When many events contributed to an outcome, people consistently judge some more causal than others, based in part on the prior probabilities of those events. For instance, when a tree bursts into flames, people judge the lightning strike more of a cause than the presence of oxygen in the air—in part because oxygen is so common, and lightning strikes are so rare. These effects, which play a major role in several prominent theories of token causation, have largely been studied through qualitative manipulations of the prior probabilities. Yet, there is good reason to think that people’s causal judgments are on a continuum—and relatively little is known about how these judgments vary quantitatively as the prior probabilities change. In this paper, we measure people’s causal judgment across parametric manipulations of the prior probabilities of antecedent events. Our experiments replicate previous qualitative findings, and also reveal several novel patterns that are not well-described by existing theories. When something happens, people commonly ask: What caused that? Making causal judgments is often deceptively easy. We naturally conclude that the lightning strike caused the forest fire; the last-minute goal caused the sports team’s victory; or the scandal caused the political candidate’s defeat. These kinds of causal judgments are also very important, structuring how we understand and interact with our environments.Yet, there are certain things that people are reluctant to label causes of an event, even though the event clearly depended on them. For instance, it is less natural to conclude that the forest fire was caused by the presence of oxygen, or by the lack of rain, or by the arsonist’s birth, even though the all of these events were necessary for the fire to happen.This phenomenon is widespread: When an outcome occurs, people consistently judge certain seemingly-relevant events more causal than others –4. This graded [When attempting to understand why some causes stand out while others recede, a key fact is that this distinction is not dichotomous, but rather graded . Rather graded , the gragraded , 12–14.quantitative relationship between those parameters and the resulting judgments.  produce qualitative shifts in causal judgment . HerSeveral qualitative manipulations are known to affect which events people consider causal. We do not attempt an exhaustive review; rather, we follow Icard et al.  and highrarer—i.e. if, a priori, it was less likely to occur.We first consider conjunctive causal systems, where multiple antecedent events were counterfactually necessary for an outcome—e.g. there was a fire which required both oxygen and a lit match. In these systems, classic normative analyses of causation would, roughly, label the necessary events as equally causal . Yet, peThis effect has been demonstrated in two ways. First, when asked to select which of several necessary events was the cause of an outcome within some naturally occurring series of events, people tend to pick the rarer event. For instance, people say the match, not the oxygen, caused the fire. They do so in part because oxygen is always present, and a lit match is rarer . This baSecond, researchers have directly manipulated the prior probability of antecedent events, and found that making an event rarer causes people to assign it more causality. For instance, Icard et al. gave people vignettes like the following :Professor Smith works at a large university. At this university, in order to get new computers from the university, faculty like Prof. Smith must send an application to two administrative committees, the IT committee and the department budget committee. Prof. Smith will be able to get her new computers if the IT committee AND the department budget committee approve her application.Icard et al. manipulated the prior probability of one of the causes by telling participants that the budget committee either “almost always” or “almost never” approves applications. Then, participants were told that both of Prof. Smith’s applications were approved, and she received a computer. Participants were asked the extent to which they agreed that the focal event—the budget committee’s approval—caused Prof. Smith to receive the computer. Icard et al. found that people agreed substantially more when the budget committee almost never approves applications. This result, along with many similar results, demonstrates that rarer events are often considered more causal. Following others, we refer to this effect as “abnormal inflation”.less causal if the other necessary events are rare [A less thoroughly explored effect is that, in conjunctive systems, people also judge an antecedent event are rare . For insare rare , 21.less rare—an effect labeled “abnormal deflation” [In disjunctive causal systems, where multiple events are each sufficient to produce an outcome, these effects change. Here, people appear to judge events more causal if they are flation” . For insflation” , 21. TheIn sum, we’ve highlighted four causal selection effects in token causal judgment: abnormal inflation and supersession in conjunctive systems, and abnormal deflation and no supersession in disjunctive systems . There aSecond, these causal selection effects have typically been studied with qualitative manipulations of the rarity of the antecedent events. Relatively little is known about how token causal judgments vary quantitatively with the probability of antecedent events. This is our point of departure.To examine the quantitative nature of token causal judgment, we consider a deterministic causal system in which two binary variables combine, either conjunctively (Experiment 1) or disjunctively (Experiment 2), to produce a binary outcome . In two C1 and C2) to ten values: 0.1, 0.2, …, 1. By independently manipulating Prob(C1 = 1) and Prob(C2 = 1) across the ten values, we produce one hundred different parameterizations of the causal system. We present these hundred parameterizations to people, and ask them to what extent they agree with the statement that C1 caused the outcome.As discussed above, previous research has established that the prior probabilities of the antecedent variables matter for causal judgment. However, prior work has largely restricted itself to qualitative manipulations—e.g. comparing cases where an antecedent event is rarely present to cases where it is almost always present. Here, we map the prior probability of each antecedent variable , but we find a “reverse” form of supersession in disjunctive structures. Moreover, our experiments reveal nonlinear effects which, to our knowledge, are not well captured by existing theories.https://github.com/adammmorris/causality.The data and analysis code for both experiments can be found at In Experiment 1, we presented participants with the following vignette and an associated image :A person, Joe, is playing a casino game where he reaches his hand into two boxes and blindly draws a ball from each box. He wins a dollar if and only if he gets a green ball from the left box and a blue ball from the right box.Joe closes his eyes, reaches in, and chooses a green ball from the first box and a blue ball from the second box. So Joe wins a dollar.Please tell us much you agree or disagree with this statement: Joe’s first choice  caused him to win the dollar.focal) and blue as the “alternate” variable . We divide the continuous space of probability into ten values: Prob = 0.1, Prob = 0.2, …, Prob = 1. We divided the probability of alternate similarly. .The images presented the two boxes, illustrating the percentage of green balls in the left box and blue balls in the right box. By manipulating these images, we manipulated the prior probability that Joe draws a green ball or a blue ball. Since we ask people to rate the causality of drawing the green ball, we will refer to green as the “focal” variable , and showed the participant the corresponding image. We then told the participant that Joe wins the dollar, and asked them to rate the extent to which they agree that Joe drawing the green ball caused him to win the dollar. Participants responded on a scale from 1  to 9 . . There are 100 different probability settings , which gives an average of about 50 individual ratings per probability setting.We employ a within-between subjects design. Each participant was given the vignette five times, each time with a randomly chosen probability setting. (We ensured that a participant did not see the same probability setting twice). We ran shape of the predicted judgment patterns , rather than the absolute value of the predictions . The former is more essential to each model, while the latter is influenced by many extraneous factors , which are difficult to predict.In addition to the qualitative predictions described above , several contemporary theories of token causation make more precise quantitative predictions in our task. In our treatment of these models, we sometimes make additional assumptions to adapt them to the present task. Our intent is not to critically evaluate the models in their exact original setting, but rather to examine the extent to which plausible instantiations of them can capture quantitative variation in the situations we study. Also, we focus primarily on the One influential theory comes from Halpern & Hitchcock . HalpernC was counterfactually necessary for an outcome, we consider an alternate world—a “witness” world—in which the variable is absent  there exists a witness world in which C was necessary for the outcome, and (b) if that witness world—wC—was more normal than the actual world. Moreover, if two variables C1, C2 meet these criteria, then C1 will be considered more of a cause if Halpern & Hitchcock argue that this observation can explain the dependence of causal judgment on the prior probabilities of the variables. To determine whether a variable ils, see , 23). Cawfocal–the world in which green is absent, which is the sole witness world for green –is less likely than the actual world.  > .5, then, all else being equal, wfocal is less likely than the actual world). This result is depicted in the bottom part of wfocal is more likely than walternate. This is depicted in the top part of Halpern & Hitchcock’s model is not committed to a particular normality ordering over possible witness worlds . In ourfocal = 1 was a token cause of Joe’s win is:xmid < xhigh ≤ 1.Formally, then, the degree to which xhigh and xmid. Here, for simplicity, we assume that xhigh = 1 and Halpern & Hitchcock’s model is ordinal , and so focal = 1 was the cause of Joe’s win, they sample a world in which focal = 0  in proportion to Prob, and a world in which focal = 1  in proportion to Prob. Then, crucially, people evaluate a different counterfactual depending on what they sampled. If they sampled a world in which focal = 0, they evaluate whether focal was necessary ; if they sampled a world in which focal = 1, they evaluate whether focal is, in general, sufficient .Another theory we consider comes from Icard, Kominsky, & Knobe . Icard efocal = 0 is monotonically related to Prob, the two are not necessarily equal; there may be other factors that influence which worlds come to mind. This feature makes it difficult to derive precise quantitative predictions in our experiment. Nonetheless, if we make the simplifying assumption that the probability of sampling counterfactual worlds comes directly from the prior probability of green, then we can derive a predicted response profile:According to Icard et al., while the probability of sampling a counterfactual world with focal was certainly necessary , and, allowing other variables to vary, would be sufficient if alternative were present. Hence, this equation reduces to:In the structure of Experiment 1, The predictions of this simplified version of Icard et al.’s model are shown in Finally, we consider three classic “causal strength” measures, some of which were designed primarily to capture other flavors of causal judgments (e.g. type causation). Nonetheless, these measures make plausible quantitative predictions in the causal systems we test, and so we include them in our analysis.TCSP = Prob(E ∣ C) − Prob(E) [TCSP = Prob ⋅ Prob  . For ins .TCDelta−P = Prob(E ∣ C) − Prob(E ∣ ¬C) [TCDelta−P = Prob  , 27. On ternate) .TCPPC = Prob (Prob(outcome ∣ cause). In both of our experiments, this alternate formula predicts a constant rating of 1 across all conditions. Hence, we do not include it in our analysis).The third measure builds on the second, but normalizes the rating by the probability that the event is absent when the cause is absent. This model, called Power-PC, takes the form: b(¬E∣¬C) . It assiternate) .  as more causal when drawing green was less likely. They also exhibited supersession; on average, they rated drawing green as less causal when drawing blue  was less likely.b = −0.02, SE = 0.002, t = −11.8, p < .001), and when the probability of drawing blue was higher . ).To demonstrate both of these effects statistically, we estimated a linear mixed effects model, regressing the causal ratings of green on the prior probabilities of drawing green and blue, with random intercepts and slopes for each subject. On average, people indeed rated green as more causal when its probability was lower , and remained significant when restricting the analysis to the first trial (p′s < 6E − 9), suggesting that the observed influence of Prob and Prob on causal judgment is not due to order effects. . Future research might examine whether there are consistent effects of repeated measures on causal judgment in these scenarios).To rule out that these basic patterns were driven by order effects in our repeated-measures design, we analyzed how they changed from the beginning to the end of the experiment. The results did not differ much between the first and second half of trials see , remaineProb = 0.9 to Prob = 1 , excluding trials where Prob = 1. We then used this regression model to predict people’s causal ratings for Prob = 1, and test whether their actual ratings when the focal variable is certain fall outside the 95% confidence interval of the regressions’s predictions. Indeed, people’s average causal rating when Prob = 1 is lower than what would be expected based on the ratings in the other conditions .More interesting, however, are the nonlinear patterns. We highlight four such patterns, all revolving around what happens when drawing one of the balls is certain. First, people exhibit a large jump in abnormal inflation from cal) = 1 . In otheProb = 0.9 to Prob = 1  = 1 . In otheProb = 1—except for a large decrease when focal also becomes certain , including only trials in which Prob = 1 and excluding trials in which Prob = 1. Here, the probability of drawing green had no effect on people’s judgment . Moreover, there was a significant interaction: People showed more abnormal inflation when Prob < 1, compared to when Prob = 1 .Third, people do not exhibit abnormal inflation when Prob = 1 , including only trials in which Prob = 1 and excluding trials in which Prob = 1. When the focal variable was certain, the probability of the alternate variable had no effect on people’s judgment . Moreover, there was a significant interaction: People showed more supersession when Prob < 1, compared to when Prob = 1 .Fourth, there is an analogous effect for supersession; people do not exhibit supersession when Prob and Prob  and Prob. For example, when Prob = .7 and Prob = .4, the Icard model predicts a causal rating of 0.58, and people’s average causal rating was .47. Since there were 10 possible settings of Prob and 10 possible settings of Prob, this gives us 100 pairs of data points  for each model. We then estimated a Pearson correlation coefficient between the predicted and empirical ratings across these 100 conditions, giving us an estimate of how well each model predicted variation in people’s average ratings across conditions. Icard et al.’s model and SP showed the highest correlation with people’s ratings (r′s = .73), and did not differ significantly from each other (p = .9). Icard et al.’s model was significantly more correlated with people’s ratings than the Halpern & Hitchcock model or the Delta-P/Power-PC model . SP was significantly more correlated with people’s ratings than the Halpern & Hitchcock model (p < .05), and marginally more correlated than the Delta-P/Power-PC model (p = .08).To compare the shape of people’s response patterns to those predicted by the models, we computed the correlation between the empirical ratings and each model’s predictions, across settings of ternate) . ConcretThe Icard model and SP both scored well by predicting the linear trends of abnormal inflation and supersession. However, neither correctly characterized the overall nonlinear patterns in people’s ratings. In particular, none captured the effects of certainty described above.We characterized people’s quantitative causal ratings across the free parameters of a conjunctive causal system. As expected, people exhibit both abnormal inflation, where their causal rating of the focal variable (green) increased as it became rarer, and supersession, where their causal rating of the focal variable decreased as the alternate (blue) became rarer.Moreover, people showed clear nonlinear patterns in judgment when the candidate causes were certain to occur. Specifically, they showed an unexpectedly strong drop in their causal rating of the focal variable when it was certain to occur, and an unexpectedly strong increase when the alternate was certain to occur. Moreover, the effect of abnormal inflation disappeared when the alternate was certain to occur, and the effect of supersession disappeared when the focal variable was certain to occur. These patterns are not well-described by extant models.or a blue ball was drawn from the right box ) and blue ) from 0.1 to 1, and asked people the extent to which they agreed that Joe’s drawing the green ball caused him to win the dollar. .On each trial, participants were told that Joe drew both a green and blue ball, and won the dollar. As before, we parametrically manipulated the prior probabilities of drawing green . The experiment again took about 3 minutes to complete, and participants were paid $0.35 each.All participants were recruited through Amazon Mechanical Turk, and gave informed consent. We ran deflation , and (b) that participants would not show supersession  that participants would show abnormal ent; see , 21). Fowfocal—in which neither a green nor a blue ball was drawn, because it is only when alternate = 0 that focal could have been necessary. . Hence,Prob and Prob are less than 0.5. In this case, wfocal  is surely more normal than the actual world , and the model predicts that focal will be judged causal. Moreover, as long as Prob < .5 and Prob < .5, the extent to which focal is judged a cause is not predicted to vary with either probability; the only other candidate cause, alternate, has exactly the same witness world, and so the relative causality of focal will not vary as the normality of that witness world changes. Hence, in this case, the model assigns focal a constant causal rating, which we label xhigh.To determine the precise predictions, there are three cases to consider. First, consider the case where both Prob and Prob are both greater than 0.5, then wfocal is less normal than the actual world, and the model predicts that focal will be judged noncausal. Hence, in this case, the model assigns focal a causal rating of zero.Similarly, if Prob < .5 and Prob > .5, or when Prob > .5 and Prob < .5. Here, there are at least two ways of instantiating Halpern & Hitchcock’s model. From one perspective, the actual world and wfocal are, in this case, incomparable; one variable takes on its more typical value, but another takes on its less typical value, and the model offers no machinery for integrating those two facts. This is the view adopted in the original paper [focal will be judged noncausal.The third and hardest case is when one of the two variables is likely, and the other is rare—that is, when al paper . When thtotal probability of its variables. On this view, focal will only be considered causal if the total probability of the actual world — Prob ⋅ Prob—is less than the total probability of the witness world — Prob ⋅ Prob. This condition reduces to Prob + Prob < 1.But it is plausible that, in our experiment, people would rank the normality of a world according to the This leaves us with two potential response profiles, HH1 and HH2: xhigh = 1.Below, we consider both variations of the model. Their predictions are depicted in Recall that, according to the model of : TCIcarfocal is not necessary in the actual world, and would always be sufficient. Hence, this equation reduces to:In the structure of Experiment 2, The Icard model’s predictions are shown in In the disjunctive structure of Experiment 2, the three causal strength measures considered above reduce to : TCSP. However, contrary to previous work [focal as less causal when alternate was more common . , and remained significant when restricting analysis to the first trial (p′s < .02)).People’s average ratings are shown in ous work , 21, theials see , remaineinflation in conjunctive structures, here there is no large jump from Prob = 0.9 to Prob = 1  = 1 were significantly lower than expected based on a linear model of the other conditions . Noteably, the reverse supersession effect, though small, was still significant after excluding the condition when drawing blue was certain .We dissect these effects further in cal) = 1 . The revcal) = 1 . We testProb or Prob = 1 . In Experiment 2, we find an analogous pattern: People stop exhibiting abnormal deflation when drawing blue is certain , and stoProb = 1, there was no effect of Prob on people’s ratings ; moreover, the effect of Prob was significantly stronger when Prob < 1 compared to when Prob = 1 . Similarly, when Prob = 1, there was no effect of Prob on people’s ratings ; moreover, the effect of Prob was stronger when Prob < 1 compared to when Prob = 1, although in this case the interaction was marginal .These visual observations were confirmed statistically. When r = .64, p < .00001); It was significantly more correlated with the empirical ratings than the next-best model (p = .0005). As in Experiment 1, however, it did not capture all the major patterns in people’s responses—most notably, it did not capture the reverse supersession effect in The correlations between the model predictions and people’s ratings across conditions are shown in We characterized people’s quantitative causal ratings across the free parameters of a disjunctive causal system. As predicted by the Icard model , people Moreover, people’s nonlinear patterns mirrored those in Experiment 1: They stopped exhibiting abnormal deflation when the alternate variable was certain to occur, and stopped exhibiting reverse supersession when the focal variable was certain to occur. These nonlinearities are not well-described by extant models.We presented people with two basic causal systems, and elicited people’s causal judgments across parametric manipulations of two key parameters in those systems: the prior probability of the focal event , and the the prior probability of an alternate event (that was either necessary or sufficient for the outcome). These experiments replicated three previously established qualitative effects—abnormal inflation and supersession in the conjunctive structure, abnormal deflation in the disjunctive structure—and revealed a novel effect of reverse supersession in the disjunctive structure. Moreover, people exhibited a constellation of nonlinear patterns that were strikingly consistent across the two experiments. We discuss these results in turn . Given the logic of supersession, this reversal is unsurprising. In disjunctive structures, events become causally preferred when they are more common . It makes sense, then, that if the alternate event were to supersede the focal event, it would do so when it was common, not rare.is surprising is that, in contrast to previous work, the alternate event supersedes the focal event at all in the disjunctive structure. This result should be interpreted with caution. The effect is mostly driven by the condition where the alternate event is certain to occur . Nonetheless, it is useful to consider how this result might be reconciled with the null findings in previous papers [What s papers , 21. We Across most of the parameter space, the effects of prior probabilities on causal judgment appear largely linear and additive. That is, the prior probabilities of the focal and alternate events each have a mostly-linear influence on causal judgment, and do not interact much. The main exception is when the probability of either event approaches 1. When one of the events is certain to occur, people exhibit a constellation of departures from the linear, additive baseline.Prob = 0.9 to Prob = 1 is much larger than the drops between other conditions  is considered less and less causal as it becomes more common, the drop in causal rating from ions See .somewhat causal , and why people judged the certain events to be highly causal in the disjunctive structure.Discontinuities in causal judgment when an event is certain have been discussed before. Perhaps most prominently, Cheng & Novick  argued tAnother possibility is that these nonlinearities result from a domain-general feature of people’s probabilistic reasoning: People, in general, treat the jump from almost-certain to certain as more important than other equivalently sized jumps in probability , 36. Thiother event is certain to occur. Concretely, the prior probability of the focal event stops influencing people’s causal judgment when the alternate event is certain to occur, and the probability of the alternate event stops influencing people’s judgment when the focal event is certain to occur. These results, which are consistent across both experiments should be further examined in future research.Both of these perspectives, however, have trouble accounting for the second set of departures from the linear, additive baseline: People stop exhibiting all the effects described here  when the not to occur, or when the probability gets close to zero. This is a ripe question for future research.The nonlinearities observed in our experiments are primarily about what happens when one of the events becomes certain to occur. It is also possible that there are similar nonlinearities when one of the events becomes certain Out of the models analyzed here, the response profile predicted by Icard et al. was consistently the most similar to people’s ratings . This isPeople’s causal judgments are influenced by the prior probabilities of the candidate causes. Here, we find that people show systematic patterns of judgment across parametric manipulations of those probabilities, in a way that is not fully characterized by existing theories. We hope this spurs further research into the quantitative nature of token causal judgment.S1 FigProb and Prob is not primarily due to order effects. , which got slightly stronger in the second half .)(A-B) The effects in Experiment 1; (C-D) the effects in Experiment 2. People show roughly similar patterns across the two halves, suggesting that the influence of (TIFF)Click here for additional data file.


Schistosoma mansoni, the histone methyltransferases (HMTs) and the histone demethylases (HDMs). Amongst these, a S. mansoni Lysine Specific Demethylase 1  homolog was selected for further analyses. Homology modelling of SmLSD1 and in silico docking of greater than four thousand putative inhibitors identified seven (L1 – L7) showing more favourable binding to the target pocket of SmLSD1 vs Homo sapiens HsLSD1; six of these seven (L1 – L6) plus three structural analogues of L7 (L8 – L10) were subsequently screened against schistosomula using the Roboworm anthelmintic discovery platform. The most active compounds (L10 - pirarubicin > L8 – danunorubicin hydrochloride) were subsequently tested against juvenile (3 wk old) and mature (7 wk old) schistosome stages and found to impede motility, suppress egg production and affect tegumental surfaces. When compared to a surrogate human cell line (HepG2), a moderate window of selectivity was observed for the most active compound L10 . Finally, RNA interference of SmLSD1 recapitulated the egg-laying defect of schistosomes co-cultivated in the presence of L10 and L8. These preliminary results suggest that SmLSD1 represents an attractive new target for schistosomiasis; identification of more potent and selective SmLSD1 compounds, however, is essential. Nevertheless, the approaches described herein highlight an interdisciplinary strategy for selecting and screening novel/repositioned anti-schistosomals, which can be applied to any druggable (epigenetic) target encoded by the parasite's genome.Schistosomiasis endangers the lives of greater than 200 million people every year and is predominantly controlled by a single class chemotherapy, praziquantel (PZQ). Development of PZQ replacement (to combat the threat of PZQ insensitivity/resistance arising) or combinatorial (to facilitate the killing of PZQ-insensitive juvenile schistosomes) chemotherapies would help sustain this control strategy into the future. Here, we re-categorise two families of druggable epigenetic targets in   •Schistosoma mansoni contains 27 histone methyltransferases (HMTs) and 14 histone demethylases (HDMs).•S. mansoni lysine specific demethylase 1 (SmLSD1) is a druggable target.•Schistosomes treated with the putative SmLSD1 inhibitor pirarubicin or siRNAs targeting SmLSD1 are less fecund. Currently, the drug of choice used in schistosomiasis control throughout endemic or emerging areas is praziquantel, a pyrazino-isoquinolone compound developed over 40 years ago as part of Merck's effort in developing a new tranquilizer class (reviewed in ). While Over the past decade, we, and others, have been involved in developing and applying high-throughput methods for selecting targets or compounds integral to early stage schistosome drug discovery projects . Our speSchistosoma mansoni, the histone methyltransferases (HMTs) and histone demethylases (HDMs). Amongst these epigenetic enzymes, a S. mansoni lysine specific demethylase 1  homolog was selected for an early stage drug discovery investigation based on the importance and role of Homo sapiens lysine specific demethylase 1 (HsLSD1) in essential biological processes  adhered to the United Kingdom Home Office Animals (Scientific Procedures) Act of 1986 as well as the European Union Animals Directive 2010/63/EU and were approved by Aberystwyth University's (AU) Animal Welfare and Ethical Review Body (AWERB).2.2Schistosoma mansoni was used throughout the study and passaged between Mus musculus  and Biomphalaria glabrata  for 1 h and used to percutaneously infect M. musculus (180 cercariae/mouse for the generation of 7 wk old adult worms or 4000 cercariae/mouse for the generation of 3 wk old juvenile worms)  of d hybrid ) hosts. e worms) . Cercarie worms) . Adult  hosted in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and Wormbase-Parasite (S. mansoni genome (v. 7.0). S. mansoni sequences were recognized as HMTs or HDMs only if they demonstrated high sequence similarity  to human HMTs/HDMs and were observed  to contain conservation over the catalytic domain as defined by InterPro , 1 PR domain containing methyltransferase (PRDM), 1 DOT1 Like Histone Lysine Methyltransferase (DOT1L) and 5 Protein Arginine Methyl Transferases (PRMTs). The HDMs were grouped into 3 Lysine Specific Demethylases (LSDs) and 11 Jumonji domain-containing proteins (JMJDs). The most closely related Schistosoma haematobium and Schistosoma japonicum homologous sequences for these 27 SmHMTs and 14 SmHDMs were also identified by BLASTp interrogation of WormBase-Parasite. For the BLASTp searches, full-length protein sequences of each SmHMT and SmHDM were used as queries against the S. haematobium and S. japonicum predicted protein datasets. S. haematobium and S. japonicum sequences were recognized as the most closely related HMT and HDM homologs respectively (Sh/SjHMTs and Sh/SjHDMs) only if they demonstrated high sequence similarity . Only the top S. haematobium or S. japonicum hit (as defined by score) was included (The combination of these approaches led to the identification of 27 HMTs (three novel members) and 14 HDMs (three novel members) in included .2.4Alignments of the catalytic domain amino acid sequences within the identified SmHMTs and SmHDMs were performed with MUSCLE (Multiple Sequence Comparison by Log Expectation) using the default parameters . The mul2.5S. mansoni HMT and HDM domains having InterPro, SMART and Pfam identifiers were extracted from Wormbase-Parasite. These catalytic domains were defined as follows: SET, Su(var)3–9, Enhancer-of-zeste and Trithorax domain for the PKMTs; DOT1, Disruptor of the telomeric silencing 1 for DOT1L; PRMT core, Protein Arginine (R) Methyltransferase core domain for PRMTs; AOL, Amine Oxidase-like domain for LSDs; JMJC, Jumonji C domain and JMJN, Jumonji N domain for the JMJDs. The start and end position of these domains were confirmed, or modified if needed, according to the multiple sequence alignments.The amino acid positions of The architecture of these proteins was then populated with other N-terminal and/or C-terminal domains known from the literature to be associated with the aforementioned catalytic domains. Graphical representation of these domains within the schistosome HMTs and HDMs was prepared using Illustrator for Biological Sequences .2.6S. mansoni long-oligonucleotide DNA microarray studies of Smhmts and 14 Smhdms across 14 different lifecycle stages. Here, Log2 normalised gene expression data were subjected to hierarchical agglomerative clustering (Euclidean distance and complete linkage) using the R statistical programming language and the Bioconductor package gplots  was prepared with the Molecular Operating Environment (MOE) 2015.10  (http://mordred.bioc.cam.ac.uk/∼rapper/rampage.php).An induced fit option was selected to take into account the presence of the substrate (histone H3) and cofactor . Ten different intermediate models were built and minimised using Amber94 before refining the final model, which generates a Cartesian average of the 10 generated intermediates. The final model was confirmed by I-TASSER (inement) , Robettainement)  and SWISinement)  with the2.8www.specs.net, natural compounds and fragment-based library) in sdf format. These compounds  were then processed by Maestro's LigPrep tool to include all the partial charges, the appropriate protonation state at the defined pH, the proper tautomeric states, the bond lengths and angles. Secondly, the crystal structure of HsLSD1 was downloaded from PDB (PDB code 2V1D) and the three-dimensional structure of SmLSD1 was prepared by homology modelling as described above. The two structures were processed with the Schrödinger Protein Preparation Wizard tool using the OPLS_2005 force field to assign partial charges, bond orders, cap residues, add hydrogen atoms, fill in missing loops as well as side chains and to minimize the protein structure relieving any steric clashes. Thirdly, a 12 Å docking grid (inner-box 10 Å and outer-box 22 Å) was computationally built using, as a centroid, the structure of the histone H3 that was used for the co-crystallisation of HsLSD1 as well as for the induced-fit homology modelling of SmLSD1.Compounds capable of binding to SmLSD1 were identified using Glide docking software within Maestro v10.1 . To init2V1D) docking. The compounds with a more favourable docking score (XP score) for SmLSD1 compared to HsLSD1 were considered hits and the top 100 were selected for further processing.Docking simulations for virtual screening of Specs compounds with SmLSD1 were next performed in Glide using the standard precision function  and the results were refined using the more accurate extra precision (XP) function. In all cases, default parameters were selected and ten output poses (conformations) per input ligand were included in the solution. From the 2500 solutions (conformations) ranked according to the Glide XP scoring function, 500 distinct compounds were identified and retained for HsLSD1  of the compounds. From this iterative approach, seven putative SmLSD1 inhibitors (L1-L7) and three structural L7 analogues (L8 - L10) were identified (SmLSD1 docking results (Glide XP) of the 100 selected compounds were saved in a single entified .2.9S. mansoni screens included praziquantel  and/or auranofin , which were also diluted in DMSO to a stock concentration of 10 mM and working concentration of 1.6 mM.Six of the seven putative SmLSD1 inhibitors (L1-L6) were obtained from Specs . As L7 w2.10Mechanically-transformed schistosomula were screened on the Roboworm platform as previously described . Primary°C in an atmosphere containing 5% CO2 for 72 h at which time worm motility was scored between 0 and 4: 0 = dead, 1 = movement of the suckers only and slight contraction of the body, 2 = movement at the anterior and posterior regions only, 3 = full body movement but sluggish and 4 = normal movement. DMSO  and praziquantel  were included in these juvenile worm assays.Juvenile worm screens (15–20 worms/well) were performed in a 96 well plate format (flat bottom) containing a final volume of 200  μL of media  supplemented with 10% v/v Hepes , 10% v/v Foetal Bovine Serum , 0.7% v/v 200 mM L-Glutamine  and 1X v/v penicillin-streptomycin ). To each well, L8 and L10 were added at the following concentrations: 20 μM, 15 μM, 10 μM, 5 μM and 2.5 μM and 1.25 μM (in 1.25% DMSO). Parasites were cultured at 37 2. As controls, schistosome pairs co-cultivated in DMSO (1.25%) and praziquantel (10 μM in 1.25% DMSO) were included in these assays. After 72 h, motility was scored according to defined WHO-TDR metrics (Adult worm screens were initiated as described previously . Briefly metrics  and eggs2.11Adult schistosomes were cultivated (as described above) in sub lethal concentrations of L10 (12.5 μM) for 72 h. Afterwards, schistosomes were prepared for SEM as previously described .2.124 cells/well were seeded in black walled 96-well microtiter plates  and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. To each well, L8 and L10 was subsequently added to obtain final concentrations (in 1.25% DMSO) of 200 μM, 100 μM, 75 μM, 50 μM, 20 μM, 10 μM and 1 μM; negative  and positive (1% v/v Triton X-100) control wells were also included. Following a further incubation for 24 h, the MTT assay was performed as previously described  siRNA duplexes were purchased from Sigma ; numbers of eggs produced at this time point were also quantified. Separate worms were maintained in continuous culture for up to 168 h, after which time, deposited eggs were also quantified.Following the perfusion of 7-week infected mice, adult worms were recovered and RNAi performed as previously described . Smlsd1 2.14Smlsd1 and siLuc, mixed-sex adult worms were incubated for a total of 48 h before processing them for RNA isolation. Briefly, worms were homogenised using a TissueLyser LT  in TRIzol Reagent  before isolation of total RNA using the Direct-zol RNA Kit . cDNA was then generated, qRT-PCR performed and data analysed as previously described  or histone deacetylase- (HDAC) activities has been the predominant focus of such investigations to date  and the histone demethylases (HDMs) , should be considered as viable next-generation anthelmintic targets . The ide to date . Howevers (HDMs) . The juss (HDMs) . TherefoS. mansoni HMT and HDM family members 3–9, Enhancer-of-zeste and Trithorax) domain  and PRD domain, ) contain) domain B. In con) domain B.Fig. 1RS. mansoni PKMTs in Wormbase parasite  to query the S. mansoni genome, only these 20 SmPKMTs were found .The use of the SET domain signature  PKMT and 5 PRMTs  C Cabeza. A detaiE value = 2e−155) as a putative SmLSD1 and Smp_160810  and Smp_162940  as putative SmLSD2s  domain was found in all three Smps, but a TOWER domain was only present in Smp_150560. In addition, both Smp_160810 and Smp_162940 contained two zinc finger (zf-C4H2C2 and zf-CW) domains. Based on these features , which are FAD-dependent amine oxidases and the Jumonji C domain-containing demethylases (JMJDs), which are Fe(II) and 2-oxoglutarate-dependent enzymes. For identification purposes, the catalytic domain signatures of these two subfamilies are the Amine Oxidase-Like (AOL) domain (LSDs) and the Jumonji C terminal domain  . In the  SmLSD2s . Here, afeatures , Smp_150S. mansoni members; three of these also contain the JmjN domain , when found, were also recorded Fig. 2). 3.3in silico binding of potential SmLSD1 inhibitors was performed.Secondary structural analysis of SmLSD1 predicted a 1164 AA protein containing a N-terminal SWIRM domain  as well as a C-terminal amine oxidase like (AOL) domain  A. Similain silico molecular docking of 4532 compounds  to the ructures . While wructures , we idenructures . These aructures . All 10 ructures .Fig. 4In3.4in vitro cultivated schistosomula  available compounds against schistosomula on the Roboworm platform , only L8 and L10 affected both parasite phenotype and motility metrics A; these ed to L8 . This aned to L8 . The greed to L8 , structued to L8 ) or its ed to L8 . Further50 = 14.18 μM, L8 EC50 > 50 μM). While egg production was also affected by co-cultivation with both compounds, L10 again demonstrated a more striking effect in inhibiting this critical process involved in host immunopathology and lifecycle transmission (50 recorded for adult parasites (14.18 μM) compared to schistosomula  . Consistsmission B. The hin adults , suggest defects , further50 concentration of this compound (12.5 μM) was performed to assess if L10 also affected surface worm morphology  in affecting schistosome phenotype or motility, regardless of lifecycle stage examined. Therefore, SEM analysis of adult male schistosomes co-cultivated in a sub-ECrphology C. Here, rphology . While wrphology  and, thurphology ). Indeedrphology . Anthracrphology . Neverth3.5Smlsd1 siRNAs, led to a significant reduction  in transcript abundance after 48 h when compared to worm pairs treated with siluc siRNAs (Smlsd1 treated worms (Smlsd1 transcript reduction (56%) and egg production inhibition (50%) at 48 h is only correlated, the magnitude by which both processes were affected suggests a role for SmLSD1. After 7 days, siSmlsd1 treated worms recovered slightly, but were still producing far less eggs than their siluc counterparts (Smlsd1 vs siluc treated worm pairs) would be observed if greater than 56% knockdown in Smlsd1 was achieved and allow for these two scenarios to be resolved. Nevertheless, these functional genomics results, targeting Smlsd1, support the egg production defects mediated by L10  can be leveraged to identify next-generation schistosome chemotherapeutic targets. By re-classifying two opposing enzymatic families responsible for histone (and other protein) methylation and demethylation, we have identified SmLSD1 , or non-financial interest  in the subject matter or materials discussed in this manuscript.


In particular, a specific capacitance of 1209.1 F g−1 was found for the cauliflower-like Co0.75Ni0.25(OH)2 electrode material using DTAB as the surfactant at a current density of 1 A g−1, whose structure boosted ion diffusion to present a good rate ability of 64% with a 50-fold increase in current density from 1 A g−1 to 50 A g−1. Accordingly, the asymmetric supercapacitor assembled by current LDH electrode and activated carbon electrode showed an energy density as high as 21.3 Wh kg−1 at a power density of 3625 W kg−1. The relationship between surfactant and electrochemical performance of the LDH electrode materials has been discussed.Tailoring the morphology of cobalt–nickel layered double hydroxide (LDH) electrode material was successfully achieved via the process of cathodic electrodeposition by adding different surfactants  or cetyltrimethylammonium bromide). The as-prepared Co The s2+ is a divalent ion including Ni2+, Zn2+, Mn2+; M3+ is a trivalent ion, i.e. Co3+, Al3+, Cr3+; −, Exploring electrode materials with high specific capacitance (Sc) is necessary to improve the energy density of supercapacitors. Carbon materials, conducting polymers and transition metal oxides/hydroxides with high surface area are commonly used as supercapacitor electrode materials . Carbon-2− etc.)  has neve2− etc.) . Nickel-2− etc.) .There are various techniques used to fabricate LDH electrode materials such as co-deposition , hydrothIn this paper, we proposed a facile route to synthesize cobalt–nickel LDH films of different morphologies by electrodepositing from aqueous solution in the presence of different surfactants (hexamethylenetetramine (HMT), dodecyltrimethylammonium bromide (DTAB) or cetyltrimethylammonium bromide (CTAB)). Inductively coupled plasma–optical emission spectrometry (ICP), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were applied to investigate the structures and morphologies of the as-prepared samples. Asymmetric supercapacitors (ASCs) based on current cobalt nickel LDHs as positive electrodes and activated carbon (AC) as negative electrodes were fabricated. The electrochemical performances of the ASC devices were also investigated.2.2.1.3)2·6H2O), cobalt nitrate hexahydrate (Co(NO3)2·6H2O), thiourea (CH4N2S) and HMT were acquired from China National Medicine Corporation Ltd. DTAB and CTAB were purchased from Chengdu Kelong Chemical Regent Co. Ltd (China). All chemicals were of analytical grade and used without further purification. The nickel foam was washed in 6M HCl and acetone with ultrasonication for 10 min, then washed with de-ionized water and alcohol several times, and dried in an oven at 60°C. The nickel foam substrate was rolled to a thickness of 0.2 mm before use.Nickel nitrate hexahydrate 2·6H2O, 2 mM CH4N2S with vigorous stirring, without or with 2 mM HMT, DTAB or CTAB. The samples were prepared firstly by using a multi-potential-step technique in a three-electrode configuration with Ag/AgCl as the reference electrode, platinum as the counter electrode and nickel foam as the working electrode at −2 V for 50 s under constant magnetic stirring. The obtained samples were rinsed with de-ionized water and dried at 60°C for several minutes. Then the above procedure was repeated five times. The precursors were annealed at 200°C for 2 h. The mass of active material was determined by the weight difference of the nickel foam substrate before and after deposition, and the mass loading of active material was about 0.6–1 mg per square centimetre.Electrochemical cathodic deposition was carried out for the synthesis of the LDH samples in an aqueous solution; the solution was prepared by dissolving 2.5 mM Ni(NO)2.3.α radiation λ = 0.15406 nm), SEM (JSM-7500F), TEM (JEM-2010) and X-ray photoelectron spectroscopy (XPS) (XSAM 800). The electrochemical performance was measured by electrochemical workstation (CHI660E) and LAND test system (CT2001A).The composition and structure of the samples were characterized by ICP (ARCOS SOP), FTIR spectroscopy (Nicolet 6700), XRD 2 (no surfactant), Co0.75Ni0.25(OH)2: H (HMT as surfactant), Co0.75Ni0.25(OH)2: D (DTAB as surfactant) and Co0.75Ni0.25(OH)2: C (CTAB as surfactant).0.75Ni0.25(OH)2 LDHs includes an electrochemical reaction and subsequent precipitation of mixed hydroxide, as described by the following two equations:The cathodic electrodeposition of the CoKsp) at room temperature of Co(OH)2 (2.5 × 10−16) is close to that of Ni(OH)2 (2.8 × 10−16) [× 10−16) .0.75Ni0.25(OH)2 LDHs supported on Ni foams is shown in − ions [+, CTA+) can be electrostatically adsorbed on the negatively charged surface of working electrode and expected to serve as templates [figure 1a, +, DTA+ has a shorter carbon chain length [0.75Ni0.25(OH)2: D is shorter than that (500–600 nm) of Co0.75Ni0.25(OH)2: C , which tends to aggregate to reduce the surface energy from a thermodynamic perspective [The possible formation mechanism of CoOH− ions . Meanwhiemplates . The diftructure , and reln length , so the (OH)2: C c,d, whicspective . Thus, t3.2.θ range of 10°–80°. The background peaks of nickel foam are very strong, which would disturb the diffraction peaks of electrode materials; so, cleaned stainless steel was employed to act as a substrate. As shown in θ = 11.1°, 22.2°, 38.2° are indexed as (003), (006) and (015), which originate from the characteristic lattice planes of hydrotalcite-like LDH phase [XRD analysis is a powerful tool to clarify the structure of the as-electrodeposited sample in the 2DH phase ,31. The DH phase ,33.Figu−1 and 649 cm−1, respectively [−1 is due to a small amount of intercalated sulfate [−1 is contributed by carbonate resulting from the adsorption of atmospheric CO2 and the intercalation of carbonate caused by electrodeposition process from the electrolyte dissolving CO2 in air. There is a sharp and intense band at 1384 cm−1 corresponding to the −1 and 1627 cm−1 can be assigned to stretching vibration and deformation vibration of the adsorbed water molecules as well as the interlayer water molecules [ectively . The wea sulfate . It is c sulfate . The bantructure . The banolecules . These d0.75Ni0.25(OH)2: D as a representative example was characterized by XPS analysis 2: D. The peaks can be identified as the contribution from Ni, Co, O and C, which manifest the existence of these elements in tested samples. The Co 2p spectrum (b) can be mainly split into two peaks centred at 794.9 eV for Co 2p1/2 and 779.3 eV for Co 2p3/2. Also, the spin–orbit splitting value of 15.6 eV and weak Co 2p3/2 satellite line intensity confirm the coexistence of Co2+ and Co3+ [figure 4c, the peaks located at 872.5 eV for Ni 2p1/2 and 855.2 eV for Ni 2p3/2 accompanied with two shake-up satellites are the trait of Ni2+ [figure 4d) result from O in metal–O–metal and metal–OH groups, respectively [To ascertain the chemical bonding states of every element in cobalt–nickel LDH, Coanalysis . Figure spectrum b can be and Co3+ ,38. As e of Ni2+ ,40. The ectively .Figure 0.75Ni0.25(OH)2 film obtained by electrodeposition without adding any surfactant exhibits a smooth surface with few wrinkled paper-like sheets covered on the nickel foam substrate (cf. a). The wrinkles become more pronounced and uniformly continuous on the surface of Co0.75Ni0.25(OH)2: H electrode (b). Moreover, in figure 5c for the Co0.75Ni0.25(OH)2: D sample, a single nano-sheet is grown, and the nano-sheets form a cauliflower-like 3D structure by self-assembling simultaneously. The significant 3D pores cause a loosely packed structure, which further boosts electrolyte ions to access the active materials during the fast Faradaic reaction process [0.75Ni0.25(OH)2: C sample can be found in figure 5d; the structure of a serried net-like (the inset of figure 5d) interconnected surface with inter-flake pores offers passageway for intercalation and de-intercalation of ions and electrolyte diffusion in the bulk. The TEM characterization was done by separating the Co0.75Ni0.25(OH)2: D active material from Ni foam; there are some faint nano-sheets on the grid in figure 6a–c manifesting its ultrathin nature [figure 6d) possesses broad and diffused halo, suggesting its polycrystalline feature [SEM and TEM provide further insight into the morphology and microstructure. As revealed by detailed morphologies in the images of rate cf. a. The wrlectrode b. Moreov process . The pren nature . The cor feature , which i3.3.a, it is apparent that the curves almost overlap; no significant differences of the Nyquist plots of the cobalt–nickel LDHs can be found. From the enlarged high-frequency region shown in the inset of figure 7a, by virtue of the binder-free and layered structure, no semicircles are visualized in this region, which is correlated to a low charge transfer resistance [Figure 7b exhibits the cyclic voltammetry (CV) curves of these electrodes in 2 M KOH electrolyte at a fixed scan rate of 50 mV s−1 in the potential range of 0–0.6 V. The area of the CV curve of Co0.75Ni0.25(OH)2: C is larger than that of the others, manifesting better Sc. The typical CV curves of Co0.75Ni0.25(OH)2: D at different scan rates are displayed in figure 7c, where all the curves maintain the same shape with the change in scan rates. It is associated with the improved mass transportation [The electrochemical performances of the cobalt–nickel LDHs were evaluated by a typical three-electrode experimental cell equipped with a working electrode, a platinum foil counter electrode and an Ag/AgCl reference electrode. Electrochemical impedance spectroscopy (EIS) was employed in the frequency range from 100 kHz to 0.01 Hz to understand the electrochemical reaction kinetics of the as-synthesized samples. From figure 7sistance . Figure ortation . It is eortation :3.3Ni, applied current load (A), discharging time(s) and mass of active material (g), potential window (V), respectively. The obtainable high values of Sc for Co0.75Ni0.25(OH)2: C (1318.2 F g−1), Co0.75Ni0.25(OH)2: D (1209.1 F g−1) and Co0.75Ni0.25(OH)2: H (1120 F g−1) electrodes at the current density of 1 A g−1 are in contrast to the low value for the pure Co0.75Ni0.25(OH)2 electrode (713.1 F g−1). It is thus evident that 3D structure fabricated through the use of surfactant promotes the electrochemical performance. Figure 7e displays the Scs of the cobalt–nickel LDHs synthesized with surfactants when the charge–discharge rate increases from 1 to 50 A g−1. The electrode of Co0.75Ni0.25(OH)2: D retains about 64% of the Sc at 50 A g−1, while 62% and 57% capacitance retentions for Co0.75Ni0.25(OH)2: H and Co0.75Ni0.25(OH)2: C electrodes are observed, respectively. These consequences imply that the electrodes of cobalt–nickel LDHs possess high rate capabilities. Figure 7f depicts the cycling stability tested at a current density of 8 A g−1. Seventy-five per cent of initial Sc is kept after 2000 cycles for Co0.75Ni0.25(OH)2: H, while 62% for Co0.75Ni0.25(OH)2: C and 61% for Co0.75Ni0.25(OH)2: D are retained.To further explore the electrochemical performance of the cobalt–nickel LDH electrodes, the galvanostatic charge–discharge technique was performed at the current density of 1 A gequation :3.6C=IΔt3.4.mac/mcobalt nickel LDH = 2.4 according to the following equation [+m and −m are the mass of electrode materials, c+ and c− are the specific capacitances of the positive and negative electrodes, Δ+V and Δ−V represent the potential window for the electrodes, respectively.The practicability of these materials was investigated by assembling ASCs using the cobalt–nickel LDHs as positive electrodes and AC as negative electrodes in 6M KOH aqueous electrolyte. It is found that the optimal mass ratio to fabricate the ASC device is equation :3.7m+m−=a, all EIS plots of these ASCs contain a semicircle in the high-frequency region and then a straight sloping line; the smallest diameter of the semicircle of Co0.75Ni0.25(OH)2: D//AC is associated with a smallest charge transfer resistance (RCT) [RCT is pretty vital to high electrical conductivity and outstanding rate capability [figure 8b were obtained in the window of 0–1.45 V at a scan rate of 50 mV s−1. The CV curves of the Co0.75Ni0.25(OH)2: D//AC keep the same shape at various scan rates, which means the fast charge–discharge property of the device (figure 8e). From the galvanostatic charge–discharge curves at the current density of 0.2 A g−1 (figure 8c), the discharge time of Co0.75Ni0.25(OH)2//AC is shortest among these ASCs. Energy density (E) and power density (P) were estimated using the following equations [As can be seen from figure 8ce (RCT) . The lowpability . Benefitquations :3.8E=12C−1 (Co0.75Ni0.25(OH)2: C//AC), 28.4 Wh kg−1 (Co0.75Ni0.25(OH)2: D//AC) and 26.7 Wh kg−1 (Co0.75Ni0.25(OH)2: H//AC). This result further implies that the use of surfactant during the electrodeposition process is advantageous to the electrochemical performance. As the current density increases from 0.2 to 5 A g−1 shown in figure 8f, the calculated Sc value of the Co0.75Ni0.25(OH)2: D//AC ASC device decreases from 97.3 to 73.1 F g−1. As shown in figure 8d, the trend in rate capabilities of Co0.75Ni0.25(OH)2: D//AC (75%), Co0.75Ni0.25(OH)2: H//AC (69.3%), Co0.75Ni0.25(OH)2//AC (65%) and Co0.75Ni0.25(OH)2: C//AC (54.8%) is similar to the trend in the diameters of the semicircles in the inset of figure 8a. This observation supports the fact that Co0.75Ni0.25(OH)2: D//AC has better interfacial faradic reaction kinetics; the Co0.75Ni0.25(OH)2: D//AC device also keeps a better cycle life (65%) after 1000 cycles in figure 8h. The two important results are owing to the preferable electronic conductivity and unique surface structure; the cauliflower-like structure of Co0.75Ni0.25(OH)2: D has many interspaces between the particles, which allow the volume expansion to take place easily, and promote the high distribution of active sites as well as high speed pathway for continuous electronic and ionic transportation [figure 8g. A maximum power density (3625 W kg−1) at an energy density of 21.3 Wh kg−1 was achieved by the Co0.75Ni0.25(OH)2: D//AC ASC device, which is in line with the conclusion that a high rate ability of the electrode material favours supercapacitor providing a high power density [0.75Ni0.25(OH)2: D//AC electrode does not present the highest Sc, it still seems more suitable for practical application on grounds of its preferable rate capability and cycle life.The energy densities of the devices can be further calculated to be 28.6 Wh kgortation . Ragone  density . Althoug4.0.75Ni0.25(OH)2 active material having a Sc of 713.1 F g−1, the modified Co0.75Ni0.25(OH)2 LDHs with distinct surface morphologies showed superior Scs: wrinkle-like, cauliflower-like and net-like Co0.75Ni0.25(OH)2 electrodes exhibited Sc of 1120 F g−1, 1209.1 F g−1 and 1318.2 F g−1, respectively. Among ASC devices fabricated using Co0.75Ni0.25(OH)2 LDHs as positive electrodes, the Co0.75Ni0.25(OH)2: D//AC ASC device showed a preferable energy density at the same power density. These results demonstrated that morphology modification by surfactant enhanced the electrochemical performance in cobalt–nickel LDH electrode materials.The morphologies of cobalt–nickel LDHs were successfully modified by using surfactants during the process of cathodic electrodeposition. In comparison to the pristine Co


Contributions to plasticity from hard matrix and binder metal in nanocrystalline cermets were studied by molecular dynamics simulations. Using the typical WC–Co cemented carbide as an example, the interactions of dislocations within the ceramic matrix and the binder metal, as well as the possible cooperation and competition between the matrix and binder during deformation of the nanocrystalline cermets, were studied by molecular dynamics simulations. It was found that at the same level of strain, the dislocations in Co have more complex configurations in the cermet with higher Co content. With loading, the ratio between mobile and sessile dislocations in Co becomes stable earlier in the high-Co cermet. The strain threshold for the nucleation of dislocations in WC increases with Co content. At the later stage of deformation, the growth rate of WC dislocation density increases more rapidly in the cermet with lower Co content, which exhibits an opposite tendency compared with Co dislocation density. The relative contribution of Co and WC to the plasticity of the cermet varies in the deformation process. With a low Co content, the density of WC dislocations becomes higher than that of Co dislocations at larger strains, indicating that WC may contribute more than Co to the plasticity of the nanocrystalline cermet at the final deformation stage. The findings in the present work will be applicable to a large variety of ceramic–metal composite materials. Then, the uniaxial compression was performed along the Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS)  to overall plastic deformation. In this period the density of Co dislocations increases rapidly with strain in each material, indicating the important contribution of Co to the plastic deformation of the cermet. The strain threshold for the nucleation of WC dislocation increases with the increase of Co content in the cermet. The WC dislocation occurred at strains of 7.2, 8.3 and 8.8% for WC–8Co, WC–12Co and WC–16Co, respectively.In the fourth period, the growth rate of the Co dislocation density decreases with loading. In addition, this period starts later in the cermet with higher Co content. For example, the growth rate of the dislocation density in Co slowed down at a strain of about 0.135 in the WC–8Co sample, whereas the corresponding strains are 0.165 and 0.185 for the WC–12Co and WC–16Co samples, respectively.Obviously, in the later periods of deformation, at the same level of strain, the dislocation density in Co increases with the Co content in the cermet. This implies that the dislocations in Co develop faster in the cermet with higher Co content.3.2.2.a)–4(c). This indicates that Co has a larger contribution to the plastic deformation of the cermet with a higher Co content. This can probably be attributed to the mean free path of Co and the contiguity of WC in the cermet. Table 2et al., 2012The aforementioned dislocation activities in Co become straightforward in the three-dimensional distribution of dislocations. Fig. 4In contrast, the contiguity of WC grains decreases with the increase of Co content, meaning the WC/Co phase boundaries increase as well. As a result, stress transfer from WC to Co during deformation becomes more significant in the cermet with higher Co content. This leads to a higher degree of plastic deformation of Co. Besides, as the Co content increases in the cermet, the Co mean free path increases and the mean length of the WC/WC grain boundaries decreases. It is thus possible that some of the Co dislocations propagate bypass the local short WC/WC grain boundaries. Consequently, the deformation capacity of Co increases with the long-distance propagation of the dislocations in Co.a), in the WC–8Co cermet, the dislocation lines are mainly single and short with a few simple multi-junctions due to the low density. The multi-junction in the dislocation lines locks the mobile dislocations, acting as the obstacle for the dislocation mobility. In the cermets with higher Co content, where the dislocation density is higher, the probability of the dislocation interactions increases. In addition, the structure of the dislocations becomes more complicated at higher Co content in the cermet. Many claw-like multi-junction dislocations started to form in the WC–12Co cermet, as seen in Fig. 5b). And lots of dislocation tangles and complex dislocation networks were observed in the WC–16Co cermet [Fig. 5c)] due to the high density of dislocations.Furthermore, the local structure and configuration of the dislocations in Co were investigated for the three samples at a strain of 0.15. As shown in Fig. 53.2.3.Fig. 63.2.4.Fig. 7a) and 8(b), the Shockley partial dislocations were locked due to the reactions, e.g. 1c) and 8(d). In this period, the proportion of mobile dislocations decreases while that of sessile dislocations increases  The content of Co clearly affects the mechanical properties of the nanocrystalline cermets through modulating the deformation process. With higher Co content, the ratio between mobile and sessile dislocations in Co becomes stable earlier in the cermet. However, this ratio stabilizes to a value of 3:1 at the final stage of deformation regardless of Co content, owing to the dynamic equilibrium between pinning and depinning of Co dislocations through reactions.(ii) The Shockley partial dislocations are distributed widely in Co and make a major contribution to the plastic deformation. Independent of Co content, the densities of the stair-rod and Hirth dislocations are close to each other and count for about 1/5 of that of the Shockley partial dislocations. The stair-rod and Hirth dislocations of Co hinder the continuation of plastic deformation of the cermet.(iii) Nucleation of dislocation in WC has a smaller strain threshold in the cermet with lower Co content. Moreover, in the low-Co cermet, the growth rate of WC dislocation density increases rapidly at the late stage of deformation, which shows an opposite tendency to that of Co dislocation density. As a result, the density of WC dislocations may be higher than that of Co dislocations at larger strains in the cermet with lower Co content.(iv) Co is responsible for the plasticity of the cermet at the beginning of the plastic deformation. As WC starts to participate in the plastic deformation of the cermet, its dislocation density is much lower compared with that of Co. However, with loading the relative contribution of Co and WC to the plastic deformation of the cermet varies with the deformation stage. With the WC dislocation density exceeding the Co dislocation density in the low-Co cermet, WC plays a more important role in the plasticity of the cermet at the later stage of deformation.6.The authors declare no conflict of interest.


Solitary, persistent wave packets called solitons hold potential to transfer information and energy across a wide range of spatial and temporal scales in physical, chemical, and biological systems. Mechanical solitons characteristically emerge either as a single wave packet or uncorrelated propagating topological entities through space and/or time, but these are notoriously difficult to control. Here, we report a theoretical framework for programming static periodic topological solitons into a metamaterial, and demonstrate its implementation in real metamaterials computationally and experimentally. The solitons are excited by deformation localizations under quasi-static compression, and arise from buckling-induced kink-antikink bands that provide domain separation barriers. The soliton number and wavelength demonstrate a previously unreported size-dependence, due to intrinsic length scales. We identify that these unanticipated solitons stem from displacive phase transitions with periodic topological excitations captured by the well-known  Mechanical solitons are notoriously difficult to control. Here, the authors report a theoretical framework for programming static periodic topological solitons into a metamaterial, and demonstrate its implementation in real metamaterials computationally and experimentally. Localizations associated with solitons explain many fundamental features of dislocations, ferroelectric domain walls, surface diffusion, conducting polymers, intrinsic localized modes, and nonlinear excitations in biological molecules12. Although they have been observed in solitary excitations of mechanical metamaterials14 and domain-wall formations of van der Waals layered materials17, they have previously been stochastic and scale-independent discrete and/or single-soliton systems. A longstanding goal has been development of programmable, localized topological structures to control solitons18. This technology would hold prospects not only in tunable photonic and phononic rectification19, but also in designing smart soft robots and high sensitivity devices20.The localized deformation underlying kink-excitations drives numerous nonlinear physical and mechanical phenomena including fault zones in earthquake22, but also on designing desired patterns and interfaces in architected metamaterials24. The central challenge is the widely-recognized problem that localized deformations are sensitive to inherent or externally induced defects, rendering the location and dimension of localization zones hard to predict26. Successful previous efforts include ordered configurations achieved by introducing long-range interactions27, geometric frustration28, periodic dopants or macro-defects29. However, generation of programmable, ordered and robust static solitons and ensuing localized instabilities in nominally defect-free mechanical systems still remains largely unexplored.Extensive work has been focused not only on engineering localizations into civil structures 30, here we develop a general framework for analyzing this entire class of materials through the formalism of static periodic solitons. Many have previously addressed the bi-stability of metamaterials through conventional snap-though equilibrium techniques32. The general framework presented here is based on an approach to the underlying physics whereby interactions between double-well on-site potentials of the unit cell and the strong coupling energies of adjacent unit cells determine how metamaterial architecture and loading produce buckling-induced kink and antikink deformation bands that provide domain separation barriers.Inspired by recent numerical work indicating that excitations of ordered localizations can be achieved via carefully designed architectures34. This unanticipated behavior originates from a structural phase transition analogous to mechanical soliton-lattices of magnetic systems35. We theoretically illustrate that the period of the soliton-lattice in fact introduces an intrinsic length scale perpendicular to the compression direction, which leads to novel size-dependence of the soliton number and wavelength in the metamaterial.We therefore explored the instability of cellular metamaterials subjected to quasi-static compression. We found that, beyond a critical loading, deformation symmetry breaks down, leading to two distinct macroscopic buckling modes separated by localized topological interfaces that are static periodic solitons (kink-antikink pairs) governed by the well-known 37. Between these two states, highly localized topological domain walls emerged to accommodate the incompatible deformation . To verify the structural origin of this order, we performed 2D plane strain finite element (FE) simulations that reproduced experimental observations  are excited in the metamaterial, inducing an orderly localized configuration. A discrete size-dependence of the wavelength of the periodic solitons caused by an intrinsic length scale is also unveiled. We emphasize that the double-well potential and the strong coupling energy are pivotal factors that ensure the displacive phase transition and, evidently, can be programmed into the unit cell geometries to prescribe soliton-lattice configurations.43, atomic-scale frictions16, and domain switching44. This holds potential to be leveraged for a range of new metamaterials with programmable localized deformations for fields such as smart molecular robotics. Besides, the alternative kinks and antikinks count the reversals of the topological mechanical polarizations along the quasi-1D metamaterial chain. By encoding the inward and outward domain-states as mechanical bits which can be identified by probing the local order parameters, we envisage such robust reversal features are promising for enabling information storage and retrieval on static soliton-lattices45. More broadly, the results enable design of unit cell topologies with multistable states that can pave novel pathways to prescribing ordered localization excitations and controllable geometric phase transitions across a range of physical contexts.The striking size-dependent property of kink-antikink pairs, denoted by a ceiling function in our metamaterial systems, applies across multistable systems including molecular transport in restricted spacesWe fabricated our samples via a double-molding process. Firstly, we cast polyurethane solution into a 3D printed mold . Before testing, sample surfaces and loading platens were covered with white Vaseline to reduce friction. Tests were conducted at a constant nominal strain rate of entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaThe commercial finite element software Abaqus/Standard  was employed in our static finite element simulations. Based on the uniaxial testing results, the Mooney-Rivlin hyperelastic constitutive model was adopted to characterize the metamaterial. Material constants were taken as mentclass2pt{minimSimulation of full samples: we conducted nonlinear FE simulations on the full metamaterial, sandwiched between two rigid plates, with frictionless contact between the sample and the plates (Supplementary Fig. entclass1pt{minimaOn-site potential of the unit cell: two static steps were employed to obtain the on-site potential of the unit cell. First, a static compressive displacement, i.e. Hinge stiffness: the hinge stiffness of the simplified model is dependent on the geometry of the elliptical holes and neck thickness46. In the ideal case such as necks of constant cross-section, or circular holes with the slender limit The general framework for predicting solitons in metamaterials was verified by considering a range of classes of metamaterials. For all of these see Fig. –12, sampSupplementary InformationSupplementary Movie 1Supplementary Movie 2Description of Additional Supplementary Files


Mucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways. Accordingly, knowledge of MUC5B structure and its interactions with itself and other proteins is critical to better understand airway mucus biology and improve the management of lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). The role of an N-terminal multimerization domain in the supramolecular organization of MUC5B has been previously described, but less is known about its C-terminal dimerization domain. Here, using cryogenic electron microscopy (cryo-EM) and small-angle X-ray scattering (SAXS) analyses of recombinant disulfide-linked dimeric MUC5B dimerization domain we identified an asymmetric, elongated twisted structure, with a double globular base. We found that the dimerization domain is more resistant to disruption than the multimerization domain suggesting the twisted structure of the dimerization domain confers additional stability to MUC5B polymers. Size-exclusion chromatography-multiangle light scattering (SEC-MALS), SPR-based biophysical analyses and microscale thermophoresis of the dimerization domain disclosed no further assembly, but did reveal reversible, calcium-dependent interactions between the dimerization and multimerization domains that were most active at acidic pH, suggesting that these domains have a role in MUC5B intragranular organization. In summary, our results suggest a role for the C-terminal dimerization domain of MUC5B in compaction of mucin chains during granular packaging via interactions with the N-terminal multimerization domain. Our findings further suggest that the less stable multimerization domain provides a potential target for mucin depolymerization to remove mucus plugs in COPD and other lung pathologies. Polymeric mucins are the major structural component of mucus; a hydrogel that plays a pivotal role in protecting mucosal surfaces. In healthy airways, mucus in conjunction with cilia, protect the airway epithelium from inhaled pathogens, particles, and toxins , 2. The 3–MUC5B and MUC5AC are the major polymeric mucins in respiratory tract mucus –9. The f–O-glycosylation of the central mucin domain followed by D3-domain disulfide-mediated N-terminal multimerization in the Golgi apparatus  homotypic interaction between C-terminal dimerization domain and (b) heterotypic interaction with recombinant N-terminal multimerization domain.Here, using recombinant protein (D4-B-C-CK), we describe the detailed structure of the C-terminal dimerization domain of MUC5B using single particle cryo-electron microscopy (cryo-EM) and small-angle X-ray scattering (SAXS). We demonstrate that the C-terminal dimerization domain of MUC5B has similar structure to that of the related glycoprotein, vWF , 29. MorA). We also expressed the previously published N-terminal dimerization domain of MUC5B (NT5B protein) consisting of D1-D2-D′-D3 domains (B) and showed that CT5B was expressed mainly as a disulfide-linked dimer with a small proportion of monomer. The molecular mass for the CT5B dimer (∼245 kDa) and monomer (∼147 kDa) was determined by size exclusion chromatography multi-angle static light scattering (SEC-MALS) (C).To investigate the structure and further define the role of the C-terminal dimerization domain of MUC5B, we stably expressed a C-terminal construct of MUC5B (CT5B), consisting of D4-B-C-CK domains A. We als domains . The exp domains B and shoEC-MALS) C.Figs. S1 and S2). 2D classification of CT5B showed an elongated shape consisting of a globular base connected to an extended stalk region . There appeared to be flexibility between the globular base and the stalk as visualized in the 2D classes . As CT5B is a dimer, the data were processed using either C1 or C2 symmetry. The C1 structure refined to a resolution of 9.3 Å, where the twist in the stalk was clearly visible (A), and the C2 structure refined to a resolution of 8.9 Å where the base benefitted from the application of 2-fold symmetry, but the flexible stalk structure deteriorated when symmetry was applied (B). Therefore, the stalk was subtracted from the C2 refined structure and locally refined resulting in a gain in resolution to 7.5 Å .We investigated the structure of dimeric CT5B using cryo-EM and SAXS . Based on the similarity to the vWF structure and crystal structure of the vWFD domain . A resolution of 7.5 Å was achieved despite the heterogeneity contributed by complex N-glycosylation and conformational flexibility through the stalk region.Analysis of the SAXS data confirmed the flexible nature of CT5B, with the normalized Kratky plot indicating a biphasic system with both globular and elongated regions connected by limited flexibility D. Based D domain , 29, it D domain . The D4 C and BFig. S1) and 3D reconstruction (A and C) that the two molecules within the stalk region twist around each other. This may increase the interface and the number of domain-domain interactions between the monomers in the stalk region, potentially increasing the stability of this C-terminal region . Results showed no evidence of a change in CT5B structure at different pH values. Taken together, our data suggest that the twist in the stalk of the molecule, which represents the B-C domains, could be a feature that provides enhanced stability to the CT5B dimer.The B-C-CK domains form the stalk structure. It was apparent from the 2D class averages   or with 1 μg/ml of trypsin at room temperature  and samples collected at different time points were analyzed by SDS-PAGE. Dimer and monomer bands are indicated by arrows and trypsin inhibitor (∼50 kDa) is indicated by a dashed arrow. Results showed that CT5B dimer was resistant to limited proteolysis and partial reduction, maintaining a dimer throughout the time course of the experiment (B and D). In contrast, NT5B dimer was sensitive to both treatments, being reduced to monomer in the presence of DTT after ∼0.5 min (A) and lower molecular weight species by the action of trypsin within 1 min (C).MUC5B forms linear polymers via disulfide-linked N-N and C-C–terminal interactions. We compared the relative stability of the expressed protein representing the N-terminal multimerization domain of MUC5B (NT5B) B 19) wi wiB) . Western blot analysis before and after proteolysis was performed and blots were probed for D3 domain (5BVIII) in the N terminus, and B domain (CC1) in the C terminus, and total glycoprotein was determined by PAS stain (B). Results for the unreduced samples showed ∼70% loss of N-terminal antibody signal following proteolysis, whereas the C-terminal antibody signal was only decreased by ∼15% (C). A similar trend was observed for the samples that were reduced prior to electrophoresis. These data are consistent with results following trypsin digestion of the recombinant MUC5B proteins (C and D) and demonstrate that in native MUC5B polymers the C-terminal dimerization domain is more stable than the N-terminal multimerization domain.To assess whether this differential stability demonstrated with the N- and C-terminal recombinant proteins was observed with native polymers, we analyzed purified MUC5B following proteolysis. SEC-MALS analysis showed that proteolysis caused a reduction in the average molecular mass of MUC5B from 6.8 to 3.5 MDa A. WesterAS stain B. Result by ∼15% C. A simiproteins , C and Dm) at pH 7.4 or 6 and analyzed by SEC-MALS . Results in the presence of EGTA at pH 7.4 and 6  showed a peak of dimer (peak II) and monomer (peak I). In the presence of calcium at pH 7.4, 6, or 5, the profiles of CT5B were similar to the data for EGTA and showed that calcium and pH did not facilitate noncovalent interactions between CT5B . These results were also confirmed using analytical ultracentrifugation, which showed monomer (I) and dimer (II) peaks with similar sedimentation profiles for CT5B under all conditions studied . These data show that unlike the N-terminal multimerization domain, the C-terminal dimerization domain of MUC5B does not form homotypic calcium or pH-dependent multimers and therefore, does not have the same role as the N-terminal multimerization region in the organized packaging of MUC5B.We next investigated the potential role of CT5B in MUC5B intragranular packaging. Prior to secretion, linear disulfide-stabilized MUC5B polymers are condensed and packaged within secretory granules that have an acidic pH and high calcium content , 19. We m dimeric NT5B was analyzed in the presence of 5 mm CaCl2 over a range of pH (A). This pH scouting assay showed that there was some low background binding between NT5B and CT5B in the presence of calcium at pH 7.4. Binding was shown to increase as a function of decreasing pH, the most binding was at pH ∼ 6.2, and binding was abolished following EDTA treatment. Single cycle kinetic assays were performed with increasing concentrations of NT5B (5-40 nm) flowed over immobilized CT5B in the presence of 5 mm CaCl2 at pH 6 and 7.4 . Analysis showed a strong binding interaction between NT5B and CT5B at pH 6, with a DK value of ∼1 ± 0.04 nm. At pH 7.4, NT5B flowed over immobilized CT5B with 5 mm CaCl2 and produced some binding with a DK value in a similar range (∼3 nm), however, the binding response was 5-fold lower; suggesting the same binding affinity, but a much lower amount of NT5B bound.Although the presence of calcium and acidic pH did not engender homotypic interaction between CT5B, we hypothesized that the C-terminal multimerization domains of MUC5B may interact with N-terminal dimerization domains during packaging to further facilitate the organization of the mucin polymer inside the secretory granule. Using surface plasmon resonance (SPR), the interaction of immobilized dimeric CT5B with 50 nge of pH A. This pDK model of fit using the NT Affinity Analysis software (C and D). The results confirmed that NT5B and CT5B interact in calcium at pH 6, with a DK of 3 ± 1.4 nm , which was comparable with the result obtained from SPR. The results also showed an interaction at pH 7.4 with a similar binding affinity . Importantly, there was no interaction detected between NT5B and CT5B in the presence of EGTA at either pH . These results establish that MUC5B dimerization and multimerization domains form reversible pH-sensitive and calcium-dependent intermolecular associations between polymeric mucins.We investigated the heterotypic binding further by performing in solution analysis of the interaction between NT5B (fluorescently labeled) and CT5B  using microscale thermophoresis (MST). The data were analyzed using a software , C and DDetermining the nature of polymeric mucin chain interactions during packaging into secretory granules and their subsequent uncoupling post-secretion is critical to understanding the mechanisms that control mucus gel formation in health and disease. Abnormal mucus gel properties may arise due to mucin chains not fully expanding upon secretion, resulting in altered molecular structure. Indeed, airway mucins with aberrant macromolecular structure are a feature of hyper-concentrated mucus that obstructs the CF airways . We haveDuring packaging of MUC5B within secretory granules, the mucin chains are organized around protein nodes formed from pH and calcium-dependent, noncovalent, reversible, homotypic interactions between N-terminal multimerization domains , 21. In BFig. S1) and by the normalized Kratky plot (D), and the large proportion of heterogeneity as a result of glycosylation across the molecule . The N-terminal construct (NT5B consisting of D1-D2-D′-D3) has been described previously . A Superose 6 10/300 GL column was used for size fractionation. Protein identity and purity were confirmed by tandem MS. Samples, with and without treatment with 10 mm for 4 min at 95 °C, were analyzed by SDS-PAGE using 4–12% BisTris gels (Life Technologies) and stained with InstantBlue (Expedeon).MUC5B (UniProtKB accession number Q9HC84) C-terminal construct consisting of D4-B-C-CK   was creaNA cells , 43, 44 NA cells A. The N-eviously  . Analysis was performed using ASTRA version 6 software.For determination of molecular mass by SEC-MALS analysis, recombinant proteins were applied to a Superose 6 10/300 GL column and MUC5B was applied to a Shodex OHpak SB-806 M HQ column, in 0.2 m CaCl2 overnight at 4 °C in either HEPES or MES buffer . Samples were analyzed using velocity experiments on an Optima XL-A ultracentrifuge (Beckman Instruments), as previously described (Recombinant CT5B protein was incubated in the presence of 5 mescribed . The sedescribed .Quantifoil R1.2/1.3 holey carbon grids were cleaned in 2× chloroform soaks on filter paper in a glass Petri dish and then glow discharged for 2 min at 25 mA. 3 μl of purified CT5B at 0.25 mg/ml was applied to the grids and blotted for 8–10 s at 22 °C in a Vitrobot Mark IV (Thermo Fisher Scientific), before plunge freezing in liquid ethane.e− Å2 were recorded on the detector in counting mode at a calibrated magnification of 93,000 corresponding to a magnified pixel size of 1.043 Å. Defocus was maintained at −0.5 μm as suggested previously (Cryo-EM data were collected on a Titan Krios electron microscope operating at 300 kV equipped with a K2 Summit direct detector (Gatan) and a FEI Volta phase plate (Thermo Fisher Scientific) at the Electron Bio-Imaging Centre (eBIC) . EPU (Thermo Fisher Scientific) was used to automate the collection of 1441 movies, comprising of 40 frames, 10 s exposures, and a total dose of 40 eviously .ab initio model for further refinement. Refinement was carried out using C1 and C2 symmetries resulting in structures with resolutions of 10.0 and 9.4 Å, respectively. Heterogeneous refinement was then applied to each result with modest differences between the classes. The particles from the best classes were then used to re-refine the structure resulting in lower resolution structures. Non-uniform refinement was then used to refine the C1 and C2 refined models resulting in a resolution of 9.3 and 8.9 Å, respectively. Particle subtraction was then employed to enhance the resolution of each of the base or stalk regions using masks around the base and stalk. Further refinement of both subtracted symmetry models were completed yielding a base with a resolution of 7.5 Å. The stalk, in the absence of the base, was refined but resulted in a much lower resolution map.The recorded movies were processed in cryoSPARC v2.8 , 48. Mov5BPU (www.cbs.dtu.dk/services/NetNGlyc/).The sequence of CT5B residues 4955–5762) was submitted to the HHpred server (55–5762 w–I(q) versus q (q = 4π. sin 2θλ), of CT5B were collected using SEC-SAXS on beamline B21 . 50 μl of BMPER was loaded onto a Superdex 200 Increase 3.2/300 column  at 0.075 ml/min. SAXS data were collected at 3-s intervals on an Eiger X 4M detector (Dectris) at a distance of 2.7 m and wavelength of 0.95 Å.SAXS intensity data, gR) and the integral of intensity ratio to background were plotted. The data were scaled, merged, and averaged for each frame with a consistently similar gR. All further processing and analysis of data were carried out using ScÅtter (www.bioisis.net/scatter)5 (Data were reduced using in-house software. Subtractions of the SEC-SAXS data were completed for each frame across the elution peak and the radius of gyration (catter)5 .m DTT on ice between 0.5 and 180 min. At each time point, 2.5 mm iodoacetamide was added and incubated for 15 min in the dark at room temperature. For limited proteolysis, NT5B and CT5B proteins were incubated with 1 μg of sequencing-grade trypsin (Promega) at room temperature for 1 to 240 min. At each time point, 1 μl of trypsin neutralizing solution (Lonza) was added to stop the reaction. Samples were analyzed by SDS-PAGE using 4–12% BisTris gels (Life Technologies), staining with InstantBlue (Expedeon).For partial reduction experiments, NT5B and CT5B proteins were incubated with 1 mm NaCl, as previously described . The nonreduced bands were quantified, and a ratio of the signal was determined for 5BVIII and CC1 relative to PAS signal.MUC5B was purified from A549MUC5Bm (A549 cell line in which MUC5AC was knocked down by gene editing) cell-conditioned medium using cesium chloride isopycnic centrifugation in 0.1 escribed . The MUCntiserum , which dntiserum , and banm sodium acetate, pH 5.5, was immobilized onto a CM5 sensor chip by amine coupling using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride, N-hydroxysuccinimide, and ethanolamine-HCl . The immobilized CT5B gave ∼3400 response units. To perform a pH scan experiment, purified NT5B protein (50 nm) was injected over the immobilized CT5B in varying pH buffers containing 5 mm CaCl2 and 0.05% Tween 20 for 120 s at a flow rate of 30 μl/min and dissociation time of 60 s. The CM5 sensor chip was regenerated after each sample application with 10 mm EDTA, pH 7.4, at a flow rate of 30 μl/min for 30 s and stabilization of 1 min after regeneration. For single cycle kinetic analysis, 0, 5, 10, 15, 20, and 40 nm NT5B analyte was flowed over the immobilized CT5B in HBS containing 5 mm CaCl2 and 0.05% Tween 20, at pH 6 or 7.4. Curves were fitted using 1:1 Langmuir association/dissociation model  to obtain kinetic values of binding affinity.To investigate interactions between NT5B and CT5B proteins, a Biacore T200 biosensor with Biacore Control software was used . Initially 25 μg/ml of CT5B diluted in 50 mm HEPES, 150 mm NaCl, pH 7.4, was fluorescently labeled using the MonolithTM NT protein labeling kit RED-NHS (Amine Reactive) dye, following the manufacturers' instructions (NanoTemper Technologies). All stock protein samples were centrifuged for 10 min prior to setting up the experiment. The labeled NT5B protein was diluted to 2.5 nm final concentration, in the appropriate buffer for each experiment . The nonlabeled recombinant CT5B protein was serially diluted 1:1 in the same buffer (starting concentration of 6 μm) and an equal volume of the labeled protein was mixed with each dilution. The samples were loaded onto MonolithTM NT.115 Standard Treated Capillaries (NanoTemper Technologies) and thermophoresis was measured using a MonolithTM NT.115PICO instrument (NanoTemper Technologies) with NT Control software version 1.0.1 at room temperature with 5 s/30 s/5 s laser off/on/off times, respectively, and 15% LED power and 40% IR-laser (MST) power. Experiments were carried out in triplicate and data from three independently pipetted measurements were analyzed using NT Affinity Analysis software version 2.0.2, using the DK model of fit.Purified NT5B protein in 25 mC. R., M. P. L.-C., R. F. C., and T. A. J. data curation; C. R., M. P. L.-C., R. F. C., T. A. J., and C. B. formal analysis; C. R., M. P. L.-C., R. F. C., and T. A. J. validation; C. R., M. P. L.-C., R. F. C., and T. A. J. investigation; C. R., M. P. L.-C., and R. F. C. visualization; C. R. and D. J. T. writing-original draft; C. R. and D. J. T. project administration; C. R., M. P. L.-C., R. F. C., T. A. J., C. B., and D. J. T. writing-review and editing; D. B. S. and M. K. resources; C. B. and D. J. T. supervision; D. J. T. conceptualization; D. J. T. funding acquisition.