The Use of Beta-applicators for the Treatment of Intraocular Tumours” in 1963 during the chairmanship of Prof. Karl Velhagen. From 1976 to 1982, he was Professor and Chair of the Eye Clinic of the Municipal Hospital in Berlin-Buch. In 1980, he was a visiting Professor at the University of Iowa and in 1981, he was appointed full Professor at the Karl-Marx-University in Leipzig and Director of the University Eye Clinic. In 1992, he left university service and worked as an ophthalmologist in independent practice in Leipzig until 2005. During this time, he was also engaged as an eye surgeon in Ngaoundere (Cameroon).Prof. Peter Karl Lommatzsch died in Leipzig on 23.11.2020 at the age of 85. He was born on 20 December 1934 in Saxony, studied medicine from 1952 to 1957 at the Karl-Marx-University Leipzig together with his lifelong friend Gottfried Naumann, obtained his doctorate in 1957, and went to the Eye Clinic of the Humboldt University Berlin (Charité) as a research assistant. He finished his Ph.D. thesis on “Professor Peter Lommatzsch was one of the most innovative and internationally renowned ophthalmologists of his generation. The ruthenium applicators he developed together with G. Vormum at the former Academy of Sciences of the former GDR (German Democratic Republic) were one of the most important further developments in eye-preserving radiotherapy of intraocular tumours. Peter Lommatzsch pioneered work in basic scientific research on the radiosensitivity of the eye and its adnexae, which still defines the standard for further development of radiotherapy for ocular tumours today. The scientific work of more than 130 original papers over a period of 40 years covers the entire range of intra- and extraocular tumours and has enabled ground-breaking progress in the treatment of malignant melanomas of the uvea and conjunctiva, retinoblastomas, and intraocular vascular tumours. In addition to this scientific focus, Peter Lommatzsch was one of the first to introduce modern iris and capsular bag–supportedartificial lens surgery in the former GDR through his contacts with Barraquer (Barcelona), Fjodorov (Moscow), and Binkhorst and Worst (Netherlands), and was intensively involved in establishing modern vitreoretinal surgery in the former GDR.Whoever worked with Peter Lommatzsch found in him an overwhelmingly stimulating conversational partner and motivator, with an open mind for all questions of ophthalmology and beyond. Under his leadership, the Centre for Ophthalmic Oncology and Microsurgery was established in Berlin-Buch and later in Leipzig. Colleagues from Eastern and Western countries adopted ruthenium-beta radiation therapy for the benefit of patients in their home countries. Despite the difficult political environment, Peter Lommatzsch maintained intensive scientific and personal contacts with university hospitals in Hamburg, Essen, and Munich, and was always available with advice and experience. His high international reputation became known during his guest professorships in Iowa City with Prof. Blodi and in Leiden with Prof. Osterhuis as well as being an elected member in the Club Jules Gonin.In the middle of the Cold War period, he succeeded in bringing together almost all important ocular oncologists from the East and West for the first “International Symposium on Intraocular Tumours” in Schwerin in 1981. This congress was the first in a continuing series of congresses that led to the founding of the “International Society of Ocular Oncology”. Furthermore, Peter Lommatzsch was co-founder and president of the European Ophthalmic Oncology Group. Until the end of his life, Peter Lommatzsch remained scientifically connected to ocular oncology and actively participated in the latest developments in the field of eye salvaging therapy and molecular genetics.Peter Lommatzsch was awarded the Graefe Prize of the German Ophthalmological Society (2002) for his scientific excellence and was elected into the “Hall of Fame Ophthalmology” of the German Society of Ophthalmic Surgeons (2015).Peter Lommatzsch has not only been involved in ophthalmology. Another main focus of his commitment was the protection of birds and the preservation of rare species, for which he worked with great physical and social commitment until his death.With Peter Lommatzsch, we have lost one of the great innovators and stimulators of European ophthalmology, who never lost the joy of his subject of ophthalmology, even in politically difficult times, and who was also highly regarded as a mentor and scientist after his university appointment in Leipzig. In 2008, he writes in retrospect“On 1 April 2005 I ended my ophthalmological activity. I do not look back in anger, but am happy every day that my children and grandchildren have the great fortune to be able to live in freedom. Anyone who, like me, has lived for more than half a century only under totalitarian regimes does not long for the past times of bondage, even under the new challenges of our time such as globalisation, brutalisation through the excesses of predatory capitalism and the closely related threatening clash of civilisations. If my fate in Leipzig in 1992 was the price of living in freedom, I was happy to pay it for my children”.Peter Lommatzsch concludes his autobiographical essay with a quotation from the Leipzig anatomist Kurt Hermann Alverdes, whom he held in high esteem, who stated in his anatomy lecture on the occasion of the unrest and political reprisals of 1953: “Remember, ladies and gentlemen, the mentality of the people remains almost always the same under all political systems”.


The concept of internationalisation, when referring to the work of social scientists within academic institutions, takes on different meanings and involves different activities. This contribution aims to shed light on the international activities of political scientists across Europe and to investigate the various meanings and practices of internationalisation. The analysis relies on the PROSEPS survey, involving some 1,800 political scientists across 37 European countries. We identify three distinct profiles of international scholars: the networked researcher, the editorial manager, and the traveller. These profiles differ according to 1) the building of international research networks, 2) the involvement in the activities of the international publishing industry, 3) the research and teaching exchanges with foreign academic institutions. Determinants, such as gender, family status, career stage, availability of institutional and financial support, and geographical location, are considered as potential drivers or inhibitors of internationalisation. Our analysis shows that the internationalisation of academic practices follows contrasting paths according to the type of international profile. The idea that science is an intellectual enterprise overcoming national borders is something that we often take for granted, without requiring any further justification or specification. Although the international mobility of students and scholars is as old as universities themselves  list, Jane Knight : 6 mentiThe quest for a conceptual clarification is not just the result of the usual academic pedantry. The point is that each of these internationalising practices (and others that could be added) might appeal to different types of scholars and might require a different set of skills and resources. It is thus necessary to disentangle the meanings of internationalisation. To do so, we propose to draw on insights of existing practices rather than on a priori deliberation. The richness of the data of the PROSEPS survey allows us to investigate current practices of political scientists across Europe. Starting from self-reported scholarly activities, we look for different patterns of internationalisation and offer a parsimonious description of the actual meaning of internationalisation on the ground across European political science.The PROSEPS  project aims at building a network of scholars—mostly political scientists and political sociologists—to study the internationalisation and social impact of political science in Europe. It focuses on 1) the transformation of the academic subject, 2) the social and media visibility of its research outputs, 3) the international mobility and circulation of its researchers, 4) the applicability and concrete applications of the work of political scientists. Funded as a COST Action, the project ran between 2016 and 2020 and involved scholars from 40 European countries.2As far as internationalisation is concerned, the PROSEPS survey includes a large number of useful indicators 17), covering a broad range of internationalising activities related to teaching, research, and leadership. Table 7, coveriWe relied on a principal component analysis (PCA) with varimax rotation to identify the main types of international scholars across European political science. PCA is a statistical technique that is widely used to reduce dimensionality in cases of rich and complex information. In our analysis, PCA is useful for disentangling different dimensions of a concept while keeping as much information that is contained in the original set of the 17 indicators of internationalisation as possible. In other words, the goal of PCA is to maximise parsimony while preserving most of the richness of the original information. When several factors are considered, PCA allows to organise the maximum number of factors into the minimum number of underlying dimension(s). The first component retains the maximum possible information, the second component retains the maximum of the remaining information, and so forth.Published in a language (not English) other than the principal language of your academic system and Partner or subcontractor of a research project funded by international institutions) do not load sufficiently on either of the three components. It means that organising them together with other factors does not bring enough to any of the three main components. For this reason, they will not be considered in the following analyses.Table international networked researcher. Internationalised scholars belonging to this first type tend to publish in peer-reviewed international journals, and often with international co-authors , institutional support from the university of belonging, and career stage.For the purpose of this investigation, we identify three broad European regions: Northern Europe, the Mediterranean countries, and Central and Eastern Europe (CEE). In addition, we divide CEE into two sub-groups according to European Union membership.Family responsibilities are another source of contrasting propensity to build an international scholarly profile. Having children might be a particularly strong negative incentive in this sense. Gender is another key factor that may potentially affect international activities, and even more so when combined with family responsibilities. The PROSEPS survey provides information on both these aspects. Regarding family responsibilities, we have re-arranged the available information into four categories, integrating the family status with the presence of children in the same household. It seems reasonable to imagine four different situations, in order of increasing challenges posed to international mobility and activities. Scholars who are single, divorced, or widowed and without children are the least likely to have family responsibilities  and add another one, the career stage. This last variable is operationalised as a simple dichotomy that distinguishes between tenured and non-tenured scholars. As discussed above, family responsibilities can have differentiated effects on men’s and women’s career trajectories. To take this into account, we introduce an interaction term between gender and career stage in the modelling.Figure The first, quite surprising, insight is that neither gender nor family responsibilities seem to have any significant effect on the levels of internationalisation among the PROSEPS respondents. The interaction term is also statistically non-significant, and this for all the three types of international profiles. The only exception is represented by family responsibilities in the case of the international editorial manager. When compared to the absence of family responsibilities (scholars that are single/divorced/widowed and do not have children), the highest level of family burden (single/divorced/widowed with children living at home) has a negative impact on this specific set of activities. It does not exert any effect on the other two sets of activities. This result meets the 95% confidence interval statistical standard. As pointed out earlier, this may also be partially due to self-selection for responding to the survey. Scholars who are juggling between work and family commitments are probably among the least likely to answer a survey about the profession. While care responsibilities are unlikely to be the only driver for self-selection, it is certainly an important one. There are numerous accounts in the literature about how international activities are not always gender- or family-friendly. This said, a recent study from Norris  on the gfrom the three other areas.On the contrary, the variables capturing the career stage, the level of institutional support, and the regional location are all statistically relevant. Tenure is a strong predictor of internationalisation when this refers to editorial management activities. This most probably indicates that experienced scholars are more likely to take on (and to be offered) such responsibilities. International travellers are more commonly found among non-tenured (and often at an earlier career stage) scholars. This is also understandable. Tenured positions often imply teaching duties and institutional responsibilities that cannot be easily bypassed and normally require a physical presence at home university. Institutional support for international activities is also a significant predictor of internationalisation. This time, however, it is more relevant for networked researchers and travellers, and less so for editorial managers. This makes sense, as the four possible types of support included in the PROSEPS survey primarily refer to research activities to be carried out abroad, such as, participation in international conferences, teaching/research fellowships, and support for cooperation in international projects. This finding echoes the argument made by Norris  about thThis article aims at advancing our knowledge on the internationalisation of European political scientists. Drawing on the PROSPEPS survey, we have first disentangled three different and complementary set of practices regarding internationalisation and identified three ideal typical profiles: the networked researcher, the international editorial manager, and the traveller. Our investigation about the determinants of the propensity of European scholars to resemble one of the mentioned profiles reveals that some factors  exert differentiated directions for each type of international political scientist. While the PROSEPS survey covers the entire area of Europe and the response rate is satisfactory, we nevertheless have to keep in mind potential effects linked to self-selection and self-reporting. Our findings are thus best considered as illustrations of trends in practices rather than an iron law about any successful internationalisation of political science.Contrary to our expectation, gender and family responsibilities seem not to have a significant impact on internationalisation. Again, we should be careful in our phrasing. Gender and family responsibilities do not exert any significant impact among the PROSEPS respondents who were included in this specific set of analyses. A self-selection effect may blur the gender picture of international activities. Women academics are also more likely to be single or to have no children compared to men academics (for a recent overview of the gender barriers in European academia, see Engeli and Mügge Last but not least, geography matters too. The uneven distribution of resources between universities belonging to different European areas represents another obstacle for political scientists of southern and eastern countries to fully take part in the international debate, and even more so for countries outside the EU. The fact that scholars from the disadvantaged areas travel more than their colleagues from Northern European universities does not contradict this finding. There are certainly benefits from a circulation of political scientists for long-term visiting positions in foreign institutions. Nevertheless, problems arise for building a truly European Political Science when circulation goes one-way only (see also Norris


Except for the control group (no contamination), zirconia specimens were contaminated with either human saliva (five groups) or blood (five groups), and then subjected to one of five cleaning methods: water rinsing, 38% phosphoric acid etchant , 70% isopropanol alcohol , Ivoclean  and Katana Cleaner . The resin–zirconia SBS was tested at 24 h and after thermocycling (10 k cycles). Three-way ANOVA followed by Tukey’s multiple comparisons test were utilized to analyze the SBS data. Failure modes were evaluated using a scanning electron microscope. Results: Both blood and saliva significantly affected resin–zirconia SBS as contaminants. After thermocycling, there was no statistically significant difference between SBS obtained after decontamination with the Katana Cleaner  or Ivoclean , and the control group . Adhesive and mixed failures were the most frequent among the tested groups. Conclusion: Both 10-MDP-containing cleaner (Katana Cleaner) and zirconium oxide-containing cleaner (Ivoclean) could eliminate the negative effect of saliva and blood contamination on resin–zirconia SBS.Contamination of zirconia restorations before cementation can impair the resin–zirconia bonding durability. The objective of this study was to evaluate the effect of human saliva or blood decontamination with 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP)-containing cleaner on the resin–zirconia shear bond strength (SBS). Methods: A total of 220 zirconia specimens were prepared and air-abraded and randomly distributed into 11 groups ( Zirconium dioxide (zirconia) ceramic is a biocompatible material with excellent mechanical properties and a wide range of clinical applications in esthetic anterior and posterior indirect restorations . DespiteThe zirconia bonding and testing conditions applied in laboratory studies are ideal, but in certain clinical situations, contamination of zirconia restorations by saliva and/or blood may occur during the try-in step . This coSince water rinsing is ineffective for zirconia decontamination , severalA recent study investigated the effect of a 10-MDP-containing cleaner on the bond strength saliva-contaminated zirconia , and altThe materials used in the study and their compositions are described in 2O3 with 2 bar air pressure for 20 s according to the manufacturer instructions, followed by ultrasonic cleaning in distilled water for 5 min, and subsequently air-dried.Pre-sintered zirconia blocks  were milled into a total of 220 smaller blocks with the dimensions of 8 mm length, 8 mm width and 3 mm height using a dental milling machine . The prepared zirconia blocks were sintered according to the manufacturer’s instructions and embedded in a self-curing acrylic resin. The top surface of zirconia specimens was polished for 2 min with 600-grit silicon carbide paper mounted on a polishing machine at a speed of 300 rpm under a water coolant and subjected to air-abrasion using 50 μm Aln = 20 per group) according to the contamination and decontamination protocols detailed in Under the Institutional Review Board (IRB) approval (PSAU2020028) obtained from the College of Dentistry, Prince Sattam Bin Abdulaziz University, a total of 10 mL of saliva and 10 mL of venous blood were obtained from a healthy donor (one investigator), collected in plastic tubes, and used within 24 h. Zirconia specimens were randomly divided into 11 groups  was applied to specimens of each group and air-dried for 5 s according to the manufacturer’s instructions. Then, a silicon mold with the dimensions of 3 mm diameter and 2 mm height was fitted on the zirconia surface and filled with a dual-cure resin cement . The resin cement was subsequently light-cured for 40 s to form the resin build-up. One half of the bonded specimens of each group were stored in distilled water at 37 °C for 24 h; thus, adequate polymerization of the dual-cured resin cement was achieved before the evaluation of resin–zirconia SBS, while the other half of specimens were tested after being subjected to thermocycling (10 k cycles) as a form of artificial aging before SBS testing. Each cycle involved immersion of the specimens in distilled water with a temperature of 5 and 55 °C for 30 s each, while the transfer time was 5 s.2. The study design and specimens’ preparation steps are illustrated in The cross-sectional diameter of the bonded interface was measured with a digital caliber. A metal chisel mounted on a universal testing machine  was utilized to apply a shear force perpendicular to the resin–zirconia adhesive interface at a cross speed of 0.5 mm/min until failure or debonding. SBS in MPa was calculated by dividing the force recorded at failure or debonding in Newton (N) by the cross-sectional area of the bonded interface in mmFailure modes were evaluated using a scanning electron microscope (SEM)  at ×25 magnification after gold sputtering using a sputter coater . Failure modes were classified into adhesive interfacial failure, cohesive failure in resin cement, cohesive failure in zirconia and mixed failure, which involved adhesive and cohesive failures.p < 0.05).Three-way analysis of variance (ANOVA) was used to statistically analyze the SBS data of all tested groups for overall significance, evaluating the effect of contamination , cleaning method  and Katana Cleaner (Kuraray Noritake)), artificial aging (24 h or TC) and their interactions on resin–zirconia SBS. The difference between specific groups’ means was statistically analyzed with Tukey’s multiple comparisons test (SBS means and standard deviations (SDs) of all experimental groups, at 24 h and after 10 k TC, are graphically illustrated in In the first step of the statistical analysis, a first-order interaction for each of the three study variables: ‘Contamination’, ‘Cleaner’, and ‘Aging’, was individually tested. All the three study variables were found significant, indicating that each individual variable should have influenced the resulted SBS. Second-order interaction analysis revealed that the ‘Contamination × Cleaner’ interaction was statistically non-significant, indicating that the effect of the cleaner (decontamination method) should not have been affected by changing the type of contamination, on the resultant SBS. However, the other two second-order interactions were found significant, meaning that the resulted SBS would have been affected by changing the cleaner and the type of contamination. The third-order interaction, combining all three variables, was found non-significant and hence proposed as the final statistical model, based upon which data was analyzed using Tukey post-hoc testing analysis to accurately detect the individual differences between the experimental groups .p < 0.001) reduced the resin–zirconia SBS, with no statistically significant difference between them at 24 h and after thermocycling. The SBS means following decontamination of zirconia using phosphoric acid etchant or alcohol were significantly (p < 0.001) less than those of uncontaminated groups, with no statistically significant difference compared to water rinsing. The SBS means following decontamination of zirconia using the zirconium oxide-based cleaner (Ivoclean) and 10-MDP-based cleaner (Katana Cleaner) were statistically comparable to those of the uncontaminated group, with no significant difference between the two cleaning methods, at 24 h and after 10 k thermocycling. Thermocycling (10 k TC) significantly affected the resin–zirconia SBS of all groups. The results of the three-way ANOVA are described in Both saliva and blood contamination significantly  reduced the resin–zirconia SBS, at 24 h and after thermocycling, with no statistically significant difference between them. Therefore, the first null hypothesis was accepted. Rinsing with water or immersion in alcohol cannot eliminate the organic elements such as carbon or nitrogen retained on blood- or saliva-contaminated zirconia surfaces [Both saliva and blood contamination significantly  group. Ivoclean is composed of a hyper-saturated solution of zirconia particles. Upon application of Ivoclean onto contaminated substrates, it adsorbs contaminants such as phosphate; in this way, contaminants can be removed from the surface of the contaminated ceramic surface. Previous X-ray photoelectron spectroscopy (XPS) analysis proved the efficacy of Ivoclean as a ceramic cleaner as it eliminates the residue of organic contaminants such as saliva or blood ,30,31. MThe SBS statistical analysis of this study revealed that there was no statistically significant difference between Ivoclean and Katana Cleaner, at 24 h and after artificial aging by thermocycling, which is in accordance with a recent study in which the effect of the two ceramic cleaners on resin–zirconia SBS was evaluated . The 10-The resin–zirconia SBS of all tested groups was significantly reduced after thermocycling (10 k TC). Thermocycling deteriorates the bond strength of the two adhesively bonded materials . The temThe predominant adhesive failures in the groups decontaminated with phosphoric acid etching or alcohol may be an indicator of the decrease in the SBS. Mixed failures in the groups decontaminated using Ivoclean or Katana Cleaner may represent the relatively high bond strength achieved. No cohesive failures in zirconia ceramics were recorded in any group, and this can be explained by the high mechanical properties of sintered zirconia . CohesivThis study tested the cleaning effect of human saliva and blood on resin–zirconia SBS. Both zirconium oxide-containing (Ivoclean) and 10-MDP-containing (Katana Cleaner) cleaners were able to overcome the negative effects of saliva and blood contamination on resin–zirconia SBS. There was no statistically significant difference in the resin–zirconia SBS means obtained following decontamination using these two types of cleaning agents. The use of phosphoric acid etchant or alcohol cannot be recommended for cleaning blood- and/or saliva-contaminated zirconia restorations. Both blood and saliva contamination can result in a significant reduction in the resin–zirconia SBS.


The MS and LS kibbles exiting the extruder were denser and less expanded (p < 0.05) than the HS treatment. Resistant starch, starch cook, and raw:cooked starch RVA AUC increased linearly as shear decreased. These results confirmed that lower mechanical energy processes led to decreased starch gelatinization and greater retention of in vitro RS.The objective of the present work was to produce dog foods from a single recipe at three levels of resistant starch (RS). The low (LS), medium (MS), and high shear (HS) foods were produced on a single-screw extruder at target screw speeds of 250, 375 and 460 rpm, respectively, and with increasing in-barrel moisture as shear decreased. Post-production, kibble measurements and starch analyses were conducted. Kibble parameters were compared by ANOVA with significance noted at  Extrusion cooking is the most common process used to produce pet foods worldwide . Prior tCorn is a starch ingredient commonly used in pet foods. Its initial gelatinization temperature was reported to range from 60 °C to 70 °C, and endothermic peak temperature to be close to 78 °C . GelatinAlthough starch molecular transformations are important for matrix development and kibble expansion, these become readily available for enzymatic digestion in the animal’s small intestine, which in their extreme may not be desired nutritionally. Rapidly digestible starches may contribute to a sharp increase in blood glucose , whereasThe pet food industry is in constant search of innovation. In the present work, we propose to modify the nutrition of starch from corn to create a new product with higher amounts of RS, with the ultimate goal to benefit dog colonic health. Corn was chosen as the sole starch ingredient in the recipe for a few reasons. This cereal and its co-products are known to be well utilized by dogs, they are widely available for pet food companies in the US, and can promote desirable processing characteristics such as matrix development and expansion of the kibble . Kibble In a previous study, we explored the effects of changing processing parameters to create a surface response model to predict RS content in a dog food using a small-scale twin-screw extruder . The tre©; CFC Tech Services Inc., Pierz, MN, USA) to meet the nutrient requirements for adult dogs at maintenance [A single diet was formulated  experiment. Ration was preconditioned  and extruded in a single screw extruder  equipped with a 100 hp drive motor and an integrated operating system which provided real-time calculations for specific mechanical energy (SME), specific thermal energy (STE) and measurement of system temperatures and pressure readings. The extruder barrel was approximately 1.495 m long (11.41 cm screw diameter) with solid flight conveying screws and spiral liners, and a low shear configuration screw profile composed of 7 heads: head 1 was the inlet head with a tapered single flight, heads 2–5 were single flight conveying screws, head 6 was a double/solid flight, and 7 was a double/solid flight cone screw. The extruder die plate had 4 die holes of 0.82 cm diameter each. The treatments SS were set at either 250, 385, or 525 rpm, and IBM was targeted for moisture addition to being considered at low, medium and high levels with the expectation to produce diets with three levels of RS  at high,Post extrusion, kibbles from each replicate were dried in a forced convection dryer , which was equipped with load cells and programmed to track loss in weight for moisture control . Each treatment transition lasted approximately 20 min. Photographs of the extruder panel were taken at the same time of sample collection, and each replicate data was averaged to provide representative processing parameters. Bulk density  and flow rate (kg/s) were measured at the same time as sample collection. Post extrusion, a fraction of each subsample was weighed, combined, mixed, and ground prior to laboratory analysis. This allowed for representative data per replicate.® Instruments, Inc. Winooski, VT, USA) to calculate the amount of starch digested. Rapidly and slowly digestible starch were the starch fractions digested after 20 min and 120 min of incubation, respectively. Total digestible starch was the total starch digested within 4 h of incubation, and RS was the undigested fraction after 4h. Insoluble and soluble dietary fibers (IDF and SDF) were determined on extruded replicates using an enzymatic kit .Wet finished kibbles collected after the extruder die were subjected to RVA as described by . Data we2e/cm2d, where cm2e is the squared kibble diameter, and cm2d is the squared die diameter. Lastly, the longitudinal expansion index (LEI) was calculated as a function of VEI divided by SEI.Texture analysis was performed on 20 dried kibbles per replicate using a 25 mm cylindrical compression probe equipped with a 50 kg load-cell  at 50% strain level. Prior to the analysis, kibbles were equilibrated in an oven at 40 °C overnight. The endpoints measured were kibble toughness (kgxmm) and hardness (kg) which was considered the highest significant fracture force per compression. The diameter and length of 15 wet and dry kibbles were measured twice with a digital caliper and averaged, then these were weighed on an analytical balance  to calculate kibble density and expansion indices. Kibble volumetric expansion index (VEI) was calculated on wet kibbles according to : VEI =  equipped with a digital camera.p < 0.05. Analysis of variance of kibble measurements and expansion indices were performed by the GLIMMIX procedure  with replicate nested within the diet, and the means were considered significantly different at a p < 0.05. Multiple testing was adjusted by the Tukey–Kramer post hoc test.The experiment was conducted as a complete randomized design (CRD). Single degree of freedom orthogonal contrasts for extrusion outputs, starch analyses, and viscosity (measured by RVA) were performed using the generalized linear mixed model (GLIMMIX) procedure from statistical analysis software , and linear (L) and quadratic (Q) relationships were considered significant at a p < 0.0001; Each experimental diet was produced three times on a single day in a randomized order as follows: medium shear (MS), high shear (HS), low shear (LS), HS, MS, LS, MS, HS, and LS. The HS diet was produced with the highest extruder shaft speed (SS), while the LS was extruded with the lowest SS, and MS was intermediate . The dryp < 0.05) due to the PC water additions in the MS and LS treatments. Extrusion motor load and SME had a quadratic decrease (p < 0.05) from HS to LS driven by the decrease in SS and increase in IBM in each treatment level. Although STE was not significant among extrudates, the sum of total specific energy (TSE) decreased in a linear fashion (p < 0.05). As a result of extrusion inputs, both kibble wet and dry bulk densities increased linearly (p < 0.05) and moisture lost at the drier was greater in the treatments with less intensive cooking  and more expanded volumetrically (VEI) and longitudinally (p < 0.05) than the MS and LS treatments, which had similar expansion indices to each other (p < 0.1) to be harder and tougher than the other treatments  the extrudate temperature inside the die was not recorded due to a system failure. To estimate steam flash-off at the die for kibble expansion calculation, die temperature was one of the factors needed. Hence, it was estimated by Equations (2) and (3): e; temperature of the product in the extruder just before the die (°C), Qe; total mechanical and thermal energy rate inside the extruder (kJ/h), mex; total mass flow of water and dry material inside the extruder (kg/h), Cpe; specific heat of the extrudate (kJ/kg·°C), Tref; the reference temperature (0 °C), Pe; extruder motor power (kW), Qse; steam energy (kJ/h), Qwe; water energy (kJ/h), Qp; preconditioner discharge energy (kJ/h), calculated by the weighted average of the mass fraction of carbohydrate, protein, ash, fat, and moisture inside the barrel.Tp < 0.05; p < 0.05) driven by the cold swollen starch AUC results.Processing diets at various levels of thermomechanical energy led to different starch transformations. The RVA curve of the HS showed a more pronounced increase in viscosity from 0.4–6.0 min relative to the other treatments. This suggests that starch underwent more extensive chain scission and greater damage . Conversp < 0.1) to decrease linearly from the HS to LS diet. Both the slowly digested starch (SDS) and RS increased linearly (p < 0.05) among treatments (p < 0.05) consistent with the thermomechanical energy that each treatment received. Fiber analysis was not different among treatments in any of the fiber fractions (Starch enzymatic assays confirmed the extent of starch that was available for digestion. Rapidly digested starch (RDS) had a marginal significance . This is usually achieved by fully cooking the starch under a high thermomechanical process known as food extrusion. While effectively cooked kibbles are sufficiently expanded , durableStarches are primary ingredients in extruded foods due to their functional properties to bind particles, form a matrix, and assist with kibble expansion upon exiting the extruder die. Since this nutrient is not considered to be a dietary essential, it has been deemphasized regarding nutrition benefits until recently. Less cooked starches may partially retain native starch granules with greater RS compared to their highly cooked forms, which provides substrate for beneficial saccharolytic bacteria in the large intestine and act as a prebiotic ,6,7. ThuIt was the goal of the present study to produce diets with three levels of RS. This was achieved, although with less RS than anticipated. The corn used to produce the diets was ground through a hammermill using a 1.59 mm sieve size twice, which led to a smaller corn particle size that contributed to an increase in corn surface area (relative to mass) and starch gelatinization than what was targeted.  used a 2The modification of extrusion SME can have a large effect on starch gelatinization. Altering process settings can affect SME and consequently TSE. The preservation of partially gelatinized starches during the mild extrusion process has been shown to retain more RS  that actThe HS food in the present study had a 22% greater TSE compared to the LS, and this difference led to changes in kibble expansion, density, and extent of starch gelatinization. However, the change in total energy input was not equally distributed across treatments; wherein, TSE decreased by 15% from HS to MS, and only by 8% from MS to LS. Leading these last two treatments to be more closely related in starch cook levels and kibble characteristics. The kibbles produced at lower SME and higher IBM were denser, less expanded, and had a tendency to be tougher and harder than the food produced under high shear conditions. Other studies have also reported that a greater process moisture was positively correlated with hardness and negatively correlated with expansion . In regaBesides altering kibble characteristics, the differences in extrusion parameters in the present study also modified the starch digestion profile. The physical-viscosity method exemplified by the RVA methodology was performed to confirm starch transformations due to limitations in the RS and starch cook enzymatic assays. The main limitations of the RS and starch cook procedures are that starch digestion in vivo is dependent on physiological responses in the animal-like nutrient transit time, enzyme activity, intestine motility, presence of mucins, and hormones, which are not accounted for in vitro. Moreover, the digested starch from the starch cook procedure also includes some raw starch that can be digested due to the presence of pores and channels in corn and other cereals that can be sites for enzymatic adhesion ,29. In tThe RVA profile of the HS diet had a slight increase in cold temperature viscosity (below 25 °C), due to the high mechanical shear profile used to produce this treatment. This increase in cold viscosity reflects the presence of dextrins and the interactions among them . ConversThe last method explored in the present study to detect RS in dog kibbles was the total dietary fiber procedure (TDF). According to the Codex Alimentarius Commission (2010), dietary fiber includes carbohydrate polymers that are not hydrolyzed by the endogenous enzymes of humans. This may be extrapolated to other monogastric animals. Resistant starches from enzymatic digestion as part of the TDF procedure may be captured in the insoluble fiber component . In the The modifications in extrusion processing mechanical energy had an impact on kibble characteristics and starch transformation. The low shear (LS) and medium shear (MS) were more dense and less expanded than the high shear food (HS) and were more closely related due to their common IBM with little difference in the total specific energy imparted to the product. Physical and chemical starch analyses complemented and strengthened one another. The RVA profile indicated that high mechanical shear led to more starch damage than the other treatments. The starch raw: cooked ratio and RS of each food increased as mechanical energy decreased. The starch% cook assay corroborated the RS analysis. In conclusion, we were successful in producing diets with more RS by imparting less mechanical shear. The effects these diets have on overall dog health will be determined in a future study.


Potamanthellus edmundsi and Pulchephemera projecta, of this family. The lengths of two mitogenomes were 15,274 bp and 16,031 bp with an A + T content of 73.38% and 73.07%, respectively. Two neoephemerid mitogenomes had a similar gene size, base composition, and codon usage of protein-coding genes (PCGs), and the sequenced gene arrangements were consistent with the putative ancestral insect mitogenomes as understood today. The most variable gene of Furcatergalia mitogenomes was ND2, while the most conserved gene was COI. Meanwhile, the analysis of selection pressures showed that ND6 and ATP8 exhibited a relaxed purifying selection, and COI was under the strongest purifying selection. Phylogenetic trees reconstructed based on two concatenated nucleotide datasets using both maximum likelihood (ML) and Bayesian inference (BI) estimations yielded robust identical topologies. These results corroborated the monophyly of seven studied families and supported the family Leptophlebiidae as being of the basal lineage of Furcatergalia. Additionally, the sister-group relationship of Caenidae and Neoephemeridae was well supported. Methodologically, our present study provides a general reference for future phylogenetic studies of Ephemeroptera at the mitogenome level.Mayflies of the family Neoephemeridae are widespread in the Holarctic and Oriental regions, and its phylogenetic position is still unstable in the group Furcatergalia (mayflies with fringed gills). In the present study, we determined the complete mitogenomes of two species, namely  The mitochondrion is an important organelle in metazoan cells as it is mainly involved in life cycle, apoptosis, and metabolism . In mostEphemeroptera (mayflies) is one of the most archaic of extant winged insects, originating in the late Carboniferous or early Permian periods (about 300 Mya) . MayfliePotamanthellus edmundsi and Pulchephemera projecta. Subsequently, we performed comparative analyses of the mitogenome features among Furcatergalia species concerning genomic structure, nucleotide composition, and the secondary structure of tRNAs. In addition, we incorporated the new mitogenome sequences into the Furcatergalia dataset to obtain a more reliable and robust phylogeny to understand the phylogenetic relationships within Furcatergalia.Mayflies of the family Neoephemeridae are widespread in the Holarctic and Oriental regions . So far,P. edmundsi and P. projecta were generated by next-generation sequencing on the Illumina platform. The sequencing qualities were high for both mayflies and the Q20  base percentage of two samples were 98.41 and 98.17, respectively. Both complete mitogenomes were circular double-stranded structures (GenBank accession numbers: OK272542 and OK272543) and the sequences were 15,274 bp (P. edmundsi) and 16,031 bp (P. projecta) in size  were coded on the L-strand . In the togenome . Both Netogenome . All PCGP. edmundsi) and 3732 (P. projecta), and the codon AGG was not found in P. edmundsi. Relative synonymous codon usage (RSCU) values for the 13 PCGs of two mitogenomes are summarized in The total numbers of amino acids (without the termination codons) were 3739  were situated between trnL (CUN) and the control region in the L-strand, separated by trnV . The lenP. projecta) to 71 bp (trnV of P. edmundsi). All tRNA secondary structures of two mayflies were inferred and, among all the tRNA genes, 21 could be folded into the regular clover-leaf secondary structures (P. edmundsi and 27 (three U-U) in P. projecta. The pair of G-U could be corrected through the editing process and should not affect the transport function . Tw. Tw65]. Potamanthellus (P. edmundsi) and Pulchephemera (P. projecta). Results showed that the sequenced gene arrangements were consistent with the putative ancestral insect mitogenomes as understood today. Two neoephemerid mitogenomes generated in this study had a similar AT nucleotide bias, AT and GC-skews, and a codon usage of PCGs, and were comparable overall to other sequenced Furcatergalia mayflies. The nucleotide diversity of the PCGs of Furcatergalia mitogenomes showed that the most variable gene was ND2, while the most conserved gene was COI. Meanwhile, the analysis of the selection pressures on each gene showed that ND6 and ATP8 exhibited relaxed purifying selection, while COI was under the strongest purifying selection. The comparative analysis could improve our understanding of the evolution of mayfly mitogenomes. Phylogenetic analyses based on PCGs from the mitogenomes of 26 species clarified the phylogenetic relationships of Furcatergalia. The results corroborated the monophyly of the seven families and supported the family Leptophlebiidae as the basal lineage of Furcatergalia. Additionally, the sister-group relationship of Caenidae and Neoephemeridae was well supported at the mitogenome level. Our findings also suggested that the mitogenome sequences were effective molecular markers to study the phylogenetic relationships within Furcatergalia.In this study, two neoephemerid mitogenomes were newly sequenced, among which were representatives from the genus


After recalling the history and the concept underpinning soins-études in psychiatry, we performed a systematic review of the literature based on the PRISMA statement via a search for quantitative studies on soins-études facilities. Eleven quantitative studies on 10 different samples of young people hospitalised in psychiatry in FSEF were identified between the opening of the first unit in 1956 and 2016. The young people involved were mostly aged 16–20 years, which reflects the curricula covered in the FSEF establishments. These young people generally presented severe chronic psychiatric disorders. Their previous care trajectory had lasted for more than 3 years and 24–55% of them had attempted suicide at least once. Their stays lasted more than 6 months. Depending on the severity of the disorders, 44–63% of the young people were considered to have improved at discharge. The contribution of soins-études appears valuable for these young people, since there was a clinical improvement for 54–74% of them 1–15 years after their hospitalisation, with resumption of schooling, professional training or entry into employment in 60–75% of the cases. These results are compared with data in the international literature concerning therapeutic residential care, and lines for future research are identified.Early psychosocial rehabilitation of young people presenting mental disorders is a major challenge. In France, the therapeutic residential care called “ Most chronic psychiatric disorders have an onset before the age of 18 years . Care prsoins-études” were developed in order to enable inpatient care and schooling for severely ill adolescents and youg adults in one and the same place. This is one of the systems engaged in psychosocial rehabilitation  nor are soins-études units part of juvenile delinquency prevention. Young people who are admitted were often previously invested in their studies, and are motivated to continue but have dropped out because of their health problems. The system could be seen as a supported education program applied to inpatient care  was established by the Union Nationale des Etudiants de France . The FSEF first created establishments in which students could pursue their studies while at the same time receiving the long-term hospital care required by tuberculosis , the units being attached as pedagogical annexes to the national authority , the Conservatoire des Mémoires d'Etudiants , the Bibliothèque de Documentation Internationale Contemporaine in Nanterre University (a library-museum covering the history of the twentieth and twenty-first centuries), and the Bibliothèque Sigmund Freud run by the Société Psychanalytique de Paris. We also consulted the archives of the FSEF general management, those stored by the National Director of Studies, and archives in the FSEF clinical libraries.The data collection was completed by interviews with psychiatrists and physicians formerly in charge of FSEF clinics so as to identify further references. The search was also extended to caregivers and teachers working in FSEF. Finally we performed a manual search by analysing the bibliographies of the different documents retrieved so as to identify any references that had not been previously found.soins-études in psychiatry, and the evolution observed among young people in the course hospitalisation in FSEF soins-études and thereafter, since the opening of the first unit (02/04/1956 up to 31/01/2019), were included. Studies excluded were those that were case studies, qualitative studies and opinion papers .soins-études are grouped in soins-études, since the indication was based on enlistment in a higher education course The second study  varied across FESF studies see .soins-études. In France the socio-economic level of upper secondary school pupils and young people in higher education is generally above that of the general population  most often at discharge see . The stuIn the earliest study, published in 1974, Péraud  describePages  describeIn the study by Condé-Diaz , dedicatThe study by Halfon  found 27The cross-sectional study by Gasquet and Choquet  describeIn the study by Levitchi and Botbol , only thThe study by Gibert  found 35All the young people included in the study by Pinel  met the The study by Chandellier  found 27In the cohort studied by Flais  [from whsoins-études had severe, chronic psychiatric disorders, starting in adolescence and continuing into adulthood.In all, on the basis of the use of international classifications, it appears that the very large majority of the young people hospitalised in Certain studies set out to assess the impact of the disorders on the earlier schooling. They frequently observed schooling delays or dropout , 45–47. soins-études system at discharge .soins-études appear to be 10–20 times higher than in the general population. They also appear higher that the 0.2–0.6% death rates reported among adolescents following hospitalisation in a psychiatric facility . For the young people who did not return to school, their period in soins-études system enables social and school reintegration while at the same time providing healthcare. This is why soins-études is considered as third-level care and recommended in French guidelines for adolescents and young adults with psychiatric disorders  and more frequently male than in the populations cared for in FSEF –80. BehaThe meta-analyses on the efficacy of public residential care include studies that were mostly conducted in the USA and Europe (and did not include France). They noted a decrease in symptoms among the young people in care, although the improvement appeared to wane after discharge –84. Favovia therapeutic activities, and family therapies  are oriented towards fostering the autonomy of the young people, allowing them to go out freely during the day to attend training outside the facility  , 92, butsoins-études in psychiatry. Nevertheless, these first evaluations are only partial. Studying systems of this sort amounts to assessing complex systems (soins-etudes but were not admitted).The data we have appears to support the usefulness of  systems . Future The original contributions presented in the study are included in the article/FH and NG performed the selection process for the systematic review. FH wrote the paper. CH, DD, and NG contributed to the critical revision of 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.


User preference has the potential to facilitate the design, control, and prescription of prostheses, but we do not yet understand which physiological factors drive preference, or if preference is associated with clinical benefits.Subjects with unilateral below-knee amputation walked on a custom variable-stiffness prosthetic ankle and manipulated a dial to determine their preferred prosthetic ankle stiffness at three walking speeds. We evaluated anthropomorphic, metabolic, biomechanical, and performance-based descriptors at stiffness levels surrounding each subject’s preferred stiffness.Subjects preferred lower stiffness values at their self-selected treadmill walking speed, and elected to walk faster overground with ankle stiffness at or above their preferred stiffness. Preferred stiffness maximized the kinematic symmetry between prosthetic and unaffected joints, but was not significantly correlated with body mass or metabolic rate.These results imply that some physiological factors are weighted more heavily when determining preferred stiffness, and that preference may be associated with clinically relevant improvements in gait.The online version contains supplementary material available at 10.1186/s12984-021-00916-1. The field of assistive robotics has become remarkably adept at tailoring device design and control to maximize application-specific performance. Because new designs are validated according to a limited set of metrics, the field’s understanding of what “works” is inherently tied to the details of those metrics. Even if resultant design biases are not intentional, the integration of knowledge from the literature and decades of shared experience inevitably drives design in the direction of historical and current measures of success. Some recent approaches, such as human-in-the-loop optimization, go as far as making objective-driven design an explicit part of the tuning process; in these approaches, system parameters are manipulated with the express goal of minimizing a carefully-constructed but highly simplified cost function –3. In obMeaningful efficacy criteria are especially important in the design, evaluation, and prescription of clinical assistive technologies. Despite their promise for restoration of normative gait following amputation, injury, or other limb pathologies, robotic lower-extremity prostheses and orthoses have yet to see widespread adoption. Recent advancements in mechatronic hardware –7 and coUnfortunately, practical constraints—including equipment requirements, measurement time, and analytical overhead—make it difficult to integrate and interpret efficacy criteria that are derived from several simultaneous metrics. This is especially true at the prescription stage, as clinicians do not typically have access to the resources available in the research setting, such as motion capture, force plates, or metabolic monitors. In addition, many patient-specific priorities  can be difficult to quantify with certainty. As a result of these challenges, researchers tend to motivate and validate their new devices with a narrow set of metrics; criteria that have been used include muscle activation –19, joinWe propose to introduce user preference as an alternative criterion to assess efficacy for designing, tuning, and prescribing prostheses, orthoses, and exoskeletons. It stands to reason that user input should play a substantial role in characterizing an assistive device’s clinical impact, irrespective of underlying pathology. In fact, patients are regularly asked to anecdotally describe their feelings of comfort and stability when walking with a new prosthetic leg, as part of the current clinical prescriptive process . Users lAlthough user feedback currently plays an informal role in clinical prescription, two key roadblocks have prevented the formal incorporation of user preference as a measure of efficacy for clinical assistive technologies. The first of these roadblocks is practical: until recently the field has lacked rigorous, repeatable methodologies to quantify user preference of control parameters for wearable robotics , 43, 44.We also do not know what factors users might prioritize when developing preference, or what measurable quantities correlate with preference in a way that would permit objective-driven design, control, and fitting. If, for instance, consistent across-user trends exist between preference and a simple biomechanical variable , designers could target improvement of this variable, and clinicians could focus more time honing device parameters towards this variable. More broadly, these gaps in knowledge create uncertainty around how preference should be used in the assessment of assistive technologies, and may have led the field to underutilize user preference as an indicator of success.To address these deficiencies in understanding, we leveraged a quasi-passive prosthetic ankle–foot in a systematic evaluation of user preference with respect to a single, intuitive control parameter. The Variable Stiffness Prosthetic Ankle (VSPA) Foot has the unique ability to rapidly adjust the stiffness of its ankle joint during continuous walking. We gave control of this parameter to each of seven persons with unilateral below-knee amputation, and asked them to identify their preferred stiffness during treadmill walking at different speeds. We then evaluated gait biomechanics and metabolic expenditure at several stiffness values above and below the preferred stiffness. Our objectives in this analysis were to (i) understand how behavioral and anatomical factors, such as walking speed and body mass, affect preference; (ii) quantify how kinematic, kinetic, metabolic, and performance outcome measures vary across prosthetic ankle stiffness levels; and (iii) explore biomechanics factors that may be important to the user by identifying kinematic, kinetic, or metabolic descriptors that have local minima or maxima at or near the preferred stiffness.The primary hypotheses investigated in this study are that (i) preferred prosthetic ankle stiffness varies significantly across walking speeds, and (ii) kinematic, kinetic, metabolic, and performance outcome measures vary significantly across prosthetic ankle stiffness values, with at least one measure having a local optimum at or near the preferred stiffness. The experiments described herein were designed to highlight specific measurable factors that contribute to user preference. This was a crossover study design, in which each subject was exposed to several experimental conditions . Seven subjects  with unilateral below-knee amputation participated in the study. Subjects were recruited from the University of Michigan Orthotics and Prosthetics Center, and constitute a representative sample of different body masses (range 58.6 to 99.2 kg) of persons with amputation. All subjects were community ambulators (K3 activity level or higher) without complicating lower-extremity injury, who regularly use conventional passive prostheses. All experiments were carried out with informed consent at the University of Michigan, with approval from the Institutional Review Board of the University of Michigan Medical School (IRBMED).This study was performed with the Variable-Stiffness Prosthetic Ankle (VSPA) Foot, a quasi-passive lower-extremity prosthesis that supports complete specification of the ankle joint’s torque–angle relationship , 47. The−1 and 23.3 Nm deg−1 respectively, producing a total range of 5.9 times the minimum stiffness.Despite its relatively low-mass design, the VSPA Foot’s fully customizable torque–angle relationship enables the device to closely approximate the passive components of biological ankle–foot mechanics , 48. StePrior to donning the VSPA Foot, each subject’s self-selected overground walking speed was measured as they walked on their daily-use prosthesis . A certified prosthetist then disconnected each subject’s daily-use prosthesis from their prosthetic socket, and affixed the VSPA Foot in its place; each subject’s daily-use socket was used for all study experiments. Alignment was adjusted according to standard clinical practice, with VSPA stiffness set to a nominal value based on each subject’s weight . After fSubjects were instructed to use a sensorized dial to identify their preferred stiffness during treadmill walking at three different speeds . The dial, which directly controlled VSPA stiffness via a microcomputer, was designed with infinite rotation and no absolute reference. This made it impossible for subjects to rely on direct external indicators of VSPA stiffness while identifying their preference. The dial could be freely rotated beyond the minimum and maximum of VSPA stiffness; however, the VSPA Foot saturated at these extrema, such that any supramaximal changes to the dial were ignored by the controller. This method of adjustment converges faster to the user’s preference than the two-alternative forced choice methods we have previously implemented , 49, andMetabolic rate and gait biomechanics were recorded simultaneously at different VSPA stiffness values and treadmill speeds. These trials were carried out during a second experimental session, scheduled at least 2 days after the preference identification experiment. To ensure continuity across trial days, each subject’s prosthetic alignment was carefully preserved between sessions. On the morning of the metabolic trials, subjects were instructed to eat a light breakfast, then to refrain from eating for at least 2 h prior to the start of data collection. Metabolic rate was calculated from instantaneous measurements of inspired oxygen and expired carbon dioxide, measured via a portable pulmonary gas exchange unit .Each subject’s resting metabolic rate was first collected during 5 min of quiet standing. This resting rate was subtracted from all subsequent metabolic measurements, yielding net metabolic rate. Net metabolic rate was measured on the daily-use prosthesis, while each subject walked for 6 min at the self-selected, slow (− 30% self-selected), and fast (+ 30% self-selected) treadmill speeds, in pseudorandom order. Once these baseline net rates were established, the study prosthetist removed the daily-use prosthesis from each subject’s socket, affixed the VSPA Foot in its place, and gave each subject approximately 10 min to re-acclimate to the device. Subjects then returned to the treadmill and walked at each treadmill speed , while VSPA stiffness was set to each of five different stiffness values, in random order. The experimental VSPA stiffness values for these trials were selected to be multiples  of each subject’s average preferred stiffness for the given treadmill speed, as measured during the preference identification trials. VSPA stiffness was changed remotely between trials without interruption to subject gait. Stiffness modulation occurred during the swing phase, and was always completed within two steps.For each treadmill speed, all five trials were completed as a single, consecutive block. Remote, near-instantaneous changes to VSPA stiffness ensured a step-change in metabolic rate between trials in a single block. This experimental design enabled us to estimate steady-state metabolic rate using dynamic modeling techniques, rather than waiting for each subject’s metabolic rate to reach steady-state after each stiffness change . Specifimentclass2pt{minimeader to ) to be cMatlab, MathWorks, Natick, MA, USA).Biomechanics data were collected simultaneously with metabolic rate, during the second trial day. A standardized set of 36 reflective spherical markers were affixed to each subject’s pelvis, legs, and feet according to a modified Helen Hayes model . Lower-egait10dof18musc model. We modified the model to fit our experimental marker set, and changed affected-side scaling parameters to (i) use femoral segment length estimates from the sound side, which avoids relying on socket markers to estimate prosthetic knee joint center, (ii) allow the location and orientation of the modeled VSPA Foot’s joint axis to differ from that of the unaffected ankle joint, and (iii) reflect the mass properties of the prosthetic device and socket. Bone dimensions were scaled for each subject using OpenSim’s Scale Tool, based on the subset of markers placed over easily-identifiable anatomical landmarks. Mass properties for each segment were scaled relative to body mass, and segment inertias relative to both mass and limb segment lengths. Model marker locations were adjusted for each subject based on a static pose trial, in which the subject stood with feet at shoulder width. We determined joint angles during walking using OpenSim’s Inverse Kinematics Tool, which constrains joint motion to modeled joint kinematics, and seeks to minimize the squared errors in resultant marker trajectories [Inverse kinematic and dynamic analyses were performed in OpenSim v4.0 , using tectories . We thenMatlab. Kinematic and kinetic trajectories were split into gait cycles based on GRF data, and time normalized to percent gait cycle. Stance phase was also identified as the portion of each gait cycle during which there was a sustained, positive vertical GRF. Gait cycles were excluded from analysis if the GRF data showed that stance-phase foot plate was not isolated to a single force plate. From these gait-cycle-normalized trajectories, the biomechanical descriptors in Table All subsequent analyses of gait biomechanics data were performed in In this part of the experiment, we measure the effect of VSPA stiffness on self-selected overground walking speed; note that this is the experimental inverse of the preference identification trials (described above), in which we measure the effect of walking speed on preferred stiffness. At the start of the first experimental session, subjects completed three standard overground 10 Meter Walk Test (10MWT) trials  with theMatlab. We hypothesized that preferred stiffness varies with treadmill walking speed and body-mass. To evaluate this hypothesis, we first assessed a linear fit between walking speed and preferred stiffness, using a first-order linear mixed effects model (LMEM). When this model did not show significance, we assessed a quadratic fit, using a second-order LMEM. A simple linear regression was used to evaluate the relationship between body mass and preferred stiffness.All statistical analyses were performed in p < 0.05 for the second order coefficient), we used the delta method to generate a 95% confidence interval for VSPA stiffness at which model’s vertex was located. After the second-order LMEM failed to show a significant quadratic relationship between overground self-selected walking speed and VSPA stiffness, we carried out additional post-hoc t-tests to further understand the effect of stiffness on self-selected walking speed.To evaluate our second hypothesis—that biomechanical, metabolic, and performance-based descriptors vary significantly across prosthetic ankle stiffness levels, and are locally maximized or minimized at or near the preferred stiffness—each outcome measure was treated as a dependent variable and analyzed separately using a second-order LMEM. For a descriptor to be considered a potential indicator of preference, it was necessary that it (i) show a significant second order trend with preference-normalized VSPA stiffness, and (ii) have a predicted vertex at or near the preferred stiffness (within 10% of preferred stiffness). We chose a threshold of 10% as this approximates the difference in mechanics that stem from a change in prosthesis “category” for common commercially available prosthetic feet . On the p = 0.081). However, a post-hoc quadratic LMEM did show a significant second-order effect of treadmill speed (p = 0.0046), indicating an underlying nonlinear relationship between walking speed and preference  showed no significant linear relationship between walking speed and preferred stiffness  or quadratic  effect of preference-normalized VSPA stiffness on metabolic rate  or quadratic  trends in metabolic rate as a function of weight-normalized VSPA stiffness. These results indicate that metabolic rate is not measurably affected by stiffness changes on the scale considered in this study, independent of subject preference. Treadmill speed had a significant linear effect on metabolic cost .There was no observable linear , with nine showing a linear trend (p < 0.05 for the first-order term), and six showing a quadratic trend . Second-order polynomial fits to the inter-subject mean values for a subset of these descriptors highlight each type of trend . Notably, there was no significant second-order trend between Cross-leg RMS diff. ankle angle and weight-normalized stiffness , indicating that preference illuminates this underlying relationship between ankle kinematic symmetry and prosthetic foot stiffness . However, we did observe a significant first-order relationship (p < 0.0001), and a post-hoc analysis showed that subjects chose to walk at slower speeds when VSPA stiffness was below their preferred stiffness values  value of the descriptor exists but was not obvious or known  The third category includes those descriptors that showed a quadratic trend, with a vertex predicted at stiffness settings higher or lower than the preferred stiffness. Because descriptors in this category had extrema far from the preferred stiffness, they are unlikely to hold substantial influence in the selection of preference. The final category, which was the target of our analysis, includes descriptors that have a significant second-order relationship with preference-normalized stiffness, Cross-leg RMS diff. ankle angle describes the kinematic asymmetry between a subject’s prosthetic and unaffected ankle joints. Although the underlying second-order relationship between kinematic asymmetry and prosthetic joint stiffness was not unexpected, due to the sensitive linear relationship between prosthetic-side kinematics and stiffness [negative outcomes, such as elevated metabolic cost, reduced 10MWT performance, or increased pelvic tilt. In future longitudinal studies, we will directly measure the long-term health implications of prostheses that are designed and controlled according to user preference.tiffness , 66, thetiffness , 71, 72.tiffness . HoweverCross-leg RMS diff. ankle angle was only observed when viewed through the lens of preference-normalized stiffness  may preclude the level of patient-specific preference optimization performed in our study. Additionally, because we have shown that aggregate patient preference may correlate with improvements in walking performance according to relatively “accepted” metrics such as self-selected walking speed, providers may be more inclined to include patient feedback in the prescription process.It is noteworthy that the optimum in ess Fig. . This reThis study was limited by several practical considerations inherent to the VSPA Foot. We chose to use the VSPA Foot for our experiments because it is capable of producing a wide-range of highly-repeatable mechanics along a single, intuitive, continuous axis, in a lighter package than powered prostheses. However, the geometry of the foot and the overall shape of the torque–angle relationship did not perfectly reflect the behavior of typical passive prostheses. In addition, although none of the subjects preferred stiffness values at or above the VSPA’s maximum stiffness, two subjects did prefer stiffness values (at the fast speed) that were within 30% of the maximum, such that we were forced to restrict the range of experimental stiffness values to less than + 30%. Our results also showed that metabolic cost was generally higher for the VSPA Foot than for the daily-use prosthesis, and 10MWT speed generally slower. Although this is likely attributable to experience and training, it may also point to limitations of the experimental device.Our analysis in this study was focused to a subset of all the possible factors that may contribute to user preference. For instance, we did not record electromyography, which would have provided targeted estimates of muscle activity, or upper body kinematics, which would have enabled center of mass calculations. We also did not take comprehensive surveys of patient-reported comfort and stability. Further, with robotic or quasi-passive devices that vary other parameters , prosthesis users may prioritize a different combination of factors, and outcome measures may be more or less sensitive. As such, our results are not intended to be comprehensive; rather they provide preliminary evidence that preference is associated with some measurable physiological factors (and not others), and highlight the importance of further research in this area.The subjects in this study were not representative of the population of persons with below-knee amputation, due primarily to the amount of walking required for participation. However, these subjects were representative of community ambulators, which are the target population for most ankle–foot prostheses. Although it is possible that preference manifests differently in people who ambulate at lower activity levels, this falls outside the scope of the present study. Additionally, the relative number of men and women  in our sample is not representative; unfortunately, we were not able to find a more balanced cohort within a reasonable time frame. Finally, adaptation times may not have been long enough to allow preferences to settle, and both preferences and gait biomechanics may continue to refine over much longer time periods.Rigorously-measured user preference has the potential to inform the design, validation, and prescription of clinical assistive devices that increase user satisfaction and improve health outcomes. Our study sheds light on the factors that contribute to preference, which is an essential first step toward understanding its potential role in clinical practice. User preference is not a perfect metric, in that it is noisy and unlikely to encode all possible factors that determine the efficacy of a clinical device; however, it does provide a direct measurement of what users want. Additionally, we have shown its correlation with potentially meaningful biomechanical and performance-based outcomes. In the future, our methodology for measuring preference may provide a rapid means of selecting design or control parameters from a multi-dimensional space, which currently poses a substantial challenge for the field. For this to be possible, new techniques for efficiently optimizing for user preference will need to be developed , 43, andOur objective in this study was to understand the biomechanical indicators and potential clinical benefits of patient-preferred prosthetic ankle stiffness. We found that preferred stiffness was lowest at the self-selected treadmill speed, and that metabolic cost of walking was not minimized at the preferred stiffness, but ankle kinematic symmetry was maximized. Our results showed that self-selected overground walking speed was highest at stiffnesses at or above the preferred stiffness. This study introduces a formalized approach to identifying the metabolic and biomechanical descriptors that contribute to patient preference in design and control of assistive technology. When applied more broadly, this approach opens the door to a new preference-driven paradigm for development and prescription of rehabilitation devices.Additional file 1: Table S1. Secondary biomechanical descriptors and their trends with stiffnessAdditional file 2: Movie S1. Design and function of the VSPA FootAdditional file 3: Movie S2. Sample gait kinematics across VSPA stiffness values


These results are consistent with previous experimental data and provide a better insight into conditions that promote FN fibril assembly. We have also investigated two distinct phenotypes of FN fibrils that we have previously identified; we show that the ratio of the two phenotypes depends on both substrate stiffness and contractile phenotype, with intermediate contractility and high substrate stiffness creating an optimal condition for stably stretched fibrils. Finally, we have investigated how re-stretch of a fibril affects cellular response. We probed how the contractile phenotype of the re-stretching cell affects the mechanics of the fibril; results indicate that the number of myosin motors only weakly affects the cellular response, but increasing actin velocity results in a decrease in the apparent stiffness of the fibril and a decrease in the stably-applied force to the fibril. Taken together, these results give novel insights into the combinatorial effects of substrate stiffness and cell contractility on FN fibril assembly.Assembly of the extracellular matrix protein fibronectin (FN) into insoluble, viscoelastic fibrils is a critical step during embryonic development and wound healing; misregulation of FN fibril assembly has been implicated in many diseases, including fibrotic diseases and cancer. We have previously developed a computational model of FN fibril assembly that recapitulates the morphometry and mechanics of cell-derived FN fibrils. Here we use this model to probe two important questions: how is FN fibril formation affected by the contractile phenotype of the cell, and how is FN fibril formation affected by the stiffness of the surrounding tissue? We show that FN fibril formation depends strongly on the contractile phenotype of the cell, but only weakly on  Fibronectin (FN) fibrils are the primordial extracellular matrix assembled by fibroblasts during wound healing and embryogenesis. In embryogenesis, FN fibrils are essential for some of the earliest developmental steps: in Xenopus embryos, gastrulation fails in the absence of FN, and the embryos have significant cardiovascular defects . In wounFibronectin is a 250-kDa glycoprotein that is present in a soluble form at high concentration in the blood plasma . Stretchin vitro, while epithelial and endothelial cells exhibit negligible fibrillogenesis [Since FN fibrils require cell-derived traction forces to expose cryptic binding sites , and sinogenesis . There iogenesis . One wayogenesis . These sOver the past two decades, there has been an increased understanding of the role that substrate stiffness plays in driving cellular mechanoresponses –17. It iin silico FN fibrils from first principles [The mechanism of FN fibril assembly is still poorly understood. This is partially due to the fact that, as opposed to actin or tubulin which spontaneously assemble in cell-free environments, FN fibril assembly requires cell-generated forces, and is thus difficult to replicate in cell-free environments. While several groups have attempted to generate FN fibrils in cell-free environments by shearing soluble FN –21, thesinciples . This moinciples  and predinciples ). We thePrevious studies suggest a potentially complex relationship between cell phenotype, substrate stiffness, and FN fibril formation. In the current work, we investigate these relationships by using our existing FN fibril assembly model to investigate the following questions: first, how does substrate stiffness affect FN fibril formation in our model? Our prior experimental work would suggest that the model should predict FN fibril assembly across a range of stiffness values. Second, how does cellular contractile phenotype, as characterized by the unloaded actin velocity, affect FN fibril assembly? Prior studies from our group and others have suggested that the contractile phenotype of the cell strongly affects FN fibril assembly, regardless of substrate stiffness. Finally, how does the contractile phenotype of a cell that is re-stretching a previously assembled fibril affect its mechanosensing response? In other words, if two cells of differing contractile phenotype migrate over an existing FN fibril and re-stretch it, do the cells “feel” a different fibril? By answering these questions, we can gather significant insight into the mechanism of FN fibril assembly in response to a range of mechanical environments and cellular phenotypes.fsub is the force acting on the substrate, fstall = Nmyofmyo is a myosin stall force for which vact approaches 0 and that is proportional to the number of myosin motors Nmyo and individual myosin stall force fmyo, and vu is the unloaded actin velocity.To simulate fibronectin fibril growth, we used a hybrid stochastic-deterministic model that we have previously developed . A schemksub.The focal adhesion “clutch” is represented by Hookean springs in parallel, each of which has a force-dependent unbinding rate, which drives integrin/FN rupture proportional to force generation. The FN molecule is represented by 30 springs in series, which represent the elastic Type III domains in FN, which have previously been shown to unfold in response to force . Finallyvu, a parameter of the simulation. The springs that comprise the clutch stochastically bind and unbind, attaching to the FN molecule, which in turn transmits force to the spring representing the substrate. The resultant motion generates a reaction force. This reaction force alters both the speed of the actin filament and the binding/unbinding kinetics of the focal adhesion clutch. The force also unfolds Type III domains in FN; once a domain has exceeded a length threshold  FN molecule, and the actin filament moving at its unloaded actin velocity  domains ), it is  domains .in silico FN fibrils as follows: the result of the simulations above is a stretched FN fibril, with many Type III domains stretched open and binding to other FN molecules. To probe the mechanics of the FN fibrils, we “relax” these fibrils by resetting all FN Type III springs and the substrate spring to their resting lengths. This fibril is “re-stretched” by simulating the same myosin force, actin filament speed, and focal adhesion clutch binding as in the previous assembly simulations. Importantly, these parameters can differ from those used in the original simulation, allowing us to simulate a fibril assembled with one set of actomyosin parameters, but re-stretched with a different set of parameters. Note that in these re-stretching experiments, the addition of new FN molecules is inhibited.In this study, we have conducted new “re-stretching” simulations of Our prior studies have demonstrated two distinct FN fibril “phenotypes”: one in which the simulation stabilizes at a constant FN fibril length with a constant applied force, which we have termed “Stably Stretched Fibrils (SSFs)”; and one in which the simulation never stabilizes, but stochastically fluctuates around a fibril length and applied force, which we have termed “Fluctuating Stretched Fibrils (FSFs)”. ksub; 2) the unloaded actin velocity vu, which is the speed at which myosin pulls the actin filament in the absence of any resistive force, is changed to represent different contractile phenotypes. A higher unloaded actin velocity represents a larger pulling force by mysoin; previous experimental studies have demonstrated that unloaded actin velocity varies by several orders of magnitude from approximately 7 to 5000 nm/s, depending on cell type [Nmyo was varied. For each combination of parameters, 100 simulations were run.In the current work, we investigate two parameters: 1) the stiffness of the Hookean spring is changed to represent tissues of varying stiffness ell type . In a suksub) and the unloaded actin velocity of the pulling myosin motor (vu), where a higher value represents a cell with a stronger contractile phenotype. Results from these simulations are shown in u, but little to no dependence on substrate stiffness. The one exception is the FN fibril assembly time, which shows negligible dependence on either substrate stiffness or actin velocity.We first investigated the effects of substrate stiffness and cellular phenotype by varying the stiffness of the spring that represents the underlying substrate . The resulting displacement and force generation were calculated to generate a force-displacement curve for each fibril. Data from a representative re-stretch simulation is shown in Stress in the fibril was calculated by dividing force by the maximum fibril cross sectional area, and strain was calculated as fibril stretch divided by the unstretched fibril length. The slope of the stress-strain curve gives a measurement of the fibril elastic modulus or “stiffness”. For analysis, we compared the stress-strain curve slope at the initiation of stretch. Given this predominance of actin velocity on FN fibril mechanics, we again analyzed the effects on FN fibril mechanics as solely a function of actin velocity, regardless of substrate stiffness . ResultsTaken together, there are three conclusions that can be drawn from the results presented so far: first, substrate stiffness has minimal effects on FN fibril morphometry and FN fibril mechanical properties; second, increasing actin velocity yields FN fibrils that are smaller and softer; and third, an intermediate unloaded actin velocity exists at which stress and strain in the fibril are maximal. We next sought to determine the effects of substrate stiffness and actin velocity on FN fibril phenotype. As discussed above, we have previously identified two distinct FN fibril phenotypes: SSFs, which remain statically stretched, and FSFs, which fluctuate around a value of force and stretch length. Cells can assemble FN fibrils, and then migrate away from them, leaving them to be re-stretched by other migrating cells. We next investigated whether the apparent properties of an assembled fibril changed depending on the contractile state of the cell. For these experiments, we used a population of 100 fibrils that were generated under the same conditions: an unloaded actin velocity of 300 nm/s, a substrate stiffness of 1000 pN/nm, and 100 myosin motors. These fibrils were released of any tension, and then re-stretched as explained above. The re-stretching of fibrils was performed over a range of values for the number of myosin motors and the unloaded actin velocity. Results, shown in In silico experiments demonstrate that as contractility increases, assembled fibrils are smaller, with a shorter length, fewer FN molecules, and a lower fraction of attached integrin clutches. Our results also demonstrate that increasing contractility decreases the stiffness of the fibril. Both stress and strain within the fibril exhibit a maximal value at intermediate contractility, with both low and high contractile states resulting in lower stresses and strains.In the current work, we have used a previously-developed computational model of FN fibril assembly to investigate the effects of both substrate stiffness and cellular contractile phenotype on fibril growth and mechanics. We have found that our model, based on first principles of force-driven FN stretching and assembly, predicts that the morphology and mechanics of FN fibrils depend strongly on the contractile phenotype and only weakly on substrate stiffness. We have also examined how substrate stiffness and contractility alter the proportion of two previously identified FN fibril phenotypes: SSFs and FSFs. Our data demonstrates that the proportion of fibrils in each phenotype depends on both substrate stiffness and contractility. At high or low contractility, FSFs are the predominant phenotype. At intermediate contractility, the percentage of SSFs increases with increasing substrate stiffness.Finally, we have examined the effects of cellular contractile phenotype on the re-stretch of previously assembled FN fibrils. Results indicate that the unloaded actin velocity of the re-stretching cell affects the fibrils: a more strongly contracting phenotype results in fibrils that are less stretched and have a lower steady-state force magnitude.u = 0, and shows that as cell contractility increases in the model, cells assemble fibrils that are smaller and shorter. Results demonstrate that as actin velocity increases, the fraction of cluthces bound to the fibril decrease, suggesting that decreased FN assembly at higher actin velocities is driven by more frequent rupture events at the integrin/FN interface.One significant impact of the current work is that it provides a mechanistic explanation of two counter-intuitive experimental observations. First, previous studies have shown a bi-phasic response of FN fibril assembly to contractile force magnitude: knocking out traction forces with either myosin inhibitors or actin disrupters results in no FN fibril assembly ; but, inGiven that cellular traction forces increase with substrate stiffness , 30, andWhile the current work recapitulates several key experimental findings, it fails to accurately predict certain aspects of the FN/force/stiffness relationship. For example, while we have previously demonstrated that FN assembly is not dramatically affected by substrate stiffness, we and others have shown that traction forces still increase in response to increasing substrate stiffness when FN fibril assembly occurs , 31. HowOf particular interest is the role that the two identified FN fibril phenotypes play in mechanotransduction, and how these phenotypes are affected by contractility and substrate stiffness. One could imagine that the cellular response to an FSF, in which the strain and force transmitted to the attached cell are fluctuating randomly, and an SSF, in which the fibril is tonically stretched and a constant force is transmitted, could be dramatically different. Our findings that: SSFs are only assembled in substantial number at an intermediate contractility; re-stretched fibrils exhibit SSF behavior at an intermediate contractile phenotype; and the fraction of SSFs increases with increasing stiffness, suggest that FN fibrils may be optimally tuned to provide mechanical cues at a given contractile state. Cells that cannot maintain this state, either by generating less force or by generating more force, do not assemble SSFs, and thus may lose a key mechanism to sense substrate stiffness. Investigation of FN fibril mechanics and phenotype, both experimentally and computationally, represents an active area of future work for our group.Using a computational model of fibronectin fibril formation, we demonstrate that actin velocity, and in turn contractile phenotype, have a much more profound effect on fibril formation than substrate stiffness. Our work also demonstrates that both substrate stiffness and actin velocity/ contractility affect the ratio of two FN fibril phenotypes. At both low and high contractility, cells predominantly assemble fluctuating stretched fibrils (FSFs), while at intermediate contractility, cells assemble more stably stretched fibrils (SSFs), with the percentage of SSFs increasing with substrate stiffness. Together, these results give new insights into how substrate stiffness and cell contractility govern the assembly of FN fibrils.Understanding the complex relationship between substrate stiffness, contractility, and FN assembly is key to understanding states for which FN assembly is desirable, such as wound healing and tissue engineering applications, as well as understanding conditions for which FN assembly is not desirable, such as the desmoplastic extracellular matrix of a growing tumor, the fibrotic response to implanted material within the body, and scar tissue in fibrotic disease.