Retrogradation enthalpy (∆Hret) and retrogradation percentage (R) of vitreous and floury endosperm flours ranged from 6.23 to 7.92 J/g and 52.72% to 73.62%, and from 5.46 to 6.45 J/g and 45.70% to 56.58%, respectively. In conclusion, vitreous and floury endosperm flours had significantly different physicochemical properties. Results of this study provide a foundation for better and valid utilization of different endosperm section during grain processing.Three maize cultivars with different endosperm types  were studied to characterize vitreous endosperm flour properties compared with those of floury endosperm flour from the same maize kernels. Vitreous endosperm flour had higher amylose and protein contents, and lower starch content, higher percentage of large starch granule, bigger mean diameter of starch granule, higher iodine capacity, higher trough viscosity and final viscosity and setback, lower swelling power, lower peak viscosity and breakdown, and higher peak time and pasting temperature than the counterpart floury endosperm flour. X‐ray diffraction analysis indicated typical A‐pattern for starches of vitreous and floury endosperm flours. Floury endosperm flour showed the presence of greater crystallinity and higher enthalpy change ( The experiment was a randomized complete block design with three replications. Every plot comprised of ten rows, 0.60 m spacing between rows and 8.0 m long. Maize kernels at the middle position on an ear from the same hybrid plants were collected.2.2Vitreousness was determined according to the method of Zhang et al. . To prep2.3Amylose content was measured using the colorimetric method described by Knutson . Starch 2.42‐KI, starch granule diameter was measured by 15× eyepiece and 40× objective with Olympus microscope. More than 100 starch granules were determined for every field. Every sample was made for three fields. Starch granule mean diameter and size distribution were calculated.After the flour samples were dyed with I2.5max) and blue value of flours were determined as described by Fiedorowicz and Rebilas  as described by Lu and Lu . 3.5 g f2.8The X‐ray diffraction (XRD) patterns of the samples were achieved using an X‐ray diffractometer  as described by Lu and Lu . The dif2.9To), peak temperature (Tp), conclusion temperature (Tc), and enthalpy change (ΔgelH) of flour samples were calculated automatically. After thermal analysis, the samples were kept at 4°C for 7 days for retrogradation analysis. The sample pan containing the flour was reheated at the rate of 10°C/min from 20 to 100°C to measure retrogradation. The retrogradation enthalpy (ΔHret) was evaluated automatically, and retrogradation percentage (%R) was calculated as %R = 100 × ΔHret/ΔHgel.The thermal characteristics of the flours were determined by differential scanning calorimetry  as described by Lu and Lu . 5 mg sa2.10The data were subjected to analysis of variance by LSD test at the 5% probability level using the DPS software.33.1Amylose level was significantly higher in samples from vitreous endosperm than those from corresponding floury endosperm Table , which iAs previously shown in opaque and inbred maize line  than vitreous endosperm flour  of vitreous and floury endosperm flour ranged from 4.67 to 4.73 min and 71.00 to 72.55°C, and from 4.00 to 4.47 min and 69.35 to 70.25°C, respectively  and enthalpy change (∆Hgel) of vitreous endosperm flour from three maize cultivars ranged between 63.70 and 64.32°C, 69.16 and 69.70°C, and 75.03 and 76.61°C, and between 8.57 and 12.58 J/g, respectively, while floury endosperm flour had a range of 64.39–65.64, 69.72–69.87, and 75.64–77.02°C, and 9.65–12.90 J/g, respectively  of vitreous and floury endosperm flours ranged between 6.23 and 7.92 J/g and between 5.46 and 6.45 J/g, respectively. Their retrogradation percentage (R) ranged between 54.06%–73.62% and 44.54%–56.59%, respectively. And the higher ∆Hret and R were recorded in the vitreous endosperm flour, and the lower in floury endosperm flour (Table In this study, the transition temperatures of the endotherm (onset temperature, ur Table .4The findings in the current study revealed that the physicochemical properties of floury endosperm flour samples of three maize cultivars differed from their counterpart vitreous endosperm flour samples. Due to poor agronomic traits and nutritional quality, floury endosperm is usually undesirable. In this study, the higher swelling power, higher pasting peak viscosity, and lower setback and lower pasting temperature and less peak time, higher crystallinity, and lower retrogradation level compared to vitreous endosperm flour make floury endosperm flour a potentially new functional ingredient. Therefore, either floury endosperm or vitreous endosperm flour has great potential to play the special role in the food industry.The authors declare that they do not have any conflict of interest.This study does not involve any human or animal testing.


Human language processing must rely on a certain degree of abstraction, as we can produce and understand sentences that we have never produced or heard before. One way to establish syntactic abstraction is by investigating structural priming. Structural priming has been shown to be effective within a cognitive domain, in the present case, the linguistic domain. But does priming also work across different domains? In line with previous experiments, we investigated cross-domain structural priming from mathematical expressions to linguistic structures with respect to relative clause attachment in French . Testing priming in French is particularly interesting because it will extend earlier results established for English to a language where the baseline for relative clause attachment preferences is different form English: in English, relative clauses (RCs) tend to be attached to the local noun phrase (low attachment) while in French there is a preference for high attachment of relative clauses to the first noun phrase (NP). Moreover, in contrast to earlier studies, we applied an online-technique . Our results confirm cross-domain priming from mathematics to linguistic structures in French. Most interestingly, different from less mathematically adept participants, we found that in mathematically skilled participants, the effect emerged very early on (at the beginning of the relative clause in the speech stream) and is also present later (at the end of the relative clause). In line with previous findings, our experiment suggests that mathematics and language share aspects of syntactic structure at a very high-level of abstraction. Abstract syntactic structures enable us to produce and understand sentences that we have never heard or produced before. Beyond these syntactic representations, human sentence processing requires cognitive resources in varying amounts depending on complexity of the sentences. A question that is still debated is how specific these abstract syntactic structures and the required cognitive capacities are to language processing . To answ(1)Sharing of structure building resources: Are abstract representations and/or procedures in language different from other domains, such as mathematics or music? Or do the domains share representations and/or operations for processing? If there are shared representations and/or procedures, which level of representation do they concern? Do they concern syntactic  representations of linguistic, mathematical, or musical representations or do they concern their meaning, i.e., semantic or conceptual representations?(2)Sharing of processing capacity: Independent of linguistic representations, does the linguistic domain share cognitive resources with other domains like music or mathematics, meaning that domains require the same additional cognitive resources in cases of cognitive load ?Much of the literature in psycholinguistics and cognitive psychology focused on the second research question (shared cognitive capacity) or some combination of the two questions. The experiment presented in this paper focused on the sharing of structure building resources. It showed that linguistic and mathematical expressions shared abstract structural representations or structure building procedures leading to mathematical priming effects on sentence processing. Priming studies within  or even A very strict separation between the linguistic domain and others claiming the absence of any shared resources has been suggested by [1] a.A finite left-invariant measure over a compact group is bi-invariant.b.In finite measure, the series expansion of the roots of a holomorphic map is reflexive.[2] a.In ancient Greece, a citizen who could not pay his debts was made a slave.b.The Greek mythology is the smallest alcohol derived from the VAT.The results showed that left-hemispheric brain regions generally associated with language competence were not activated for meaningful mathematical statements in professional mathematicians. Conversely, regions associated with mathematical capacities were not activated during meaningful non-mathematical statements. shared syntactic resource hypothesis. This hypothesis implies that domains like music and language demand syntactic rules for processing . Even though these rules themselves are probably specific to their domains, they might employ shared basic structure building resources, e.g., common operations to be executed in the same neural areas. An example for this may be operations to reactivate preceding elements in order to integrate new ones. With respect to shared cognitive capacities, In favor of shared structure building resources,  in English. Before presenting our experiment, we describe these findings, and the rationale behind them, in more detail.Some of the clearest evidence established so far can be found in la criada) or “low” to the second noun phrase (la actriz). Interestingly, different languages have different basic attachment preferences: the relative clause in French  will more often attach to the first noun phrase (high attachment), whereas the relative clause in other languages like in English will more frequently attach to the second noun phrase (low attachment).[3]Algúien disparó contra la criada de la actriz [que estava en el balcón].Someone shot the maid of the actress [that was standing on the balcony]. .Attachment preferences for relative clauses can be quite different across and within languages depending on the particular construction, showing that they can be influenced by a variety of factors: the anaphoric status of the relative pronoun , length In this paper, we aimed to find out whether these preferences can be changed via structural priming, particularly via priming from non-linguistic structures. The hierarchical structure ambiguity involved in relative clause attachment, i.e., integrating some structural element locally (low-attachment) or non-locally (high-attachment) is not specific to linguistic processing and can be found in other domains like mathematics, music, and possibly in a variety of other cognitive domains see also . Therefo∗5 versus 90-9 + 1∗5 on the other hand. Apart from giving different results, these two mathematical expressions have hierarchical structures comparable to, respectively, high versus low attachment of a relative clause in a sentence like [3], and can actually be represented in similar ways. For example, in Figure ∗5 [where the final multiplication operator takes scope over a complex expression (9 + 1) on its left] is analogous to high attachment of a relative clause , while in Figure ∗5  corresponds to low attachment of a relative clause .∗5 and 90-9 + 1∗5 are not ambiguous at all. By contrast, sentences with relative clause attachment like Someone shot the maid of the actress that was standing on the balcony remain ambiguous on the surface. The main research question of Figures To answer this question, they set up off-line sentence completion experiments in English. Participants were presented with a questionnaire composed of equations to solve and sentence preambles to complete . Another subsample of participants (psychology students) did not show reliable cross-domain structural priming effects, presumably due to a lack of knowledge of the arithmetic operator-precedence rules, as suggested by a high number of mathematical errors in that group see also .To address this problem, Thus, the experiments from While being fairly conclusive in terms of shared structural representations across different cognitive domains, the previous research leaves many questions unanswered. The one we are interested in is whether this cross-structural priming effect can also be generalized to on-line language comprehension, which is less prone to metalinguistic or strategic effects.visual-world eye-tracking  shows a general high-attachment preference. Indeed, for the notion of cross-domain structural priming to bear substance, it is important to demonstrate that it is independent from language-specific structural biases.For the current experiment, we expected early as well as late effects of priming from mathematical expressions to relative clause attachment, particularly for participants with good mathematical knowledge and less so for mathematically less adept participants. Similar to different types of relative clauses depending on the verb in the main clause, and this peculiarity (which does not exist in English) can have a modulating influence on relative clause attachment preferences . They were recruited via the RISCThe items consisted of 30 sentences containing an ambiguous relative clause, followed by two possible interpretations of the relative clause (see [6]). The interpretations were either about the first noun  or the second noun .[6]Voici le cuisinier de l’ingénieur qui va finir ce sur quoi il travaillait.Here we have the cook of the engineer who will finish what he was working on.a.Le cuisinier va finir ce sur quoi il travaillait.The cook finished what he was working on.b.L’ingénieur va finir ce sur quoi il travaillait.The engineer finished what he was working on.Orders of interpretation-paraphrases (NP1-related first or NP2-related first) were counterbalanced across the 30 items per presentation list. Two lists were created with order of paraphrases swapped across lists. Thirty filler sentences of an independent experiment were added to the lists. The order of presentation was randomized individually for each participant.IbexFarm . All participants gave written informed consent before taking part and the study was approved by the College of Science and Engineering Committee at the University of Glasgow (Application Number: 300150090) as well as by the Comiteì d’Ethique pour les Recherches en Santeì at the University Paris Sorbonne Cité .3 (more information in the procedure section): a mathematical knowledge group and a no mathematical knowledge group, see also Participants were divided into two groups4.For this experiment, we used the Here we have the cook of the engineer who will finish what he was working on”). Relative clauses were constructed to be “semantically neutral,” i.e., they were constructed to be equally plausible modifiers of NP1 or NP2. The sentences were spoken by a female native French speaker using neutral intonation and digitized for later presentation using Audacity and checked in PRAAT /2) and low-attachment  – which were paired with one of the pictures (composed of four objects and two human characters) plus a spoken sentence as target for visual-world trials. See Table /2) and lHere we have the gardeners who will offer a necklace to the sculptor,” Figure 5.Fifty six fillers were also added. These comprised 26 equations  were presented to participants following a Latin square design: Two counterbalanced presentation lists were generated such that (a) half of the experimental picture-sentence combinations (serving as targets) were preceded by high-attachment prime equations and the other half by low-attachment prime equations and (b) item-condition combinations were swapped across the two lists. As our dependent variable, we looked at fixations on the NP1 object vs. the NP2 object, meaning fixations on the NP1 object out of fixations on both objects.The materials per list were pseudo-randomized, ensuring that each experimental pair of prime equation and picture-sentence combination was separated from the others by at least one filler trial, randomly chosen from the pool of filler equations and filler visual-world trials. Because of the latter, there was no regular sequencing of mathematical versus visual-world trials . To ensure that participants paid attention to the pictures and sentences, comprehension questions were included after 16 of the  visual-world trials. Eight concerned the spoken sentence , and eight referred to the picture itself .The incorrect answer option for the mathematical equations always corresponded to the result of linear processing of the equation without taking operator-precedence rules into account . Positioning of correct/incorrect answer options was counterbalanced across items.qui va finir ce sur quoi il travaillait” (“who will finish what he was working on”) in the sentence, we expected participants to look more at the NP1-related object (roast chicken) in Figure Based on our experimental design, upon hearing “∗3 = ? Depending on the answer (7 or 9), they were either in the mathematical knowledge group (7) or the no mathematical knowledge group (9).Before the experiment started, participants had to solve the following equation: 1 + 2The eye-tracking experiment was run with an SR Research Eyelink 2 system in a sound attenuated booth. We recorded eye fixations from the dominant eye based on the Test 6. Each picture-sentence trial was preceded by the presentation of a fixation dot for drift-correction. On mathematical trials, participants saw an equation on the screen with two possible answer options below it  mathematical trials and (ii) picture-sentence trialsIn picture-sentence trials, they had to look at pictures while listening to related spoken sentences. Participants were informed that after some of the picture-sentence trials, a question related to either the picture or the sentence would appear, which they were to answer with either the right-hand button (for “yes”) or the left-hand button (for “no”) on the gamepad. Eye movements were recorded throughout the presentation of each picture, which stayed on screen for 5000 ms. The speech recordings for the sentences started playing right after the onset of each picture presentation. Figure Importantly, participants remained unaware about the pairing of critical prime-target trials until debriefing at the end of the experimental session.7). Random variables were participants and items.We used the R package EyetrackingR  to analyWe chose growth curves due to the fact that eye fixations on the target objects should change depending on what part of the sentence participants are listening to at a certain moment. Thus, time is a very strong predictor that needs to be taken into account. Growth-curve analyses included time as a continuous predictor with the dependent variable being categorical (binary).Growth-curves for the visual world data were analyzed with Bayesian generalized linear mixed models . BayesiaOur hypothesis was that a priming effect would show up especially for the mathematical knowledge group. In other words, looks to the NP1-related object would be more frequent in the high attachment condition for the mathematical knowledge group. The opposite pattern was expected for the NP2-targeted object.Fixed effects were Time, Group, and Prime Condition as well as their interactions. Random effects were participants and items, and we also included random slopes for the effects of Prime Condition and Group for items, as well as their interactions. For participants, we only included random slopes for Prime Condition.As for the coding, the group factor was coded 1 for the mathematical knowledge group and -1 for the no mathematical knowledge group. The Prime Condition factor was coded 1 for high attachment and -1 for low attachment. Normal distributions with μ = 0, SD = 10 on a logit scale were used as weakly informative priors.8. Then, we ran Bayesian models with 4 chains and 6000 iterations each. Model convergence was verified graphically and by inspecting model coefficients in R.To determine the shape of the fixed effect of time, we ran three linear mixed models with simple intercepts to test which polynomial function corresponds best to our data . We therefore analyzed the data per participant group in two separate time-windows: (i) 200 ms before the beginning of the relative clause until 200 ms after the beginning of the relative clause, and (ii) 500 ms before the end of the sentence until the end of the sentence.∗3 = ?), 20 participants were initially attributed to the mathematical knowledge group and 16 participants to the no mathematical group. However, after inspecting the results more closely, we found that the accuracy rate for the condition equations within the experiment was lower than 70% for two participants from the mathematical knowledge group. We decided to put them in the no mathematical knowledge group, leading to two balanced groups with 18 participants each. We present the analysis with this change in the results section9.Concerning the pre-test (1 + 2Did you see an umbrella on the picture? / Was the little girl sick?). Participants from the mathematical knowledge group answered 87% of the comprehension questions correctly, while participants from the no mathematical knowledge group answered 85% of the comprehension questions correctly. This difference was not significant.To be sure that participants were engaged to the task, we included comprehension questions on the pictures .10. Probabilities of looks in this section are looks to the NP1-object out of NP1- and NP2-object fixations. We only show looks to the NP1-related object because looks to the NP2-object would be their mirror image.As explained before, we focus on fixations on the NP1- and the NP2-object in the results section. Fixations to other objects can be found in supplementary materialsmathematical knowledge group , and from the no mathematical knowledge group  are presented. The blue line refers to fixations to the NP1-related object under the low attachment condition while the red line concerns fixations to the NP1-related object under the high attachment condition.Figures  = 8.20, 95% CrI = , P < 0 < 0.006) and (less so) 500 ms before the end of the sentence until the end of the sentence . This means that there was an effect of mathematical attachment over time and that the difference between high attachment and low attachment differs across mathematical knowledge groups over time.As shown, respectively, in Figures mathematical knowledge group and the no mathematical knowledge group in the two time-windows.When sub-setting the data by group, we found different patterns for the  = 6.43, 95% CrI = , P < 0 < 0.02)11 for the mathematical knowledge group. We also found an interaction for the no mathematical knowledge group but with a different pattern over time: the initial difference between high attachment and low attachment disappears over time .At the end of the sentence Figures , 16, themathematical knowledge group of participants compared to the no mathematical knowledge group.Overall, it can be concluded that cross-domain structural priming effects emerged very early on (even before the onset of the relative pronoun itself) and were especially robust and persistent in the on-line sentence comprehension: indeed, the visual-world eye-tracking paradigm allowed us to pin down more precisely than previous studies at which point during processing of an attachment-ambiguous relative clause potential cross-domain syntactic priming effects would emerge. Another potentially important aspect of the present study is that the visual world paradigm is arguably less prone to strategic effects, as it requires no metalinguistic judgment or explicit syntactic attachment decisions by the participants . It may be argued that participants leave interpretations underspecified when no explicit interpretation is required as The present study replicates and extends aspects of earlier off-line results by no mathematical knowledge group of participants in our study: although there was evidence of cross-domain structural priming in this group as well, effects were less pronounced and more distributed over time than in the mathematical knowledge group who were much more likely to solve the prime equations correctly, and who showed cross-domain structural priming effects during the earliest stages of integrating the ambiguous relative clause into the preceding sentence context .Given that structural priming relies on the accurate processing of the structural primes , clear structural priming effects were in fact only expected for mathematically adept participants – in line with incremental-procedural account they proposed that, just as the processing of linguistic materials, processing of mathematical equations structured like A + (B + C)∗D, respectively, A + B + C∗D would proceed in an incremental “left-to-right” fashion. This would trigger a form of mild (monotonic) revision as soon as the final multiplication or division operator is encountered at the end of the equation, because the operator-precedence rules require that this operation is applied before the preceding term  is added; in “high-attachment” equations like A + (B + C)∗D, the final operator would combine with a complex expression on its left (B + C), whereas in “low-attachment” equations like A + B + C∗D it would combine with the most recent number (C) on its left. In analogy to this, a form of mild revision is also likely to take place when encountering a relative pronoun after incrementally processing a partial sentence such as The tourist guide mentioned the bells of the church… (that) because (i) the sentence is obviously not complete at this point and (ii) the relative pronoun needs to be integrated with a preceding noun phrase. In analogy to the mathematical primes, this relative pronoun can either combine with a complex noun phrase on its left  or with the most recent noun phrase on its left . incremental-procedural view: Evidence for structural priming became manifest as soon as the relative pronoun (qui) was encountered in the speech stream, i.e., in a time window from 200 ms before to 200 ms after the onset of the relative pronoun. This early onset of effects is in line with the hypothesis that cross-domain structural priming influences the immediate  syntactic integration of relative pronouns during sentence processing. However, one probably needs to be cautious with this interpretation, as a different treatment of the response proportions in a supplemental analysis  suggested more delayed effect onsets relative to the relative pronoun, while still showing clear support for cross-domain structural priming.The early effect is interesting in terms of when exactly during sentence processing cross-domain structural priming effects would emerge. specialized for linguistic and/or mathematical processing. Indeed, it is conceivable that potentially shared representations may be processed at some separate, domain-general level that is independently accessible by brain networks that are more specialized for mathematics and language. Such a view also concurs with findings from What are the more general implications of cross-domain structural priming between mathematics and language processing? In the introduction, we discussed seemingly conflicting evidence suggesting a strict separation between language and mathematics in the brain e.g., . We woulincremental-procedural account of hierarchical structure priming.To conclude, in line with previous findings e.g., , the preCP and CS conceived the idea and designed the experiments. CP and BH designed the pretest. CP analyzed the data. CP, BH, and CS interpreted the results and revised the content. CP wrote the initial draft.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.


African swine fever (ASF) has one of the highest case-fatality rates among pig diseases. Europe was considered ASF-free for about two decades until 2007, when the virus was introduced into Georgia. Since then, it has been identified throughout Eastern Europe, and reached Belgium in late 2018, increasing the risk of ASF being introduced into neighboring countries—namely Germany, Luxembourg, the Netherlands, and France. French authorities have therefore reinforced surveillance measures to improve the probability of detecting ASF rapidly if it emerges in France. Predictive modeling may help to anticipate the extent of virus spread and evaluate the efficiency of these surveillance measures. A previously published and well-documented model that simulates ASF virus spread was therefore tailored to realistically represent the French situation in terms of the geographic distribution of swine production sites and the commercial trade between them on the one hand, and the implementation of surveillance protocols on the other. The outcomes confirmed the moderate spread of ASF through the swine trade network, a situation that had been previously highlighted for the case of Denmark. However, the diversity of the French pig production landscape has revealed a huge potential for the geographic dispersal of the virus, especially should the index case occur in a low-density area, with a median source-to-case distance reaching 300 km. Free-range herds, which are more likely to have interactions with wild boars, were also identified as potential entrance gate for the virus. Transmissions from conventional herds were quasi-exclusively due to swine movement on the commercial network, representing 99% of transmission events. In contrast, 81% of transmission events occurred in the neighborhood of the index herd when the virus was introduced in free-range herds. The current surveillance measures were found relatively efficient for detecting the virus in large herds, leading to detection rates of 94%. However, infections on smaller production sites—which often have free-range herds—were more difficult to detect and would require screening protocols specifically targeting these smaller herds. African swine fever virus is a DNA virus belonging to the Asfarviridae family . With a To date, sub-Saharan Africa is seriously affected by ASF, the virus being endemic in several countries . DespiteASF is a notifiable disease according to European legislation, and strict containment and eradication policies are implemented in the European Union in the event of its suspicion. However, such policies require substantial human and financial resources, which drive the ability to detect the emergence of the virus in free countries and to control its spread in the meantime (hot period). Predictive modeling may therefore help to anticipate the extent of the virus's spread and to evaluate potential surveillance and control measures tailored to specific incursion scenarios. Halasa et al. recently developed such a model to analyze the epidemiological and economic consequences of ASF introduction on the swine production network in Denmark , 17. TheThe modeling work consisted in tailoring the DTU-DADS-ASF model fully described in the literature by Halasa et al. . Only a S), exposed but not infectious , not clinically affected but infectious , clinically affected , and not playing a role in transmission . Transmission is assumed to be either through direct contact between susceptible and infectious animals, or through environmental contamination due to infected leftovers. The environmental reservoir (E) is proportional to the number of ASF-related dead animals, and balanced by an exponential decay representing the virus's capacity for survival in the environment. Using the transmission rate (β) estimate of Guinat et al. (t (days) is expressed byThe herd is considered as a homogenous, randomly-mixed population, independent of its physiological status. The pigs evolve through five health statuses: susceptible . The duration of the latent, subclinical and clinical stages of newly-infected animals are randomly drawn from probability distributions derived from Halasa et al. . For direct contact modeling, the number of animals involved in the movement is next evaluated depending on the herd size and type of production site. The destination herd is then randomly selected based on (i) the distance between the seeder and receiving herd, (ii) the type of production site, and (iii) the herd size, which has to be large enough to receive the incoming animals. Finally, the probability of transmission is evaluated based on both the prevalence of infectious individuals on the seeder farm and the size of the incoming batch.For medium- and low-risk contact, the number of outgoing animals is again evaluated using a Poisson distribution with respective frequencies of contact derived from the literature , 21, 22.Transmission caused by indirect contact through travel to the slaughterhouse is represented in the same way with the exception of the number of potential contacts, which follows an exponential distribution where the average path length (mean number of herds en route to the slaughterhouse) is used as the distribution mean .Finally, to represent local spread due to shared material or fomites, all herds within a 2-km radius of an infected herd are subjected to a distance-based probability of infection related to the within–herd force of infection (Equation 1).Different introduction scenarios were evaluated depending on the density zone and herd type of the initial case (nucleus/multiplier or randomly-selected herds). For each scenario in this study, 2,000 simulations were run to achieve convergence of the outputs with a stable mean behavior and variability. Equations and epidemiological parameters are detailed in the supplementary material of the original paper describing the modeling framework .In the context of the imminent threat of AFS being introduced into France, the surveillance measures described by Halasa et al. have been reinforced . MortaliAll animal movements from and to these zones are prohibited for 45 (SZ) or 50 days (PZ), and all the herds in these two zones have to be monitored during that period. Clinical surveillance as described above is applied to both zones except for the second point: mortality notification is compulsory and systematically induces clinical inspection leading to further laboratory analyses (virology and serology). All herds in PZ are subject to mandatory serological surveillance visits, for which the sample size depends on the herd size and type . BreedinThe National Swine Identification Database (BDporc) has been registering all swine movements in France since 2010. Salines et al.  performeBased on pig trade network analysis, different scenarios regarding the type of herd in which ASF could be introduced were evaluated. We first considered introduction into breeding herds  having large outdegrees and movements mainly toward production herds . Seeder 2 in the four zones. The highest densities were observed in the western and south-western parts of France . When transmission occurred, the number of infected herds could reach 15, but 95% of these simulations resulted in the infection of eight herds or less. Sixty per cent of transmission occurrences involved small production herds with fewer than 80 pigs, and 37% concerned free-range herds. In contrast with previous results, local transmission was clearly identified as the major transmission route, accounting for 81% of events, whereas animal movements only represented 11% of transmissions . Taking The previous results clearly evidence the failure to detect infections occurring in small herds. We therefore assessed the impact of additional screening measures focusing specifically on these herds. We assumed that these herds were systematically subjected to clinical inspection as soon as they declared more than 15 dead or clinically-infected animals within two successive weeks , or when the whole population was affected during the same period. This latter assumption factored in the inspection of very small herds, which could have been easily omitted when considering only the previous surveillance measures.The results obtained with this additional screening procedure are provided in African swine fever has been spreading throughout Eastern Europe since 2007 and recently emerged in Western Europe . AlthougThe first scenario considered an introduction into herds at the top of the pyramidal structure of the French swine production system: nucleus, multiplier and farrowing herds. Although these kinds of herds are expected to have the highest biosecurity measures, hence the lowest probability of virus introduction, the structural analysis of the pig trade network highlighted these herds as having the highest outdegrees and outgoing contact chains , so theyThe second scenario considered free-range pig herds as index cases, assuming this type of production site to be more susceptible to viral exposure through potential contact with wild boars. Keeping the same model structure and assumptions, the results obtained here were quite different from those obtained with the previous scenario. Indeed, the infection was contained within seeder herds in 87.5% of simulations. When transmission did occur, however, up to 15 herds could be infected, clearly identifying a real risk of transmission even from herds with little network connectivity. Furthermore, local transmission was clearly the main transmission route in this case, corresponding to transmission via material shared between neighboring herds, whereas pig movements played a major role in the previous scenario. Although the tracing of pig movements is well-documented  and may Based on these results, we decided to evaluate the impact of additional screening focusing on small herds with <300 animals. We therefore assumed a systematic clinical inspection of these small herds when either the number of clinically infected or dead animals exceeded the threshold value of 15 pigs over a 14-day period (on average more than one affected or dead pig per day), or the whole population was affected during the same period. With this additional surveillance measure, the detection rate exceeded 95% for all scenarios with the same number of visits. Although the hot periods were similar when the index case involved a free-range herd, the higher detection rate allowed a faster reaction time, thus avoiding further secondary cases (only 4% of simulations in this case were affected by secondary infections). Furthermore, in this specific case, shortening the infection process through enhanced screening of small herds also enabled the number of herd visits to be made during the epidemic to be reduced. These results clearly show the beneficial impact of targeted surveillance on small herds in addition to the emergency surveillance protocol already in place.Although specific surveillance and control measures were integrated to represent the current situation in France, the model's outcomes were in close agreement with the results of Halasa et al. , 27. AfrThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.TH developed the model. MA and NR designed the study and analyzed the results. MA performed data analysis, implemented the study, and wrote the manuscript. All the co-authors revised and approved the final manuscript.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.


Because of the generation of protons by intermediary metabolism as well as the negative membrane potential, protons accumulate within the cytosol. Extracellular signal-regulated kinase (ERK)-mediated regulation of NHE1 is important in several human pathologies including in the myocardium in heart disease, as well as in breast cancer as a trigger for growth and metastasis. NHE1 has a N-terminal, a 500 amino acid membrane domain, and a C-terminal 315 amino acid cytosolic domain. The C-terminal domain regulates the membrane domain and its effects on transport are modified by protein binding and phosphorylation. Here, we discuss the physiological regulation of NHE1 by ERK, with an emphasis on the critical effects on structure and function. ERK binds directly to the cytosolic domain at specific binding domains. ERK also phosphorylates NHE1 directly at multiple sites, which enhance NHE1 activity with subsequent downstream physiological effects. The NHE1 cytosolic regulatory tail possesses both ordered and disordered regions, and the disordered regions are stabilized by ERK-mediated phosphorylation at a phosphorylation motif. Overall, ERK pathway mediated phosphorylation modulates the NHE1 tail, and affects the activity, structure, and function of this membrane protein.The human Na The cytosolic C-terminal tail of NHE1 serves as a regulator of transport of the membrane domain, but more than that, it is a focal point for regulation of the protein that occurs principally by protein binding and phosphorylation (and lipid binding). ERK mediated phosphorylation of the C-terminal occurs at multiple sites and affects the structure and function of the protein. The regulatory tail itself possesses both structured and unstructured intrinsically disordered domains. The intrinsically disordered domains are important in the function of the protein and phosphorylation by ERK affects their structure, inducing helical structure in regions adjacent to the phosphorylation site. We suggest that this conformational change in the tail affects the membrane domain, altering its affinity for protons and increasing activity at more alkaline intracellular pH (It is clear that there are a number of regulatory mechanisms and physiological effects of regulation of NHE1 in several tissues. Perhaps the most significant to mankind is the role the protein plays in heart disease and breast cancer, which affect relatively large percentages of the whole population and of women, respectively. Elevated activity of the NHE1 isoform of the Nalular pH .+/H+ exchanger are of great interest. Clearly, they are involved in the regulation of the NHE1 protein and can alter their structure upon phosphorylation; however, the mechanism by which ERK phosphorylation alters activity of the Na+/H+ exchanger still needs to be further elucidated. It is also not clear how and where other proteins bind to the intrinsically disordered region and how they alter its structure, and how this can affect function. It has been suggested that a gate exists in NHE1 that is closed upon autoinhibitory interactions with the region of the C-terminal tail [The intrinsically disordered regions of the cytosolic domain of the Nanal tail . Howevernal tail . Neverth


Caspase-8 is an apoptotic protease that is activated by a proximity-induced dimerization mechanism within the death-inducing signaling complex (DISC). The death effector domain (DED) of caspase-8 is involved in protein-protein interactions and is essential for the activation. Here, we report two crystal structures of the dimeric DEDs of the F122A mutant of caspase-8, both of which illustrate a novel domain-swapped dimerization, while differ in the relative orientation of the two subunits and the solvent exposure of the conserved hydrophobic patch Phe122/Leu123. We demonstrate that mutations disrupting the dimerization of the DEDs abrogate the formation of cellular death effector filaments (DEFs) and the induced apoptosis by overexpressed DEDs. Furthermore, such dimerization-disrupting mutations also impair the activation of the full-length caspase-8 and the downstream apoptosis cascade. The structures provide new insights into understanding the mechanism underlying the activation of procaspase-8 within the DISC and DEFs. Procaspase-8 exists in the cytosol within the cell as an inactive monomer, which is characterized by an N-terminal tandem death effector domains (DEDs) and a C-terminal catalytic protease domain4. The activation of procaspase-8 is proposed to occur through an induced proximity mechanism7. Upon engagement with the death ligand such as Fas Ligand (FasL), the clustering death receptor Fas recruits the adaptor protein Fas-associated death domain (FADD) to its cytoplasmic tail8. The inactive procaspase-8 is then recruited to FADD via homotypic interactions between the DEDs of procaspase-8 and the adaptor protein FADD, leading to the dimerization and activation of procaspase-89. During this process, dimeric FasL trimers potentially serve as a minimal unit fully capable of activating the Fas pathway10.Caspase-8 is a cysteine protease that initiates the extrinsic apoptotic pathway in response to cell surface death receptor activation12, or chimeric caspase-8 that Fpk3 or FKBP is fused to truncated procaspase-8 without the DEDs13. In these early studies, kosmotrope-induced dimerization of the caspase domain of procaspase-8 leads to its activation in vitro12. Moreover, the cell-permeable ligand-induced dimerization of the FKBP domain also drives the activation of the chimeric procaspase-8 in the intact cell9. However, the function of the DEDs in the activation of procaspase-8 cannot be fully addressed based on these studies using the truncated procaspase-8 that lacks the DEDs. It remains unclear whether the DEDs of procaspase-8 forms dimer, if true, how the DEDs forms the dimeric structure within the DISC.The current induced proximity model is proposed based on early studies using drug-induced dimerization of a truncated procaspase-8 without the DEDs14. Through mutant screening, we recently solved the structure of the monomeric DEDs of caspase-8 for a soluble F122A/I128D mutant (DEDF122A/I128D)15, which closely resembles the structure of viral FLIP MC15917. However, the monomeric DEDF122A/I128D mutant provides limited clues about the homotypic interactions between the DEDs of caspase-8. More recently, the cryo-EM structure of caspase-8 tandem DED filaments was determined18, which provides an elegant structural framework to understand interactions between the DEDs of caspase-8 itself and that between caspase-8 and other proteins such as FADD, cFLIP, and vFLIP. This structure also takes a significant step forward in understanding the mechanism underlying death-inducing signaling complex (DISC) assembly. On the other hand, the filament formation generally occurs under high concentrations of DEDs in vitro18 or intracellularly22, it is unclear how the engagement of FasL triggers the downstream filament formation of caspase-8 under physiological condition. Moreover, further studies are still needed to address how the filament structure accommodate the requirement for dimerization in the activation of caspase-8, and how this structure reconciles with the observation that dimeric FasL trimer is a minimal unit fully capable of activating the Fas pathway10.To address these questions, we tried to determine the structure of the DEDs of caspase-8, but the structural study of the DEDs is markedly hindered by the low solubility and high aggregating propensity for the tandem DEDs of caspase-8In this study, we determined the crystal structures of the DEDs of caspase-8 illustrating a novel domain-swapped dimer, which adopts either open or closed conformations in terms of the solvent-exposure of the conserved hydrophobic patch Phe122/Leu123 that is proposed to bind with the DED of FADD. Together with the biochemical data and the computational modeling, the domain-swapped structures of the dimeric DEDs of caspase-8 provide an alternative model to understand the dimerization-driven activation of caspase-8 in the Fas pathway mediated by a minimal dimeric FasL trimer.18, suggesting that dimeric DEDs rather than filamentous DEDs could be obtained under low expression level. However, further SDS-PAGE analysis showed that the DEDs dimer mixed with a bacterial folding helper protein slyD23, highlighting that the solubilization of the insoluble wild-type DEDs must be facilitated by slyD , which is soluble and less aggregated , while runs as the monomer in the presence of βME, suggesting the disulfide bond formation between the two subunits in the absence of reducing agent. In contrast, F122A mutant runs as mainly a monomer in the absence or presence of βME  and the Q125C (DEDQ125C) mutant. No dimer staining for both WT and Q125C DEDs no matter in the absence or presence of βME, which might be attributed to the low concentration of the proteins and the potential interference of the mixed slyD.To further validate that the domain-swapped dimeric structures are not the artifact by the crystal packing, we generated a structure-based Q125C mutation on the helix α2b to facilitate the formation of an intermolecular disulfide bond between the two subunits of the domain-swapped dimer  upon transient overexpressionion Fig. . Compareion Fig. , highligern Fig. , which ites Fig. . The DEFin vitro, in which Phe122 mediates the type I interaction between different DEDs units in the filament  and wild-type (WT), F122A and I128D mutant procaspase 8 Fig. . Transfe18 and the recruitment of caspase-8 to the DISC31, but it is unclear how Phe122 mediates the interaction between caspase-8 and FADD via their DEDs at the DISC, and how the dimerization of the DEDs of caspase-8 involves in this interaction. The previous study32 has indicated that FADD self-association via the DED is required for stable interaction of FADD with Fas and with caspase-8. The replacement of the DED of FADD by a dimeric leucine-zipper coiled-coil maintains the capability of the death domain (DD) of FADD to bind with the Fas DD, suggesting that the dimerized DEDs of FADD might be the functional unit for the interactions between the DDs of FADD and Fas. In line with this hypothesis, the dimerized DEDs of FADD might serve as the docking site of the domain-swapped dimer of the DEDs of caspase-8 and facilitate the recruitment of caspase-8 to the DISC.Phe122 of caspase-8 has been shown to be important for interaction with FADD DEDTo understand the potential binding mode between FADD and caspase-8, we used molecular modeling to generate a complex model between the DEDs of FADD and caspase-8. Given a small cluster including Phe25, Leu28 and Lys33 critical for the self-assembly of FADD, we first built up the dimeric structures of the DEDs of FADD with this cluster as the interfacial residues. Using Phe122 of the opened domain-swapped dimeric DEDs of caspase-8 as the anchoring points, a potential complex model was generated for the dimeric DEDs of FADD and caspase-8 Fig. . The sol7. Extensive previous studies have provided significant insights into understanding this process, but the underlying mechanisms still need further investigation and continuing refinement34. In this study, we found that the tandem DEDs of caspase-8 form a novel domain-swapped dimer, in which the domain-swapping involves the exchange of the C-terminal helices α4b-α6b between the two subunits. Employing functional studies we demonstrated that dimerization of the DEDs is essential for activation of caspase-8. Furthermore, though the conserved residue Phe122 is not necessary for the dimerization, it is essential for the activation of caspase-8 and the downstream apoptosis cascade.DISC formation and subsequent caspase-8 activation are critical initial events in the extrinsic apoptotic pathway7. The domain-swapped dimerization of the DEDs brings the protease domains into proximity and facilitates the dimerization and activation of caspase-8, which closely resembles that occurs in the activation of the chimeric FKBP-caspase-8 induced by the dimerizing agent FK10129. Moreover, upon engaging with the DED of FADD, the domain-swapped dimer of the DEDs of caspase-8 are also easily integrated into the clustered Fas/FADD complexes in which trimeric Fas assemble into arrays bridged by dimeric FADD  of NLRP14 also forms a homodimer by a rearrangement of the α5/6 stem-helix of the C-termini40. In addition, domain swapping has been well described for the dimerization/oligomerization of both the pro-apoptotic proteins Bax/Bak42 and the pro-survival protein BCL-XL43 in the intrinsic apoptosis pathway, in which the transition from the monomer to the domain-swapped dimer is facilitated by the detergents or membrane lipid. We speculated that the domain-swapped dimerization of the DEDs of caspase-8 might also be facilitated by the membrane lipid when recruited to the membranous DISC, or possibly assisted by yet identified chaperones46.It is not uncommon to observe the domain swapping of functional proteins in the death domain superfamily. For example, the N-terminal caspase-recruitment domain (CARD) of Nod1 forms a domain-swapped homodimer by swapping the H6 helices at the carboxy terminiin vitro at the high concentration of DEDs22. The recent cryo-EM structure of caspase-8 tandem DED filaments has revealed that the DEFs formation likely occurs through repeated self-assembly by type I, II, and III interactions of the DEDs, in which both DED1 and DED2 are critical for the self-assembly18. This structure does not provide a clear understanding for the previous study that only DED2 but not DED1 still forms fine cytoplasmic filaments19, while this discrepancy can be explained by the domain-swapped filament model in which only DED2 is involved in oligomerization 47. This observation can be easily explained by our model in which the domain-swapped dimer of the DEDs of caspase-8 bind with the dimeric DEDs of FADD in the membranous DISC, the fluorescent signal of caspase-8 therefore reflects the array of FADD. In contrast, upon a high level of overexpression of the DEDs of caspase-8, the cytoplasmic DEFs were detected even without FasL stimulation22.DEDs can form death effector filaments (DEFs) intracellularly or In summary, we reported the crystal structures of a domain-swapped dimer of the DEDs of caspase-8. Together with functional analysis, we demonstrated that the domain-swapped dimerization is critical to the activation of caspase-8. Furthermore, we generated a potential complex model for the homotypic interactions between the DEDs of FADD and caspase-8, which would shed new light on the DISC in the extrinsic apoptotic pathway. Meanwhile, the domain-swapped dimerization represents the fourth homotypic interaction type, together with other three known interaction types, for understanding the functions of proteins in the death-fold superfamily.The monoclonal agonist antibody against CD95 (Anti-Fas) was purchased from Millipore . Caspase-8 deficient Jurkat cells were kind gift from Dr. Junying Yuan at Harvard University. HeLa cells were obtained from Cellbank . Jurkat cells were cultured in RPMI1640 and HeLa cells were cultured in DMEM , supplemented with 10% fetal bovine serum . All the other biochemical reagents were from Sigma Aldrich.The DNA encoding the death effector domain (DED) of human caspase-8  were subcloned into a pET-21b vector with a C-terminal His6 tag or subcloned into a pET-28a vector with an N-terminal His-SUMO tag by a standard PCR method. The relevant mutants of Caspase-8 DED (residues1-188) were generated by Fast Mutagenesis System . All the Caspase-8 DED constructs mentioned above were transformed into Rosetta (DE3) pLysS cells (Novagen). All of the recombinant protein expression were induced with 0.1 mM IPTG at 20 °C overnight. His-tagged proteins were purified with Qiagen Ni-NTA agarose according to the manufacturer’s instructions. After the first step purification, His-SUMO tag was removed by ULP-1 enzyme, and the proteins were re-chromatographed on a Ni-NTA column. The His-SUMO-tag-cleaved protein was further purified by gel-filtration chromatography .F122A was collected and concentrated to 6–8 mg/ml in buffer A  for crystallization. Using hanging drop vapor diffusion method, crystals of DEDF122A in two crystal forms grew in a few days in condition A  and condition B , respectively. For cryo-freezing of the crystals, 20% glycerol in mother liquor was used as Cryo-protectant by multi-steps transferring. The crystals were flash frozen in liquid nitrogen for a further X-ray diffraction data collection.The dimeric peak of DED48. The detailed statistics are shown in Extended Data Table The diffraction data for the crystal form A were collected in homesource Rigaku micromax-002+. The diffraction data for the crystal form B were collected at beamline BL17U of Shanghai Synchrotron Radiation facility. Data were integrated, scaled and processed by HKL200050, the monomer of DEDF122A/I128D (PDB accession code 4ZBW15) was used as the initial search model. The final models were manually built in Coot51 and refined by Refmac53 in CCP4. The refinement statistics were listed in Supplementary Table 54.The crystal structure was determined by molecular replacement carried out by Phaser-PHENIX program suiteHeLa cells were cultured on coverslips in 12 well plates for 24 hr before transfected with 1.4 μg/well of vectors using Lipofectamine® 2000 Reagent (Invitrogen) according to the manufacturer’s instructions. 18 hr later, cells were fixed in Formalin solution for 30 min at room temperature and the nuclei was stained with DAPI for 20 min. Cells were washed in TBS buffer  3 times. Images were taken with AxioImager A1 microscope and AxioCam digital camera .5) were transfected with plasmids (4 μg) as indicated in figure legends using TransIn EL Transfection Reagent . For Jurkat cells, 2 μg plasmids were electroporated into 1 × 106 cells with Amaxa SE Cell Line 4D-Nucleofector X Kit . 18 hr post transfection/electroporation, cells were further stimulated with anti-Fas (1 μg/ml) for 20 hr before harvest. The cell sample were then analyzed for apoptosis via Annexin V staining (eBioscience) using FACSCalibur flow cytometer . Apoptosis rates were quantified as the percentage of Annexin V-APC positive versus GFP positive cells.For cell-based apoptosis assay, the wild type (WT) and F122A, I128D mutants of full length Caspase-8 (residues 1–479) and DEDs (residues1–209) were subcloned into pEGFP-N1 vector. HeLa cells (3 × 10Supplementary information


Staphylococcus aureus, the development of novel antimicrobials has been called for, with bacteriophage endolysins having received considerable attention as alternatives to antibiotics. Most staphylococcal phage endolysins have a modular structure consisting of an N-terminal cysteine, histidine-dependent amidohydrolases/peptidase domain (CHAP), a central amidase domain, and a C-terminal cell wall binding domain (CBD). Despite extensive studies using truncated staphylococcal endolysins, the precise function of the amidase domain has not been determined. Here, a functional analysis of each domain of two S. aureus phage endolysins (LysSA12 and LysSA97) revealed that the CHAP domain conferred the main catalytic activity, while the central amidase domain showed no enzymatic activity in degrading the intact S. aureus cell wall. However, the amidase-lacking endolysins had reduced hydrolytic activity compared to the full-length endolysins. Comparison of the binding affinities of fusion proteins consisting of the green fluorescent protein (GFP) with CBD and GFP with the amidase domain and CBD revealed that the major function of the amidase domain was to enhance the binding affinity of CBD, resulting in higher lytic activity of endolysin. These results suggest an auxiliary binding role of the amidase domain of staphylococcal endolysins, which can be useful information for designing effective antimicrobial and diagnostic agents against S. aureus.In response to increasing concern over antibiotic-resistant  Staphylococcus aureus is a Gram-positive facultative anaerobe that frequently colonizes the nose, skin, or gastrointestinal tract . Bi. BiS. auositions A. As in  LysSA97 A. The bi LysSA97 B,C, whic LysSA97 D–G. ThesS. aureus cells, while LSA12AMICBD and LSA97AMICBD did not. These findings raised questions about the function of the central amidase domain of endolysins.Improvement of endolysin’s antibacterial activity by the increased binding ability of CBD has been reported in several previous studies ,30,31. IInterestingly, the removal of the amidase domains from both LysSA12 and LysSA97 reduced their lytic activity, suggesting that the amidase domains are required for full lytic activity. Similar results have been reported for LysK; amidase deletion reduced the activity of LysK as measured in the turbidity reduction assay using equimolar amount of proteins . To specS. aureus peptidoglycan (S. aureus phage endolysins facilitate binding of each CBD to host cell walls, leading to maximal lytic activity of these endolysins. Considering that the amidase domain alone did not show any cell binding capacity, it could be that the amidase domains help with and stabilize the folding of the CBD or act as a spacer between the CHAP domain and CBD, enabling the CBD to interact with their cognate target cells efficiently. Further structural and biophysical studies will be needed to fully understand the molecular mechanism of enhanced binding activity provided by the amidase domain.Several studies have shown that the central amidase domain of endolysins has little or no activity and have proposed potential roles of the amidase domain. Donovan and Foster-Frey pointed out that the apparent lack of activity of the amidase domain may be due to inherent differences in cleavage between lysis from within and lysis from without . Indeed,doglycan  while noS. aureus infections. Furthermore, the newly constructed fusion proteins containing the amidase domain and CBD that have higher binding ability to S. aureus can be used as promising materials for the development of diagnostic agents.In summary, several truncated derivatives of LysSA12 and LysSA97 were generated to identify the function of each domain. The amidase domains of both staphylococcal endolysins enhanced the overall exolytic activity of enzymes by helping CBD bind its target cells more efficiently. These molecular studies will be helpful in the creation of novel chimeric proteins for preventing and treating


A computationally efficient method is described for simulating the dynamics of the left ventricle (LV) in three dimensions. LV motion is represented as a combination of a limited number of deformation modes, chosen to represent observed cardiac motions while conserving volume in the LV wall. The contribution of each mode to wall motion is determined by a corresponding time-dependent deformation variable. The principle of virtual work is applied to these deformation variables, yielding a system of ordinary differential equations for LV dynamics, including effects of muscle fiber orientations, active and passive stresses, and surface tractions. Passive stress is governed by a transversely isotropic elastic model. Active stress acts in the fiber direction and incorporates length–tension and force–velocity properties of cardiac muscle. Preload and afterload are represented by lumped vascular models. The variational equations and their numerical solutions are verified by comparison to analytic solutions of the strong form equations. Deformation modes are constructed using Fourier series with an arbitrary number of terms. Greater numbers of deformation modes increase deformable model resolution but at increased computational cost. Simulations of normal LV motion throughout the cardiac cycle are presented using models with 8, 23, or 46 deformation modes. Aggregate quantities that describe LV function vary little as the number of deformation modes is increased. Spatial distributions of stress and strain change as more deformation modes are included, but overall patterns are conserved. This approach yields three-dimensional simulations of the cardiac cycle on a clinically relevant time-scale.The online version of this article (10.1007/s10237-019-01168-8) contains supplementary material, which is available to authorized users. The mechanical pumping performance of the left ventricle (LV) depends in a complex way on the ventricular geometry, the passive mechanical properties of the myocardium, the arrangement of cardiac muscle fibers, and the fibers’ contractile force generation. Quantitative understanding of the effects of these characteristics on ventricular function requires the use of theoretical models to simulate the dynamics of the LV. Such models typically employ a continuum mechanics approach in which the active and passive components of stress at each point in the myocardium are expressed as time-dependent functions of local myocardial strain. The equations of equilibrium of mechanical stresses are then solved using approximate methods, subject to boundary conditions that include the external forces acting on the tissue.Three-dimensional models of the LV are typically developed through the application of the finite element method (FEM)  througha defines the focal length of the ellipse. These coordinates are illustrated in Fig. Prolate spheroidal coordinates mentclass2pt{minimWe define the myocardial domain in the reference configuration in terms of its endocardial, epicardial, and basal boundaries. The endocardial and epicardial surfaces are given by f in prolate spheroidal coordinates is described in the supplementary material Section S2. We also define a function The formulation of the bicubic spline functions The myocardium may be deformed by an arbitrary displacement from reference coordinates In this section, we show how three-dimensional volume preserving deformations of the LV wall can be derived, given specified displacements of the endocardial surface. It is emphasized that this analysis is independent of the choice of functions used to describe the shape and deformation. The constraint of incompressibility is directly imposed on the deformation modes. While this assumption is not strictly true  determine the number of basis functions in the Fourier series, and therefore the overall deformable freedom for the coordinate under consideration  is introduced with base vectors in the fiber direction The helical arrangement of cardiac muscle fibers is defined according to observations  representation isMyocardial elasticity is represented using a transversely isotropic material law, referred to as the “Guccione law,” , a length–tension relationship The active contractile force of the cardiac muscle is assumed to act only in the fiber direction Nash :31\docum Sollins . We use ted from .The traction at the endocardial surface generated by LV chamber pressure isFor the system to conserve energy, the LV chamber must be closed to define the cavity volume and work done by motion at the base must be incorporated into the virtual work equations. An appropriate closing surface is difficult to define in prolate spheroidal coordinates, and so the surface entclass1pt{minimat. The total PK2 stress can be expressed asUnder the above assumptions, the virtual work equations  may be wWith these definitions, the virtual work system  yields41Under equilibrium conditions, the time derivatives vanish and the pressure E system  to a nonm. The reference myocardial domain The system ,42 is anficients . The volTo validate the proposed approach, we describe two sets of simulations. In the first, we validate the model by comparison to analytic equilibrium solutions to the strong form equations Malvern :44\documWe verify the variational method solutions using two types of analytic solutions. In the supplementary material Section S12, we evaluate the ability of the model to solve for the deformation of an infinitely extended hollow cylinder. Here, we test the ability of the variational method described in Sect. k system  includinTo illustrate the degree of kinematic freedom that is required for describing cardiac dynamics, we simulate the cardiac cycle in two cases. First, we simulate the full cardiac cycle. Second, we examine the fiber stretch and stress distributions under end-systolic and end-diastolic equilibrium conditions. In both sets of simulations, we compare models that use the Fourier-series deformation modes  with 8, We use simplified lumped-parameter models Below is the link to the electronic supplementary material.


In striated muscles, molecular filaments are largely composed of long protein chains with extensive arrays of identically folded domains, referred to as “beads-on-a-string”. It remains a largely unresolved question how these domains have developed a unique molecular profile such that each carries out a distinct function without false-positive readout. This study focuses on the M-band segment of the sarcomeric protein titin, which comprises ten identically folded immunoglobulin domains. Comparative analysis of high-resolution structures of six of these domains ‒ M1, M3, M4, M5, M7, and M10 ‒ reveals considerable structural diversity within three distinct loops and a non-conserved pattern of exposed cysteines. Our data allow to structurally interpreting distinct pathological readouts that result from titinopathy-associated variants. Our findings support general principles that could be used to identify individual structural/functional profiles of hundreds of identically folded protein domains within the sarcomere and other densely crowded cellular environments. Sarcomeric filaments in skeletal and cardiac muscles are one of the most complex and ordered cellular structures, which enable them to perform their extraordinary function of alternating contraction and relaxation. Various proteinaceous filaments that constitute the sarcomeric ultrastructure are formed either by repetitive arrays of identical subunits, as found in actin and myosin filaments, or by very long polypeptide chains that are composed of extensive arrays of domains with an identical fold ,2. Howevin vitro conditions  polyethylene glycol 4000. M4 crystallized in 0.2M Na-citrate (pH 4.0), 1.2M ammonium sulfate and 0.3M magnesium sulfate at a protein concentration of 10mg/mL  and in 0.1 M MES (pH 6.5), 2.0 M RbBr at a protein concentration of 20mg/mL . For M7, the precipitant solution was 0.1M Tris (pH 8.0), 3.2M ammonium sulfate and 1mM TCEP at a protein concentration of 8 mg/mL. M10 was crystallized in 0.1M Bis Tris (pH 6.8), 55% [v/v] MPD, 0.2M magnesium sulfate at a protein concentration of 40 mg/mL.(M4) and Aimless  glycerol, immediately before flash freezing. The final M4 model was obtained from a subsequent X-ray data set to 1.60 Å resolution. The absence of RbBr was confirmed by the lack of a measurable X-ray fluorescence signal at the corresponding elemental absorption edges. Interestingly, a broader X-ray fluorescence scan revealed the presence of zinc in the crystal.The structure of the M4 domain was solved in two different crystal forms. The first one crystallized in a trigonal space group and experimental phases were determined with the single anomalous dispersion (SAD) method, using the anomalous signal of bromide ions from the crystallization buffer. The heavy atom structure was determined with SHELXD  and phas, plus 210 water molecules and seven sulfate ions.The M4 domain structure in the tetragonal crystal form was solved by molecular replacement using Phaser  and the The structure of M7 was solved by molecular replacement with Phaser , using t, with the final model composed of the complete domain fragment of M10, four sulfate ions and 152 water molecules.The structure of M10 was solved by molecular replacement with Phaser , using t Ramachandran statistics are calculated using MolProbity [Detailed X-ray data collection and refinement statistics are summarized in lProbity S2 Table.Suitable structural templates for homology models were initially searched by using the template search engine, provided by SWISS-MODEL protein structure homology-modeling server . For allThe level of sequence conservation of titin M-band domains was estimated by comparing the sequences of human titin (Q8WZ42) with representative sequences from mouse (A2ASS6), horse (F6VG02), Atlantic salmon (A0A1S3PNI2), and zebrafish (A5X6X5) by generating a multiple sequence alignment using Clustal (version 1.2.4) . As a se, Table 1. At this resolution, the estimated coordinate error is generally less than 0.2 Å [. The structure of the M4 domain was determined in two different crystal forms, with one showing a trimeric M4 domain assembly and the other revealing a disulfide-mediated dimeric state (Tables 2). For the M10 domain, there are multiple structures available, in the absence and presence of the obscurin domain 1 (Obs-1) and obscurin-like 1 domain 1 (Obsl1 (Ig1)), both of which bind M10 specifically [The aim of this study was to unravel common and diverse individual structural signature patterns of the ten-fold array of identically folded Ig domains of the C-terminal M-band segment of titin. To achieve this goal, we have solved the crystal structures of five titin M-band domains: M1, M3, M4, M7, and M10. All five structures have been determined to a resolution of less than 2.1 Å, allowing structural interpretation at the atomic level an 0.2 Å . The higifically ,16,20. Sifically ,19 and a. I-set Ig domains contain a split β-strand A/A’, of which the first part A is associated with the β-sheet composed of strands ABDE and the second part A’ is associated with the opposite β-sheet composed of strands A’CC’FG. In addition to the ABDE/A’CC’FG β-sheet sandwich core, all M-band domain structures comprise two characteristic tip regions, which we refer to as “N-tip” and “C-tip” due to their proximity to the N-termini and C-termini of the respective domains . The N-tip is formed by three short loops. Two of them are intra-sheet loops connecting β-strands D and E (slightly below the tip), and F and G, originating from the two opposite sheets ABDE and A’CC’FG, respectively. The third loop connects β-strands B and C, originating from the two different β-sheets. The C-tip is solely formed by three sheet-connecting loops: A’B, C’D, and EF. The EF loop is the longest of all the tip loops and contains a highly conserved 310 helix. The C’D loop, slightly below the very C-tip, is also relatively long and transverses across the surface of the ABDE β-sheet.As expected, all M-band domains investigated belong to the I-set Ig domain category, which is most common in muscle filament proteins ,11,49,50. Most of these domains could be reliably modeled, with the exception of the I-set β-sheet association of the split β-strands A and A’ that is less robust in the respective models. In the absence of further experimental evidence, it is however unlikely that these M-band domains fall into a different Ig domain category other than the I-set observed in all experimental structures.For the remaining four M-band domains , homology models were built using the most closely related Ig domain structures from the titin M-band segment , due to the different experimental structural determination approach by NMR spectroscopy. Based on these data, for the available M-band domain X-ray structures  the numerical structural diversity can be estimated in the order of 0.5–0.8 Å, after subtraction of the estimated coordinate error and conformational diversity of surface residues due to specific crystallization conditions. The calculated pairwise sequence identities of these M-band domains based on respective structure positions are in the range of 18% (M1/M5) to 30% (M4/M5), which is quite low in light of the relatively high level of structural conservation. When the high level of sequence identity of residues forming the hydrophobic core of the respective Ig domains is taken into consideration (details shown below), the sequence diversity of the surface of these M-band domains is even more pronounced. In line with these observations, we found no obvious correlation between the level of overall structural and sequence similarity in different M-band domain pairs . A phylogenetic analysis did not reveal any regular pattern of conservation that could provide clues about the evolvement of M-band domains, unlike other parts of titin [All experimentally determined M-band domain structures superimpose with r.m.s. deviations of 1.0 to 1.8 Å, except for the pairwise comparisons with the M5 domain that show significantly larger r.m.s. deviations of 2.2 to 2.3 Å of titin .To identify structural signature features that may generate the distinct functional properties of individual M-band domains, we used three different approaches. Firstly, we analyzed the structural conservation of the cores of all M-band domains with known structures by directly comparing the underlying β-sheet structures. Next, we identified hot spots in terms of individual structural diversity by plotting pairwise spatial differences based on structural superimposition. Thirdly, we characterized conserved and divergent sequence features using a structure-based sequence alignment.. The majority of these are at structurally identical positions in five of the six available structures. As expected, diminished structural hydrogen bond pattern conservation is found in the peripheral areas of both sheets close to the N-tip and C-tip of the overall structure and in interactions involving the split β-strand A/A’. In conclusion, structural diversity in these M-band domains is restricted to the loop regions at the two tips, close to the N- and C-termini of each domain, whereas the core of the six M-band structures is highly conserved.Direct comparison of the β-sheet hydrogen bond pattern of the six M-band domain structures provides insights into the level of structural conservation. Both opposite β-sheets ABDE and A'CC'FG indeed comprise a highly conserved pattern with more than 20 specific hydrogen bonds per sheet , S4 Fig. The N-terminal split A/A’ β-strand segment shows a generally elevated level of spatial differences. This finding reflects the conformational variability observed for this segment in the respective crystal structures, in which the extent of the split A/A’ β-strand is considerably more variable than those of other secondary structural elements of the M-band domains . The second segment in the pairwise analysis of spatial deviations ranges from the A’B loop to the DE loop. This region features several significant individual M-band domain deviations for M1 (BC loop), M3 , and M7 (DE loop), indicating potential M-band domain signature motifs (discussed in more detail below). Finally, the C-terminal segment, ranging from the DE loop to the C-terminus, presents the most structurally conserved area. Taken together, the data demonstrate that potential signatures of individual M-band domains have specific localizations mostly in loop segments spanning over large parts of the respective Ig domain fold.Plots of structural deviations per residue along the different M-band domain crystal structure sequences, using the structures of M7 and M10 as reference, reveal three distinct segments . For clarity, we have established revised numbering schemes for each M-band domain to facilitate direct comparison of structurally equivalent residue positions ( rather than using residue numbers of the complete titin sequence (UniProt ID Q8WZ42). As there are deviations due to insertions/deletions within the structure-based alignment, we always quote the residue position of the M1 domain when discussing specific residues from other M-band domain structures. The alignment reveals six residue positions, which are identical both between different M-band domains and among different species for the same M-band domain , S5 Fig, whereas another five residue positions are highly conserved. Most of these residues are involved in forming a cluster as part of the hydrophobic core of each of these M-band domains, with a particularly strong contribution to the lower part close to the C-tip .We have generated a structure-based sequence alignment from the superimposition of all M-band domains with known experimental structures Figs  and 3B has the specific role of generating an invariant hydrogen bond to the main chain carbonyl group of D73 (M1) at the conserved 310 helix situated at the C-tip, connecting β-strands E and F from the two opposite β-sheets. In four of the six structures , D73 (M1), also invariant in all M-band sequences, forms an additional salt bridge with a conserved lysine/arginine (K52 in M1), located at the CD loop that connects two β-strands from the two opposite β-sheets . In the M5 domain NMR structure, a defined interaction of this residue pair is missing, probably due to a lack of experimental data supporting the interaction. The conserved lysine/arginine position in the M3 domain is replaced by an asparagine , and hence no interaction with the equivalent aspartate  is found. As N52 in M3 is close to an M3 structural motif that significantly deviates from the other structures , it is plausible that the loss of this specific interaction is of functional relevance and part of a specific M3 signature. These conserved C-tip interactions are embedded in various non-conserved interactions, adding structural diversity to the C-tip. Direct comparison of the C-tip areas of all six M-band structures also reveals substantial differences in the interaction network complexity, which is particularly high in the structures of M4, M7, and M10 .Within this core, Y77 (M1) from β-strand F Figs  and 3B. This asparagine connects the two intra-sheet loops BC and GH via several hydrogen bonds in all M-band domain structures apart from M1, and thus seems to be crucial for establishing a conserved and compact N-tip structure. In the M1 domain, the BC loop has a distinct sequence composition and structure that significantly differs from the other M-band domains Figs  and 2A. In this structure, no residues from this loop interact with N83 (M1), possibly establishing an M1 domain-specific signature.The invariant residue N83 (M1) is located at the N-tip GH loop and so is structurally separate from the other invariant residues in the M-band domains Figs , S5 Fig. Hence, these residues are likely to contribute to specific structural and functional signature patterns across different M-band domains. Two of them are located at an exposed loop, connecting the two split β-strands A and A’ (positions 12 and 14 in M1); the third one is located at β-strand B (position 27 in M1). The side chain of residue 14 in particular is highly exposed in all structures and thus likely to contribute to specific structural and functional signatures in all M-band domains. This proposal is supported by known structures of the M10 domain with Obs-1 or Obsl1 (Ig1), in which the equivalent residue D15 (H14 in M1) is close to a major interaction site in the respective protein/protein interface [.The structure-based alignment has also identified three residues that are distinct in different M-band domains but invariant among different species for each individual M-band domain nterface  . Most cysteines are oriented towards the hydrophobic core and are solvent inaccessible , Table 2 By contrast, the three cysteines of the M3 domain , which are located in different β-strands A’, B, and G, are all at least partially surface exposed . Two other M-band domains each contain a single exposed cysteine: C20 in the M4 domain (E19 in M1) located at the A’B loop at the C-tip, and C42 in the M10 domain (F42 in M1) located at the tip of the CC’ loop near the C-tip.The sequences of the six M-band domain structures investigated have one to three cysteines each. Our structure-based alignment, however, shows no indication of a conserved cysteine pattern . The distance of the sulfhydryl groups of the respective face/face cysteines is 4.0 Å. Interestingly, the homologous models of the domains M6 and M8 are suggestive of two further cases of potential disulfide bridge formation , one providing another connection between the two opposite β-sheets (M6) and the other establishing an additional connection within one of the two β-sheets (M8). Taken together, the non-conserved cysteine pattern in the structures of the M-band domains could generate further functional diversity among different M-band domains with respect to specific redox conditions and potential post-translational modifications such as S-glutathionylation [In the M1 domain, we found the only case where two cysteines–C27 from β-strand B and C79 from β-strand F–in the hydrophobic core are properly positioned for potential disulfide bridge formation, connecting the two opposite β-sheets ABDE and A'CC'FG nylation ,53.Tables , S1 Fig.Many Ig domains from filamentous, multi-domain proteins, including titin, are known for their unique capacity to form either specific non-covalent self-assemblies or heterodimeric assemblies with other Ig domains . Howeverin vitro conditions but the M-band segment has not yet been investigated in detail [, domains M3, M4, and M10 are candidates for such linkages. We validated our structural findings by measuring the free thiol content for domains M3, M4 and M10 by titrating them with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), using M7 (which has two buried cysteines only) as a negative control. As expected, we found one free cysteine equivalent for domains M4 and M10, and almost three free cysteine equivalents for M3 .An alternative way of covalent Ig domain assembly under non-reducing conditions may be via intermolecular disulfide bridge formation involving surface-exposed cysteines. For titin, such potential has been shown for several sections of the entire sequence under n detail –58. Base2+-binding site with tetragonal association of D68 (D68 in M1) and H70 (T70 in M1) from the two interacting M4 domains . This interaction generates an elongated C-tip/C-tip M4-SS-M4’ complex. This result is in line with our cysteine surface accessibility analysis, showing that C20 of the M4 domain is the most exposed (92% surface accessibility) of all M-band domain cysteines analyzed .In one of the two crystal structures of the M4 domain, we found evidence for an intermolecular disulfide bridge across a crystallographic two-fold axis. It is mediated by two exposed cysteines, both C20 from two M4 domains at the C-tip coupled with an intermolecular Zn, S6 Fig, rendering it unlikely that any of these domains play a role in longitudinal titin self-assembly. The data also indicate that the non-covalent assemblies found in the crystal structures of M3, M4, and M10  are most likely artifacts due to specific crystallization conditions. Nevertheless, they underline the potential of β-strands with unused valences for hydrogen-bond intermolecular-mediated β-sheet formation to form assemblies with other protein-binding partners.Based on these structural findings, we also analyzed the potential for self-assembly of the three M-band domains with exposed cysteines M3, M4 and M10, again using M7 as a negative control. Under reducing conditions, all of the M-band domains tested were monomeric , S6 Fig. By contrast, for domain M4, we found an additional higher molecular peak when using size exclusion chromatography, indicating limited dimerization. Interestingly in the presence of Zn2+, which is found in the structure of the disulfide bridge-mediated M4 tandem arrangement , we detected a further increased level of M4 domain oligomerization . In correlation with our size exclusion data, SDS-PAGE analysis under reducing and non-reducing conditions revealed a covalent higher molecular weight species, indicating intermolecular disulfide bridge formation , S6 Fig. For the M10 domain, a residual higher molecular peak could be detected by size exclusion chromatography, but this was not detectable by SDS-PAGE analysis. Unlike our findings for M4 and M10, size exclusion chromatography of the M3 domain under non-reducing conditions revealed a mixture of different higher molecular weight species. We attribute this to possible combinatorial linkages through all three exposed cysteine residues under the given experimental conditions . In the corresponding SDS-PAGE analysis, higher molecular weight species were only faintly detectable . Taken together, our data indicate a significant potential for intermolecular disulfide bridge formation, especially in domains M3 and M4, with one or more highly exposed cysteines in the respective structures. Whether the potential to link to other domains with exposed cysteines takes place under physiological conditions within the M-band region of muscle sarcomeres remains a highly interesting topic for future research.Under non-reducing conditions, as expected, the M7 domain remained entirely monomeric . In addition, the split β-strand AA’, which is associated with both Ig fold β-sheets in all M-band structures determined, shows diversity in terms of the conformations observed . Where structural variability is detected, it is frequently reflected in a distinct sequence pattern when compared with those of other M-band domains .Long filament proteins of muscle sarcomeres comprise extensive arrays of identically folded domains, which we refer to as “beads on a string”. One of the main challenges in understanding the structural and ultra-structural organization of muscle sarcomeres is how these domains have developed an individual structural and functional profile at the single domain level. Indeed, how false-positive functional readouts from identically folded neighboring domains can be avoided has not been thoroughly investigated to date. Here, we focus on the C-terminal M-band segment of titin that harbors an array of ten Ig domains and establish an atomic resolution structural repository of more than half of these domains. Our data allow general principles of both structural domain conservation and diversity to be dissected. We found structural diversity hotspots in three exposed loops, with two at the N-terminal tip (loops BC and DE) and the third located at the C-terminal tip (loop C’D) Figs , 2 and 3 [Many of the structural properties shared by the M-band Ig domains investigated here present general features found in the I-set of the Ig superfamily . Example 2 and 3 –61. OtheFigs S5). Our data, together with those previously published [Figs  and S4. The C-tip region of all M-band domains investigated follows previous findings in terms of sequence and structure for other titin Ig domains characterized. Whereas the 310 helix-containing EF loop is comprised by a DxGxY motif that is highly conserved in other titin Ig domains [Fig 1B) [.Structural data for an increasing number of Ig-like domains from other parts of titin are also presenting a rich repository for comparative analysis. As part of a structural investigation of repetitive arrangements of Ig domains from the titin I-band region, a “N-conserved type” was identified showing correlated occurrence of three conserved features within the N-tip region: a PP motif within β-strand A, a PxP/PPx motif within the BC loop and a NxxG motif within the FG loop . Whereasublished , reempha domains , in cont[Fig 1B) . In our . Our structural data mirror an earlier notion that cysteines at positions conserved in many other titin Ig domains on β-strands B, F, and G, are less frequently found in M-band domains but are complemented by various additional non-conserved cysteine positions  [Another key contributor to structural diversity is in the non-conserved distribution of highly exposed cysteines, which are of specific interest as they are highly reactive, polarizable, and redox-active in response to specific physiological conditions such as pH and redox environment . Hence, sitions  . Finallysitions  ,62,65.. In the M10–Obs/Obsl1 assembly, an extensive intermolecular β-sheet is formed by M10 β-strand B and obscurin/Obsl1 β-strand G. However, as these β-strands are structurally highly conserved Figs  and 3 and therefore do not explain the lack of binding with other M-band Ig domains, we focused instead on amino acid-specific interactions observed in the corresponding interface. We identified three residues located at M10 domain β-strand B–T25, A27, A29 –and one on β-strand A  that are all highly exposed in the separate M10 domain but buried in the M10–Obs-1/Obsl1 (Ig1) complex . Two of these residues  were previously shown to substantially reduce binding affinity to both Obs-1 and Obsl1 (Ig1) [, these four residue positions are replaced almost exclusively by large and hydrophilic amino acids in other M-band domains, suggesting that these cumulative changes are critical for avoiding unwanted binding to Obs-1/Obsl1 (Ig1) by other M-band domains except for M10.Available structural data on functional titin M-band domain assemblies with other filament proteins are limited to structures of the C-terminal M10 domain with the N-terminus of another long filament protein Obs and Obsl1. Therefore, we used those as a test case to identify possible molecular parameters for M10 functional specificity ,16,20  the role of the residue within the structure, with possible structural consequences of the mutation; ii) the involvement in known binding sites to the applicable extent; iii) the conservation of the residue positions within sequences across different species and across different M-band domains . Based on these criteria, there are three TTN variant residue positions all found in the M10 domain, which are likely to have major structural/functional effects. Two of them  belong to the conserved residue cluster that forms a common hydrophobic core in all M-band domains with known structures . The third M10 variant is associated with a cysteine  that points towards the hydrophobic core of M10 . As described above, two of its adjacent neighbors  are directly implicated in Obs-1/Obsl1 (Ig1) binding.In light of these existing complexities, we limit our analysis to those functionally annotated ications ,69,70 Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigS2 Table.Presentations are as in (TIF)Click here for additional data file.S4 Figcf.  are indicated.Color codes: M1, yellow; M3, orange; M4, magenta; M7, blue. The positions of secondary structural elements and sequence numbers ((TIF)Click here for additional data file.S5 FigFig 1B and indicates the level of sequence conservation among sequences of the same M-band domains from different species. Color codes: orange, invariant; yellow, conserved. Other graphical elements have been adopted from Fig 1B.The alignment has been taken from (TIF)Click here for additional data file.S6 FigRow annotations 1 and 2 refer to dimeric peak (1) and monomeric peak (2). Data for M4 has already been shown in (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableHomology models were calculated using SWISS-MODEL protein structure homology-modeling server . Sequenc(DOCX)Click here for additional data file.S3 TableWhere multiple protein chains are found, chain A was used.(DOCX)Click here for additional data file.S4 Table2. Cysteine surface accessibility was calculated using pymol . Where applicable, mean and S.D. were calculated with program Excel  from different domain copies available in the pdb file .Maximum accessible surface area of cysteine side chain: 46.6 Å(DOCX)Click here for additional data file.S5 TableMean and standard deviations of the free thiol titrations were calculated with program Excel  from three independent repeats of an experiment.(DOCX)Click here for additional data file.S1 Coordinate File(PDB)Click here for additional data file.S2 Coordinate File(PDB)Click here for additional data file.S3 Coordinate File(PDB)Click here for additional data file.S4 Coordinate File(PDB)Click here for additional data file.S5 Coordinate File(PDB)Click here for additional data file.S6 Coordinate File(PDB)Click here for additional data file.S7 Coordinate File(PDB)Click here for additional data file.S8 Coordinate File(PDB)Click here for additional data file.S9 Coordinate File(PDB)Click here for additional data file.S10 Coordinate File(PDB)Click here for additional data file.S11 Coordinate File(PDB)Click here for additional data file.S12 Coordinate File(PDB)Click here for additional data file.S1 Validation(PDF)Click here for additional data file.S2 Validation(PDF)Click here for additional data file.S3 Validation(PDF)Click here for additional data file.S4 Validation(PDF)Click here for additional data file.S5 Validation(PDF)Click here for additional data file.S6 Validation(PDF)Click here for additional data file.