both PROM and MFWDiff (Hypothesis 2 / H2). Finally, we asked how VECDI-derived values could be used to classify an individual as having decreased, normal or increased STJ stiffness and how that classification could influence the previously described relationships. We hypothesized that different relationships would be observed within the different classification groups, based on stiffness differences across the groups (Hypothesis 3 / H3).In order to address the study purposes, we first aimed to establish VECDI intra- and inter-rater reliability when assessing STJ stiffness in vivo data collection was approved prior to initiation by the TTUHSC Lubbock/Odessa Institutional Review Board for Protection of Human Subjects (#-L14-036). Informed consent was obtained from each participant in accordance with TTUHSC Lubbock/Odessa Institutional Review Board for Protection of Human Subjects’ policies and procedures. In addition, all procedures followed the Helsinki Declaration’s ethical principles.All cadavers used in this study were obtained from the Willed Body Program at the Institute of Anatomical Sciences of the Texas Tech University Health Sciences Center (TTUHSC). None of the cadavers were from a vulnerable population and all donors or next kin provided written informed consent that was freely given. All cadaveric specimens were handled in accordance with TTUHSC policies and regulations as determined by the Texas State Anatomical Board. All human subjects’ Moreover, the individuals in this manuscript  have givin vivo, eight un-embalmed cadaveric STJ’s were used for preliminary testing. Such testing was meant to test if the planned study procedures could be transferred to an in vivo investigation described below. To determine if VECDI could detect STJ stiffness changes, measurements were completed before and after STJ fixation using two 7mm cannulated surgical screws  in a delta configuration. Such surgical screw delta configuration produces the greatest STJ stability [Prior to VECDI testing tability . Prior tPrior to VECDI testing, two ELGONs were used to collect cadaveric ankle-foot complex PROM. One ELGON was applied to the ankle-foot’s dorsum spanning from the distal tibia to the second metatarsal. A second ELGON was adhered on the ankle complex’s posterior aspect from a level approximately 5cm proximal to the lateral malleolus to the posterior distal calcaneus. Ankle-foot inversion / eversion PROM values were collected by passively moving the calcaneus to joint end range three times, where each of the ELGONs measured a different joint system–STJ and midfoot inversion / eversion, respectively.Investigators then removed the ELGONs and the table was lowered in order to contact the posterior calcaneus on the top plate of the vibration energy shaker apparatus  while sustaining the 10 degrees of ankle plantar flexion . Vibratitalus vs. TUcalcaneus, respectively). The calcaneal threshold units  were subtracted from the talar threshold units , resulting in the difference in vibration threshold at the two boney structures (ΔTU). Such ΔTU values were used as a measure of STJ stiffness.Vibration energy was detected by an ultrasound transducer  spanning from the talar neck to the distal lateral calcaneus . VibratiThis procedure for measuring ΔTU was repeated two additional times and a mean value was calculated for analysis. In order to begin addressing H1 using cadaveric specimens, one rater performed all TU measures three times each. Once a measurement was taken, the US probe was removed for 10 seconds before it was again placed in the same position on the cadaver’s foot and the next measurement was taken. No other factors were adjusted between the measurements. The rater was a physical therapist with 4 years of musculoskeletal ultrasound experience.A priori defined criteria of success and, thus, justification for conducting an in vivo follow-up study were (1) VECDI intra-rater reliability of at least 0.75 and (2) statistically significant lower ΔTU and ankle-foot PROM values, respectively, following cadaveric STJ screw fixation compared to the unscrewed condition. Because these criteria were met  was used. Exclusion criteria were: 1) known, unhealed calcaneus or talus fractures; 2) known tumor in the ankle-foot bony structures; 3) current formal healthcare for a lower extremity injury; 4) open wounds in the ankle-foot region; and 5) inability of the investigators to discriminate VECDI readings between the subject’s talus and calcaneus. A history of lower extremity injuries did not exclude subjects from participation if the subject did not present with current pain at the time of data collection.Based on a moderate effect size (d = 0.5), an alpha level of .05 and 80% power, a sample size of 28 was required for final analysis . Thus, fin vivo data collection, all subjects signed an approved informed consent form. Next, the subject’s dominant weight-bearing leg, defined as the leg that the subject would stand on while kicking a ball, was identified. The subject was asked to lie supine on a high/low table. The dominant leg was rested in a custom-made jig designed to position the test lower extremity in approximately 70 degrees of hip and knee flexion. The ankle was positioned at approximately 10 degrees of plantar flexion for all testing procedures. The subject's non-dominant limb was placed in a hook-lying position and the hands were placed in a resting position at the subject's side. In order to address H1 for the in vivo subjects, two separate raters performed TU measures three times each using the VECDI procedure as previously described. No other factors were adjusted between the measurements. Each of the two raters was blinded to the other rater’s TU values, their own TU values, and the PROM values. Both raters performed all TU measurements on the first 10 subjects while the second rater exclusively performed all TU measurements for the remaining 18 subjects. Both raters were physical therapists with musculoskeletal ultrasound experience (one was the same as in the cadaveric study and the other with 2 years of experience).Prior to In order to address H2, subjects were instructed to stand on the ground with both shoes and socks removed and feet positioned approximately shoulder-width apart with an estimated even weight distribution between the lower extremities. A digital caliper  was used to measure foot width three times at the mid-point (50% of foot length) in the standing position. The subject was then asked to sit on the high/low table with both feet suspended off the ground. A lightweight rigid platform was raised to the plantar surface of the subject’s foot with care to not deform the foot’s resting position. Three additional foot width measurements were performed in this position. For all midfoot width measurements, the measuring rater was blinded to the measurement values. Midfoot-width-difference was calculated by subtracting the non-weight bearing width from the weight bearing width for each subject and the resulting values were used as a measure of foot mobility.3,1) were used to calculate intra-rater reliability for VECDI and PROM measures. The root mean square standard deviation (RMS-SD) values were calculated for VECDI.Data analysis was completed using IBM SPSS version 21.0 for Windows . For descriptive data from the preliminary cadaveric study, mean, standard deviation (SD), and 95% confidence interval (95% CI) were calculated for ΔTU and PROM. Intra-rater reliability of VECDI and PROM measurements was assessed using data collected from three repeated measures from all eight cadaveric specimens. Investigators performing PROM and VECDI measurements were blinded to measurement values. Two separate intraclass correlation coefficients  and PROM values were assessed between the two mobility conditions (screw fixation versus no-screw fixation) using paired t-Tests. Two-tailed paired t-tests were performed to test for a difference between conditions of normal stiffness and screw fixation for both ΔTU and ankle complex PROM. For statistical significance, alpha was set at .05. A Pearson product moment correlation was calculated to determine the relationship between STJ VECDI values and ankle complex PROM in the same sample.in vivo study, descriptive statistics were calculated to check for data normality. Normal distribution was considered present when at least two out of the three following criteria were met: Shapiro Wilk test p-value >.05, skewness and or kurtosis each between -2.0 and +2.0. In addition, descriptive data were calculated to summarize the sample’s demographic characteristics. Group descriptive data in terms of mean, standard deviation (SD), and 95% confidence interval (95% CI) were derived using each subject’s three-trial ΔTU average.For the 3,1) representing relative agreement were performed to determine intra-rater reliability of STJ stiffness (ΔTU), PROM, and MFWDiff measures and one ICC2,3 was performed to measure relative inter-rater reliability for stiffness measures. In accordance with other authors we considered values between .75 and .90 representing good reliability and values greater than .90 being indicative for excellent reliability [In order to address H1, intraclass correlation coefficients  and foot mobility (represented by MFWDiff).n vivo, we used a subsequent exploratory analysis to better understand data variance patterns and how those patterns affected the same relationships between ΔTU and our reference standards. To do this, we first placed each subject into one of three classification subgroups based on ΔTU dispersion from the sample mean in accordance with previous investigators [In order to address a noted large ΔTU variance and further support proof-of-concept that VECDI could detect STJ stiffness changes itigators ,34: incrWith regards to H1, good to excellent reliability values were established for all preliminary cadaveric study measurements. Intra-rater reliability for cadaveric VECDI measurement was found to be 0.80 (95%CI = 0.60–0.92). Root mean square standard deviation was 1.34 ΔTU for both conditions together, 1.59 ΔTU for normal stiffness, and 1.03 ΔTU for simulated hypomobility. Additionally, intra-rater reliability for ankle complex PROM measurements was found to be 0.99 (95%CI = 0.98–1.0).in vivo, based on appreciable changes in the VECDI values between before and after STJ screw fixation, as well as respectable changes in correspondence between VECDI and PROM values.Paired samples t-tests comparing mean ΔTU across structural stability conditions exhibited significant differences between conditions. First, a significantly lower ΔTU at the STJ was observed during the screw fixation versus normal condition . Thus, the STJs that were stabilized by screw fixation showed a greater energy transfer across the joint. Second, paired samples t-test results revealed that ankle complex PROM was significantly lower in the screw fixation versus normal condition . Results demonstrated that increases in STJ stiffness (represented by ΔTU) corresponded with decreases in overall sample ankle PROM. This was reflected by a fair correlation between ΔTU and ankle complex PROM (r = 0.660). These preliminary findings served as precedence for continuing test procedures in vivo subject demographics, STJ stiffness (represented by ΔTU) and ankle complex PROM can be found in Mean and standard deviation values for All reliability data can be found in r = .440, p = .019,adjusted R2 = 0.163) and posterior PROM . No significant relationship was found between ΔTU and MFWDiff  or between MFWDiff and either dorsum PROM  or posterior PROM .With regard to H2, a significant positive low to moderate correlation was found between ΔTU and both dorsum PROM  versus poor, nonsignificant relationships found in normal  or decreased stiffness  groups. No significant correlations were found within the classification groups between ΔTU and MFWDiff or between ΔTU and posterior PROM (p > .05).In response to these findings, correlations were conducted between the same variables within each group. A correlation between ΔTU and dorsum PROM revealed a significant and meaningful relationship in the increased stiffness group  and ankle-foot PROM. This suggests that foot mobility as measured by MFWDiff may be unrelated to STJ stiffness and ankle-foot PROM in this asymptomatic sample. Moreover, the MFWDiff values of 7.1 (SD = 2.6) mm as measured in this sample are similar to results of a previous study demonstrating a mean MFWDiff of 9.3 (SD = 3.2) mm. ContrarWe were able to divide subjects into three groups based on ΔTU mean and standard deviation values. After dividing the sample, analyses results demonstrated significant differences between the three groups, thus supporting H3. Within this classification system, dorsum PROM values were significantly different between increased and normal stiffness and between decreased and increased stiffness, but no difference was found between normal and decreased stiffness. This lack of PROM difference between the normal and decreased stiffness groups can have two plausible explanations. First, given the difficult nature of clinically measuring STJ mobility, it is possible that VECDI may be a more sensitive tool for representing STJ stiffness than PROM alone. Secondly, based on a visual analysis of a simple scatter ΔTU and PROM value plot , it is p2 = 0.163), PROM may only explain approximately 16% of STJ stiffness variance. While the two measures are related to a moderate degree, using one measure to infer the other would be inappropriate since 84% of STJ stiffness variance is unexplained by PROM alone. As a result, it is important to not only assess total distance moved through PROM, but also assess the movement resistance within the available range. Since vibrations applied during VECDI assessment are very small (<0.05mm) and stay within the unconstrained region of movement , it is apparent that VECDI is not measuring PROM, but rather another non-angular characteristic influencing joint mobility, such as joint stiffness. This observation is consistent with previous investigations using VECDI at the first tarsometatarsal joint [Considering the moderate relationship between ΔTU and PROM Click here for additional data file.


To evaluate graft properties on magnetic resonance imaging (MRI) after superior capsular reconstruction (SCR) to help improve our understanding of postoperative imaging.We identified consecutive patients who underwent SCR by a single surgeon and who had postoperative MRIs available. MRIs were analyzed to look for common postoperative findings on imaging.Ten consecutive patients with an average age of 58 years who underwent SCR by a single surgeon had postoperative MRIs on average 404 days from surgery. Eight patients had completely intact grafts on follow-up MRI. All intact grafts were similar with homogenous appearance on all coronal images. All patients displayed some trace fluid with mild heterogenous signal at the level of the glenoid, which could represent failure of the graft to completely incorporate at the level of the glenoid or could be normal in the postoperative setting since all eight intact grafts displayed this finding. None of the patients with intact grafts had bony edema noted on either the glenoid or humeral side. Four of 8 patients were noted to have trace bony edema at the level of the lateral acromion. One patient had complete disruption on the glenoid side. One patient had partially intact graft that revealed heterogenous appearance of graft.An intact graft displays a more homogenous signal on consecutive postoperative MRI coronal images than disrupted grafts or partially intact grafts. This suggests that intact grafts have better clinical outcomes than a partially disrupted or completely disrupted graft. However, the finding of heterogenous signal/fluid at the glenoid graft interface in all intact grafts could not be explained in this study.Level IV, therapeutic case series. SCR originally was described using fascia lata autograft and has shown promising results clinically.Even though there has been an explosion in literature about SCR, little is known about findings on the postoperative magnetic resonance imaging (MRI). In the primary papers on dermal allograft for SCR, only 34 MRI scans were obtained postoperatively.The purpose of this study was to evaluate graft properties on MRI after SCR to help improve our understanding of postoperative imaging. Our hypothesis was that postoperative MRIs of intact SCR grafts would show homogeneous signal on 3 consecutive coronal images.The study included consecutive patients who underwent arthroscopic SCR by a single surgeon using dermal allograft . Inclusion criteria were patients who had full-thickness rotator cuff tear (supraspinatus and infraspinatus tendons) who were found to be irreparable at the time of surgery. Exclusion criteria were patients with full-thickness retracted subscapularis tears, full-thickness cartilage defects, patients who were smokers, had uncontrolled diabetes defined as A1C >8, or who were unable to comply with postoperative protocol for any reason.,The surgical technique for the SCR has been described by multiple authors.All included patients had preoperative and postoperative MRI on affected shoulder. The pre- and postoperative MRIs were analyzed to look for any changes that occurred after SCR was performed. Preoperative MRI evaluation included number of tendons torn, size of tear in both the coronal and sagittal planes, and Goutallier score. Subscapularis integrity was noted at the time of surgery as well as the number of arthritic changes that was not seen on preoperative imaging. All patients had both preoperative and postoperative MRIs performed. Graft integrity was defined as completely intact , partially intact , and completely disrupted . Postoperative MRI was used to evaluate the integrity of the graft on multiple consecutive coronal images, as described by Burkhart et al.Postoperatively, all patients were placed in sling immobilization with abduction pillow for 8 to 10 weeks. No active range of motion of above chest level until after 3 months. No strengthening or lifting until after 6 months postoperatively.Ten consecutive patients were included in the study. Seven patients were male and 3 were female. The average age was 58 years. Three patients were involved in workers compensation claims. No patients were smokers or used any type of tobacco products. No patients had more than 1 to 2 drinks per week. Only 1 patient had had previous rotator cuff surgery. Interestingly, 7 of 10 patients had subscapularis repair done at the time of surgery. Only 2 patients were found to have grade 1 to 2 changes on the humeral head at the time of surgery. No patients experienced any postoperative complications.The average time from preoperative MRI to surgery was 127 days. The average time from surgery to postoperative MRI was 404 days. Nine of 10 patients had preoperative tangent sign. The average preoperative Goutallier score for the supraspinatus was 3.3, infraspinatus was 2.5, and subscapularis was 0.6. The average tendon retraction in the coronal plane was 4.2 cm. The average tendon tear size in the sagittal plane was 4.07 cm .Table 1PThe average number of anchors used on the glenoid side was 2.6. The average number of anchors used on the humeral side was 4.1. The average postoperative Goutallier score for the supraspinatus was 3.8, infraspinatus was 3.2, and subscapularis was 0.5. Eight patients had completely intact grafts, 1 patient had a partially intact graft, and 1 patient had a completely disrupted graft .Table 2PAs noted previously, 8 patients had completely intact grafts on follow-up MRI. All intact grafts looked similar with homogenous appearance within the graft body on all coronal images. All patients displayed some trace fluid with heterogenous signal at the level of the glenoid . This flThe patient with the partially intact graft also had subscapularis repair at the time of the procedure. Interestingly, the subscapularis failed as well as the anterior half of the graft. Also, the MRI revealed a heterogenous signal within the body of the graft on coronal images . This waThe patient with complete disruption of the graft occurred within the first 6 months. The graft failed on the glenoid side . The patient was revised to a reverse total shoulder arthroplasty and has done well. At the time of surgery, the graft was noted to be incorporated on the humeral side and all anchors were still intact on glenoid side. The failure was at the suture–graft interface.All patients with intact grafts demonstrated a homogenous signal at humeral attachment with no evidence of fluid at the graft–bone interface between the medial and lateral rows. This likely represents a quiet interface demonstrating a stable humeral attachment. All intact grafts displayed minimal fluid in the joint or in subacromial space. The partially disrupted graft revealed increased fluid in subacromial space compared to intact grafts. It also demonstrated bony edema on the humerus and glenoid, an MRI finding only identified in the single, partially-intact graft. This may represent abnormal contact forces secondary to failure or abnormal strain on the remaining graft that is stressing the graft-bone interface. There were 3 patients with intact grafts who displayed edema on the undersurface of the lateral acromion. This is likely secondary to continued abnormal contact between the humeral head and acromion. No acromioplasty was performed in any of the patients in the study. The Goutallier scores increased in respect to the supraspinatus and infraspinatus and remained stable for the subscapularis. Therefore, even with an intact graft, muscle atrophy of the torn tendons continued.The findings from our study demonstrate that intact grafts display a more homogenous signal on consecutive coronal images than disrupted grafts or partially intact grafts. The fact that there is some signal noted at the glenoid graft interface could be a normal finding or could represent incomplete incorporation of the graft. However, in our study all patients with an intact graft were happy with their results to date and have had no revision surgery. The graft that was partially intact displayed a more heterogenous signal throughout the body of the graft and likely represents graft breakdown, resorption of the graft, or incomplete vascularization of the graft. The patient with the partially intact graft had not been revised at the time of publication but was not as happy with his outcome when compared to our other patients with intact grafts. The patient with graft failure on MRI was unhappy with result and thus revised to a reverse total shoulder arthroplasty.Our study adds 10 additional MRI studies to the current literature to give a total of 44 postoperative MRIs in the current literature at time of publication. Burkhart and HartzlerBased on our observations and the aforementioned studies, an intact graft appears to have better clinical outcomes than a partially disrupted or completely disrupted graft. Our imaging data show intact graft demonstrate a more homogenous appearance on both T1 and T2 coronal imaging with the partially intact graft demonstrating a more heterogenous appearance. Also, all intact grafts in our study displayed a trace amount of fluid with heterogenous signal at the glenoid attachment. This finding needs further research to be certain that this is a normal finding with intact grafts or a sign of incomplete incorporation at the level of the glenoid.This study adds to the existing literature regarding SCR and provide a framework for interpretation of postoperative MRIs. It is our hope that this paper will prompt other surgeons currently performing SCR to review their patients’ MRI findings to help strengthen the current understanding of MRI findings and their correlation with clinical outcomes.Limitations of this study are noted with sample size being an obvious weakness; however, providing free MRIs with radiologist reading is difficult in the clinical setting. Another limitation is the lack of correlation of imaging findings to clinical outcome scores. An additional limitation is that only one of our patients was found to have a partially intact graft on postoperative MRI.An intact graft displays a more homogenous signal on consecutive postoperative MRI coronal images than disrupted grafts or partially intact grafts. This suggests that intact grafts have better clinical outcomes than a partially disrupted or completely disrupted graft. However, the finding of heterogenous signal/fluid at the glenoid graft interface in all intact grafts could not be explained in this study.


Santalum album Linn., a hemiparasitic tree, using morphological and physiological methods. Our findings showed abundant starch in the stem and root of S. album at the early seedling stage. Although S. album seedlings attached to the host showed no significant changes in starch levels throughout the experiment, unattached and host-removed seedlings exhibited a gradual decrease in the starch content over time. When the starch content of unattached seedlings was less than 1%, they started to die. Starch accumulated to high levels in developing and active haustoria; however, starch levels were low in the inactive haustoria. The present study suggests that starch may provide energy to seedlings that have no host, allowing them to survive during the unattached phase, thus increasing their chance to attach to host roots by extending their survival duration. In addition, we speculate that storage starch is potentially involved in the development of haustoria and in the physiological processes of S. album related to the absorption and transportation of water and nutrients from its host.Allocation dynamics of stored starch plays essential roles in the development and growth of trees. Previous studies focused on the dynamics and the characteristics of starch in autotrophic trees. However, although starch granules have been detected in the organs or tissues of some parasitic plants, studies on the allocation dynamics and roles of storage starch in them are limited. Therefore, we determined and estimated the allocation dynamics and roles of starch in  Starch is the most critical long-term energy storage carbohydrate in plants. Under abiotic stress conditions, such as low/high temperature or drought, starch is degraded, generating osmotically active compounds and energy, which are used to ensure plant survival . DeciduoSalix viminalis  of S. album and on the potential roles of starch in this parasitic plant. The qualitative visualization of starch of stems, roots, and haustoria of S. album was analyzed by light microscopy and scanning electron microscopy. In addition, we quantified the amount of starch and soluble sugar in these organs and assessed the plant morphological characteristics, as well as sap flow, net photosynthesis, and transpiration rates, under different conditions.d tissue . However2 (18 m × 3 m) in size and was further divided into three plots of 18 m2 (6 m × 3 m). The three blocks were adjacent to each other, and the field was flat. No buildings or other plants disrupted the incoming sunlight in this field. Each plot contained 36 potted S. album seedlings, arranged at 1 m row-to-row spacing and 0.5 m plant-to-plant spacing (within each row). The potted seedlings were subjected to three treatments: US, where seedlings were unattached to hosts throughout the experiment; AS, where seedlings were attached to hosts throughout the study period; and RS, where seedlings were attached to hosts from February 2015 to November 2015, and the host was removed in early December 2015. These three treatments were randomly assigned to plots within a block such that each block consisted of one replicate of each treatment.This study was conducted at the field site of the Research Institute of Tropical Forestry , Chinese Academy of Forestry between January 2015 and March 2017. Field trials were laid out using a randomized complete block design with three blocks and three treatments. Each block was 54 mS. album seeds was induced by gibberellic acid, and three germinated seeds were planted in each pot. After approximately 15 days, when all seedlings had emerged, only one seedling was retained in each pot. Then, 324 potted seedlings were randomly assigned to the three blocks in each plot. In early February 2015, two host (Kuhnia rosmarnifolia) seedlings were transplanted near each S. album seedling in the AS and RS treatment plots. In early December 2015, the hosts were removed from the RS plots.In early January 2015, the germination of Santalum album seedlings were planted in pots (25 cm wide × 27 cm deep) filled with a mixture of sterilized pastoral soil and river sand . Each pot was fed weekly with 50 ml of all-nutrient solution, which was formulated according to S. album.S. album seedlings were determined at 3-month intervals during a 2-year period (March 2015 through March 2017). Sampling was conducted in either sunny or slightly cloud weather. The sap flow rate was measured using a sap flow meter . Briefly, the area of the stem was calculated, and then the stem was wrapped in sap flow sensors to quantify its sap flow rate. The net photosynthesis and transpiration rates were measured between 9:00 am and 14:00 pm using a portable photosynthesis analyzer . In the US treatment plots, some seedlings withered or died before March 2016, and almost all seedlings died by December 2016, owing to the lack of a host. Because of the limited number of plants remaining in the US treatment, no measurements were taken after September 2016.In each plot, the sap flow, the net photosynthesis, and the transpiration rates of three randomly selected Three plants were randomly selected from each plot for destructive sampling at 3-month intervals from March 2015 through March 2017. Lignified stems and roots (2–6 mm in diameter) were used for conducting a morphological analysis and measuring the starch content. Different types of haustoria  were carefully identified and collected for morphological observation and starch quantification. Samples for morphological observation were cleaned, placed in formaldehyde, acetic acid, and ethanol (FAA) fixative, and stored at 4°C. Materials for determining starch levels were first thoroughly rinsed with running water and immediately dried in an oven for 2 h at 90°C. Then, these samples were dried at 65°C for 72 h and stored in a desiccator in the dark.Materials fixed in FAA were rinsed first with tap water and then with distilled water for 1 h. Then, stem, root, and haustorium sections were prepared using a new Gillette razor blade. Free-hand sections were stained with an iodine-potassium iodide (I-KI) solution or a solution of I-KI and phloroglucinol hydrochloride. These sections were observed and photographed under a stereomicroscope .Paraffin embedding and sectioning were conducted according to 2. The dried samples were glued to the upper surface of an aluminum stub with silver paste and then covered with gold powder in a gold sputter coating unit. Finally, the coated samples were observed under a scanning electron microscope  and photographed.The fixed samples were immersed in distilled water for 2 h to thoroughly wash off the fixative. The rinsed materials were cut into small pieces (2–5 mm long × 2–4 mm wide × 2–4 mm thick) appropriate for observation, while maintaining the characteristics of the material. These pieces were dehydrated using a gradient of alcohol and isoamyl isovalerate and then dried in a critical point dryer  using liquid COStorage starch and soluble sugars were measured as described preciously . All ove−1α-amylase  and 20 U ml−1 amyloglucosidase . This reaction system was incubated in a 50°C water bath for 24 h and then centrifuged at 3,000 rpm for 15 min. The supernatant was transferred into a new test tube and diluted with 0.05 M sodium acetate buffer; the volume of the buffer was dependent on the amount of the supernatant. Five hundred microliter of diluted sample solution was transferred into a colorimetric tube containing 2 ml of peroxidase-glucose oxidase  and o-dianisidine dihydrochloride  reagent (PGO-color solution). The reaction system was incubated in the dark at room temperature for 45 min. Then, 500 μl of 75% sulfuric acid was added to the reaction system and mixed, and the absorbance of the solution was determined at 525 nm after 10 min. The starch concentration was determined by multiplying the glucose content with the conversion factor of 0.9  was used to build the compound bar and multiline graphs and for conducting all statistical analyses.All data, presented as mean ± SD, were first tested for normality and variance constancy. One-way ANOVA and the least significant difference (LSD) multiple comparison test were used to detect differences between the analyzed traits of different samples and at different determination times and between soluble sugar and starch content of various haustoria types. One-way ANOVA or paired K. rosmarnifolia seedlings (host) were transplanted near an S. album seedling (parasitic plant) in early February 2015, successful parasitization of the host seedling roots by S. album was observed after 10–20 days. In the first 4 months after planting, S. album seedlings in the US treatment showed similar growth indicators as those in the AS and RS treatments , there was no significant difference in the soluble sugar content among the other types of haustoria (p > 0.05; The allocation dynamics of starch in haustoria varied with the developmental stage of the haustoria and host roots; however, the soluble sugar content almost showed no significant difference among the different types of haustoria. At the early developmental stage, when haustoria had not yet begun to penetrate a root, starch granules were abundant in the zone between the collapsed layers and core meristematic regions . While tst roots . The stast roots . Howeverst roots . Starch S. album . The resS. album seedlings, promoting their growth and development.In the AS treatment, the net photosynthesis, sap flow, and transpiration rates gradually started increasing in March 2015, reaching or approaching the maximum before December 2016 . When thAll storage starch granules were distributed in the parenchyma cells of stems, roots, and haustoria. These granules exhibited a wide variety of shapes, such as spherical, hemispherical, tetragonal, polygonal, and irregular shaped . The diaS. album seedlings in the AS, US, and RS treatments were rich in starch at the early stages of development. However, the starch content of US seedlings started declining from September 2015 onward until the plants died. The storage starch content of seedlings in the RS treatment also decreased when the host plants were removed. By contrast, the starch levels of AS seedlings remained stable throughout the experiment. As described above, plants disturbed by frequent external stresses usually stored more starch than their undisturbed counterparts did. Starch provides energy and substances to plants under stress conditions, such as low temperature, drought, wildfire, and disturbance. In this context, the lack of a host may be considered an external stressor to the hemiparasitic tree; therefore, it is reasonable that a vital role of storage starch is to supply energy and substrates for S. album seedlings under conditions where no host is present.Previous studies confirmed that storage starch serves as an energy source for the sprouting, extension, and thickening of branches at the early growth stages of autotrophic plants . In fireS. album seedlings stopped increasing in a no-nutrient substrate. However, seedlings grown in an all-nutrient substrate continued increasing in height and leaf number, suggesting that these seedlings obtain water and nutrients from the substrate via their roots at the early stage of development (S. album seedlings in the US treatment gradually stopped increasing after 6 months. Seedlings in the US treatment accumulated a similar amount of starch as seedlings in the AS treatment during the first several months of growth. These results support the speculation of via their roots from the cultivation substrate at an early developmental stage.The study of elopment . In the S. album and C. japonica seedlings showed that abundant starch granules are accumulated in their haustoria at an early developmental stage (Previous studies on al stage . Yang etal stage . In addial stage . The preS. album seedlings in the US or RS treatment decreased, the soluble sugar content remained stable. Therefore, storage starch may supplement osmotic substances in the US and RS treatments through a degradation process, thus contributing to maintenance of a low water potential in S. album.Some studies reported that parasitic plants have a lower water potential than their hosts . OsmoticS. album. The results also indicate that storage starch of S. album may not only provide energy and osmosis-related substances when the hemiparasitic tree lose its host but also be related to the physiological activities of haustoria, such as root penetration, water absorption, and nutrient absorption. From an evolutionary perspective, storage starch in S. album may have acquired novel roles under selective pressure. Overall, this study provides a comprehensive understanding of the roles of starch in parasitic plants.In autotrophic plants, storage starch plays a crucial role in providing energy for growth and development and in offering active compounds for osmotic regulation under stress conditions. Consistent with previous studies , our resAll datasets generated for this study are included in the article/supplementary material.XZ, NZ, and DX conceived the project and designed the experiments. NZ, LD, GX, and JT performed the experiments and analyzed the data. XZ and YZ drafted the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.