This paper addresses the relationship between age and international research collaboration. The main research question is: do younger researchers collaborate more internationally than their senior colleagues? A common assumption is that younger generations are generally more internationally oriented than older generations. On the other hand, senior researchers may have larger international networks compared to younger colleagues. The study is based on data for 5,600 Norwegian researchers and their publication output during a three-year period . Two indicators for international collaboration are used: The share of researchers involved in international collaboration measured by co-authorship and the average proportion of publications with international collaboration per researcher. These indicators reflect two different dimensions of international collaboration. Although the findings are not consistent across age cohorts and indicators of internationalization, the overall trend is that international collaboration tends to decline with increasing age. This holds both at aggregate levels and within groups of academic positions. However, the generational differences are not very large, and other variables such as the field of research explain more of the differences observed at an individual level. In the present study we compare international collaboration rates across age groups using co-authorship data for Norwegian university researchers in the period 2015–2017. The aim of the study is to investigate whether younger and older researchers are differently engaged in international research collaboration.The extent of international research collaboration has increased significantly in recent decades , 2. In mempirically been demonstrated that so-called international papers  generally are more cited than national papers [theoretically plausible reasons to assume that engaging in international research collaboration is beneficial beyond what can be measured by bibliometric indicators. Such factors may be considered as intermediary steps towards higher productivity and research quality, due to expansion of networks, knowledge sharing, and coverage of necessary academic diversity needed for both publications and proposals .From a policy perspective, and certainly within the individual higher education institutions themselves, the understanding of how different generations contribute to internationalization of research is important. Bibliometric indicators play an (increasingly) important role in most European countries either in research evaluations or in performance-based funding systems at national levels , 8. Herel papers , especial papers . Severall papers , 11. Secscientific publications is the common approach in studies of research collaboration [Studying how international collaboration varies by age is not new (see overview of former studies in the next section). Although international research collaboration may take many forms, and scientific publishing may not necessarily be one of them, studying boration , despiteboration . Here, pFirst, most studies with a bibliometric approach rely upon data from the Web of Science or Scopus. The poor coverage of Social Sciences and Humanities in these databases is well known [ll known . In our Second, unlike former studies that have been restricted to certain academic positions, our study includes all researchers from the youngest doctoral students to the oldest professors.Third, studies differ in whether they measure international collaboration according to whether they have collaborated internationally or not (a binary response) or whether they measure the degree of international collaborations . In our study we will present results for both measures, thus eliminating the chance of presenting results for one of the two indicators, which may be reversed for the other.Finally, unlike the survey approach often being used in non-publication-based studies, where respondents’ claims about international cooperation are self-reported and with inevitable questions about content validity, we rely upon verifiable publication data from a total population of researchers with no selection bias or validity concerns. Previous studies on the relation between age and international collaboration have not combined the entire spectrum of age groups, all scientific and scholarly fields, and publications in a similar manner to our study, presenting both binary and continuous measures for international collaboration.productivity. The average production of publication increases with age and reaches a peak at some point during the career and then declines [see e.g., citation impact, some studies have found, in line with Merton & Zuckerman’s [creativity, such as Liu et al. [age playing a part in both preferences, processes and outcomes of academics: research performance may improve/increase or worsen/diminish (or quite simply change) by age, or it may be accentuated at certain points during the career. There are different theoretical models that may be relevant for explaining why one might expect generational differences in international collaboration, with two main contrasting lines of research. One emphasizes changes in individual behavior , while another line of research focuses on structural changes in the science system .The literature on how different attributes of researchers  are associated with bibliometric performance measures (foremost publication and citation indicators) is extensive. The focus of this study is the age dimension, where many previous studies have investigated how the age of scientists influences on their scientific performance. In particular, the issue has been addressed in relation to publication productivity. The findings from these studies are not entirely consistent, but it seems to be quite firmly established that there is a curvilinear relationship between age and kerman’s  claim ofkerman’s . Others kerman’s . For exakerman’s , identifu et al.  who founu et al.  concludeu et al.  study ofThe behaviors of individuals are influenced by the generation they belong to , 22. ThiSeveral studies have pointed at an “age-creativity relationship” , claiminThe increasing internationalization of higher education and international mobility of researchers, and perhaps even more importantly, students in their younger and formative years, may be stimulating to the willingness for future international collaborations , 28. ButElaborating on ‘utility maximizing theory’, Kwiek  makes armain hypotheses and possible explanations: Younger researchers are more internationally oriented than their senior colleagues are.Against this background, based on a dataset of Norwegian researchers, we will assess our Older researchers are more internationally oriented than their younger colleagues are.Despite generational differences plausibly leading younger researchers towards more international collaboration, both empirical and theoretical arguments for a reversed association are easy to find, i.e., the alternative hypothesis: A plausible explanation for older researchers’ potentially higher degree of international cooperation, is related to the possibility for external funding , which mSeveral studies investigating Italian data have found a correspondence between cooperation and productivity , 38, andTo some extent, we may describe older researchers’ international collaborations of today as driven by the past. There is a certain ‘Matthew effect’ pertaining to this: as a researcher’s curriculum vitae expands over the years based on number of publications, number of grants received and projects led, and the attractiveness of the researcher as a collaborating partner grows. Working in the opposite direction, from the outset of the researcher, is the self-organized willingness to engage in international collaboration that may also increase the level of internationalization, cf. an ever increasing and continuous search for recognition, rewards and visibility, so-called ‘preferential attachment’ , where pAs showed by the discussion above, the previous findings on the relation between age and international collaboration are not entirely consistent. There are also studies showing less of a gradient, but more complex relationships. For example, in a Norwegian study where academic staff were asked to report if they had collaborated with foreign colleagues during a three-year period , the proIn the present study, we are analyzing the issue of generational differences in international research collaboration using bibliometric data. The study is based on a dataset consisting of 5,554 Norwegian researchers at the four largest traditional universities in Norway: University of Oslo, University of Bergen, The Arctic University of Norway and The Norwegian University of Science and Technology. The study sample includes professors (full-professors), associate professors, postdocs and PhD-candidates with at least one publication during the years 2015–2017 .Cristin. This is a current research information database used for storing and managing publication metadata, as well as for the purpose of performance-based funding of the institutions [The study is based on a Norwegian bibliographic database called itutions . The datitutions ). Based itutions .The database contains structured data which can be applied for bibliometric analysis. For example, all authors and addresses are indexed, including country as a controlled term, which is important for the study of international collaboration. All publications are attributed to individuals and these data are verified by the authors and institutions. Thus, there is no need for author disambiguation, which is often a problem in studies based on other databases . In the Norwegian Research Personnel Register as a supplementary data source of the individual attributes of the researchers analyzed . The individuals were classified in 10-year age cohorts defined as the aggregate of individuals who were born in the same time interval. Although we might lose some of the data variation, age intervals simplify the descriptive presentation and interpretation at an aggregated level. However, in the regression analysis, we use age as a continuous variable.In addition, we have used the The distribution of researchers by academic position, fields and age groups is shown in The researchers are not equally distributed across positions and age group. Moreover, the distribution of publications differs significantly from the distribution of researchers . This is a measure of whether the individuals have collaborated internationally or not [cf. indicator A. Second, the average proportion of the researchers’ publications involving international co-authorship. This is a measure of the intensity of the international collaboration [indicator B. In the regression analysis, indicator A will have value 1 for those researchers who have had international co-authors, or 0 for those who have only had Norwegian co-authors. Indicator B will have continuous values in the range from 0 to 1.Two indicators of international collaboration are applied. First, the share of researchers who have been involved in international collaboration measured by co-authorship . Group 2 is researchers with two publications (14 per cent). Group 3 is researchers with three and four publications (21 per cent). Group 4 is researchers with five to seven publications (18 per cent). Group 5 is researchers with 8 to 12 publications (15 per cent), and group 6 includes researchers with more than 12 publications and account for 15 per cent of the researchers in total. Group 1 and 2 can be categorized as researchers with low production, while group 3 to 5 can be considered intermediate productive, while group 6 is the most prolific researchers. By this classification we will be able to provide further insight into how international cooperation relates to productivity.Previous studies have shown that researchers involved in international collaboration tend to publish papers with more co-authors than other researchers . HoweverIn addition to descriptive statistical analyses, we have conducted regression analyses on both dependent indicators: researchers that collaborates internationally or not (A) and their proportion of papers with international co-authors (B). The purpose of the regression analyzes is to see whether there is an independent association between age and international collaboration, taking into account other compositional factors that have been demonstrated in former studies to influence on international collaboration. A negative correlation between age and the two dependent variables will tell if the degree of international collaboration decreases with increasing age. For indicator A, every researcher is assigned a value of either 0 or 1, where 1 means that the researcher has collaborated internationally and 0 means that he/she has not. For this binary indicator, we have conducted a logistic regression analysis. For indicator B , we conducted an ordinary least squares linear regression, where the dependent variable  was log transformed, due to the lack of normal distributed data. Because of this transformation, all researchers without international collaboration were excluded from the analysis (since log of 0 is undefined). Hence, the analysis of indicator B includes only researchers who have collaborated internationally. This implies that we are measuring how age is correlated to international collaboration for researchers who are internationally collaborative.Since researchers in different scientific fields have different publication patterns, we have analysed this issue separately by major fields. For the regression analyses, the variables have been recoded: all academic positions were recoded to categorical variables and then to dummy variables (1 if present and 0 if not present); the gender variable recoded in the same way .There are large differences in international collaboration across fields: it is most common in the natural sciences and in medicine and health sciences, and at its lowest in social sciences and humanities . OverallWhen we compare international collaboration by age and academic position, we also observe generational differences. Overall  the two indicators still show a contrary pattern, but within each position, except postdocs, also Indicator A displays that the youngest researchers are more internationally collaborative then the older ones.For the full professors, associate professors and PhD-candidates, the propensity to collaborate internationally (indicator A) is declining with age, albeit with some variations in the age patterns. For instance, 78 per cent of the full professors in the age group 30–39 years were involved in international collaboration, while the corresponding figure for professors above 60 years was 66 per cent. For the postdocs, the proportion is highest in the 30–39 age group, and then declines with age.As noted above, the total bar charts in The alternative indicator (B) measuring the intensity of the international collaboration also shows that the rates of international collaboration generally tend to decline with age. The pattern is most distinct for the full professors and associated professors, while the post docs show a more heterogeneous picture.We have included additional descriptive statistics in tables in the . These tFor all fields, except social sciences, younger professors have on average a higher proportion of their publications with international co-authorship then their older colleagues (indicator B). For the other academic positions, the patterns are mixed. In medical and health sciences, the declining rate of international collaboration applies to all academic positions Appendix .We have so far analysed the issue using descriptive statistics combining the two dependent variables with independent variables of age intervals, academic position and major fields. In order to provide further insight into the issue, we have conducted a regression analysis where all variables are considered.As an initial analysis we calculated the correlation coefficients between all variables included in the regression analyses, except the productivity intervals . When coOverall, the selected independent variables  explain 30–38 per cent of the variance dependent on scientific field. The value is lowest for social sciences and highest for the natural Sciences.We find substantial differences across academic positions where beta coefficients tend to increase by academic rank. This means that higher-position staff are generally more internationally collaborative than PhD-candidates (which is the reference group for the dummy variable). This corresponds with findings presented in Age of the researchers is negatively correlated to international co-authorship, i.e., younger researchers collaborate more internationally. This holds for all major fields. The coefficient levels are rather low, but here it should be noted that we have used age as continuous variable, and not age groups . Age is only statistically significant correlated with Indicator A for the natural sciences and humanities. In the other fields, the relationship is also negative, but without significant coefficients.The regression analysis of the proportion of internationally co-authored publications (indicator B) shows that the selected variables in total explain 17–37 per cent of the variance . The figSince this analysis only includes researchers who have published at least one internationally co-authored publication, the findings will reflect whether the variables contribute to a high degree versus a low degree of international co-authorship. For productivity there is a negative correlation. This might seem counterintuitive and is also in conflict with the results of Of more importance for the research question of this paper are the results concerning the age variable. The regression analysis shows that age is negative correlated with international collaboration in all fields—and regardless of academic position. However, the association is only statistically significant  for Engineering and technology and Medical and health sciences (-0.008). For the remaining three major fields, the association between age and international collaboration, is weaker, and not significant.B-1) for small values. A beta-value of -0.008 for age means that the average proportion of international co-authored papers per researcher is reduced with 0.8 percent from the initial value per year. As an example, this implies that for researchers within Engineering and technology the proportion of internationally co-authored publications is reduced with 16 percentage points from the age of 40 to the age of 60 (20 years).Since the dependent variable is log-transformed, the change caused by a variable is given by the change in B and can be expressed as 100*.The main finding of this study is that there other types of international collaborative work such as committee work, network participations, various leadership tasks, etc. Thus, bibliometric data–limited to just one aspect of research collaboration–can give different results on generational differences than e.g., survey data covering all forms of collaboration. Methodological differences also appear in the specific indicators used, age classification systems and personnel analysed. For example, the study by Abramo, D’Angelo & Murgia [positively with age, which is indeed an ecological fallacy as the within-groups associations by academic position are all negative.Previous studies on the relation between age and international collaboration have reached different conclusions. There might be several reasons for this. One is that generational differences tend to be relatively small and may depend on particular circumstances of the research systems analysed. Another might be that there are methodological differences in the way international collaboration is analysed. For example, in the study by Kwiek  internat& Murgia  includedwithin the same academic position, younger staff are more international collaborative than older staff members.As emphasized by Bozeman & Gaughan  and ZengIt is, however, none of these factors that matters the most for the inclination to co-author internationally. Rather it is the field of research that is the most decisive factor. Scholars in the humanities have relatively limited engagement in international collaboration, at least in a way which is reflected through bibliometric co-authorship patterns. In the natural sciences, on the other hand such cooperation is very frequent and the large majority of the researchers collaborate with colleagues in other countries.We acknowledge that, the generational differences in the propensity to collaborate internationally are not very large. Our dataset does not enable us to provide any detailed  explanations for this. If younger researchers are indeed more international, one plausible explanation to why the age differences are not bigger is that many publications have both younger and older researchers contributing as co-authors . Thus, the research process and its outcome in terms of publications are often intertwined across generations. Even a more detailed study taking first- and corresponding author position into account would not have been able to overcome this, although we acknowledge that especially for the youngest researchers, the international network of co-authors that they engage with are most likely set up by their older (professor) supervisors.a priori reasons why this should be the case. It seems more likely to characterize the archetype of a research group as pyramid-like with age increasing towards the top-level of the group. If so, this may help explain the increased international collaboration in young age groups, as they are found at the bottom of the hierarchy with the highest n. The younger researchers’ inclination to collaborate internationally was identified regardless of academic position and was found in all major fields–also in terms of publication activity.Most researchers work in groups, but we do not know of any studies addressing the age distributions of individuals in research groups. There is therefore no available empirical evidence about whether research groups have skewed age-distributions, i.e., older researchers are drawn to some groups; while younger are drawn to other groups . We do within a country.While our findings are based on a large dataset, they are limited to just one country: Norway. It is important to emphasize that the proportions of internationally co-authored publications vary greatly across nations , generalNorway also has a large and increasing volume of international academic staff. This pattern is seen across all fields and academic positions, particularly the lower ranks. The influx of foreigners is generally much higher in engineering and the natural sciences than in the other fields (54). One might assume that the foreigners are more internationally oriented than the Norwegian-born researchers and some of them might continue collaboration with researchers from their home institutions abroad. In turn, this might influence on the findings obtained. We do not have data available to assess to what extent this is the case. However, we note that the generational differences are observed across all fields analysed , also in the fields where there has been less recruitment from abroad.We see our results as examples of certain global trends in academia where increased international mobility of both students and researchers is the starting point. Numerous studies point at how those who go abroad to study or have research stays sets up future research collaboration from home later on –57; or aAnother explanation is less focusing on the researchers themselves, but on the research system. Globally, research teams are getting bigger, partly due to large scale infrastructure involving many nations e.g., , e.g. thteams (or ‘research groups’), working across borders, it will on an aggregate level move the younger researchers more than the older researchers towards being international on our two indicators .In the generation of global ubiquity , transacS1 Appendix(DOCX)Click here for additional data file.


Escherichia coli. This study confirmed the successful utilization of fenugreek oil as a very effective antimicrobial agent in preparing edible films.In this study, cross-linked pearl millet starch and fenugreek oil was used to develop active starch edible films to overcome the limitations of native starch and to substitute artificial preservatives with natural one. The starch was cross-linked at three levels  using sodium trimetaphosphate (STMP), and physicochemical properties were studied. Moreover, a comparative study was conducted among four samples of films prepared using native starch, cross-linked starch, and native and cross-linked starch loaded with fenugreek oil for physical, thermal, mechanical, morphological, and antibacterial properties. The solubility, swelling, and amylose content of native and modified starch varied from 11.25–12.75%, 12.91–15.10 g/g, and 8.97–16.55%, respectively. The values of these parameters were reduced as the concentration of STMP increased. Cross-linked starch films showed lower moisture, solubility, water vapor permeability(WVP), and elongation at break (EB) values while having higher thickness, opacity, thermal, and tensile strength values. The microscopic images of cross-linked starch films showed smooth surfaces and the absence of ridges, pores, and cracks. The films loaded with fenugreek oil showed different results; the moisture content, water solubility, and tensile strength were decreased while thickness, opacity, WVP, and EB were increased. The onset temperature and peak temperature were lower, while enthalpy of gelatinization was increased to a greater extent than films without oil. The addition of fenugreek oil to films showed a good inhibition area of 40.22% for native+oil films and 41.53% for cross-linked+oil films % against  There is a rising concern for using petroleum-based plastics for food packaging as they are non-biodegradable, thus leaving long-lasting environmental problems. The degradation of these polymers on earth typically leads to the toxicity of the environment. Their waste in water bodies may have new or unintended harmful environmental consequences . The emi2, O2, and moisture [The availability and ease of starch extraction make pearl millet grain a good source, as about 59–80% of its overall components is starch . The stamoisture . Using nmoisture ,12. Starmoisture ,14.3), epichlorohydrin, and sodium trimetaphosphate (STMP) [Modification can be achieved by various means such as chemical, physical, genetic, and enzymatic . These me (STMP) . Among ae (STMP) .Trigonella foenum-graecum is an annual legume commonly known as Fenugreek, which belongs to the Fabaceae family. It has been proven that fenugreek oil’s components such as phenolics, alkaloids, anthracene glycosides, flavonoids, saponins, tannin, volatile oils, and phenolics make it an astonishing antimicrobial agent [Synthetic preservatives are mostly used today compared to natural ones due to their easy availability. Researchers have reported that the use of artificial preservatives such as nitrates, sorbates, parabens, formaldehyde, benzoates, sulfites, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and several others leads to serious health hazards such as asthma, hyperactivity, hypersensitivity, allergy, neurological damage, and cancer ,19,20. Tal agent .T. foenum-graecum) has potential preservation effects against various microbe strains that cause food-borne illness, and therefore, can be substituted for chemical preservatives. Alwhibi et al. [E. coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Staphylococcus aureus, and Salmonella Typhi. In the past, a limited study was conducted on the effect of cross-linking on the properties of the starch film. Little data is available on incorporating fenugreek seed oil in edible film preparations and its effect on different functional and microbiological properties. Thus, the present study explored the preparation of edible films using STMP cross-linked pearl millet starch and comparatively studied its different properties with native starch film. Further, the investigation also explored the effect of the incorporation of fenugreek seed oil on different properties of the obtained film.Fenugreek oil  was extracted by following the method of Akbari et al. [Fenugreek oil SwellCalculations SolubiliLight transmittance (%) of pastes of pearl millet starch was calculated following the method of Perera and Hoover . An aqueThe film was formed by following the procedure of Sharma et al.  with sliThe moisture content of the films was measured by the oven-drying method. Films were kept at 100 °C in a hot air oven  for 24 h. Samples were evaluated at least in triplicate, and results were calculated as (%).The thickness of the films was calculated using a digital micrometer with an accuracy and precision of ±0.001. Thickness was measured at ten different locations for individual film, and the mean value was calculated.Film opacity was calculated by the method explained by Ramos et al. . RectangThe water solubility of film samples was evaluated following Romero-Bastida et al. . Strips 2  as a desiccant in the cup and sealed the mouth with film using a sealant. A temperature of 30 °C and RH of 75% was maintained using a saturated solution of NaCl inside the permeation cell. The weight-gain measurements were recorded at 3 h for 48 h. Measurements were taken in triplicate, and their mean value was evaluated. After measuring the permeation, WVTR was calculated using the following equation [2).WVP of films was determined by the gravimetric modified cup method, based on ASTM E96/E96M . We tookequation :(6)WVTR −1 s−1 m−2) was calculated using the following equation [WVP (g m Paequation :(7)WVP =g) and enthalpy of melting (ΔHm) were determined from the resulting thermograms.Differential scanning calorimetry of film samples was performed on DSC . The film sample (8–10 mg) was cut into small cuts, weighed in an aluminum pan, and sealed hermetically. An empty pan was used as a reference. The samples were analyzed over a temperature range of 20 to 200 °C with a heating rate of 10 °C/min. The glass transition temperature . The film samples were mounted on specimen holder stubs with double-sided carbon adhesive tape; next, a thin layer of gold was sputtered. SEM images were taken at an accelerating voltage of 10 kV and magnifications of ×2000 and ×4000.The antimicrobial activity of films was evaluated by the disc diffusion method after seven days of the film elaboration . The bacStatistical analyses of the mean of triplicate values data were carried out using MinitabStatistical Software version 14 .The amylose content of native and sodium trimetaphosphate cross-linked starch is tabulated in The solubility and swelling power of the native and STMP cross-linked starches are tabulated in p < 0.05) in the transmittance values between native and cross-linked starch. Initially, the light transmittance value was almost doubled after 1% STMP addition. However, it was first decreased then increased slightly with 3% and 5% STMP.The value of starch paste clarity for native and cross-linked starches was observed for the storage period of 0 to 5 days at 4 °C, and the results are shown in p ≤ 0.05) higher than the corresponding native pearl millet starch. Liu et al. [Further, during each storage day, the transmittance values for all cross-linked pearl millet starches were significantly , peak temperature (Tp), conclusion temperature (Tc), and melting enthalpy (ΔHm). Native starch films showed To, Tp, Tc, and ΔHm of 165.99, 168.39, 172.04°C, and 328.34 J/g, respectively. CL starch films showed To, Tp, Tc, and ΔHm of 169.84, 172.63, 178.34°C, and 358.20 J/g, respectively. The CL starch film showed significantly (p ≤ 0.05) increased Tp and ΔHm. A similar increase in Tp and ΔHm in cross-linked corn starch films was also observed by Xu et al. [o, Tp, Tc, and ΔHm values for the native starch film were 167.84, 184.99, 197.39°C, and 468.64 J/g, respectively. For a cross-linked starch film with oil addition, values of To, Tp, Tc, and ΔHm were 179.52, 185.68, 197.47 °C, and 498.38 J/g, respectively. The results showed that adding fenugreek oil to films increased To, Tp, and Tc and majorly increased ΔHm for both native and CL starch films. Among oil-added films, cross-linked starch with oil (cross-linked+oil) films had higher To, Tp, Tc, and ΔHm values than native starch with oil (N+oil) films. Suput et al. [o, Tp, Tc, and ΔHm of films by facilitating chain mobility.Thermal properties of films were measured using a differential scanning calorimeter (DSC). The resulting thermograms and values are shown in u et al. . It was t et al.  also repThe scanning electron microscopy (SEM) images of the surfaces of pearl millet starch films are shown in E. coli. The results proved that even at a minimum concentration, fenugreek oil would be an effective antimicrobial agent without altering the sensory characteristics of packaged food. Sulieman et al. [E. coli. Limited studies have been conducted on the antimicrobial characteristics of starch and essential oils films. Nevertheless, no information has been presented about the effect of fenugreek oil on the addition of starch films.The inhibition areas showed by native and modified starch film disks with and without fenugreek essential oil against studied bacteria are shown in n et al.  also repThe present study contributes to ongoing research on oils derived from plants  with potential antimicrobial properties against some common fungi. Their incorporation into different films (such as edible starch films) has been gaining increasing attention within the food industry and other industries ,65,66,67E. coli compared to native and CL starch films without oil, confirming the inhibitory effect of fenugreek oil, even at low concentrations. In summary, CL starch improved the barrier and mechanical properties, while fenugreek oil addition enhanced the antimicrobial potential of films. This suggests different applications, such as flexible packaging of food products and as a good preservation material to maintain quality attributes and extend their shelf-life. Further, being an underutilized crop, pearl millet can be utilized by starch-based industries as a good alternative for conventional cereal starches.Cross-linking of starch and oil incorporation significantly affected the physicochemical, thermal, mechanical, morphological, and antimicrobial characteristics of pearl millet starch films. Significant differences were observed for moisture, thickness, opacity, water-solubility, mechanical, thermal, and antibacterial characteristics of films. Water vapor permeability rate(WVPR) was decreased in cross-linked (CL) starch films and increased after oil addition. The tensile strength of CL starch film was highest, while a native film with added oil showed the highest value for elongation break. The CL starch film showed higher thermal transition temperatures  and melting enthalpy (ΔHm), indicating that modified starch films have a more compact internal structure. CL starch also enhanced the morphological properties of films to a greater extent, resulting in a compact, uniform, and homogeneous structures with improved surface smoothness. This indicated a closer interaction of matrix molecules induced by cross-linkers. Oil-added films showed an inhibition zone against


The values of stiffness increased with age, with a correlation coefficient of about 0.35 and a p < 0.0001. Age was a significant influencing factor of masseter muscle stiffness. The left and right masseters had similar stiffness. We conclude that stiffness values are significantly lower in women than in men with a difference of 9%. Age significantly influences the stiffness of masseter muscle, and the values of stiffness increase significantly with age, particularly in men. However, further studies are required to determine the precise ranges of stiffness accounting for age and sex in healthy subjects and people with disorders and conditions of the masticatory system.Shear wave elastography (SWE) is an objective and reliable method for the assessment of muscles and internal organs. Every organ exhibits its own stiffness characteristics and hence requires individual reference values. We aimed to determine the reference values of stiffness of the masseter muscle in healthy adult individuals using SWE. We analyzed the data of 140 participants  with a median age of 50 years. The overall mean elasticity was 10.67 ± 1.77 kPa. The average values were lower by 2.25 kPa (9.15%) in women compared to men (9.48 ± 1.47 kPa vs. 11.73 ± 1.27 kPa;  Up to date, several ultrasound elastography techniques have been used for the evaluation of muscle stiffness. These include transient elastography, acoustic radiation force impulse elastography, real-time tissue elastography, and real-time shear wave elastography (SWE). Although SWE has been recently introduced to clinical practice, it has been validated and is gaining attention among practitioners. An SWE device produces mechanical vibration sources which radiate low-frequency shear waves inside tissues, creating two intense plane shear waves . These wClinical applications of SWE primarily include the evaluation of muscles and internal organs. However, every organ exhibits its own stiffness characteristics and therefore requires individual reference values. The reference values for muscles have been mainly established for healthy people. Ewertsen et al.  determinTaking into account the characteristics of SWE, we decided to use it for the assessment of the masticatory muscles. Patients with temporomandibular disorders (TMD) often suffer from hypertrophy and increased tension of the masseter muscle ,10; howeWe enrolled a total of 140 healthy adult volunteers (164 volunteered for this study and 140 were included based on the inclusion criteria). The percentage of excluded subjects was lower than in the general population  because This study is a part of the research project titled, “Shear Wave Sonoelastography in the Diagnosis and Management of the Masseter Muscles Disorders”, funded by the National Science Centre, Poland (funding ID: PREL.B160.18.007).During the study, each participant was first interviewed by a trained dentist and examined for the fulfillment of the inclusion criteria. If qualified for the study, the participant was referred for the SWE examination. All the SWE assessments were conducted by a single trained radiologist with seven years of experience in the technique. For each participant, three measurements each were taken in the right and left masseter muscle and averaged. In the previous pilot phase (unpublished data), measurements were made with a transducer placed longitudinally and transversely to the masseter muscle; however, no significant differences were found between measurements. Based on our experience, we recommend a longitudinal  placement as shown in A circular, 4 mm ROI was positioned in the center of the muscle tissue. The ROI of 4 mm was chosen to reflect the size of the masseter, avoiding the deep and superficial fascia of the muscles. It was located in an area of relatively uniform elasticity as guided by an SWE image and standard deviation (SD) of less than 30% of the mean elasticity value. During the SWE examination, the patients were asked to lie in a supine position, remain relaxed and comfortable, and refrain from swallowing. Before the examination, the probe was covered with an ultrasound gel to reduce the passage of air between the probe and the skin, which enabled good visualization. The tissues were not compressed during the examination.The stiffness of the masseter muscle was measured with the Aixplorer Ultimate device  using a high-frequency linear probe SL 18-5 (5–18 MHz). The obtained measurements were validated using the elasticity QA Phantom model 049A .Data were statistically analyzed using MedCalc v. 19.5.3 . Means and SDs were calculated. The Shapiro–Wilk test was used to analyze the data distribution. The hypothesis of normal distribution was rejected for all variables. The stiffness values were compared using the Mann–Whitney U test. Correlations between age and stiffness were checked with Spearman’s rank correlation coefficient. Regression models were built for predicting the stiffness value of the masseter based on age. A probability value lower than 0.05 was considered statistically significant.We analyzed data obtained from 140 healthy volunteers (74 men and 66 women) with a median age of 50 years : 45.9–55). For women, the median age was 45 years (95% CI: 40–53) with a range from 22 to 65 years. Men were older with a median age of 54 years (95% CI: 47–57.9) and a range from 25 to 65 years. The age distribution is shown in r = 0.353, 95% CI: 0.199–0.490, p < 0.0001). Similarly, a moderate correlation was found between age and the stiffness value of the right masseter muscle . As the comparison between left and right masseter muscle did not show any significant difference, we analyzed the stiffness values of left and right masseter together to calculate the correlation coefficients by sex. In women, a moderate correlation was found between age and the stiffness of the masseter muscle . On the other hand, a weak correlation was found in the case of men .We investigated the relationship between age and stiffness values of the masseter muscle. Taking into account the side of the body, a moderate correlation was found between age and the stiffness value of the left masseter muscle . The unadjusted R-squared was calculated as 0.1870, and the model thus explained approximately 18% of the variability. For men, the regression equation was as follows: y = 10.2745 + 0.02881 × x. The average stiffness value regardless of age was 10.27 kPa, which increased by 0.03 kPa for every additional year. Similar to women, age significantly influenced the masseter muscle stiffness in men (p < 0.0001). The unadjusted R-squared was 0.0709, and the model thus explained approximately 7% of the variability.Regression models were built for predicting the stiffness value of the masseter based on age separately for men and women. In each model, the stiffness values were set as a dependent variable (Y), and age as an independent variable (X). For women, the regression equation was as follows: p-value below 0.0001. Thus, age was identified as a significant influencing factor of masseter muscle stiffness.The reference values of the masseter muscle stiffness have not been established so far in the literature. Our study showed that the mean stiffness value in men was 11.73 ± 1.27 kPa, and in women was 9.48 ± 1.47 kPa. Age and sex had a significant impact on muscle stiffness. The average stiffness values were lower by 2.25 kPa (9.15%) in women compared to men. The values increased with age with a correlation coefficient of about 0.35 and a SWE is gaining importance in oral and facial imaging. Despite the insufficient evidence for its use in determining the stiffness of masticatory muscle, SWE has been widely applied over the past few years. However, there is still a need for large studies to establish the reference values of stiffness. For this reason, we conducted this study on 140 healthy adult individuals using SWE. Few reports providing stiffness values can be found in the literature . NeverthReports from the literature regarding the relationship between the stiffness of the masseter muscle and age are inconclusive. Some researchers report a positive correlation ,20 and sThe effect of age on stiffness has been reported in the literature for other muscles but not for the masseter muscle. Ekby et al.  examinedp-value of < 0.001, but they also found that the correlation between age and stiffness was not significant (r = 0.173 for men and r = 0.018 for women).On the other hand, Alfuraih et al.  reportedp = 0.3). In addition, the correlation between age and masseter muscle stiffness was weak (p = 0.50).In the present study, the stiffness values were found to be higher in men, and therefore it is worth comparing the masseter muscle stiffness of men and women. For the masseter muscle, Arda et al.  reportedp-value of 0.032. However, they did not examine the correlation as they did for age, which may suggest that this relationship might be not linear or could be confounded by outliers. Hence, it is difficult to draw conclusions about the sex-related differences in stiffness.Yoshida et al.  reportedThe present study has some limitations to be addressed. Firstly, SWE measurements were taken by only one experienced radiologist on a limited number of subjects. A question regarding the reliability of SWE might arise. However, SWE has already proven to be a reliable and accurate method for measuring the hardness/elasticity of soft tissues. It is also widely used in other therapeutic areas. Reports in the literature indicate that the measurements obtained using this technique are reliable and repeatable . The resOlchowy et al. assessed the reliability of SWE measurements for the masseter muscle using intraclass correlation coefficients and observed excellent results . Other rp < 0.0001). The masseter muscle on the left and the right side of the body had similar stiffness. Age significantly influenced the stiffness of the masseter muscle, and the value of this parameter increased significantly with age, particularly in men. However, further studies with larger samples are required to determine the precise ranges of stiffness accounting for age and sex in healthy subjects and people with disorders and conditions of the masticatory system.The present study showed that the stiffness of the masseter muscle was about 9% lower in women compared to men (9.48 ± 1.47 kPa vs. 11.73 ± 1.27 kPa;


Euglena gracilis. We show that the peculiar non-planar shapes of its beating flagellum, dubbed 'spinning lasso', arise from the mechanical interactions between two of its inner components, namely, the axoneme and the paraflagellar rod. The spontaneous shape of the axoneme and the resting shape of the paraflagellar rod are incompatible. Thus, the complex non-planar configurations of the coupled system emerge as the energetically optimal compromise between the two antagonistic components. The model is able to reproduce the experimentally observed flagellar beats and the characteristic geometric signature of spinning lasso, namely, traveling waves of torsion with alternating sign along the length of the flagellum.We propose and discuss a model for flagellar mechanics in  Flagella and cilia propel swimming eukaryotic cells and drive fluids on epithelial tissues of higher organisms . The innChlamydomonas reinhardtii, the flagellar beat is, to a good approximation, planar. For these organisms, beat planarity is thought to be induced by the inter-doublet links between one pair of MTs, typically those numbered 5 and 6 . We first analyze the experimental data from the 3d reconstruction of the beating euglenid flagellum obtained in E. gracilis’ flagellar beat, we observed stationary cells trapped at the tip of a capillary. In this setting, the flagellum is not perturbed by the hydrodynamic forces associated with E. gracilis’ rototranslating swimming motion. The beat can then manifest itself in its most 'pristine’ form. We recorded trapped cells during periodic beating. We rotated the capillary and recorded the same beating cell from different viewpoints. Videomicroscopy images from one specimen are shown in To further investigate E. gracilis’ flagellar shapes as seen from common 2d microscopy, for either swimming or trapped organisms. Typically, the 2d outline  of a beating euglenid flagellum is that of a looping curve, see Getting back to the torsion measurement in s. The unit vector We model Ax and PFR as cylindrical structures with deformable centerlines, see Thus, We remark that the right-hand side of The active internal energy of the Ax is defined as minus the total mechanical work of the dyneinsThe active internal energy can be written in a more natural way at the level of individual MT pairs in terms of the work done by the sliding forces  strains , as disc strains . We also1 and H2 . The sinTo explain how the shear variables As mentioned above, the kinematic constraints of the Ax couple the shear variables with bending strains. We haveThe above formulas (whose detailed derivation is given in Appendix 1) underlie the essential mechanism of axonemal motility: collective sliding of MTs generates bending of the whole Ax. We point out here that there is no coupling between the shear variables The special axonemal deformation in We remark here that The PFR is modelled as an elastic cylinder with circular cross sections of radius The PFR shear strains and stretch are defined as follows. The cross-sections centers of the PFR lie on the curve The shear strains thus depend on the orientation of the cross sections with respect to the centerline (tangent), while the stretch measures the elongation of the centerline.The PFR-Ax attachment couples the kinematics of the two substructures, see For each To summarize, we have the following formulas for the PFR orthonormal frame vectorsBy replacing the expressions in The shear of axonemal MTs determines the orientation of the PFR cross-sections. In Under generic (steady) dynein actuation, that is, given elations . The torWe consider here our mechanical model in the presence of external forces. For simplicity, we ignore the possible forces exerted by the cell surface on the non-emergent portion of the flagellum, located inside the reservoir of the cell. Indeed, we suppose that the flagellum is immotile and straight in the region inside the reservoir. We can assume, therefore, that our model effectively describes the flagellum from its emergence point outwards. The only forces the flagellum is subject to come from fluid interaction, which we assume to act all along its length. We consider the extended functionalThe details of the derivation of Once we solve for We analyze the geometry of the centerline In general, the shape of a curve is determined by its curvature From the previous definitions and from ncoupled . The torUnder the assumptions Chlamydomonas. We take this feedback-based self-organization process as a given, and we consider a force pattern that produces local spontaneous bending on the plane Our working hypothesis is that MTs' sliding stretches the Ax-PFR bonding links, which, in turn, inhibits sliding of MTs’ 1, 2, and 3, and triggers a dynein organization  similar to the one present in E. gracilis is if the Ax-PFR joining line is neither parallel nor orthogonal to the spontaneous bending plane of the Ax, then the emergent flagellar shapes are non-planar.We consider here the equilibrium Indeed, suppose E. gracilis, the non-planarity of flagellar shapes is not just possible. It is the only outcome under any non-trivial dynein actuation.For Alongside the previous analysis, there is a less technical way to infer the emergence of non-planarity from our model. We look here more closely to the flagellum energy, and we think in terms of structural incompatibility between Ax and PFR, seen as antagonistic elements of the flagellum assembly, see Under the assumptions urations , must loWhen neither of the two energy components dominates, the emergence of non-planarity can be intuitively predicted with the following reasoning. In the intermediate case of Chlamydomonas-like flagellar beat . For lar beat . We takeChlamydomonas-like target bending strains. Each configuration presents a different (non-null) torsional profile when the antagonistic mechanical interaction of the PFR is absent In Appendix 3, we relaxed the planar constraint , and conOur model is able to predict the torsional characteristic of the euglenid flagellum in the static case, and in absence of external forces. We test here the model in the more realistic setting of time-dependent dynein actuation in the presence of hydrodynamic interactions.We first observe that, as in the static case, the dynamic equations for ecoupled , and thaChlamydomonas-like shapes in a flagellum with no extra-axonemal structures. The shear forces Chlamydomonas flagellar curvatures reported in We consider a dynein actuation that generates Finally, E. gracilis is at the base of the distinctive behavior of the organism, producing the typical roto-translational trajectories of swimming cells. This has been demonstrated by swimming simulations using the experimentally measured flagellar shapes in The unique flagellar beat of The application of Resistive Force Theory hydrodynamics to the waveforms shown in E. gracilis can arise because of a marked asymmetry in the Ax-bonding links-PFR complex in the euglenid flagellum, which is not found in kinetoplastids such as Leishmania  for the imaging of cells trapped at the tip of a glass capillary using transmitted brightfield illumination. The intermediate magnification changer (1.6 X) of the microscope was exploited to achieve higher magnification. Micrographs were recorded at a frame rate of E. gracilis often manifest under confinement. The observations reported in this paper are restricted to specimens with immotile cell bodies, that is, in absence of metaboly. This choice allows for a clear capture of the flagellar beat. The absence of metaboly also suggests minimal impact on cell behavior in response to capillary entrapment.Tapered capillaries of circular cross section were obtained from borosilicate glass tubes by employing a micropipette puller and subsequently fire polished. At each trial, observation a glass capillary was filled with a diluted solution of cells and fixed to the microscope stage by means of a custom made, 3d-printed holder. The holder allowed for keeping the capillary in place and rotating it about its axis, so as to image a cell specimen from distinct viewpoints. Cells were immobilized at the tip of the capillary by applying a gentle suction pressure via a syringe connected to the capillary by plastic tubing. Occasionally, large body deformations of the cells were observed, a behavior commonly known as ‘metaboly’ which  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The large photosynthetic biflagellate Euglena displays an intriguing swimming dynamics. The single emergent flagellum is held in front of the cell, where it executes a highly-3D, so-called spinning lasso beat pattern. Unusually, the euglena flagellum is flanked by a paraflagellar or para-axial rod structure, whose function is so far unknown. The authors put forward an interesting proposition (based on geometric mechanics) that a structural incompatibility between the two filaments leads to frustration and a torsional signature, consistent with the lasso. Both the static case and the hydrodynamic case were tested – the torsional feature seems to be robust to external forcing.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Aleksandra Walczak as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "The biomechanical role of extra-axonemal structures in shaping the flagellar beat of Euglena" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:The large photosynthetic biflagellate Euglena displays an intriguing swimming dynamics. The single emergent flagellum is held in front of the cell, where it executes a highly-3D, so-called spinning lasso beat pattern. Unusually, the euglena flagellum is flanked by a paraflagellar or para-axial rod structure, whose function is so far unknown. The authors put forward an interesting proposition (based on geometric mechanics) that a structural incompatibility between the two filaments leads to frustration and a torsional signature, consistent with the lasso. Both the static case and the hydrodynamic case were tested – the torsional feature seems to be robust to external forcing.The reviewers found this to be a clearly written and well-explained paper that should be of interest to a wide range of researchers in various fields of biology and biophysics. While the level of mathematics is non-trivial, it is explained well within the text, and illustrated well in the figures.Essential revisions:1) The numbering of MTs is chosen by the authors does not appear to be consistent with published literature. This is important and worth checking carefully, in order to cross-reference any previous structural or EM studies. Usually, authors adopt a certain convention when it comes to numbering of microtubule doublets, as such systems do not exhibit perfect rotational symmetry but retain many heterogeneities – and there could be distinguished associations between certain Mts and other, non-axonemal structures such as mastigonemes.T. brucei there are some longitudinal filaments and more like a lattice structure. Does this matter? The ventral flagellum also has a PFR – does this serve any purpose? Also, in euglena the rod extends all the way along the flagellum, but this is not the case in some other species. Do the authors believe that their conclusion extends across phyla, and if not, why not?2) According to the literature, e.g. Hyams, 1982, the PFR is hollow . In contrast in a) There is earlier work on the shape of spirochetes where a similar issue of two elastically competing structures appears: Dombrowski, et al., (2009).The authors may wish to comment on the similarities and differences between that study and theirs.3) In many other types of cilia and flagella which exhibit strongly non-planar beats, there is no association with a PFR-like structure, i.e. the latter is not necessary to generate non-planar bending. What's different here? Would be good to make and discuss some more cross-species comparisons 4) Is it clear that the rod assumes a consistent relationship with the numbered doublets? Could the rod itself be twisted around the axoneme? Authors assumed a fixed relationship and phi_p angle, and that this is neither perpendicular nor parallel to the beat plane… this is important according to Equations 25/26 Chlamydomonas like planar beat pattern for dynein actuation, and determined the mismatch between the rod and axoneme… however, why could the naked axonemal beat not be 3D in the first place? Is there any structural evidence (as exists in Chlamydomonas) which suggests that the euglena beat is planar?5) In order to test their hypothesis the authors implemented a 6) The authors have also assumed that the PFR, together with the AX_PFR linkages are passive. We suspect this is not the case. The fact that the PFR can be digested easily by trypsin suggests it may be contractile. The question is then could this structure participate in phototaxis – what is known about how the beat pattern changes during phototaxis – could the PFR actively enable a faster change in beat pattern or even beat frequency – via active contraction say, upon perception of photic signals? This could potentially be testable in the model.7) Finally, the para-axial rod could also provide or promote a number of other functions, for instance gliding motility. What do the authors think about this possibility? Or conversely, can they suggest experiments that would help show more definitively that the PFR facilities 3D swimming?8) The authors state  that "Classical estimations on homogeneous elastic rods, see, e.g., Goriely, (2017), show that bending and twist moduli scale with the forth power of the cross section radius, whereas shear and stretching moduli scale with the second power and hence they are dominant for small radii." That there is a crossover is clear, but numbers matter here: For realistic material properties what is the length scale of the crossover?.9) Although the mechanical description is general, the main claim of the paper that a sequence of shapes with the observed characteristics can emerge from a combination of a passive and active component with the given constrains is supported.It would be interesting to know if the application of a hydrodynamic model to the theoretical waveforms can produce swimming paths similar to the experimentally observed ones.Chlamydomonas) asymmetric sliding constrains exist and that it is still debated if those are involved in generating axonemal twist or torsional waves during the beat. Therefore, it could be interesting to also discuss the effect of a weakly constrained beat 'weak para-flagella rod'.10) The conclusion of the paper is of general nature, thus it could be noted that even in cilia with quasi-planar beats  The numbering of MTs is chosen by the authors does not appear to be consistent with published literature. This is important and worth checking carefully, in order to cross-reference any previous structural or EM studies. Usually, authors adopt a certain convention when it comes to numbering of microtubule doublets, as such systems do not exhibit perfect rotational symmetry but retain many heterogeneities – and there could be distinguished associations between certain Mts and other, non-axonemal structures such as mastigonemes.E. gracilis EM studies. Specifically, we adopted the numbering convention of Melkonian et al., (1982) and Bouck et al., (1990). MT 1 is defined as the doublet with the most prominent attachment to the PFR. In this convention, MTs’ numbering increases in the clockwise direction when seen from the distal end of the Ax. This is at odds with the MTs’ labelling typically used in structural studies of cilia (which is the one we adopted in the first submission of our paper). In the typical case, MTs’ numbering increases in the anti-clockwise direction when seen from the distal end of the Ax. Dynein arms extend from MT N and reach MT N+1. In the convention adopted in this revised version of our manuscript, dynein arms extend from MT N and reach MT N-1, as in Figure 14 in Melkonian et al., (1982) and Figure 2 in Bouck et al., (1990).We have changed the numbering of MTs to make it consistent with published literature on T. brucei there are some longitudinal filaments and more like a lattice structure. Does this matter? The ventral flagellum also has a PFR – does this serve any purpose? Also, in euglena the rod extends all the way along the flagellum, but this is not the case in some other species. Do the authors believe that their conclusion extends across phyla, and if not, why not?2) According to the literature, e.g. Hyams, 1982, the PFR is hollow . In contrast in Our model for the PFR as a homogeneous elastic rod does not capture the level of detail that the reviewer is aiming for. We consider it as an “effective' coarse-grained model giving a fair first approximation of the forces of elastic origin that the PFR is subject to and transmits to the Ax. Through a suitable choice of geometric and material parameters, it can be used to model both hollow structures and structures with a dense core. Therefore, we believe that our conclusions are robust even against uncertainty of *some* structural details. See answer to no. 3 for more comments on this issue .The question about the ventral flagellum is interesting but beyond the scope of our work.a) There is earlier work on the shape of spirochetes where a similar issue of two elastically competing structures appears: Dombrowski, et al., (2009).The authors may wish to comment on the similarities and differences between that study and theirs.B. burgdorferi and that of flagellar beating in E. gracilis. Both mechanisms rely on mechanical “competition' between substructures, which generates frustration resulting in nontrivial shapes.We thank the reviewers for pointing out the study by Dombrowski et al., to us. We have added it to our reference list, and commented on the similarities between the mechanism of shape-morphing in 3) In many other types of cilia and flagella which exhibit strongly non-planar beats, there is no association with a PFR-like structure, i.e. the latter is not necessary to generate non-planar bending. What's different here? Would be good to make and discuss some more cross-species comparisons E. gracilis specifically, where a prominent asymmetry in the PFR-Ax bonding complex is present. In our model, the PFR-Ax interaction is needed to generate the typical spinning lasso geometrical signature which defines the peculiar Euglena beating style.Yes, the association with a PFR-like structure is not necessary to generate non-planarity in flagella. We did not imply otherwise, but we have made this point clearer in our resubmission. Our main conclusion applies to Leishmania mexicana and Trypanosoma brucei, such a prominent asymmetry is lacking, see e.g. Portman and Gull, (2010). For these organisms, the angle L. mexicana and T. brucei mutants with body-detached flagellum. Following the suggestion by the reviewer, we have added a reference to Koyfman et al., (2011).In other PFR-bearing flagella, like those of 4) Is it clear that the rod assumes a consistent relationship with the numbered doublets? Could the rod itself be twisted around the axoneme? Authors assumed a fixed relationship and phi_p angle, and that this is neither perpendicular nor parallel to the beat plane… this is important according to Equations 25/26 We believe that the EM study by Melkonian et al., (1982) gives a clear answer to the question raised by the reviewer. The possibility of a systematic numbering convention of MTs strongly implies a consistent relationship between Ax and PFR. The authors define a scheme for the flagellar cross-section “based on the evaluation of 80 different cross-sections of flagella', where MTs are identified based on associations with PFR and mastigonemes. The bonding links complex is conserved through the length of the flagellum (and across flagella), and it seems hard to reconcile this observation with the possibility that the PFR was twisted around the Ax. We cited Melkonian et al., (1982) in the paper when mentioning the 5) In order to test their hypothesis the authors implemented a Chlamydomonas like planar beat pattern for dynein actuation, and determined the mismatch between the rod and axoneme… however, why could the naked axonemal beat not be 3D in the first place? Is there any structural evidence (as exists in Chlamydomonas) which suggests that the euglena beat is planar?In our model the role of the PFR is twofold: it acts as (1) a MTs’ sliding inhibitor, and (2) as a mechanically antagonistic element.Chlamydomonas and sperm cells. However, we are not yet able to model the microscopic details of this response mechanism. We take this feedback-based self-organization process as a given, and we consider a force pattern that would produce planar beat in absence of antagonistic forces on the Ax.The Ax-PFR bonding links play the central  role in MTs’ sliding inhibition. Our working hypothesis is that MTs’ sliding stretches the bonding links. This inhibits MTs’ sliding and triggers a dynein organization  similar to the one present in The agreement of the observed flagellar shapes with the ones we predict shows that our working hypothesis is consistent with the available evidence. Further work will be needed in the future to address the issue of whether the dynein actuation pattern is truly planar and, if so, which feedback mechanisms lead to the emergence of this pattern. We have clarified this point in the revised manuscript (see subsection “Dyneins’ actuation induced by sliding inhibition').6) The authors have also assumed that the PFR, together with the AX_PFR linkages are passive. We suspect this is not the case. The fact that the PFR can be digested easily by trypsin suggests it may be contractile. The question is then could this structure participate in phototaxis – what is known about how the beat pattern changes during phototaxis – could the PFR actively enable a faster change in beat pattern or even beat frequency – via active contraction say, upon perception of photic signals? This could potentially be testable in the model.This is an interesting and widely open question, but we believe that the answer is highly non-trivial. The question deserves further ad-hoc study, including a large set of new experiments and a modelling effort to address the mechanisms of active contraction, light perception, and their interplay. We consider this suggestion as a very interesting direction to pursue in future studies, and we have indicated so in Discussion section.7) Finally, the para-axial rod could also provide or promote a number of other functions, for instance gliding motility. What do the authors think about this possibility? Or conversely, can they suggest experiments that would help show more definitively that the PFR facilities 3D swimming?This is another interesting question, but we are not able to suggest an answer.E. gracilis (possibly because its genome has not been sequenced yet).In other systems, the question of whether the paraflagellar rod is “vital' for a certain form of motility has been address with the study of mutants .Genetic editing techniques are not available to us, and we are unaware of results of this nature in the general literature on E. gracilis concerns swimming motility and metaboly, and we have no experience with gliding motility. We have not been able (at least not yet) to device a single physical experiment proving or disproving that PFR facilitates 3D swimming.Our experience with physical experiments on 8) The authors state  that "Classical estimations on homogeneous elastic rods, see, e.g., Goriely, (2017), show that bending and twist moduli scale with the forth power of the cross section radius, whereas shear and stretching moduli scale with the second power and hence they are dominant for small radii." That there is a crossover is clear, but numbers matter here: For realistic material properties what is the length scale of the crossover?Consider the bending energy E. gracilis we have where .We have corrected the typo.9) Although the mechanical description is general, the main claim of the paper that a sequence of shapes with the observed characteristics can emerge from a combination of a passive and active component with the given constrains is supported.It would be interesting to know if the application of a hydrodynamic model to the theoretical waveforms can produce swimming paths similar to the experimentally observed ones.We have followed this suggestion. We have shown that the sequence of flagellar shapes obtained from our model  can generate swimming paths similar to those observed in experiments. Swimming cell simulation results are reported in Figure 8 in the main text, Appendix 5, and in Video 3. The results are briefly reviewed in the Discussion section, and more details on the implementation and physics behind swimming simulations are given in Appendix 5.10) The conclusion of the paper is of general nature, thus it could be noted that even in cilia with quasi-planar beats (like is the case for Chlamydomonas) asymmetric sliding constrains exist and that it is still debated if those are involved in generating axonemal twist or torsional waves during the beat. Therefore, it could be interesting to also discuss the effect of a weakly constrained beat 'weak para-flagella rod'.How does the spinning-lasso beat breaks down?What signature would waveforms under those conditions have?Chlamydomonas-like bending under different perturbations, see Appendix 3. We relaxed the planar constraint and considered four different weakly non-planar spontaneous configurations. Each configuration presents a different (non-null) torsional profile when the antagonistic mechanical interaction of the PFR is absent We have performed a sensitivity analysis of flagellar shapes with spontaneous