Dose-response analysis revealed 95% effective dose around 1 mg rtPA in 25 and 50 mL clots. Approximately 80% of relative clot lysis could be achieved after 15 min incubation. Lysis of aged clots was less effective. A new clot model for in vitro investigation was established. Our data suggest that current protocols for rtPA based ICH therapy may be optimized by using less rtPA at shorter incubation times.Hematoma lysis with recombinant tissue plasminogen activator (rtPA) has emerged as an alternative therapy for spontaneous intracerebral hemorrhage (ICH). Optimal dose and schedule are still unclear. The aim of this study was to create a reliable in vitro blood clot model for investigation of optimal drug dose and timing. An in vitro clot model was established, using 25 mL and 50 mL of human blood. Catheters were placed into the clots and three groups, using intraclot application of rtPA, placebo, and catheter alone, were analyzed. Dose-response relationship, repetition, and duration of rtPA treatment and its effectiveness in aged clots were investigated. A significant relative end weight difference was found in rtPA treated clots compared to catheter alone ( Spontaneous intracerebral hemorrhage occurs in 10–15% of all stroke patients and is still a major cause of stroke-related death and disability . OptimalIn consideration of these results the interest in minimal invasive procedures has grown. Stereotactic frame-based or image-guided frame-less catheter placement and rtPA lysis of ICH have shown to be safe and effective in volume reduction –12. In tIn order to establish optimal dose and timing for rtPA therapy, however high numbers of repetitive investigations are necessary to achieve valid and significant results. Studies in large ICH animal models are very expensive. An obvious alternative is an in vitro ICH model, which allows a high number of repetitions under controlled conditions. We here present an easy and robust in vitro ICH model in which we investigated optimal timing and dosing of rtPA lysis , 22.μL) in a balloon tightly closed and incubated 1.5 h in an incubator at 37°C . The application of thrombin has been adapted from an intravascular clot model 22].p = 0.222). The rtPA (3 mg) treated clots with a relative end weight of 46.01  ±  6.1% showed a significant difference compared to control (p = 0.002) and to drain plus carrier alone treated clots (p < 0.001) (The control group (drain only) showed a mean relative end weight of 64.96 ± 5.26% of initial weight. The drain plus carrier treated clots with a mean relative end weight of 69.44 ± 6.67% showed no significant difference compared to the control group (< 0.001)  21, 22], 22p = 0A dose-response relationship was evaluated in five groups each consisting of three 25 mL blood clots using five different doses of rtPA  and compared to a placebo  treated clot from the same donor. Clots were weighed before and after treatment. The rtPA treated group showed a relative posttreatment weight of 60.03 ± 1.76% when treated with 0.5 mg rtPA, 49.06 ± 0.98% when treated with 0.9 mg rtPA, 48.78 ± 2.14% when treated with 1.2 mg rtPA, 46.34 ± 4.68% when treated with 2 mg rtPA, and 46.01 ± 6.1% when treated with 3 mg rtPA. The control group had a mean relative posttreatment weight of 64.04 ± 3.67%. The 95% effective dose (ED95) was 1.2 ± 0.52 mg rtPA; the 50% effective dose (ED50) was 0.6 ± 0.1 mg  21, 22], 2221, 2The same experiment was performed in 50 mL clots. The treatment group showed a relative posttreatment weight of 54.23 ± 4.41% treated with 0.5 mg rtPA, 51.41 ± 1.79% treated with 0.9 mg rtPA, 55.23 ± 3.36% treated with 1.2 mg rtPA, 47.91 ± 6.96% treated with 2 mg rtPA, and 45.98 ± 3.06% treated with 3 mg rtPA. The control group had a mean relative posttreatment weight of 68.94 ± 6.52%. The 95% effective dose (ED95) was 0.84 ± 1.07 mg rtPA; the 50% effective dose (ED50) was 0.28 ± 0.35 mg  21, 22], 2221, 2Assuming a maximum lysis rate of 100% after 1 h, lysis rates after different exposure times to rtPA were analyzed in 25 mL clots. The normalized rate of lysis after 5 min exposure to rtPA was 53.22 ± 3.9%; after 15 min it was 79.41 ± 1.7% and after 30 min 85.38 ± 1.5%  21, 22], 2221, 2p < 0.0001) and 48 h old clots (p = 0.0002) during the first treatment (p = 0.0059) (Fibrinolytic treatment with rtPA is less effective in aged clots. There was a significant weight difference of 90 min old clots compared to 24 (reatment . During  0.0059) . Repetit 0.0059)  21, 22], 22p < 0https://doi.org/10.1155/2017/5472936). We established a novel in vitro ICH clot model [In the present study to the best of our knowledge we investigated for the first time a systematic analysis of fibrinolytic therapy of rtPA . Further we showed that 80% of the lysis occurs within the first 15 min of incubation. The data suggests that current protocols for rtPA based ICH therapy could possibly be optimized by using smaller doses and shorter incubation times. This might in the future allow a faster reduction of intracranial pressure than achieved in current clinical protocols.This flowchart illustrates the experimental workflow of establishing an in vitro clot model and the further stepwise investigations and their key results.


Journal of Fungi would like to express their sincere gratitude to the following reviewers for assessing manuscripts in 2016. The editors of https://publons.com) to receive recognition. Of course, in these initiatives we are careful not to compromise reviewer confidentiality. Many reviewers see their work as a voluntary and often unseen part of their role as researchers. We are grateful to the time reviewers donate to our journals and the contribution they make. We greatly appreciate the contribution of expert reviewers, which is crucial to the journal’s editorial process. We aim to recognize reviewer contributions through several mechanisms, of which the annual publication of reviewer names is one. Reviewers receive a voucher entitling them to a discount on their next MDPI publication and can download a certificate of recognition directly from our submission system. Additionally, reviewers can sign up to the service Publons (Journal of Fungi see the link at the bottom of the webpage http://www.mdpi.com/reviewers.If you are interested in becoming a reviewer for Journal of Fungi in 2016:Agarwal, RiteshGopinath, Shankar P.Pacioni, GiovanniÅhlin, AndersGroll, Andreas H.Pfaller, M.A.Ahmadi-Afzadi, MasoudHage, Chadi A.Ponikau, Jens U.Aimanianda, Vishu KumarHauser, Philippe M.Porollo, AlekseyAlspaugh, AndrewHoenigl, MartinPusateri, AlessandroArabatzis, MichaelHomma, TetsuyaQueener, S.F.Bahn, Yong-SunHoubraken, JosReales-Calderón, Jose AntonioBahr, Nathan C.Johnson, John A.Rolfes, RondaBensch, KonstanzeKaditis, Athanasios G.Shor, ErikaBleackley, Mark R.Kameda, HidetoSilva-Vergara, Mario LeónBuitrago, María JoséKidd, Sarah E.Smani, YounesBuzina, WalterKirschner, RolandSpecht, Charles A.Calderone, RichardKonopka, James B.Surup, FrankCoelho, CarolinaKouvelis, Vassili N.Thornton, ChristopherCrous, PerdoLaverty, GarryVatsyayan, PreetyEspinel-Ingroff, AnaLin, Hung-YinVoglmayr, HermannEzra, DavidLivermore, JoanneVoigt, KerstinFalcone, E. LianaMalo, JoshuaWang, ZheFigliuzzi, MatteoMaruyama, Ichiro N.Warris, AdiliaFisher, CynthiaMiceli, Marisa H.Wormley Jr., Floyd L.Freitas, Dayvison F.S.Moore, Margo M.Xue, ChaoyangGarcia-Diaz, JuliaMoreira, José A.S.Zaragoza, OscarGarcia-Rodas, RocioMori, MasaakiZhou, ZhichengGasch, AudreyNevez, GillesZutz, ChristophGodet, CendrineObanor, FridayZygadlo, JulioThe following reviewed for


Recently, progress has been made in the development of mechanical joints with variable intrinsic stiffness, opening up the search for application areas of such variable-stiffness joints. By varying the stiffness of its joints, the resonant frequency of a system can be tuned to perform cyclical tasks most energy-efficiently, making the variable-stiffness joint a candidate element for an advanced prosthetic device specifically designed for the cyclical task of drumming. A prerequisite for a successful variable-stiffness drumming prosthesis is the ability of human drummers to profitably employ different stiffness levels for playing different beats. In this pilot study, 29 able-bodied subjects (20 drumming novices and 9 experts) wear a cuff on the forearm, to which a drumstick is connected using changeable adapters, consisting of several leaf springs with different stiffness and one maximally stiff connection element. The subjects are asked to play simple regular drum beats at different frequencies, one of which is the resonant frequency of the adapter-drumstick system. The subject's performance of each drumming task is rated in terms of accuracy and precision, and the effort is measured using questionnaires for the perceived stress as well as electromyography (EMG) for the muscular activity. The experiments show that using springs instead of the stiff connection leads to lower muscular activity, indicating that humans are able to use the energy-storing capabilities of the springs, or that muscular activity is reduced due to the lower mass of the springs. However, the perceived stress is increased and the novices' performance lowered, possibly due to a higher cerebral load for controlling the elastic system. The hypothesis that “matching the resonant frequency of the spring-drumstick system to the desired frequency leads to better performance and lower effort” is not confirmed. Possible explanations are discussed. In conclusion, a series-elastic element appears to lower the muscular effort of drumming, while a stiff connection appears to minimize the mental load and has a positive effect on the performance of drumming novices. Series-elastic actuators (SEAs) as well as variable-stiffness actuators (VSAs) have been recently introduced into robotics  reduce impacts of collisions for motors and gears . A prosthetic drumstick holder is commercially available, including an elastic element, but without variable stiffness  that subjects play best with diagonal-type variable stiffness, and, (b) that experts are better in taking benefit of the variable stiffness, since they are familiar with the general task of drumming, which probably frees their mental capacities for adapting to the new device.This study involved 20 unimpaired drumming novices and 9 unimpaired drumming experts who wore a cuff on the forearm, to which a drumstick was attached using stiff and elastic adapters. Hence, the subjects' intact wrists were not used but replaced with an experimental prosthetic attachment, simulating the situation of a missing hand. The subjects were asked to play all combinations of adapter types and desired frequencies shown in the combination matrix in Figure The experiment setup is shown in Figure The subject wore a cuff constraining movements of the wrist . The cuff was coupled to the drumstick via a changeable adapter. The set of changeable adapters consisted of four leaf springs with different stiffness values . This sensor was used for determining the design frequency of each adapter element, which corresponds to the resonant frequency of the whole cuff including force-torque sensor, spring and drumstick. For this, the cuff was fixed in a bench vise, and the drumstick was jolted into free oscillation. From the force-torque sensor data, the frequencies of the oscillation were found to be at 3.7, 4.4, 5, and 6.3 Hz for the springs and at 27 Hz for the stiff connection. The force sensor remained between spring and cuff during the experiments to avoid changing the design frequencies. The springs were approximately 70–90 g lighter than the stiff connection, leading to a reduction of the moment of inertia around the elbow flexion axis of about 5–6%.Between spring and cuff a force-torque sensor ATI Mini45 SI-290-10 was placed . The sensor was covered with rubber mats for damping the noise and making the drumming more comfortable , pectoralis major (humerus adduction), deltoideus posterior (shoulder extension), deltoideus medius (shoulder abduction), deltoideus anterior (shoulder flexion), anconeus (elbow extension), triceps brachii long head (elbow and shoulder extension), and triceps brachii lateral head (elbow extension). The mean of the EMG values over all trials of each electrode was subtracted subject-wise to remove any constant DC offset. Furthermore, after subtracting the DC offset, the EMG values of each electrode were normalized subject-wise by dividing by the RMS value over all trials. This eliminates differences in electrodes due to location-specific tissue resistance and gives the variations between trials of the signal of each electrode the same weights.aRDnet  to read out the force-torque and EMG sensors at 1 kHz. The marker positions were recorded by the Windows computer and transferred to the Linux host using the DLR communication protocol A total of 29 healthy subjects, 25 male and 4 female, all right-handed and initially fully naive to the experiment, performed the experimental protocol as described below. 9 out of the 29 subjects had at least 1 year of drumming experience and were therefore considered as experts, and the other 20 were counted as novices. All subjects participated voluntarily and gave written informed consent to the procedures, which were conducted in partial accordance with the principles of the Helsinki agreement . Approval was received from the works council of the German Aerospace Center, as well as its institutional board for data privacy ASDA; the collection and processing of experimental data were approved by both committees. For all subjects and experiments the right hand was used, which was restricted by the design of the cuff. Subjects stood upright in all experimental conditions.The experiment session lasted between 20 and 25 min per subject. At the beginning of each experiment session, the participant was instructed about the experiment using a standardized presentation.The subjects were asked to play all 20 trials consisting of the combinations of adapter types and desired frequencies shown in the combination matrix in Figure Before each trial, subjects had the possibility to get used to the current combination of desired frequency and adapter type within a time of 10 s, followed by a phase of 15 s of collecting drumming data. The 10 s of training data were not recorded. After each trial, subjects were asked to fill in a questionnaire about the combined physical and mental stress level that they felt during playing at the respective combination of desired frequency and adapter type. The question was “How high was the perceived stress while playing of each frequency, with respect to physical and mental effort” 1: very low, 8: very highBefore and after the trials, base noise EMG during rest of the arm was measured, as well as EMG and maximum force during maximum voluntary contraction in a lifting-up and a pushing-down task. This was used for checking whether the signals look plausible. Unfortunately, for unknown reasons, the electrodes of pectoralis major and anconeus showed very noisy signals for many subjects. We therefore discarded the results of these two electrodes.The inaccuracy, i.e., the difference between the desired time interval and the mean played time interval between two drum strokes, which tells how well the desired frequency could be met; a lower inaccuracy means a better performance:the imprecision, i.e., the standard deviation of the time between two strokes, which tells how evenly the frequency was played; a lower imprecision means a better performance:the perceived stress consisting of physical and mental effort, i.e., the result of the questionnaire; lower stress means higher comfort:the measured muscular activity, i.e., the mean normalized root-mean-square (RMS) EMG signals, where normalizing means subtracting the per-electrode DC offset and dividing by the per-electrode RMS of the signals of all trials; again, a lower muscular activity means a higher comfort:To answer our research questions and evaluate our hypotheses, we predefined four outcome measures, which are gathered for each subject and trial:elt is the measured EMG signal of electrode e at time sample t in trial l, with nelectrodes = 6, ntrials = 20 and nsamples = 3,000.where EMGTo statistically analyze the results we built and applied a mixed-effects regression model with fixed and random effects. It allowed to directly include the two hypotheses as fixed effects into our model. The formula of the mixed-effects model is:yijklmn is the response variable, i.e., any of the four above-mentioned outcome measures, i is the adapter type, j is the desired frequency, k is the expert status, l is the diagonal status, which is 1 if the combination of adapter type and desired frequency is on the diagonal and 0 otherwise, m is the within-subject trial number, n is the subject number, β0 is the intercept, which is a constant term, βiadapter_type, is the fixed effect of the adapter type, βjdesired_frequency, is the fixed effect of the desired frequency, βkexpert_status, is the fixed effect of the expert status, βldiagonal, is the fixed effect of playing on the diagonal, βkldiagonal×expert_status, is the fixed effect of the interaction between the expert status and playing on the diagonal, βikadapter_type×expert_status, is the fixed effect of the interaction between expert status and adapter type, mβtrial_number is the trial-number-dependent fixed effect of learning or fatigue, ϵnsubject, is the subject-specific random effect and ϵmn is the residual random error. The random effects are assumed to follow normal distributions as follows:where The factors adapter type, desired frequency, expert status and diagonal status can assume the levels shown in Table lmer function of the lme4 library  and their interactions with the expert status, are shown in Figure The first hypothesis, that variable stiffness provides the best performance and comfort, and the second hypothesis, that experts are better able to make use of the variable stiffness, can be tested by regarding the differences between playing on or off the diagonal shown in Figure Figure Since the use of diagonal-type variable stiffness shows a beneficial effect on the accuracy of experts but the use of some of the springs shows a detrimental effect with a similar magnitude, it is interesting to see the differences in inaccuracy of experts between using the springs in diagonal-type variable stiffness mode and using the stiff connection. These effects of the springs on the diagonal are the sum of the effect of the diagonal Figure  and the In this study, we investigated whether drummers can take advantage of a variable-stiffness joint in a prosthetic wrist. We asked 20 novices and 9 experts to play different frequencies using different elastic elements and one stiff element in a connection between a cuff on the forearm and a drumstick. We hypothesized that subjects will perform best and require the least effort when playing the elastic elements at their resonant frequencies, which is one main argument for variable-stiffness actuation. Moreover, we hypothesized that such an effect will be more obvious for an expert drummer.Can drummers take advantage of a variable-stiffness joint in a prosthetic wrist? Our experimental design was unable to verify that variable stiffness is useful for a prosthetic wrist. Even if experts showed a trend for a reduced inaccuracy when playing the diagonal, they showed the opposite trend for imprecision, and it is difficult to judge which of the two is more relevant. For novices, both inaccuracy and imprecision increased when using the diagonal-type variable stiffness.The results showed the expected influence of elasticity on the EMG-measured effort, namely that elasticity reduces the effort, and that experts are better than novices in doing so. Despite that, there was a clear discrepancy between measured and perceived effort for both groups. While the muscular activity decreased with decreasing stiffness of the adapter, the perceived stress increased. We find this effect surprising.A possible interpretation is that subjects are indeed able to save muscular effort by making use of the energy-storing capabilities of the spring. However, the effort by the brain for controlling the more complex dynamics of the system involving the springs is likely higher than for controlling the system involving the stiff connection. In the answers to the questionnaire, the increased cerebral effort might therefore outweigh the reduced muscular effort.A further explanation for the lower EMG-measured effort when using the springs lies in the fact that the they are more lightweight than the stiff connection and that less muscular effort might have been necessary to accelerate and decelerate it. However, the reduction of inertia (5–6%) is considerably lower than the reduction of EMG-measured effort (16–25%). Assuming that muscular activity is roughly proportional to accelerated inertia, we therefore estimate that the influence of the different weights on the EMG-measured effort is small and that the effect of the adapter types on muscular activity is dominated by their elasticity.These findings somewhat confirm the study of Fujisawa and Miura , who fouIn interpreting the results with respect to variable stiffness, three main limitations of our experimental design have to also be considered. (a) We measured the resonant frequency of the cuff including the elastic element and the drumstick. The influence of the rebound of the drum skin as well as the elasticity of the soft tissue and the (variable) stiffness of the joints due to muscle contraction and cocontraction were not gathered when measuring the resonant frequency of the cuff-spring-drumstick combination. This possibly leads to lower resonant frequencies of the whole system including the human arm, which could explain the better performance of the stiffer springs. (b) There might be variable-stiffness strategies other than matching the resonant frequency of the system to the desired frequency. In order to potentially discover those other variable-stiffness strategies, in future experiments, one could ask subjects to play a more fine grained set of frequencies and try to discover patterns in the outcome measures. (c) Even the expert drummers were naive to the experiment and the time to become acquainted with the system for any combination of adapter type and desired frequency was limited to 10 s. While this was helpful for achieving a reasonable experiment duration, allowing a subject to practice using a variable-stiffness drumming system for weeks or months might show long-term learning effects.Future experiments into variable-stiffness drumming prostheses might use the following improvements to possibly find a beneficial effect of variable stiffness. A new device with continuously variable stiffness instead of separate adapters with different stiffness levels could be built so that subjects can better match the resonant frequency of the whole system including the rebound of the drum to the desired frequency or employ a different variable-stiffness strategy. This would also remove the problem of different masses of the stiff connection adapter and the springs. Furthermore, subjects could be given more time  to become more acquainted with the system in order to learn the more complex dynamics.Conclusively, our experimental results argue that series-elastic elements can be used to reduce the muscular activity of drumming, but that their stiffness does not need to be variable. However, the elastic elements appear to initially put an increased control burden on the user. While expert drummers seem to be able to deal with their more complex dynamics, novice drummers seem to reach a better performance with a stiff connection. Prosthesis users may benefit from this study if its results or the results of a future, improved study are incorporated into the design of an actual prosthetic device.GS, HH, and JV were involved in planning and design of the experiments. HH and GS were involved in the statistical analysis. JS was involved in setting up and conducting the experiments. HN, HH, and GS were involved in the statistical design. HH and GS wrote the manuscript. JV provided corrections to the manuscript. All authors contributed to manuscript revision, read 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. The reviewer NC and handling Editor declared their shared affiliation.


Ebolavirus (EBOV) is responsible for one of the most fatal diseases encountered by mankind. Cellular T-cell responses have been implicated to be important in providing protection against the virus. Antigenic variation can result in viral escape from immune recognition. Mapping targets of immune responses among the sequence of viral proteins is, thus, an important first step towards understanding the immune responses to viral variants and can aid in the identification of vaccine targets. Herein, we performed a large-scale, proteome-wide mapping and diversity analyses of putative HLA supertype-restricted T-cell epitopes of Zaire ebolavirus (ZEBOV), the most pathogenic species among the EBOV family.All publicly available ZEBOV sequences  for each of the nine viral proteins were retrieved, removed of irrelevant and duplicate sequences, and aligned. The overall proteome diversity of the non-redundant sequences was studied by use of Shannon’s entropy. The sequences were predicted, by use of the NetCTLpan server, for HLA-A2, -A3, and -B7 supertype-restricted epitopes, which are relevant to African and other ethnicities and provide for large (~86%) population coverage. The predicted epitopes were mapped to the alignment of each protein for analyses of antigenic sequence diversity and relevance to structure and function. The putative epitopes were validated by comparison with experimentally confirmed epitopes.ZEBOV proteome was generally conserved, with an average entropy of 0.16. The 185 HLA supertype-restricted T-cell epitopes predicted (82 (A2), 37 (A3) and 66 (B7)) mapped to 125 alignment positions and covered ~24% of the proteome length. Many of the epitopes showed a propensity to co-localize at select positions of the alignment. Thirty (30) of the mapped positions were completely conserved and may be attractive for vaccine design. The remaining (95) positions had one or more epitopes, with or without non-epitope variants. A significant number (24) of the putative epitopes matched reported experimentally validated HLA ligands/T-cell epitopes of A2, A3 and/or B7 supertype representative allele restrictions. The epitopes generally corresponded to functional motifs/domains and there was no correlation to localization on the protein 3D structure. These data and the epitope map provide important insights into the interaction between EBOV and the host immune system.The online version of this article (10.1186/s12864-017-4328-8) contains supplementary material, which is available to authorized users. Ebolavirus (EBOV), a member of the Filoviridae family. Ever since the EBOV was first discovered in 1976 [Zaire ebolavirus (ZEBOV) is observed to account for the highest mortality rate among EVD patients [Ebola virus disease (EVD) or Ebola haemorrhaging fever, although not as commonly spread as influenza, is more often deadly once contracted, and thus, making it one of the most fatal diseases encountered by mankind . The recpatients , 4. Althpatients , an effe+ and CD8+ T cells, have been implicated to be important in providing protection against the EBOV [+ T-cell epitope presentation is necessary for humoral antibody production by B cells during the breach of the immune system. Sequence changes in the epitopes (antigenic variation), even of a single amino acid, can result in viral escape from immune recognition [The adaptive immune response  plays a key role in protection against viral pathogens . The celthe EBOV . Notablyognition –11. TherA challenge in identifying T-cell epitopes is the high polymorphism of HLA, recognised as the most polymorphic human loci . As of JHerein, we applied a computational approach to map and analyse putative HLA supertype-restricted T-cell epitopes of ZEBOV, the most pathogenic species among the EBOV family. The epitopes were predicted for all representative HLA alleles of supertypes A2, A3 and B7, which provide a high coverage of the human population and are applicable to the major ethnic groups including those from Africa  , 34. TheZaire ebolavirus species. This species has been observed to have the highest mortality among human population compared to the other species of the virus, namely Sudan ebolavirus, Tai Forest ebolavirus , Reston ebolavirus, and Bundibugyo ebolavirus [Ebola protein sequences (as of April 2016) were collected through the National Center for Biotechnology Information (NCBI) Taxonomy database  by use oolavirus .The ZEBOV genome consists of a single-stranded negative sense RNA, which is approximately 19 kb long, and encodes for nine proteins, namely glycoprotein (GP), non-structural soluble glycoprotein (sGP), second secreted glycoprotein (ssGP), nucleoprotein (NP), virion structural proteins , and RNA-dependent RNA polymerase (L). Reference sequences for each protein were obtained from the NCBI RefSeq Database  (NP: NP_x) in a sequence alignment is defined as:p represents the probability of a particular nonamer peptide i at position x. The entropy value increases as the total number of peptides, n(x), present at the position x increases. The entropy value drops in cases where a certain peptide is dominant at the position x. Highly conserved regions yield low entropy values, approaching zero when there is a complete (100%) conservation. Only nonamer peptides of valid amino acids at position x were analyzed. Nonamers containing gaps were ignored in the calculation of the entropy. The entropy bias is inversely proportional to the sample size N [x was performed by randomly sampling N into smaller datasets of sequences. Entropy values were computed for all the datasets and then plotted against 1/N. Extrapolating the linear regression of the entropy values by using line of best fit to 1/N of zero will yield the estimated entropy at position x when N is large (approaching ∞).The diversity of ZEBOV proteome was measured by use of Shannon’s entropy –43. Thise size N , where aExperimentally validated MHC ligands or T-cell epitopes for EBOV were obtained from the Immune Epitope Database and Analysis Resource (IEDB) (as of January 2017). All linear human T-cell epitopes relevant to EBOV from positive assays, such as T cell assays or HLA (human MHC) ligand assays, were downloaded, analysed, and compared with the predicted epitopes., A3 , and B7 . Since a peptide may not be predicted as an epitope for every representative allele of a supertype, a 50% cut-off was set to determine a putative supertype-specific epitope . The A2, A3 and B7 supertypes were chosen because they are reported to provide a wide population coverage (86%) [T-cell epitopes were predicted by use of a local copy of NetCTLpan 1.1 , with dege (86%) . PutativAll sequences at each of the mapped, putative epitope alignment positions were extracted and analysed for antigenic diversity. The different epitope and non-epitope sequences at the position and their individual frequency were noted. The entropy of the position, individual frequency of the putative epitope sequences versus the non-epitope sequences and the amino acid substitutions between the putative epitope sequences at the same position were assessed.The known and putative structural and functional properties of the predicted epitopes were searched in the literature and by use of Pfam , InterPrZaire ebolavirus protein sequences were retrieved from the NCBI Taxonomy Database. This included 19 non-ZEBOV sequences, which were removed, and four polyprotein sequences (see Methods), which provided additional four sequences, and thus, resulting in a total of 14,098 relevant sequences. The removal of duplicate sequences reduced the number of sequences by ~91% to 1272: 208 (NP), 125 (VP35), 109 (VP40), 353 , 66 (VP30), 69 (VP24) and 342 (L) , 37 (A3) and 66 (B7) . A majority (119/125) of the positions were highly conserved, with entropy <0.5  matched reported experimentally validated HLA ligands/T-cell epitopes of A2, A3 and/or B7 supertype representative allele restrictions (Table Thirty 30) of the 125 mapped positions had a single putative epitope each that was completely conserved among all the sequences (100% incidence) and may be attractive for vaccine design Table . The rem0 of the 312 and VP40316); the dominant (% incidence) among the putative epitope sequences at the position served as the reference sequence. A total of 46 distinct amino acid substitutions (irrespective of the amino acid positions in the nonamer) were observed between the putative epitopes at these positions  of the 125 epitope positions had more than one putative epitope to allow comparison of acceptable substitution . Such positions had two to as many as five epitope sequences  analysed, isolated from different geographical regions of Africa and from as early as 1992, offered information for a broad survey of EBOV protein diversity in nature and their relevance as targets of immune responses. Although a large number of ZEBOV sequences are reported in the NCBI Protein Database, only ~9% were non-redundant. The redundant sequences were present in each of the protein datasets at about relatively similar levels, with no specific preference for any of the protein. Analysis of the duplicate sequence records showed difference in data for the geographical location, isolate and/or biosample fields. This is likely a result of ebola surveillance programs that end up identifying largely identical or highly similar circulating isolates. Although to some extent the redundancy may be accepted as a reflection of the incidence of the corresponding EBOV isolates in nature, the analysis was performed on the non-redundant dataset to minimize sampling bias. However, this introduces potential bias in the entropy values. Removal of duplicate protein sequences will reduce the number of nonamer sequences for all positions of the proteome, and thus increasing the entropy value of each position, the degree of which depends on the number of duplicates removed. The entropy bias was minimized through the correction to the estimation of entropy for large data size.Entropy analysis revealed that ZEBOV proteome is generally conserved, despite the relatively long evolutionary history of the virus, with reported data of isolates between 1992 and 2016. The lethal nature of the virus to the host and the limited geographical spread of the virus may be contributing factors to the low variability of the virus. As a comparison, the diversity of ZEBOV proteome (max. Entropy of 1.2) is on the lower side compared to other RNA viruses, such as West Nile virus (max. Entropy of ~2) , dengue flaviviruses [The availability of reliable computational tools with good reported prediction capability enable systematic screening of candidate T-cell epitopes from larger sets of protein antigens, such as those encoded by complete viral genomes. Putative T-cell epitopes for HLA-A2, -A3, and -B7 supertypes were mapped onto the aligned non-redundant sequences of each ZEBOV protein. Notably, it was observed that the putative epitopes had a propensity to co-localize at select positions of the protein multiple sequence alignment. This preferential localization of the epitopes indicates that they are structural in nature, though the individual epitope sequences may vary (i.e. structure conservation maintained). A similar observation has been made in other studies, such as localization of T-cell epitope clusters to exposed strands of HIV envelope glycoprotein (clades B and D)  and locaiviruses . A likeliviruses . Prefereiviruses . AdditioExperimental measurements as validation of computational predictions are necessary for accurate interpretation of results. Computational models that are valid, relevant, and properly assessed for accuracy are useful for planning of complementary laboratory experiments , 58. The+ and CD8+ T cells, have been implicated to be important in providing protection against the EBOV [All the nine ZEBOV proteins have been experimentally mapped of T-cell epitopes/HLA ligands, with a majority reported for NP and “All GP” . The putative HLA-A2, -A3, and -B7 supertype-restricted epitopes were predicted herein for each of the ZEBOV proteins. In contrast to the experimental data, which did not include any supertype-restricted epitopes, L was highly enriched and VP40 was most packed with putative epitopes over the protein length. The structural GP has become the main focus in many research efforts as it is the only known protein on the surface of the virus, which is presumed to mediate the viral entry into host cells . Thus, iThirty-nine (39) of the 125 epitope positions had more than one epitope to allow a comparison of acceptable substitution. A total of 46 distinct amino acid substitutions were observed between the putative epitopes at these positions: 18 for A2, 8 for A3 and 20 of B7. Several of the putative epitopes were tolerant to multiple amino acid substitutions. The amino acid substitutions reported herein at the critical peptide positions 2 and those in the C-terminus are in agreement with the amino acids recognised by the representative alleles of each of the supertypes , howeverThe EBOV is markedly one of the most fatal pathogen and thus far, there are no effective vaccines or therapeutic measures against them. The data herein offers insights into ZEBOV diversity, its evolutionary history, and provides a catalogue of mapped epitopes , matrix of acceptable epitope substitutions, and candidates for rational vaccine design, which are also attractive for a structure-based design of candidate inhibitory compounds, and improvement of the current diagnostic methods.Additional file 1: Figure S1.Average proteome entropy of each ZEBOV protein and the complete proteome. The entropy values for each protein were 0.16 (NP), 0.21 (VP35), 0.15 (VP40), 0.15 (“Pre-295 All GP”), 0.36 (“Post-295 GP”), 0.51 (“Post-295 sGP”), 0.29 (VP30), 0.30 (VP24) and 0.08 (L). “Post-295 ssGP” is not shown because it was not analysed (see Methods). The average proteome entropy was 0.16. (TIFF 81 kb)Additional file 2: Table S1.Reported human T-cell epitopes and HLA ligands of ZEBOV from the Immune Epitope Database and Analysis Resource . (TIFF 33 kb)Additional file 3: Table S2.Number of putative HLA-A2, -A3, and -B7 supertype-restricted epitopes of ZEBOV. (TIFF 163 kb)Additional file 4: Table S3.Sequence diversity at each epitope position. (TIFF 142 kb)Additional file 5: Table S4.Substitutions observed between putative epitopes. Only shown for positions that had more than one putative epitope, to allow comparison of acceptable substitution. (PDF 229 kb)


This study uses theory and experiments to investigate the relationship between the passive stiffness of series elastic actuators and torque tracking performance in lower-limb exoskeletons during human walking. Through theoretical analysis with our simplified system model, we found that the optimal passive stiffness matches the slope of the desired torque-angle relationship. We also conjectured that a bandwidth limit resulted in a maximum rate of change in torque error that can be commanded through control input, which is fixed across desired and passive stiffness conditions. This led to hypotheses about the interactions among optimal control gains, passive stiffness and desired quasi-stiffness. Walking experiments were conducted with multiple angle-based desired torque curves. The observed lowest torque tracking errors identified for each combination of desired and passive stiffnesses were shown to be linearly proportional to the magnitude of the difference between the two stiffnesses. The proportional gains corresponding to the lowest observed errors were seen inversely proportional to passive stiffness values and to desired stiffness. These findings supported our hypotheses, and provide guidance to application-specific hardware customization as well as controller design for torque-controlled robotic legged locomotion. Direct control of interaction forces or torques have been used in physical human-robot interaction to reduce interface impedance, increase reactiveness of the robotic devices and thus improves human safety and comfort  and (2), we can express the torque applied by the exoskeleton to the human ankle joint asin which the aspect ratio of transmission isKt which relates torque applied by the exoskeleton to the device joint angle is defined asand the transmission stiffness Previous work identified model-free, integral-action-free feedback control compensated by iterative learning as the most effective controller for lower-limb exoskeletons torque tracking during walking  is an anchor node in torque-angle space and Kdes is the resulted desired stiffness. S is a scaling factor on the unit curve with desired quasi-stiffness of Kdes, 0 to get different values of desired quasi-stiffness.where (θAnother type of curve tested was a piece-wise linear torque-angle curve with the format of Equation (8) as shown in Figure i,p, τi,p) defines a node in torque-angle space  marked the transition from the dorsiflexion phase, in which ankle velocity was negative, to the plantarflexion phase, in which ankle velocity was positive. Since the exact transition point varied on each stride, we used the angle and torque at the moment of transition, Here,  used for torque tracking,Due to the employment of a high-speed real-time controller and a high-acceleration servo motor, desired motor velocity is enforced rapidly, based on which we make the simplification of immediate motor velocity enforcement, i.e.,Combining Equation (10) with a linear approximation of desired torque curves, including those expressed by Equations (7) and (8), in the form ofwe have0 is maximum joint position for the device to exert torque on the human ankle, i.e., the intersection of torque-angle relationship with the angle axis.in which θN sinusoidal waves of the same frequency F, we get a profile of the ankle angle in the form ofModeling exoskeleton-assisted walking after stabilization as an oscillation process made of c is an constant denoting the offset of the profile on torque axis, dn and βn are the magnitude and phase shift of the nth sinusoidal wave, and t represents the time elapsed within one stride since heel strike. The corresponding stabilized motor position should also oscillates with the same frequency. We can therefore construct a stabilized motor position by equal number of sinusoidal waves with the same phase shifts in the form ofwhere e is a constant and fn is a complex number. Substituting Equations (13) and (14) into Equation (12), we get Equation (15).in which Equating the coefficients of the various sinusoidal waves and the offset, we haveandMotor position profile in Equation (14) can thus be expressed in terms of the ankle position profile and the controller asCombining the oscillator assumption with Equation (12), we get the expression of the torque error asIt is clear that without considering the control gains, assertingwill minimize torque tracking error. Therefore, we make the following hypothesis:Hypothesis 1. In lower-limb exoskeletons, the optimal passive stiffness of the series elastic actuator for torque tracking isAnother factor that limits torque tracking performance is the inability of the proportional gain to increase indefinitely.Reformating Equation (19), we getKt − Kdes ≠ 0, with the same step frequency F and angle profile eτ is inversely proportional to It is clear that when the passive stiffness is fixed but does not match the desired one, i.e., Meanwhile, combining the controller in Equation (9) and the assumption of perfect motor velocity tracking in Equation (10), we haveDifferentiating the expression of applied torque in Equation (3), we getTherefore, the time derivative of torque error iswhich is a first order dynamics created by feedback control with an effective proportional gain ofand a time constant ofτ, max, which corresponding to a best tracking performance regardless of the passive stiffness of the system. We therefore proposed the following conjecture:However, this dynamics does not exist independently but interacts with the human body in parallel. Therefore, in practical cases, oscillations increase when effective proportional gain increases, which impairs torque tracking performances eventually and causes discomfort or injury to the human body. In our study, motor speed limit was never hit. Thus, we suspected that there is a fixed torque tracking bandwidth limit that is dependent on the combined interactive dynamics of motor, motor drive, transmission and human body. This bandwidth limit results in a fixed maximum commanded change rate of torque error, ėConjecture 1. Assisted human walking with a lower-limb exoskeleton experiences a fixed maximum commanded tracking rate of torque error, ėmaxτ,, which limits the tracking performance of the system.R = 2.5 results in the following fact about Equation (23):In practical cases, Equation (24) can be further simplified. First, to realize real-time torque tracking, the motor velocity should be a lot faster then device joint velocity, i.e., Successful torque tracking also means a fast changing rate of actual torque compared to the desired torque, Therefore, Equation (24) can be estimated as:τ which corresponding to optimal control performance. In this case, with comparable torque error eτ, Kt are inversely proportional to each other. In other words, the application of Conjecture 1 results in a fixed time constant F and a constant angle profile According to Conjecture, there is a fixed maximum ėn instanteneous torque tracking errors at optimal conditions can be expressed asThen the root-mean-squared value of C1, C2, …, Cn are some constant coefficients. This then leads to the hypothesis below.in which Hypothesis 2. The root-mean-squared torque tracking errors under optimal feedback control conditions are proportional to the absolute difference between the desired and passive stiffness values, i.e.,Kp and Kt:Dynamics in Equation (24) directly leads to a relationship between Kdes. Previous study shows an root-mean-squared tracking error of <8% the peak desired torque under proportional control and damping injection  can be simplified asand accordingly, Equation (27) becomesKp and Kt. According to Conjecture 1, at optimal conditions, ėτ is fixed. With comparable torque error eτ, the following corollary can be made.which suggests that we can further simplify Hypothesis 3 with an approximated inverse proportional relationship between the optimal Corollary 1. des, when the passive stiffness of the series elastic actuator of the device is changed from Kt,old to Kt,new, an estimate of the new optimal proportional control, Kp,new, can be achieved byFor a fixed desired torque-angle relationship, i.e, Kp,old is the optimal at Kt,oldin which K.Although multiple approximations have been made in the derivation of this corollary, which causes inaccuracies in this estimation, it can be used to set a starting point of proportional control gain tuning when system passive stiffness is changed with only the knowledge of the old and new passive stiffness values.Furthermore, combining Equations (24), (30), (32) at optimal control conditions, we havewhich meanse,ext(t), and torque error values ėmaxτ,, across different desired stiffness, at a time of similar measured torque τ, the following hypothesis can then be drawn.With relatively invariant extreme ankle velocity values, θHypothesis 4. dThe K − t coefficient in Equation (30) is related to the desired quasi-stiffness Kdes byKin which ς, δ and ξ are constant parameters, andis linearly related to the hypothesized maximum commanded torque change rate ėmaxτ, .Combining Hypotheses 3 and 4, we can relate the optimal proportional gain to the desired quasi-stiffness and get the following corollary.Corollary 2. p,opt has an inverse linear relationship with the desired quasi-stiffness KdesThe optimal proportional control gain K.Kp,opt as either the passive stiffness Kt or the desired quasi-stiffness Kdes increases.Hypothesis 3 and Corollary 2 together suggest the monotonical decreasing trends of the optimal proportional control gain Multiple assumptions and approximations of the system dynamics were made to simplify our theoretical analysis in forming the hypotheses. Complications of the system that were not captured by the models we used are listed below.There are frictions and stictions in the Bowden cable transmission, which are also time-varying depending on the shape of the cable, conditions of the inner rope and conduit, tension in the rope and also velocity of the relative motion between the rope and conduit.The transmission stiffness is non-linear due to the existence of synthetic rope in Bowden cable, which is stretchy under a small tension and stiffer under larger. This stiffness is also subject to changes due to the slow-stretching of the rope and step-to-step gait variation.The lever arm of series spring force with respect to the device joint is not a constant.There is a communication delay and rise time during enforcement of the desired velocity. Therefore, there can never be perfect tracking.Due to step-to-step variation, even after stabilization, the assisted walking is not an exactly periodical oscillatory process as assumed.All these unfeatured complications in system dynamics can cause reality to deviate from the theoretical analysis to some extent. To ensure the guidance for hardware and controller design we provide to be meaningful practically, we conducted walking experiments to test and validate our hypotheses.We tested the hypotheses with different springs on a tethered ankle exoskeleton comprised of an off-board real-time control module and geared electric motor, a uni-directional Bowden cable transmission with a series spring, and an exoskeleton frame that interfaced with the human foot and shank Figure .In the tethered ankle exoskeleton testbed as shown in Figure The exoskeleton frame applied forces on the front of the human shank below the knee, beneath the heel, and beneath the toe, so as to generate an ankle plantarflexion torque in proportion to transmission force. Torque was measured using strain gauges  applied in a full Wheatstone bridge on the heel lever, with 1,000 Hz signal conditioning . Joint angle was measured using a digital optical encoder .Ⓡ inner rope, and was 2 m in length. A series spring was attached at the end of the rope to provide increased compliance. This spring is switched in tests to investigate the effects of changing passive stiffness on torque tracking in the device.A flexible uni-directional Bowden cable transmitted forces from the motor to the exoskeleton frame while minimally restricting leg motions. The cable was composed of a coiled-steel outer conduit  and a 0.003 m diameter VectranN = 1, female, 32 years, 1.65 m, 56 kg) was involved. The subject walked on a treadmill with a fixed speed of 1.25 m/s with a self-paced step frequency while wearing the tethered ankle exoskeleton on the right leg in all experiments. All experimental protocols were approved by Carnegie Mellon University IRB.The purpose of experiments in this study was to quantify not the human reaction but the torque tracking performance of various hardware and control conditions, in detail, the relative performance of torque tracking under different desired and passive stiffness conditions for the same subjects. In theoretical analysis, we conjected that the subject does not affect the relative relationship between desired/passive stiffness and tracking performance, but only the absolute values of the optimal control gains, and optimal tracking errors with a maximum tolerable exoskeleton control bandwidth (torque tracking rate). If this conjecture, together with the resulting hypotheses, is validated by single-subject experiments, then there is no need to test multiple subjects for the proof of the relative peroformance of different desired and passive stiffness values, which is unaffected by the subjects. If the conjecture and hypotheses do not even agree with single-sugject experiments, there is no meaning testing more subjects. Therefore, only one healthy subject  was defined by parameter values in Table S = 1 in Equation 8) was defined by the parameter values listed in Table To test the hypotheses, eight desired quasi-stiffnesses, i.e., torque vs. ankle angle relationship, were implemented, including three linear and five piece-wise linear curves. A unit linear curve  torque tracking errors averaged over the one hundred steady steps for each experimental trial was calculated as its performance indicator. For each combination of desired and passive stiffnesses, the RMS error values of all trials with different gains were compared. The lowest of them was recorded as the estimate of optimal torque error for the corresponding stiffness combination. The control gains of the corresponding data set were recorded as the estimates of optimal control gains.Then, the lowest torque tracking errors and the control gains of corresponding experimental sessions for all stiffness combinations were investigated against the difference between desired and passive stiffness values to test the hypotheses. This process is demonstrated in Figure s(t) within one stance period is achieved by firstly high-pass filtering it at 10 Hz. The filtered signal, x(t), is converted to frequency domain using Fast Fourier Transform. The resulting signal in frequency domain, X(f), is used to construct the energy spectral density as s(t) is then calculated as the integral of the energy spectral density. The level of oscillation of a signal in one experiment session is then achieved by averaging the stride-wise torque error oscillation energy.The level of oscillation included in Figure The resulting stabilized passive stiffness values are listed in Table Over five hundred successful experiment trials, each identified by a unique combination of control gains, desired curve and passive stiffness, were conducted and used for data analysis.Kt − Kdes in cases of both individual desired curves and all curves combined. Minimum torque errors for all curves combined are linearly related to a translated absolute value of Kt − Kdes, i.e,Estimated optimal tracking errors, i.e., the RMS torque errors of the data sets with minimum errors, for linear curves are approximately linearly related to the absolute difference between desired and passive stiffness values as hypothesized by Hypothesis 1 and 2 Figure . It can R2 = 0.839 at a slope of a = 0.355 for the absolute ones and R2 = 0.854 at a = 0.869 for the relative ones.with a coefficient of determinant Kt − Kdes with coefficients of determination R2 = 0.571 and R2 = 0.497 respectively. The slopes are a = 0.298 and a = 0.691. Note that for phases 1, 2, and 4, a fixed desired slopes exists in all steps of all data sets for the same desired curve. However, for phase 3, since the peak dorsiflexion angle is different for each step of each data set, the desired slope for a trial with minimum errors is defined as the phase 3 slope in its average stride.For piece-wise linear curves, the RMS torque errors of separate phases for data sets with minimum errors are also well correlated to their corresponding differences between the passive and desired stiffnesses Figure . The absFor the cases of both curve types, results Figure  agree wiR2 ≥ 0.565). For each desired curve, data were fitted into a curve with the same format as Equation (30), in which the same λ values were asserted for all curves of the same type, i.e., linear or piece-wise linear. This result agrees with Hypothesis 3, which is based on Conjecture 1.Control gains show interactions with desired and passive stiffnesses Figure . The proKp − Kt coefficient, σ, as identified in Figure The This paper investigates the existence of an optimal passive stiffness that benefits torque tracking in lower-limb exoskeletons driven by series elastic actuator during walking. Based on theoretical analysis with simple transmission model, we hypothesized that to achieve best torque tracking during walking in exoskeletons, the actuator should be designed such that its passive stiffness, defined as the slope of device torque-angle relationship with motor position fixed, matches the desired quasi-stiffness. We also hypothesized a fixed maximum commendable torque error changing rate that leads to the inverse proportional correlations between optimal proportional gains and passive stiffness values for a fixed desired curve. These hypotheses were highly agreed by a large amount of walking experiment data.Kt − Kdes, of piece-wise linear curves for full steps and present torque errors in a way similar to the linear curves in Figure Kt − Kdes. Another method tried is to calculate the difference as the area between desired stiffness vs. torque curve and passive stiffness vs. torque curve. For both cases, the relationships between torque errors and effective stiffness differences showed significantly less agreement with Equation (28) than Figure Kt should be the instantaneous values instead of a collective determined values.Although only a simplified model of the transmission sub-system was considered, torque tracking results in Figure Kt = Kdes as shown in Figure Kt = Kdes, torque errors are above zero under optimal control conditions.Meanwhile, there are other factors that add noise and complexions to the data, which causes imperfection in curve fitting and non-zero torque errors at τ, max, as a limit for proportional gain increase suggests a potential way of systematic gain tuning when desired or passive stiffness is changed for the same subject. Due to the dependence of this maximum error changing rate on full system dynamics, it is expected to be subject-dependent for the same motor system. However, since the primary goal of this paper is the identification of an optimal passive stiffness for a desired stiffness, only one subject is used. Therefore, how the maximum tolerable torque changing rate vary among subjects remains a direction of future work.Regardless of the various approximations made in various hypotheses, the results presented in Figures The results of this study can be used as guidances to more efficient hardware and controller configuration for better torque tracking perforemance in lower-limb exoskeletons. For example, with a fixed desired torque-angle relationship required by one particular application, the passive stiffness of the series elastic actuator can be changed by switching the passive compliant element to match the effective desired stiffness. Then, with optimal control gains for one desired and passive stiffness combination determined through experiments on one subject, when desired stiffness changes due to application requirments, or passive stiffness changes due to hardware limitation, the optimal control gains can be estimated based on their relationships with the two stiffness values. The applications of this study will faciliate more effective and efficient torque control in lower-iimb exoskeletons and improves the performance of these devices in experimental, rehabilitation and also assistance senarios.This paper hypothesizes and confirms by experiments that the optimal passive stiffness for lower-limb exoskeleton torque tracking corresponding to the desired quasi-stiffness, which is the slope of desired torque-angle relationship. The minimum torque tracking errors are shown to be linearly related to the difference between the desired and passive stiffnesses. This paper also hypothesizes a potential maximum torque error tracking rate and therefore a maximum control bandwidth that prevents the proportional feedback gain from increasing infinitely. This was also supported by experimental data. These results provide guidance for passive stiffness selection of lower-limb exoskeletons and other walking related robots for a fixed desired stiffness. They also provide guidance for optimal gain tuning in case of changing passive or desired stiffness.This study was carried out in accordance with the recommendations of guidelines of the Office of Human Research Protection (OHRP) and other federal regulatory agencies with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Carnegie Mellon University IRB.JZ and SC contributed equally in theory development and experiments for this study.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.


Sintered zirconia plates were air-abraded (control) or etched with 10%, 20%, or 30% HF for either 5 or 30 min. After cleaning, the surfaces were characterized using various analytical techniques. Three resin cylinders (Duo-Link) were bonded to each treated plate. All bonded specimens were stored in water at 37 °C for 24 h, and then half of them were additionally thermocycled 5000 times prior to the shear bond-strength tests (n = 12). The formation of micro- and nano-porosities on the etched surfaces increased with increasing concentration and application time of the HF solution. The surface wettability of zirconia also increased with increasing surface roughness. Higher concentrations and longer application times of the HF solution produced higher bond-strength values. Infiltration of the resin into the micro- and nano-porosities was observed by scanning electron microscopy. This in vitro study suggests that HF slowly etches zirconia ceramic surfaces at room temperature, thereby improving the resin–zirconia bond strength by the formation of retentive sites.This  Today, yttria-stabilized polycrystalline tetragonal zirconia (Y-TZP) ceramic is one of the most commonly used all-ceramic core dental materials, mainly because of its superior strength and high fracture toughness ,2. ZircoBonding to traditional silica-based ceramics is a predictable procedure yielding durable results if certain guidelines are followed . The sta2O3 particles has been found to be effective in cleaning and roughening zirconia surfaces [However, it has been reported that zirconia ceramic is not readily etched by HF owing to its high crystal1inity, making it difficult to roughen the surface for mechanical retention ,10,11,12surfaces ,14. Wegnsurfaces . Novel ssurfaces . The glasurfaces ,16,17. Asurfaces . Furthersurfaces .The reliability and long-term durability of bonding between zirconia and resin is determined by micromechanical and chemical retention ,12. Within vitro study was to test the bond strength of conventional Bis-GMA-based resin to HF-etched zirconia and compare it to that of air-abraded zirconia. In order to focus on the micromechanical retention by etching, no primers were employed prior to the direct application of the resin to the conditioned zirconia ceramic. In addition, the surfaces etched with HF were characterized using various surface-analytical techniques. The experimental groups used in this study are summarized in The purpose of this Representative scanning electron microscopy (SEM) images of the experimental groups are shown in 2O3, various particle sizes and pressures have been employed. In this study, 110-µm Al2O3 particles at a pressure of 0.25 MPa were used. These conditions may be too aggressive and may damage the surface and induce structural defects [et al. [et al. [Ra value than the HF-etched surfaces (p < 0.001). The AFM images also showed that the HF solutions roughened the zirconia surfaces in a substantially different way than did air-abrasion. The HF solutions partially dissolved the zirconia superficial grain structure (within as well as between the grains), resulting in high surface roughness at the nanometer scale, dependent on the concentration and application time. Within the HF-etched specimen groups, the mean Ra values increased with increasing concentration and application time. Rpv showed a similar tendency like Ra. However, the formation of nanoscale structures was not evident after 5 min of etching. This finding indicates that zirconia etching by HF proceeds only slowly, even when HF solutions of high concentration are used [ defects ,16. Kern [et al.  and Yang [et al.  showed tare used .p < 0.05). Within the HF-etched specimen groups, the Ra values increased  data are shown in ncreased  and the vs. the C1s peak of the hydrocarbon species at 285 eV. Aluminum at the level of 12.7 at% was detected in group APA. Fluorine at the concentration of 0.4 at% was consistently detected on the surfaces, regardless of the concentration of the HF solution used. Thus, among the HF-etched specimen groups, the differences in CA  spectra of the polished and the 30F30 specimens are shown in  surface .r > 0.999). No peaks corresponding to fluoride were detected at 0.1 ppm resolution over the entire experimental period of 30 days, indicating that fluoride ions were not significantly released from the specimen into water. Previous studies demonstrated that fluorination of zirconia converts the surface to more reactive zirconium oxyfluoride (ZrOxFy), thereby improving the chemical bonding with the resin composite [omposite ,27. Simiomposite . Thus, iomposite . HoweverThe means and standard deviations of the shear bond strengths before and after thermocycling are summarized in 2O3 particles is an essential step in achieving a durable bond to zirconia ceramics [p < 0.001), except for group 10F5 (p = 0.137). The AFM image . Moreover, all etched specimen groups, except for group 10F5, showed relatively durable bond-strength values after thermocycling (up to 29.8 MPa), as well as high initial values. Both before and after thermocycling, group 30F30 achieved the highest bond strength among all investigated groups (p < 0.001).For a given HF concentration, a longer application time (30 min) significantly increased the bond strength compared with a shorter one (5 min). In SEM observations, group 10F5 exhibited a relatively smooth surface with few signs of retentive sites formed b. The shThermocycling and water storage are the popular methods used for artificially ageing the bonded specimens, thereby testing the durability of adhesion ,35. TherHowever, thermocycling has been criticized regarding the extent to which it mimics clinical situations. The prescription of ISO 10477, namely 5000 thermocycles , may be All debonded specimens showed adhesive failure at 10× magnification, regardless of the experimental group or thermocycling conditions. However, such adhesive failure did not indicate poor resin–zirconia bonding because even the groups that produced high bond-strength values showed adhesive failure.The etched and adhesively debonded surfaces of a specimen of group 30F30 were compared after the preparation of cross-sections by focused ion beam (FIB) . Unlike HF was purchased from Sigma-Aldrich Co. . Using this acid, experimental HF etching solutions with three different concentrations of 10, 20, and 30 wt%  were produced.3) zirconia ceramic plates  were prepared according to the manufacturer’s instructions. One surface of each specimen was polished with silicon carbide (SiC) paper up to #2000 and, finally, with nylon cloths using diamond pastes of 1 and 0.5 μm grit size [2O3 particles from a distance of 10 mm perpendicular to the specimen surface at a pressure of 0.25 MPa for 13 s (control group) [Rectangular  ; 10F, 20l group) . The treOne representative specimen from each group was prepared for field emission scanning electron microscopy . The specimens were sputter-coated with platinum, and photographs of representative areas of the surfaces were taken.3N4 V-shaped cantilever (k = 42 N/m) was used. Images with 256 × 256 pixels were taken in air with scan size of 5 µm × 5 µm and at a scan rate of 0.5 Hz. Using the AFM images, the average surface roughness Ra and the peak-to-valley roughness Rpv were calculated [Three zirconia specimens of each group were prepared for atomic force microscopy . During the analysis, the microscope was operated in non-contact mode and a Silculated . Three mTo compare the surface wettability, the contact angles (CAs) of water droplets on the zirconia surfaces were measured using the static sessile-drop method by a surface goniometer . Five specimens per group were prepared for the CA measurements. All measurements were performed in a temperature-controlled room at 23 ± 1 °C with relative humidity of 50% ± 5% .−7 Pa [X-ray photoelectron spectroscopy (XPS) was used to identify possibly existing residual elements on the zirconia surfaces. All measurements were performed using an XPS system  equipped with an X-ray source providing Al Kα radiation with an energy of 1486.6 eV. The emission angle of the photoelectrons was kept constant at 45°. A 180° hemispherical analyzer with 32-channel detectors was used for the detection of the photoelectrons . Wide-sc−7 Pa .−1 at a resolution of 4 cm−1 by performing 20 scans. The spectral correction was performed employing the Kubelka–Munk algorithm [The surfaces of a polished and a 30F30 specimen were analyzed using a Fourier-transform infrared (FTIR) spectrophotometer  equipped with diffuse reflectance accessory . Far-infrared (FIR) spectra were collected over the range of 700–250 cmlgorithm .2) were covered with a composite resin , which was then light-cured using a quartz-tungsten-halogen curing light . After immersion in F30 etching solution for 30 min, the zirconia specimens were removed from the covering resin and cleaned as described above. A total of six zirconia specimens were prepared. For half of them, freshly mixed dental resin cement  covered the entire etched surfaces with a thickness of 1 mm and was light-cured for 40 s using the Elipar TriLight curing light. This light-curing was performed from each side of the resin cement to ensure optimal polymerization. All specimens were immersed and stored in individual plastic containers filled with 5 mL of distilled water at 37 °C for 5 days. After that, each specimen was removed from its container and placed in a new one, again filled with 5 mL of distilled water (37 °C). This procedure was repeated every 5 days for 30 days in total .Fluoride release from the 30F30 specimens in distilled water was tested by ion chromatography (IC). For each zirconia specimen, all surfaces except one  with a resolution of 0.1 ppm . As mobile phase, 20 mM potassium hydroxide was used. The instrument was fitted with an IonPac AS19 analytical column and IonPac AG19 guard column. The injection volume was 25 µL and the flow rate was 1.0 mL/min. Free fluoride ions have a well-defined retention time and the peak corresponding to fluoride could readily be determined from the chromatogram .n = 12/group). All bonded specimens were stored in distilled water at 37 °C for 24 h. Half of them were additionally thermocycled 5000 times between 5 and 55 °C in water baths with a dwelling time of 30 s and an exchange time of 5 s between each bath, according to ISO 10477 [Fifty-six sintered zirconia specimens were embedded in round silicone-rubber molds using an acrylic resin and their exposed surfaces were treated as described above. The zirconia surfaces to be bonded were isolated using a bonding jig . The freshly mixed resin cement (Duo-Link) was applied to the surface by packing the material into cylindrically shaped plastic matrices (Ultradent Products Inc.) with an internal diameter of 2.38 mm  and thenSO 10477 .The specimens were then perpendicularly engaged at their bonded cement cylinder bases with a round-notched custom shear blade (Ultradent Products Inc.) in a universal testing machine . Shear bond-strength testing was performed at a crosshead speed of 1.0 mm/min until bonding failure occurred. The bond strengths (MPa) were calculated by dividing the peak load at failure (N) by the bonded surface area. Following debonding, all fractured interfaces were examined under an optical microscope  at 10× magnification to determine the mode of fracture according to one of the following three types: A, adhesive failure at the zirconia–resin cement interface; C, cohesive failure within the resin cement; and AC, a combination of these failure modes.For group 30F30, cross-sectional images of the etched and debonded surfaces were examined using focused ion beam  preparation and SEM investigations. The experimental protocols were described in detail elsewhere . Brieflypost hoc test. For the roughness and bond-strength values, non-parametric statistical test procedures were used for group comparisons because the Leven’s test showed inhomogeneity of the variances between different groups (p < 0.001) [post hoc test, with adjustment of the significance level using the Sidak correction for multiple testing. Meanwhile, the Mann–Whitney test was used within each experimental group for the two thermocycling conditions of 0 and 5000 cycles. All statistical analyses were carried out using SPSS 17.0 for Windows  at a level of significance of α = 0.05.All data were examined for the normality of their distribution by the Shapiro–Wilk test and for the equality of the variances by the Levene test. The CA values of the different groups were compared using one-way ANOVA followed by Tukey’s < 0.001) . The Kruin vitro study, the HF-etching of zirconia ceramic surfaces may allow a low-viscosity Bis-GMA-based resin to penetrate and flow into the thus-formed micro- and nano-retentions, thereby creating a mechanical interlock after polymerization of the resin. The surface topography of HF-etched zirconia suggests that the application time is more critical than the concentration for the formation of retentive sites. The room temperature HF etching may have an advantage in comparison to other novel zirconia etching techniques that include a heat-treatment step. Nonetheless, it is not clear from this study whether using hazardous HF at high concentration and long exposition times in combination with Bis-GMA-based resin without adhesive monomers has a definite merit over low- or moderate-pressure air-abrasion in combination with phosphate monomer containing primers and/or luting resins. In addition, the mechanical properties of zirconia after HF etching should be further evaluated. Finally, long-term clinical investigations are necessary to ensure the clinical efficacy of HF etching of zirconia for improved resin–zirconia bonding.Within the limitations of this


Running an independent journal takes much effort, even if only focusing on managing the process of moving articles through the process of submission, review, and publication. Yet publishing an article is not the only goal. Even a great article has little impact unless it can easily be discovered for people to read and cite. Without visibility, even a journal with a terrific editorial board will not get the high quality submissions its editors seek.Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health (WestJEM) gets ten times the submissions as a decade ago, and has seen its readership climb. In 2008, WestJEM averaged 2,907 combined article views and downloads per month. In 2015, the monthly average was 130,000 [The  130,000 . WithoutOne crucial piece according to Mark Langdorf, Editor-in-Chief and UC Irvine Professor of Clinical Emergency Medicine, is getting the journal indexed in all major medical databases, and finding sufficient resources.Bibliographic databases/citation indexes are compilations of descriptive information about books, conference proceedings, reports, journals and the articles they contain. Some are discipline-specific and others cover a broad range of topics. If a journal is well indexed, readers are more likely to discover its content regardless of whether or not the journal itself is known to them.top-tier journals. Thus, it is in their interest to vet publications carefully. Even if a journal seems eligible for inclusion, it can be labor intensive to bring the journal to the evaluators’ attention and make the case that it should be added to the citation index. WestJEM applied to be indexed in MEDLINE, the major bibliographic database of the U.S. National Library of Medicine, three times over five years before it was accepted.However, getting a journal indexed can be difficult. While most citation indexes make their journal selection criteria publicly available, those criteria may be hard to meet. Citation indexes compete for users—and usually paid subscribers—the same way journals compete for readers. Most claim that they include more journals than their competitors and offer the WestJEM, its board includes one unusual member: UC Irvine Health Sciences Librarian Linda Murphy. Librarians spend a great deal of time working with citation indexes and often get asked which ones are the best to use and why. They compare the features, scope, and coverage of citation indexes before deciding which to recommend to users engaged in education, research, or patient care. With increasingly tight budgets, librarians have to determine which indexes provide the most cost-effective usage. Because of this multifaceted customer/recommender/user role, librarians are more likely to be able to get the attention of citation indexes and speak their language. Murphy, says Langdorf, has played an essential role in WestJEM’s indexing success. It is not uncommon for indexing services to ignore messages from the editor and managing associate editor, only to respond when the librarian inquires.Unsurprisingly, working with citation indexes can be a challenge for the journal editorial board. For For her part, Murphy says she’s learned much, both from her fellow board members and from the indexing evaluation process. She offers these tips for journal editors who are getting their journal ready for the index submission process:Start by identifying relevant databases to be considered for indexing. These are frequently discipline specific.Review the citation index’s requirements and its journal evaluation and selection criteria. Each has a publicly available description of their criteria with links to their journal suggestion submission form. Some forms demand more details and are more rigorous, or allow more space to justify inclusion, e.g., MEDLINE Review Application (comprehensive) vs. Thomson Reuters Electronic Journal submission form (limited) vs. Embase Journal Selection Procedures.Be persistent, even after a first rejection. Expect the acceptance process to take time. Reconsideration cycles for indexes are measured in years, not months.Citation indexes vary in journal coverage and base their selection decisions on a wide variety of criteria. Some focus only on article quality, and require journals to submit issues for them to review. Some want to see international representation on an editorial board, e.g., MEDLINE Journal Selection and Thomson Reuters Journal Selection Process. Scientific journals must also include comprehensive policies on peer-review process, full disclosure of conflicts of interest, and human and animal subjects treatment, among others.Establishing an open dialogue with the index editor is crucial. It helps to clarify questions and reasons for rejections and/or reconsideration.WestJEM editorial board was told by both MEDLINE and Web of Science evaluators that the journal had insufficient international focus and submissions by non-U.S. authors. To address this requirement while maintaining writing quality for authors whose first language is not English, WestJEM has partnered with international emergency medicine organizations to improve the quality of international submissions. MEDLINE also found the journal title too broad, and claimed it already had sufficient journal titles from emergency medicine. So WestJEM added a subtitle that clarified its niche: “Integrating Emergency Care with Population Health,” to set it apart from other specialty journals. Its third MEDLINE application specifically highlighted this population health niche with purposely placed articles demonstrating the journal’s unique role within the larger specialty.Sometimes a journal will be required to adjust the way it operates if its goal is to be accepted to a major citation index. For instance, the WestJEM continues to discuss with Thomson Reuters regarding their Science Citation Index. WestJEM has recently been accepted to a new citation index in the Web of Science Core Collection, Emerging Sources Citation Index, and currently is being reconsidered for the Science Citation Index. Inclusion in major indexes is a requirement for some submitting authors, whose university promotions are only supported by publishing in such completely indexed, peer-review journals.Journals may get frustrating rejections they might not be able to remedy, e.g., the journal contents are subjects already well represented in the citation index. This is an issue Being an open access (OA) journal may help. Many citation indexes consider OA journals to be desirable. It is easier to review the journal’s content for quality and has the potential to reach a larger audience worldwide, especially in developing countries that lack resources for expensive subscriptions or per-article purchases. There is also evidence that OA journals garner citations more quickly and broadly than traditional subscription publications, especially in the developing world. Access generates citation.WestJEM’s inclusion in MEDLINE (covering issues from 2014 onward) follows its indexing in Scopus (2011), CINAHL (2010), and PubMed (2007), among others. Langdorf and Murphy say the long hours invested have been worth it. What started as a four-page newsletter in 1999, and then became a regional journal, is now a well-established peer-reviewed international journal of some repute. WestJEM is one of the only three 100% open access MEDLINE indexed emergency medicine journals in the world. The other two are the Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine and BMC Emergency Medicine. The quantity and quality of submissions is climbing along with their pageviews . So are citations to WestJEM articles. Having achieved inclusion in major citation indexes, the editors are exploring new opportunities such as interactive issues and partnering with Altmetric, a company that provides “alternative” “social media” metrics that scour the Internet to capture article-level activity and immediate impact in blogs, tweets, reader services and news outlets. For an example, see how Altmetric provides data of attention for a recent WestJEM article, “ The San Bernardino, California, Terror Attack: Two Emergency Departments’ Response,” published in January 2016.WestJEM publicly shares and tries to minimize its acceptance-to-publication time so it can spread new knowledge quickly. It maintains a rigorous peer-review process and ethical standards as marked by membership in the Open Access Scholarly Publishers’ Association (OASPA). WestJEM also waives its article processing fee for authors from low-income countries, and for faculty of departments that support the journal through a membership/print subscription program.One thing has remained constant during this long decade of growth: The journal remains focused on serving the emergency medicine community. Part of that focus is a commitment to the OA model that ensures everyone working in the field—a specialty that deals with social injustice, health and economic disparities, violence, substance abuse, and disaster preparedness and response—can read the articles. provides a suite of open access, scholarly publishing services and research tools that enable departments, research units, publishing programs, and individual scholars associated with the University of California to have direct control over the creation and dissemination of the full range of their scholarship.” According to Langdorf, hosting WestJEM with eScholarship was crucial, especially during early implementation. As the UC Institutional Repository, eScholarship provides valuable support and free services to WestJEM. These include the peer-review platform, key to WestJEM’s initial success. Without eScholarship, WestJEM would never have been started, or continue to grow in the arena of OA publishing.eScholarship, the institutional repository of the University of California system, “Many independent journals share similar dedication and willingness to adapt, despite staff turnover, financial constraints, and technology barriers. Nonetheless, the most daunting hurdle can be building the community of authors, reviewers, and readers necessary to establish a strong journal. Perhaps some impressive new technology will come along to solve that problem. In the meantime, indexing—and working with a university librarian—is certainly a wise strategy.