We carried out three self-paced reading experiments that attempted to replicate these results. Two of our three studies found a subject-relative preference, and the third study found an object-relative advantage. Using a random effects bayesian meta-analysis of fifteen studies (including our own), we show that the overall current evidence for the subject-relative advantage is quite strong (approximate posterior probability of a subject-relative advantage given the data: 78–80%). We argue that retrieval/integration based accounts would have difficulty explaining all three experimental results. These findings are important because they narrow the theoretical space by limiting the role of an important class of explanation—retrieval/integration cost—at least for relative clause processing in Chinese.A general fact about language is that subject relative clauses are easier to process than object relative clauses. Recently, several self-paced reading studies have presented surprising evidence that object relatives in Chinese are  One of the central concerns of sentence comprehension research has been to identify universal, that is, cross-linguistically applicable, constraints that determine online parsing difficulty. The underlying assumption is that the human sentence comprehension mechanism is subject to certain universal principles which should apply regardless of the language in question. However, it is obvious that, to some extent or another, languages differ. Nevertheless, the possible existence of a core set of universal parsing constraints is a very attractive proposition and researchers in psycholinguistics have pursued it with vigor, often with remarkable success.The man who talked to the woman was a doctor.) are easier to process than object relatives (ORs) . A subject relative is a sentence where a noun  is modified by a relative clause , and the modified noun is the grammatical subject of the sentence. In an object relative, the noun modified by the relative clause is the grammatical object of the relative clause.An example of a cross-linguistically consistent result concerns relative clause (RC) processing. A robust finding in the literature is that, at least as far as sentences with animate, definite, and full noun phrases are concerned, subject relatives (SRs)  the frequency distribution of SRs versus ORs is 86% and 13% Frequencies of Relative-Clause Types Working memory accounts: As the review in These two alternative accounts of relative clause processing are interesting in the context of Chinese, because this language presents a puzzling irregularity in the universal subject preference. . The reading experiment was preceded by a plausibility norming task, in which participants were “asked to judge the naturalness in the real world of the events described in the sentences, that is, how likely they were to occur” They relied on single and double-center embedded relative clauses as shown in (1); the word (1) a. Single-embedded SR[GAP] guanyuaninvite tycoon DE official have bad intentions‘The official who invited the tycoon has bad intentions.’b. Single-embedded OR[fuhao yaoqing GAP] guanyuantycoon invite DE official have bad intentions‘The official who the tycoon invited has bad intentions.’c. Double-embedded SR[GAP[ GAP] fuhao] guanyuanxinhuaibuguiinvite conspire judge DE tycoon DE official have bad intentions‘The official who invited the tycoon who conspired with the judge has bad intentions.’d. Double-embedded OR[[fuhao yaoqing GAP] faguan] guanyuantycoon invite DE judge conspire DE official have bad intentions‘The official who the judge who the tycoon invited conspired with has bad intentions.’de or the following region  in single embeddings. In double embeddings , no significant differences were found at the first two words, but ORs (1d) were processed faster than SRs (1c) in the region containing the third and fourth words combined . The same pattern was found for the RTs in the fifth word, and the sixth word.The main results of their experiment were as follows. In single embeddings , the first two words in ORs (1b) were processed faster than the first two words in SRs (1a). No significant differences were found at The object preference in Chinese RCs that Hsiao and Gibson reported dramatically shrinks the space of theoretical explanations. Of the two classes of theories mentioned above, only the working-memory explanation can account for the object preference. The Chinese results appear to rule out the frequency-based account, and any other account predicting a subject-relative advantage, quite decisively. Hsiao and Gibson explain the results in terms of Dependency Locality Theory or DLT guanyuan in (1a) is more distant (with an intervening discourse referent fuhao) from the gap it is coindexed with than the head noun in the OR (1b) , the SR's head noun (1b) see . Integrayaoqing. Hsiao and Gibson assume that a verb-initial Chinese sentence presented out of context (as opposed to a discourse context that licenses a null subject such as a pro) should lead the reader to expect an RC structure, leading to three predicted heads: the object of the verb in the RC, the relativizer de and the predicate of the matrix clause. By contrast, in ORs (1b) the initial word is a noun, which leads the reader to predict only one head, an upcoming verb because this noun could be the subject of a matrix clause. Thus, at the first word, storage cost predicts that SRs are harder than ORs. This difference should continue right up to the relativizer de in single embeddings. As The storage cost metric can also explain the RC asymmetry. Storage cost at a given word is defined as the number of predicted heads The Hsiao and Gibson experimental materials and design have come under criticism from Lin and Bever Lin and Bever also argue that some of the verbs used in the Hsiao and Gibson experiment were ambiguous in the type of arguments they took, which could confound the results (presumably due to differing storage costs in ambiguous verbs). Lin and Bever then demonstrate through two experiments that Chinese SRs are easier than ORs, as in the case of virtually every other language examined.Lin and Bever's work was in turn criticized by Gibson and Wu In order to demonstrate the subject-relative advantage in subject-modifying cases, Gibson and Wu No differences were found between the subject and object relative at the first two words, although the SR condition was numerically slower.At the relativizer, they found significantly slower RTs in the SR than the OR, but this difference was not reliable in the by-items analysis.At the head noun, the SR was read significantly slower than the OR.Thus, compared to Hsiao and Gibson's study Although replication of published work is always desirable in experimental research, we felt it was particularly necessary to attempt a replication of studies regarding Chinese relative clauses. There are two reasons for this. First, unlike the case of comparable structures in English relative clauses, the existing reading studies on Chinese relative clauses show quite a lot of variability; second, we felt that some of the conclusions from previous studies could be problematic. We discuss both these points below.In subject relative advantage here, which directly contradicts the title of their paper, Chinese subject-relative clauses are more difficult to process than the object-relative clauses. Finally, we use total reading times at the head noun from the eyetracking study by Jäger and colleagues Some comments are necessary on how we extracted the estimated coefficients and standard errors from the various papers. Qiao et al  results , p. 177 For consistency regarding the unit of the dependent measure, we focus only on reading studies or studies that are meant to approximate reading comprehension . There is also evidence from event-related potentials studies such as The above summary of reading studies shows that previous work has reported a broad spectrum of results, from an object relative advantage of 123 ms to a subject relative advantage of 100 ms. This is in stark contrast to the English relative clause literature, where we do not see such variability for comparable structures. This uncertainty in previously published results makes it particularly important to attempt replications.A further motivation for attempting a replication is that the Hsiao and Gibson study and the Gibson and Wu study raise several worries. Regarding the Hsiao and Gibson study, as Hsiao discusses in her dissertation Another worry in this dataset is that the question-response accuracies for single center embeddings are unusually low, at 76% (OR) and 71% (OR) for single embedded relatives. This seems to be the only study on Chinese relative clauses with such low question-response accuracies. It is therefore possible that the participants were not really attending much to the sentences. The questions used in this experiment are not available with the paper or the dissertation, but the paper reports that the questions were not particularly demanding (p. 9): “The comprehension questions for target items questioned the content of the main clause or one of the RCs. For example, two possible questions for sentence (3a) would be “Did the official invite the tycoon?” (no) or “Did the official have bad intentions?” (yes). By contrast, in We also found surprising the fact that in the Hsiao and Gibson study, the question-response accuracy for double embedded object relative is approximately the same as the single embedded object relative; this is very surprising, given that Chinese double embeddings are much more difficult to process. Such a question-response accuracy pattern would be plausible if the comprehension questions were superficial. However, in the paper, it appears that questions were properly counterbalanced to target both the main clause and the relative clauses, as mentioned above. Given these potential issues in the original study, it is worth at least attempting to replicate the experiment results.The more recent Gibson and Wu results The central finding in that paper is evidence for integration cost at the head noun (they did not find any evidence for storage cost predictions). Gibson generously released the raw data, which allowed us to reanalyze their data following their analysis methodology . Estimating the variance of parameters, computing confidence intervals, and doing hypothesis testing depend on distributional assumptions being satisfied; this is one reason why the Box-Cox procedure is used in statistical data analysis. We have encountered the objection to transforming reading time data that we are no longer in the millisecond scale; however, most theories in psycholinguistics  do not make quantitative predictions about the magnitude of the effect. Rather, the predictions are only about the direction of the effect. In such a situation, the scale of the dependent measure does not play a critical role; the model assumptions do.As an aside, we note here that checking model assumptions is generally considered unnecessary in psycholinguistic research; most studies publish analyses on raw reading times, which almost always result in a violation of the normality assumption in residuals. The normality assumption is not checked because it is widely believed that statistical models are robust to violations of normality of residuals. While it is true that parameter estimation does not require distributional assumptions, and that in some cases the normality assumption can be relaxed, for the relatively large datasets used in psycholinguistics, hypothesis testing crucially requires that the normality assumption regarding residuals is satisfied. The belief in the unimportance of model assumptions has been strengthened by statements by leading statisticians, such as Gelman and Hill, who have written The above issues led us to attempt a replication of these studies. We began this investigation by carrying out a corpus count of relative clauses. This study was completed in 2006, as the diploma thesis of Gueilan Guo, “Predictability of Chinese Relative Clauses”; since 2006, several other corpus studies that have been published, among them Our corpus study serves to confirm the claims about the relative frequencies of subject and object relative clauses in Chinese. This is important as a starting point in order to determine whether the assumptions made about incremental structure building are correct. Then we present the three self-paced reading studies which present evidence consistent with a subject relative advantage. Our findings are consistent with the predictions of structural frequency-based accounts but not with working-memory based accounts.A central assumption in the Hsiao and Gibson paper was that seeing a verb-noun sequence sentence-initially would lead the reader to predict a subject relative clause. We carried out a corpus study to investigate what the most probable structure would be when a verb-noun sequence occurs sentence-initially.We searched a text corpus developed in Taiwan, the Sinica Corpus 3.0. This corpus, which contains 5 million words and is tagged for parts of speech, is a balanced corpus of texts ranging from 1990 to 1998.V NNNV de NThe corpus search yielded 639 sentences with the SR pattern These frequency patterns are consistent with Hsiao and Gibson's corpus search, which showed a higher frequency of SRs; there were 119 SRs and 45 ORs. If one restricts the noun phrases to animates only, 13 SRs and 3 ORs were found.Note, however, that the majority of the RC-like structures are not RCs, they merely have the same sequence of words as an RC. One consequence is that it is no longer obvious how the parser would use frequency information in incremental online processing. We spell out next the frequency-driven decisions of the parser.V N de NNde to form a possessive modifier of verb's inanimate object NV NFirst, consider SRs. Sequences like (2) a. tisheng [ [ qiye de ] jingzhengli ]increase company DE competitiveness‘To increase the company's competitiveness.’b. [[guyong yuangong] de] chengben…hire employee DE cost‘The cost of hiring an employee …’Neither of the structures in (2) is an SR and they are much more frequent than SRs. This has two implications for the predictions of the frequency account and the storage cost metric of the DLT.First, if frequency can arbitrate in incremental parsing decisions, when the first verb is processed in a putative SR, the parsing mechanism would not choose the (gapped) RC structure as the most likely parse. It may well be the case that a non-RC structure is posited. If this is correct, this goes against the storage cost assumption that Hsiao and Gibson Our study suggests that a non-relative clause could be the more likely prediction. Among V N de N sequences, when the first noun is animate, subject relatives occur with probability  see . If the de: it is more likely that the continuation is a non-relative.Second, once the animate first NP is seen in the SR, if a second NP is predicted at all it is more likely to be an inanimate NP in a non-RC structure: animate first NPs tend to be followed by inanimate second NPs  more often than by animate second NPs (13 instances that are RCs). The same situation holds at the relativizer The above two facts suggest that at the SR head noun, the comprehender would experience a surprise due to the falsified expectations that (i) the head noun is inanimate and (ii) the structure is a non-RC.Let us consider the situation in ORs next. In ORs, among all the sequences that had the same pattern as RCs, a majority involved inanimate nouns only ((3) chaye jinkou de shuliang…tea-leaves import DE quantity‘The quantity of imported tea-leaves…’chaye jinkou, ‘tea-leaves import’, and the de serves as a genitive modifier of the NP shuliang, ‘quantity’. The proportion of bona fide ORs was In these structures, the first NP was not the subject of the RC, but rather a compound noun de, functioning as a relativizer, is seen next, a non-RC structure is a more likely continuation than an RC. At the head noun, surprise should be experienced, due to two distinct factors: (i) there is a greater expectation for an inanimate NP rather than animate, and (ii) the structure is recognized for the first time as an RC.Here, as in SRs, once the comprehender sees the animate first NP and the following verb, the most favored expectation may not be a relative clause (this is consistent with Gibson and colleagues' assumption). Even when the character Thus, in the animate-only structures considered by Hsiao and Gibson, since SRs and ORs are ambiguous between relative and non-relative constructions up to the point when the head noun is processed, both SRs and ORs should experience processing slowdowns at the head noun, and part of this slowdown can be attributed to the unexpected animacy of the head noun  parallel? If strictly serial, only one parse possibility would be considered at each stage; the parser would be garden-pathed (however mild this garden-path may be) in both RC types until the head noun is processed. If we assume a serial parser, it is quite difficult to determine which RC type would be preferred based on structural frequencies. If the parser is ranked parallel, then the relatively high frequency of subject relatives could facilitate processing even in the pre-head noun regions, where the most probable structure is a non-RC. Although the serial versus parallel parser issue was never really resolved in the literature Under the ranked-parallel parsing view, the higher frequency of the SR structure (compared to OR) may well play a role even before the head noun is encountered. Even though an RC structure would not be the most likely upcoming structure, such a structure would be posited with a lower rank. Under these assumptions, the cost of predicting an SR would be lower than predicting an OR from the very beginning of the sentence, although this SR advantage may well express itself in later regions due to spillover. Thus, if building an SR structure is easier than building an OR, then at the head noun (and perhaps earlier) we would expect a subject-relative advantage .The next necessary step is to empirically investigate the object-relative preference in the context of the Hsiao and Gibson's and Gibson and Wu's results, and to contrast DLT's integration cost predictions with the alternative frequency based explanation.We turn next to three self-paced reading experiments that attempt to replicate the finding that object relatives are easier to process than subject relatives. All reading times were transformed  following the Box-Cox procedure; linear mixed models with varying slopes and intercepts were fit in all cases, and crossed varying intercepts were used for subject and item. Question response accuracies were analyzed using generalized linear mixed models with a binomial link function. The contrast coding in all cases was sum contrasts, with subject relatives coded as We used Hsiao and Gibson's materials from their paper for this experiment, comparing only single embedded subject and object relatives; see 4 for examples. The methods section below provides further details.(4) a. Single-embedded SR[GAP] guanyuaninvite tycoon DE official have bad intentions…‘The official who invited the tycoon has bad intentions…’b. Single-embedded OR[fuhao yaoqing GAP] guanyuantycoon invite DE official have bad intentions…‘The official who the tycoon invited has bad intentions …’The predictions made by the integration cost metric relate to an RT comparison between SRs and ORs at the head noun and possibly the region following it. We provide analyses for the regions consisting of the relativizer, the head noun, and the word following the head noun. The dependent measure is negative reciprocal reading time, and we fitted maximal linear mixed models in the sense of Barr and colleagues Subjects responded with 82.0% accuracy to all the questions. Neither the accuracies nor the question-response latencies are significantly different in the two relative clause types.In other words, this attempted replication of the claimed object-relative advantage is inconsistent with the predictions of the Dependency Locality or any other decay based account, such as the Lewis and Vasishth ACT-R based model. Rather, our results are consistent with explanations, such as the frequency-based accounts, that predict a subject-relative advantage.Next, we repeated the above result in Experiment 2 using different stimuli and participants. Given that we found a subject-relative advantage using Hsiao and Gibson's materials, it is clearly worth investigating whether we can obtain a subject-relative advantage in a second experiment with new items.This experiment used 24 sets of stimulus items that were all single-embedded RCs; the items we derived from experiment items used in Subjects responded with about 82% accuracy to all the comprehension questions after target items. Neither the accuracy nor question-response latencies was significantly different between responses to questions after SR items and those following OR items.The reading times for all regions are shown in For this experiment, we obtained the items and fillers of the Gibson and Wu paper Subject relatives had 89.3% correct responses, and object relatives had 87.7% correct responses; these differences, and corresponding question response latencies, did not reach significance.The mean reading times for the various regions are shown in At the relativizer, we see an object-relative advantage; this is inconsistent with the predictions of the frequency-based accounts but it is also inconsistent with the predictions of the storage and integration accounts developed in We also combined the original dataset of Gibson and Wu with our own replication ; the dependent measure was negative reciprocal reading time. We investigated the processing of subject and object relatives in Chinese, and demonstrated that two of the three experiments we carried out are inconsistent with the claim that Chinese relative-clause processing difficulty can be explained by dependency distance. Distance-based accounts predict increased difficulty in SRs at the head noun: in the Dependency Locality Theory subject relative advantage at the head noun in single embeddings (just as we did in Experiment 1), but their result did not reach statistical significance. The OR advantage was seen only in double-embedding items, and there the effect was found either at non-identical regions (noun versus verb), or at the second de, which was preceded by different words (noun or verb), which may lead to spillover effects at de . Why are we unable to obtain the same results as Gibson and colleagues in experiments 1 and 2? Apart from our earlier observations about their two experiments, which we think could have introduced sources of bias in their analyses and data, a fact that seems to have gone unnoticed in the literature is that the original Hsiao and Gibson study Nevertheless, a skeptical reader would be completely justified in concluding that our own studies' results may simply be a consequence of random variability (or bias on our part). Indeed, we have encountered reviewer comments to the effect that the published results show effects of both types (subject- or object-relative advantage), and the present work does not clarify the issue at all.But it is true that the previous work and ours provides no information at all about the facts about Chinese? There is a quantitative answer to that question. We can ask ourselves: what should the researcher believe given the data? That is, what is the probability of there being a subject-relative advantage given the data? A bayesian meta-analysis that synthesizes our knowledge about Chinese relative clauses would allow us to formalize our prior belief, and, more importantly, the extent to which our belief should shift given new data.Bayesian random effects meta analysis a well-known methodology in medicine and other areas and is used primarily for evidence synthesis We describe the random effects model next. Let Then, our model is:The variance parameter Plausible values of the subject/object relative clause advantage can be assumed to range between −300 and 300 ms. But we will assume three different levels of uncertainty: The 95% range is (a) We therefore use three priors for All available data were used for the analysis, including those from the present study. The modeling results are summarized in The central insight from this analysis is that the data do not lead us to believe strongly in the object relative advantage; indeed, given the data, the probability of Chinese having a subject-relative advantage is approximately 78–80%. In other words, if one believes that there is any difference at all between subject and object relative clause processing times at the head noun in Chinese, given the data one's belief should be in favor of a subject-relative advantage. One reaction to this analysis might be that a summary of the means in In sum, given the data that are currently available to investigate this issue, although it is certainly not as clear as in comparable sentences for English, the evidence weighs in favor of the subject-relative advantage. Importantly, it is not the case that the evidence available so far regarding Chinese relative clause processing is completely unclear.why subject relatives are more frequent than object relatives—surely the answer to that question would be the real explanation, not frequency, which is merely a side-effect of the preference to produce subject relatives. Thus, the frequency account is not really an explanation, but rather begs the question: why do subject relatives occur more often? As a reviewer (Roger Levy) points out, one reason that subject relatives may be more frequent is that, given that relative clauses serve to fix reference, it may be easier to fix reference by mentioning the action that an (animate) entity does than mentioning an action that is done to an entity. A related possibility is that subjects may simply be more accessible than objects We discuss here some possible explanations for the SR advantage in Chinese. The evidence from experiments 1 and 2 is consistent with frequency based explanations: the subject-relative occurs more frequently, and therefore it is easier to access that structure when a choice point is reached. This idea can be formalized within the expectation-based account of Levy Although our data do call into question dependency distance-based explanations of relative clause processing in Chinese, we do not claim that dependency distance plays no role in language processing in general or even in relative clause processing in general. Previous work on various languages with SVO order has shown clear evidence for locality effects e.g., ; we evenA final point about the role of working memory accounts in Chinese relative clause processing: Gibson and Wu In contrast, it has recently been observed that there is a reverse pattern of difficulty in Chinese: some Chinese aphasic patients cannot reliably answer comprehension questions about SRCs, although they perform well on ORCs It is true that some aphasic patients cannot reliably answer comprehension questions about SRCs. But the number of such patients is small: only two out of the six patients examined by Su and colleagues in one of two versions of their experiments showed this pattern see . In the Finally, it should not be forgotten that our work is also open to challenge. For example, it is quite possible that the local ambiguities inherent in the sentences in experiments 1 and 2 are confounding the true underlying pattern . A document certifying this is attached. Subjects did not have to sign an informed consent form because the data were collected in an anonymized manner.60 university students in Taiwan were paid 200 TWD for taking part in this self-paced reading experiment. The average age was 20 and all participants were native speakers of Mandarin Chinese using traditional characters.xinhuaibugui ‘have bad intentions’. However, a predicate-modifying adverb (i.e. hen ‘very’) could also appear at this position in other stimulus items. This arrangement prevented the sentence wrap-up effect from occurring in the verb.Experiment 1 used twenty sets of single-embedded RC items published in (5) a. Single-embedded SR[GAP] guanyuaninvite tycoon DE official have bad intentions…‘The official who invited the tycoon has bad intentions…’b. Single-embedded OR[fuhao yaoqing GAP] guanyuantycoon invite DE official have bad intentions…‘The official who the tycoon invited has bad intentions …’In addition to the stimulus items, 50 fillers with varying syntactic structures were randomly interspersed between items, with the constraint that at least one filler intervened between two items. Both items and fillers were presented in traditional Chinese characters.http://tedlab.mit.edu/sijia zhentan “private detective” and xiju yanyuan “comedy actor” (in items 11 and 12 of The experiment used the non-cumulative self-paced moving window method lmer function in R) In all experiments, a linear mixed model (the 61 college students in Dalian, China with an average age of 20 were each paid 15 RMB for taking part in Experiment 2. All participants were native speakers of Mandarin Chinese using simplified characters.Experiment 2 had a 2Experiment 2 had the same procedure as Experiment 1. The Linger software recorded RTs at each word of a presented target sentence as well as the answer to a following yes/no comprehension question.40 college students in Dalian, China were each paid 15 RMB for taking part in Experiment 2. All participants were native speakers of Mandarin Chinese using simplified characters. Experiment 3 was a replication of


The role of the collagen-platelet interaction is of crucial importance to the haemostatic response during both injury and pathogenesis of the blood vessel wall. Of particular interest is the high affinity interaction of the platelet transmembrane receptor, alpha 2 beta 1, responsible for firm attachment of platelets to collagen at and around injury sites. We employ single molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) to study the interaction of the I-domain from integrin alpha 2 beta 1 with a synthetic collagen related triple-helical peptide containing the high-affinity integrin-binding GFOGER motif, and a control peptide lacking this sequence, referred to as GPP. By utilising synthetic peptides in this manner we are able to study at the molecular level subtleties that would otherwise be lost when considering cell-to-collagen matrix interactions using ensemble techniques. We demonstrate for the first time the complexity of this interaction as illustrated by the complex multi-peaked force spectra and confirm specificity using control blocking experiments. In addition we observe specific interaction of the GPP peptide sequence with the I-domain. We propose a model to explain these observations. This firm attachment is required for the collagen to interact with the low affinity GpVI receptor which is mainly responsible for the collagen induced platelet activation. After platelet activation plasma fibrinogen and VWF may then bind to the αIIbβ3 integrin receptors of adjacent platelets, securing the platelet plug in place and preventing more blood loss.The interaction of collagen with platelets plays a pivotal role in the haemostatic response ,2. DurinIf any component of this sequential process is either absent or defective, there may be catastrophic consequences. For example, Bernard–Soulier syndrome is a bleeding disorder caused by a deficiency of GpIb receptors on platelets, and von Willebrand disease, characterised by a tendency to bleed after minor trauma, is caused by a lack of functional VWF. Other problems arise when arteries develop atherosclerotic plaques, which cause endothelial cell surfaces to become activated, initiating platelet attachment, platelet activation and thrombus formation. In some cases the plaque ruptures, revealing collagens which recruit and activate nearby platelets as described above. If thrombus formation occurs in vital blood vessels, such as the coronary artery or cerebral vasculature, then a heart attack or stroke may occur. It should be clear therefore that a thorough understanding of all these processes is a prerequisite for any potential treatment or prevention strategy.2β1, and in particular the α2 I-domain which has been shown to recapitulate the adhesion properties of the entire receptor n host sequences which impart triple helical structure to the specific, guest, sequence. These studies have identified the GFOGER peptide as being a high-affinity ligand for α2β1 5GFOGER[GPP]5GPCG. A high affinity form of the I-domain, locked open I-domain (LOI), was prepared as detailed previously [HS-.Template-stripped gold substrates were prepared using a similar process to that described by Wagner et al. ,35. BrieCovalent attachment of the I-domain and the triple helical peptides  was achieved using a similar protocol described recently . Both thSubstrate: Freshly stripped TSG samples were functionalised by incubation in 200 μL of 1mM APA in ethanol for 1 h. Samples were then washed in ethanol and dried under a stream of nitrogen. Samples were then incubated in 200 μL of 2mM crosslinker in PBS (diluted from 100mM stock in DMSO) for 1 h followed by washing using PBS to remove any unreacted crosslinker. Finally the samples were allowed to react with 200 μL of 1 μM of CLOI in TBS for 1 h. Substrates were then washed using TBS and care taken to ensure that the proteins were not dehydrated.Cantilevers: A 100 μL solution containing 0.1mM APA and 0.9mM APH (10% APA/APH ratio) was added to one well of a 96-well plate, before carefully submerging a gold coated cantilever. After incubating the lever for 1 h it was carefully washed in ethanol and allowed to dry evaporatively. Next, 100 μL of 2mM crosslinker in PBS (diluted from 100mM stock in DMSO) was added to a fresh well and the lever again carefully submerged. After a further hour the tip was thoroughly washed in PBS and any excess buffer wicked away from the chip using cleanroom wipes. Finally, 100 μL of either 4.5 μM GFOGER or GPP in TBS was added to a new well and the cantilever again submersed. After 1 h the cantilever was then washed with copious amounts of TBS to remove any unbound molecules.−1, consistent with nominal values quoted by the manufacturer. Calibration of gold coated cantilevers was always performed before any chemical modification.A PicoPlus AFM with a PicoSPM II controller from Molecular Imaging was used for all force spectroscopy work. Cantilever spring constants were estimated using the Sader Method  typicallkPEG. Thus the effective spring constant was determined by keff = (kPEG−1 + kc−1) −1, where kc is the cantilever spring constant and the loading rate found from r = keffv, where v is the retraction speed.All force spectroscopy measurements were conducted in a fluid cell in PBS. Modified tips were repeatedly approached towards and retracted away from the modified substrate in a cyclic manner whilst simultaneously monitoring the deflection signal using a four quadrant photodiode. Amplitudes were typically 200nm with a frequency of 1 Hz. Approach-retract cycles were performed 1000 times with each tip for each experiment across five different areas of the substrate. Typically an uninhibited experiment would be conducted first, followed by a blocking experiment in which free avidin was introduced into the fluid cell. Force-distance cycles were analysed using purpose written software created using MATLAB version R2007a . Briefly, an algorithm first identifies any data points that deviate significantly from the rest of the data by comparing the gradient between successive points to the standard deviation of the gradient of all points. The user then chooses an event if it is preceded by a non-linear force-displacement retraction profile that is unimpeded by any other interaction. If no such event meets this criterion, the data set is discarded. Empirical Probability density functions ,37 were 2β1 I-domain and a triple helical collagen peptide with the GFOGER binding motif using the technique of single molecule force spectroscopy. As a comparison we also measured the interaction of the I-domain with a GPP peptide sequence that lacked the guest residues. We utilised a recently developed protocol for attaching both the I-domain and the peptides to substrate and tip respectively. Control experiments in which the peptides were blocked with free I-domain were also conducted. A decrease in unbinding probability when blocking from 20% to 14% for the GFOGER interaction and from 8% to 2% for the GPP interaction indicated that specific interactions were being measured. However, although the unbinding probability was reduced when comparing the uninhibited GFOGER and GPP, it was not completely eliminated. Indeed, force-displacement curves exhibiting the characteristic delayed, non-linear retraction profile for the GPP interaction were indicative of specific unbinding. We therefore proposed a model to describe these observations in which the GFOGER peptide may interact with the I-domain not only via the hexapeptide sequence, but also at any point along the host GPP residues. Such a model would explain the observation of a multitude of force-displacement curves exhibiting both single/multiple simultaneous unbinding events as well as sequential rupture profiles. Such an array of unbinding is manifested by the complex multitude of small sub-peaks superimposed on the broad force spectrum for both the GFOGER and GPP interactions. Such observations are compatible with previous adhesion assay measurements in which it was found that the adhesion of the I-domain was much greater with GFOGER than with the GPP sequence, since we are probing only a single point on the dynamic force spectrum.We have, for the first time, measured the interaction between a recombinant form of the αWe believe the application of the recently developed protocol for the tethering of both the recombinant form of the I-domain and the peptide sequences for use in single molecule force spectroscopy is a novel approach. The significance of the GPP residues in this particular interaction under these particular conditions has also not been reported previously.


The Swedish salmonella control programme covers the entire production chain, from feed to food. All salmonella serotypes are notifiable. On average, less than 20 cases of salmonella in food-producing animals are reported every year. In some situations, the cases would be expected to cluster geographically. The aim of this study was to illustrate the geographic distribution of the salmonella cases detected in pigs, cattle and sheep.Data on all herds with pigs, cattle and sheep found to be infected with salmonella during the time period from 1993 to 2010 were obtained from the Swedish Board of Agriculture. Using the ArcGIS software, various maps were produced of infected herds, stratified on animal species as well as salmonella serotype. Based on ocular inspection of all maps, some were collapsed and some used separately. Data were also examined for temporal trends.No geographical clustering was observed for ovine or porcine cases. Cattle herds infected with Salmonella Dublin were mainly located in the southeast region and cattle herds infected with Salmonella Typhimurium in the most southern part of the country. Some seasonal variation was seen in cattle, but available data was not sufficient for further analyses.Analyses of data on salmonella infected herds revealed some spatial and temporal patterns for salmonella in cattle. However, despite using 18 years' of data, the number of infected herds was too low for any useful statistical analyses. Salmonella spp. in feed, animals or food of animal origin are notifiable and action is always taken to eliminate the infection. Whenever salmonella is isolated, restrictions on animal movements and manure are immediately put on the farm and a prompt investigation and trace-back of the infection is performed. An eradication plan is instituted by an official veterinarian and approved by the Swedish Board of Agriculture. The herd is not declared free from infection until all animals in the herd are negative in two consecutive faecal samplings one month apart, and adequate cleaning and disinfection have been completed. Culture is the detection method of choice for most samples from animals, as this is independent of serotype and has a high specificity, both important features for the Swedish situation. However, culture from faecal material has a low sensitivity [The Swedish National salmonella control programme covers the entire production chain, from feed to food . The salsitivity  and may Regular sampling of pig and cattle carcasses is performed at slaughterhouses. Furthermore, random lymph node samples are taken at slaughter. No regular sampling is performed in herds with pigs or ruminants, but a voluntary surveillance programme is in place for breeding pigs. Due to the pyramidal structure of the commercial pig production, about 60-65% of all slaughtered pigs descend directly from these breeding herds. Clinical or post-mortem suspicion of salmonella infection is notifiable for all veterinarians, who must take relevant samples in case of such suspicions. Salmonella samples are also taken at most post-mortem investigations of cattle and pigs.Only a few cases of salmonella in food-producing animals are reported every year . In someData on all herds with pigs, cattle and sheep found to be infected with salmonella during the time period from 1993 to 2010 were obtained from the Swedish Board of Agriculture (SJV).A herd was considered a case if salmonella was isolated from at least one faecal sample in the herd. Each holding was only included once even if investigations revealed that several animal species were infected. The case species was set to the animal species from which Salmonella was first identified, and thus regarded as the species of the herd. In the studied period there were a total of 267 holdings where salmonella was isolated from the animals. One large feed-borne outbreak involving 31infected pig herds occurred during the study period and only the first case of this outbreak was kept in the dataset (i.e. 30 of these herds were excluded) [Salmonella spp. as well as separately for the most common serotypes. Furthermore serotypes were grouped into cattle-, pig- and sheep-adapted types  and "other" representing mainly feed-associated serotypes, and summarised by animal species. Finally, analyses were also done for serotypes associated with small passerine birds (S. Typhimurium DT U277 and DT40) [Analysis of the data was done for all nd DT40) ,9.Because the exact coordinates of each farm from the earlier years were not available, and such detailed resolution was not deemed necessary, postal codes were used for geographical reference. A few of the postal codes had been changed since the data was first collected. Based on the address or if that was not possible, the municipality, new postal codes were obtained.Using various techniques in the software ArcGIS 10 , each case has been attributed with geographical coordinates representing the corresponding geometric centres, centroids, of the 5 digit postal code areas. Information for all cases has been stored in a geodatabase. From this geodatabase separate animated map series were generated for all serotypes or groups of serotypes isolated from pigs, cattle and sheep respectively. The maps were generated for each year and for each salmonella serotype, and subsequently amalgamated into versions conveying relevant information.Ocular inspection of the maps was made before deciding what maps would be used separately and what maps could be collapsed into one.In order to relate the distribution of cases to the geographical distribution of the animal populations, maps illustrating the animal density per 3 digit postal code area were created. For cattle, this was based on the average number of animals > 1 year of age on each holding in Sweden in 2008, as registered in the national database of individual cattle (Swedish Board of Agriculture). For pigs, the number of animals on each holding was based on data retrieved from the Swedish Board of Agriculture and the Swedish Animal Health Service in a previous study .Salmonella Typhimurium phage type 40 are shown in figure The total numbers of infected herds identified during 1993-2010 in different animal species are shown in figure Salmonella Dublin in cattle were located mainly in the southeast region of Sweden (Figure Salmonella Typhimurium cases were in the very south (Figure The cases of n Figure , while th Figure  and the h Figure . The oveh Figure .The cases in sheep herds were too few to observe any geographical clustering Figure .Seasonal variation was not investigated for sheep due to the low number of infected herds, and there was neither indication nor reason to suspect seasonal variation in pigs. Some seasonal variation was seen in cattle Figure , but avaThe species where Salmonella was first identified determined the species assigned to the herd. This may not be the same as the species that was first infected and cattle herds may be overrepresented. Complete data on how many herds that had more than one animal species infected was however not available.By establishing a geodatabase for all case data it has been possible to generate map series from almost any point of view. This facilitated the analysis. The data and the map documents generated can easily be published on a web map server giving access for others to share the results, but also for alternative analysis. The software allows for spatial statistical analysis of the gathered data. In this case, visual inspection of the generated maps proved to be enough to make the conclusions above. How GIS can add new dimensions to epidemiological work has clearly been demonstrated in other studies ,12.As the salmonella prevalence in Sweden is low, spatial and temporal analyses of data on infected herds might reveal insights not detectable in endemic regions. However, the low prevalence situation meant that full spatio-temporal analyses were not useful due to the number of herds being too low. Ocular inspection of the resulting maps and diagrams still indicate the presence of some spatial and temporal patterns. For instance, the detection of salmonella infected cattle herds appears to peak in September. The majority of cattle herds are detected through tracing form other infected herds, at necropsy or due to clinical symptoms . It coulS. Dublin or S. Typhimurium may be a reflection of the clustering of cattle movements in Sweden [S. Dublin, which is almost exclusively associated with cattle. The geographical clustering is also more pronounced for S. Dublin than for S. Typhimurium (figures The geographical clustering of cattle herds infected with n Sweden , where tn Sweden  and the  figures  and 6, tAnalyses of data on salmonella infected herds revealed some spatial and temporal patterns for salmonella in cattle. However, despite using 18 years' of data, the number of infected herds was too low for any useful statistical analyses and no patterns could be observed in pigs or sheep.Lars Skog is employed by ESRI, the company that produces the GIS software used in the study. He is conducting his PhD studies in the Royal Institute of Technology, Geoinformatics in Stockholm, on the use of GIS. Except for this, the authors have no competing interests.All authors participated in the design of the study. HW and SSL performed the data collection and data cleaning. LS produced the maps of infected herds and JF produced the population density maps. SSL wrote the manuscript and produced the graphs and table. All authors read and approved the final manuscript.


Our studies suggest that the perturbed orientation of talin relative to the membrane in the F2 mutant would be expected to in turn perturb talin/integrin interactions.Integrins are cell surface receptors crucial for cell migration and adhesion. They are activated by interactions of the talin head domain with the membrane surface and the integrin β cytoplasmic tail. Here, we use coarse-grained molecular dynamic simulations and nuclear magnetic resonance spectroscopy to elucidate the membrane-binding surfaces of the talin head (F2-F3) domain. In particular, we show that mutations in the four basic residues  in the F2 binding surface reduce the affinity of the F2-F3 for the membrane and modify its orientation relative to the bilayer. Our results highlight the key role of anionic lipids in talin/membrane interactions. Simulation of the F2-F3 in complex with the  ► Simulations and NMR define the interactions of the talin head domain with membranes ► Talin binds to anionic lipids in a bilayer via the F2 subdomain ► Mutations in the F2 lipid-binding site may perturb talin/integrin interactions Integrins are heterodimeric cell surface receptors that link the exterior and interior of a cell. They are central components of focal adhesion complexes that form during cell migration, and are crucial for a variety of signal transduction events such as proliferation, survival/apoptosis, differentiation, and gene expression . They arTalin consists of a rod domain  and a heRecent experimental studies have revealed structures for talin integrin tail complexes  and for Here, we investigate the interactions between a lipid bilayer and talin, and attempt to identify their role in integrin activation by combining experimental and computational techniques. In particular, nuclear magnetic resonance (NMR) and coarse-grained molecular dynamics (CG-MD) simulations are used to explore the position, orientation, and interactions of wild-type and mutated  talin2 F2-F3 and F2 domains , with di1H-15N-HSQC NMR spectra. Three different protein concentrations were used  liposomes , no 1H-15N-HSQC spectrum was observed, i.e., SNR ≈ 0 implying that F2-F3-WT bound tightly to the liposomes. However, it was possible to get an 1H-15N-HSQC spectrum (SNR ≈ 0.8)  B from F2sn-glycero-3-phospho-L-serine) liposomes. In this case SNR ≈ 0 for both the WT and the 4E mutant. This indicates that the strength of interaction of F2-F3 is increased as the fraction of anionic lipid is increased. In vivo the fraction of anionic lipid in the inner leaflet of mammalian plasma membranes is ∼20 mol % , implying a strong F2-WT interaction with the liposomes. In contrast, in the 1H-15N-HSQC spectra of the F2-4E with and without DMPS liposomes, the shift induced in the peaks was very small  is lower than the SNR for F2-4E (0.92) and for F2-F3-4E with 20% DMPS/PC (0.84). Given the qualitative nature of the results, this suggests that the interaction of the F2 domain with DMPS liposomes can be classified as “strong.” In contrast, SNR for the interaction of the F2 with 20% DMPS/PC liposomes is larger for both F2-WT and F2-4E than for the other systems. Therefore the interaction of F2 with 20% DMPS/PC can be classified as “weak.” By similar criteria, the interaction of the F2-F3 with 20% DMPS/PC liposome can be classified as “intermediate” and with DMPS as “very strong.” It is important to note that there is therefore some residual interaction of the protein with the bilayer for the F2-F3-4E mutant. This may reflect either nonproductive binding of F2 (see below) and/or interaction of F3 with the bilayer.We also explored the interaction of the WT and F2-4E domains with liposomes. For the F2-WT domain in the presence of DMPS liposomes no spectrum was observed at any protein concentration  lipid bilayer. Five simulations were run with the F2-F3 domain initially displaced 100 Å away from a bilayer  is consistent with the published F2-F3-β1D complex structure A. In conWe also compared the interaction of the isolated F2 WT domain and 4E mutant with a preformed POPC/POPG bilayer. The F2 WT bound to the bilayer in all five simulations carried out. The preferred binding surface (appearing in three of five simulations) C was conWe have also carried out control simulations (for both F2-F3 and for F2) in which we made the quadruple K258E, K274E, R276E, and K280E mutation but set the charge on the four glutamate side chains to zero (this may be viewed as an approximation to setting these four side chains to a protonated state). For both F2 and for F2-F3 this yielded only nonproductive complexes , but with a different orientation from the preferred orientation in the wild-type. Thus the K/R motif in F2 is needed for the productive binding mode of the protein to be adopted.Together, these four sets of simulations suggest a strong electrostatic interaction between the basic surface of the F2 subdomain and anionic lipid headgroups. The pattern of interacting lysine and arginine residues is conserved between the F2 and F2-F3 simulations and residues L262, D263, E266, Q275, G277, and R281 form the preferred surface made the largest number of contacts in both simulations.We wished to extend our analysis of talin/bilayer interactions to include the possible reciprocal effects of the interaction of talin F2-F3 with the TM domain and intracellular juxtamembrane region of integrin β. This was possible because of the known X-ray structure  of F2-F3 bound to the β1D TM helix and tail (Protein Data Bank [PDB] 3G9W). We therefore took the F2-F3-β1D structure (converted to a CG structure) and positioned it in a preformed POPC/POPG bilayer with the β1D TM helix oriented such that the positively charged patch of F2-F3 interacted with the membrane surface; this resulted in the TM helix spanning the bilayer with a tilt angle of ∼15°. This F2-F3-β1D/bilayer complex was then simulated for 0.5 μs D. DuringThe F2-F3/lipid interactions in the pre-inserted F2-F3-β1D simulations were compared . For thi3G9W) (2K9J) (We extended our study to a self-assembly simulation  of lipid3G9W) , based o) (2K9J)  of the α) (2K9J) . Note thTo test the effect of the 4E mutations on the F2 binding surface, the self assembly simulations were repeated for F2-F3-4E-αIIb-β1D. Ten separate CG-MD self-assembly simulations were carried out. However, in none of them did F2-F3-4E reach a final binding surface similar to the “correct” binding surface observed in the WT simulations of F2, F2-F3, and of F2-F3-αIIb-β1D. Thus the self-assembly simulations on the complete complex support the picture obtained from NMR and simulation studies of the talin subdomains that electrostatic interactions between the surface of F2 and anionic lipids drive the talin/integrin interaction with a cell membrane.A combination of experiment and simulation has revealed the nature of the interactions of the talin F2-F3 domain with a lipid bilayer membrane. We have defined residues within the F2 domain that play a key role in interactions with negatively charged lipid headgroups of the bilayer. Thus, four mutations  in the binding surface of F2, which switch the net charge from +4 to −4, alter the nature of the interactions between talin and the bilayer. In the presence of these mutations the F2-F3 domain binds the membrane with a modified orientation that may be anticipated to perturb the interaction of the two TM helices of the integrin heterodimer.Using NMR to characterize the interactions between talin and variously charged liposomes, it has been demonstrated that negatively charged headgroups in the bilayer promote the binding of talin to the membrane. Analysis of the simulations confirms this result because the number of contacts between either the F2-F3 domain or the F2 domain and the anionic headgroups of POPG is consistently larger than the number of contacts with the zwitterionic headgroups of POPC. In particular, the interactions of the F2-F3 domains were 3-fold higher with POPG/POPC than with POPC alone. In addition, the presence of F3 in combination with F2 seems to enhance the binding of talin to the membrane. Thus, in the presence of the F3 domain the protein is capable of binding to 20% anionic liposomes in the NMR studies, unlike the F2 subdomain alone. Therefore, anionic lipids in the bilayer and the presence of the F3 domain alongside the F2 domain are two key factors that enhance the interaction of talin with a membrane.This is of likely mechanistic significance. It has been suggested  that theThe surface of F2-F3 that binds to the membrane consists of a stretch of basic and acidic residues. Residues K258, K274, R276, and K280 appear to make a substantial number of contacts with anionic lipids in the simulations explaining why mutations in these residues disrupt interaction with the membrane. This binding surface is consistent with NMR  and receThis study raises an interesting issue of relevance to ongoing efforts in integrative structural biology. It might be contended that visualization alone could reveal the likely bilayer interaction surface of F2-F3. However, such an approach simply enables one to formulate a hypothesis that may be subsequently testing using simulation. Indeed, a more rigorous computational approach to evaluation of structural hypotheses is essential if one is to use the outcomes of integrative structural biology studies  as platfFrom a more methodological perspective, this study demonstrates that CG-MD simulations may be used to predict the position and the orientation of a protein domain bound to a lipid bilayer. This builds on previous CG-MD studies of integral membrane proteins  and of mOf course, there are limitations to the accuracy of CG-MD simulations . To impr15N-labeled F2, F2-4E, and F2-F3 domains were expressed as GST-fusion proteins in M9 minimal media using an 15N source. Cells were harvested by centrifugation and resuspended in phosphate-buffered saline. Lysozyme (1 mg/mL), 1 M MgSO4 (10 mL/mL), and 1 mg/mL DNase1 solution (10 μL/mL) were added. After three freeze/thaw cycles, 10% Triton was added and the sample was centrifuged at 40,000 × g for 10 min at 4°C. A glutathione-Sepharose 4B (Roche Applied Science) column was set up for purification and the protein was eluted in fractions of 15 mM glutathione in 50 mM Tris (∼pH 5.8). Glutathione S-transferase fusion protease 3Cpro was added to cleave the purified fusion protein. The protein was further purified by gel filtration chromatography into NMR buffer . The F2-F3-4E domain was expressed as a His-tag fusion protein and purified as mentioned above, but with Talon resin (Takara Bio) in 50 mM sodium phosphate, 300 mM NaCl, 0.035% β-mercaptoethanol, pH 7.0 buffer. TEV protease was used to cleave the polyhistidine tags and gel filtration chromatography into pH 7.0 NMR buffer was used for further purification.U-sn-glycero-3-phospho-L-serinenad and PC is L-α-phosphatidylcholine  with predominant species 16:0/18:1, DMPS and PC lipids (in chloroform) were mixed at the required ratio. The chloroform was removed using a stream of argon gas and the sample was attached to a vacuum pump overnight. One milliliter of 50 mM phosphate, 100 mM NaCl, and 1 mM DTT was added to the liposomes and the sample was agitated. At 40°C the sample was rotated for 1 hr. The large multilamellar vesicles (LMVs) were disrupted by five freeze-thaw cycles and an Avanti mini-extruder kit was utilized to extrude the sample to size the liposomes, following the protocol specified on the Avanti web site (http://avantilipids.com/) that states that unilamellar liposomal suspensions with a low polydispersity can only be prepared with membranes having a pore size of ≤200 nm. Accordingly, the suspension was extruded through a 100-nm polycarbonate membrane. The sample was maintained at a temperature of 40°C . The sample was extruded back and forth 20 times. Vesicle suspensions were stored for a maximum of 3–4 days at 4°C to avoid any significant change in vesicle size distribution. The DMPS liposomes were constructed by using the same procedure as described above.To construct the DMPS/PC 1:4 mol %, where DMPS is L-α-phosphatidylcholine/1,2-dimyristoyl-15N-labeled protein in 5% D2O/95% H2O. All samples were buffered with 50 mM phosphate and 100 mM NaCl. In addition, 5 mM DTT was added to prevent oxidation. The samples with liposomes contained 10 mM lipids. All samples were set to pH 6.5. For each sample a two-dimensional heteronuclear single quantum coherence (HSQC) spectrum was acquired. The resonance assignments of the F2 domain were made using three-dimensional (3D) gradient-enhanced f-NOESY-HSQC (τm = 100 ms) and {1H-15N}-TOCSY-HSQC (τm = 70 ms) spectra. The NMR assignments of the F2-F3 domains and F2 domain that were used in this study have been submitted to the Biomagres Bank: F2 (196-309), BMRB ID = 16930 F2-F3 (196-405), BMRB ID = 169n32. NMR experiments were processed using NMRpipe (www.cgl.ucsf.edu/home/sparky). The ratio SNR that characterizes the strength of the interactions, was calculated using the equationS is the signal to noise ration for each spectrum. The signal to noise was calculated using NMRPipe and Sparky (www.cgl.ucsf.edu/home/sparky). Individual peak SNR values were calculated using the signal/noise ratio of the peak concerned, with and without liposomes. The overall SNR value for a particular condition, was calculated as the mean of the individual peak values ± the standard deviation. For the SNR calculations, 106 and 201 peaks for F2 and F2-F3, were used respectively.NMR experiments were carried out at 40°C on 600 MHz NMR spectrometers with Oxford Instruments superconducting magnets and GE Omega consoles. All experiments with both wild and mutated F2-F3 and F2 constructs with and without liposomes were carried out using samples of 75, 100, and 150 μM of U- NMRpipe  and visu3G9W) and the structure of the αIIbβ3 complex (PDB 2K9J) were converted to a CG representation (2)   between 2); production simulations were run for 0.5 ns and 1 μs respectively for F2 and F2-F3. Five simulations were carried out for each system with all simulations starting from the same configuration but with different initial velocities. The wild-type and the mutated protein simulations of each system started from the same initial configuration. In the self-assembly simulations, POPC and POPG lipids at a ratio of 3:2 (154 POPC and 102 POPG) were randomly positioned in a cubic box along with the protein, CG water particles, and counterions. The simulation was run for 200 ns after an initial 400 steps of energy minimization.In the CG-MD simulations with the preformed POPC/POPG bilayer, the F2-F3 or F2 domain was displaced 100 or 80 Å away from a preformed bilayer containing POPC and POPG at a ratio of 3:2 (154 POPC and 102 POPG molecules). Each system was solvated with CG water particles and with CG sodium particles to neutralize the system. The system was energy minimized, and after 5 ns equilibration   . A Berenacs.org)  was used via VMD .


When competing for binding to the Grb7 SH2 domain in a complex, one synthesized nonphosphorylated ligand, containing the twenty-two amino acid-motif sequence, showed at least comparable affinity to the phosphorylated ligand of ErbB3 in vitro, and its overexpression inhibited the proliferation of SK-BR-3 cells. Such nonphosphorylated peptides may be useful for rational design of drugs targeted against cancers that express high levels of Grb7 protein.The Grb7 (growth factor receptor-bound 7) protein, a member of the Grb7 protein family, is found to be highly expressed in such metastatic tumors as breast cancer, esophageal cancer, liver cancer, etc. The src-homology 2 (SH2) domain in the C-terminus is reported to be mainly involved in Grb7 signaling pathways. Using the random peptide library, we identified a series of Grb7 SH2 domain-binding nonphosphorylated peptides in the yeast two-hybrid system. These peptides have a conserved GIPT/K/N sequence at the N-terminus and G/WD/IP at the C-terminus, and the region between the N-and C-terminus contains fifteen amino acids enriched with serines, threonines and prolines. The association between the nonphosphorylated peptides and the Grb7 SH2 domain occurred  Grb7 protein is associated with amplification and invasion of many solid cancers, including those of the breast The SH2 domain plays vital roles in signaling pathways by acting as adaptors, kinases or scaffolds SH2 domains also associate with phosphotyrosine-independent motifs. For instance, the T/S-x-x-x-x-V/I (x represents any amino acid) motif is recognized by the SAP/SH2D1A SH2 domain The Grb7 protein family, identified through cDNA expression libraries encoding phosphotyrosine receptor targets d) of the G7-18NATE peptide for binding to the Grb7 SH2 domain is approximately 13.2 µM, and that of the ErbB3 pY(1180) ligand is approximately 1.15 µM Many drugs are designed to target the SH2 domain of Grb7. Krag and colleagues have identified a nonphosphorylated peptide, designated the G7-18NATE, that binds to the SH2 domain of Grb7. The dissociation constant , the C-GIP deletion mutation (10BM) and the both N-GIPT and C-GIP deletion mutation (10CM). None of the three mutations were recognized by the Grb7 SH2 domain in the yeast two-hybrid system. This indicates that the peptide requires the conserved residues at each end to recognize the Grb7 SH2 domain.The SH2 domain of Grb14, which is another member of the Grb7 protein family, was also used to screen the random peptide library with yeast two-hybrid analysis. However, no positive clone was identified. The nine clones  that bouThe 10, 41 and 10CM sequences were fused to the mRLUC-3×Flag and co-transfected with the EGFP-Grb7 SH2 into HEK293T cells using calcium phosphate method respectively. Fusion protein expression was first verified. The expression of peptide 10 was low . In co-iThe EYFP-tagged Grb7 SH2 domain was co-transfected with the mRLUC-tagged nonphosphorylated peptides into HEK293T cells using calcium phosphate . The EYF50 = 31.83 µM) and nonphosphorylated peptide 41-A (IC50 = 18.78 µM) had at least comparable binding affinities to the Grb7 SH2 domain.To see whether the nonphosphorylated peptide could effectively compete with phosphorylated ligand for binding to the Grb7 SH2 domain, the phosphorylated ligand pY (1180) (DEEYEpY(1180)MNRRR), which was previously shown to interact with the Grb7 SH2 domain Because the peptide pY (1180) abolished the association between the Grb7 SH2 domain and nonphosphorylated peptide 41 , we specThe effect of nonphosphorylated peptide 41 on the proliferation of SK-BR-3 breast cancer cells, which express high levels of the Grb7 protein http://pawsonlab.mshri.on.ca/index.php).In this study, we identified a novel nonphosphorylated motif that bound to the SH2 domain of Grb7 protein with an affinity at least comparable to the phosphorylated ligand pY (1180). This twenty-two amino acid-motif contains conserved amino acids at the N- and C-terminus and fifteen amino residues between that are enriched with proline (21%), threosine (18%) and serosine (13%) . The binSH2 domains promote rapid and reversible signaling transduction We showed that the peptide 41 was more stable than peptide 10 , possiblGrb7: N-IHRTQLWFHGRISREESQRLIGQQGLVDGLFLVRESQRNPQGFVLSLCHLQKVKHYLILPSEEEGRLYFSMDDGQTRFTDLLQLVEFHQLNRGILPCLLRHCCTRVAL-C; Grb2: N-EMKPHPWFFGKIPRAKAEEMLSKQRHDGAFLIRESESAPGDFSLSVKFGNDVQHFKVLRDGAGKYFLWVVKFNSLNELVDYHRSTSVSRNQQIFLRDIEQVPQQP-C; ABL_1: N-SLEKHSWYHGPVSRNAAEYLLSSGINGSFLVRESESSPGQRSISLRYEGRVYHYRINTASDGKLYVSSESRFNTLAELVHHHSTVADGLITTLHYPAPKRNK-C; and Grb14: N-QSSATNMAIHRSQPWFHHKISRDEAQRLIIQQGLVDGVFLVRDSQSNPKTFVLSMSHGQKIKHFQIIPVEDDGEMFHTLDDGHTRFTDLIQLVEFYQLNKGVLPCKLKHYCARIAL-C.The ABL_1 and Grb2 plasmids were gifts from Professor Ruibao Ren , and the Grb7 (BC006535) and Grb14 cDNA (BC053559) plasmids were from the Proteintech Group. The inserted sequences of the SH2 domains were as follows: In the mutation assay, the control and mutation sequences were as follows:GIPNYTPTT PTLLLTRPLPGIPRGLRGW-C;10: N-AGVESLGIPRGLRGW-C;10AM: N-AGVESL YTPTTPTLLLTRPLPGIPNYTPTTPTLLLTRPLP RGLRGW-C;10BM: N-AGVESLand 10CM: N-AGVESL YTPTTPTLLLTRPLP RGLRGW-C.In the co-immunoprecipitation assay, the mRLUC fragment was generated by PCR amplification. The inserted sequence of the Grb7 SH2 domain was as follows: N- IHRTQLWFHGRISREESQRLIGQQGLVDGLFLVRESQRNPQGFVLSLCHLQKVKHYLILPSEEEGRLYFSMDDGQTRFTDLLQLVEFHQLNRGILPCLLRHCCTRVAL-C. This sequence was inserted at the C terminus of the EGFP tag. The sequence of peptide 10 was as follows: N-GMWEGLEGGGVAGVESLGIPNYTPTTPTLLLTRPLPGIPRGLRGWSLLRCCRLLCRNSYITAAPKHHHPLQTSPRGSPGLQEFDIKLIDTVDLEGGPGTQFAL-C. The sequence of peptide 41 was as follows: N-VGTWTTRGPWCPCVAVGIPTQPTTSSEPSPPSNPPWDPGRVLLGRIVWPGLLALGIPTHHQNDTYNSPHAHPNRDP-C. The sequence of peptide 10CM was as follows: N-AGVESLYTPTTPTLLLTRPLPRGLRGW-C. The nonphosphorylated peptide and the mRLUC fragment were inserted at the N-terminus of the Flag tag. The mRLUC fragment was fused with the nonphosphorylated peptide so that they could be easily identifiable. The empty vector containing the mRLUC fragment was used as the negative control.The phosphorylated ligand pY (1180) (DEEYEpY(1180)MNRRR) from the ErbB3 and the nonphosphorylated peptide 41-A (VAVGIPTQPTTSSEPSPPSNPPWDPG RV) from nonphosphorylated peptide 41 were synthesized by the China Peptides Co., Ltd. The HPLC purification analysis and MS quality analysis reports were obtained.For the BRET assay, the EYFP fragment was generated by PCR amplification. The ErbB3-mRLUC plasmid was constructed by the Beijing Sino Biological Inc. The SH2 domain of Grb7 was fused to the EYFP fragment, and the nonphosphorylated peptides 10 and 41 were fused to the mRLUC fragment. The Grb7 SH2 domain sequence was inserted at the C-terminus of the EYFP tag.For the cell proliferation assay, the anti-Grb7 antibody  and the anti-Flag antibody  were used for the western blot, and MTS  was used for the analysis of cell proliferation.6 clones of the random peptide library , as AD baits, were effectively transformed into yeast strain CG 1945 containing BD bait plasmids and used to be screened according to the MATCHMAKER Two-Hybrid System protocol . Plasmids of potential positive transformants selected on plates with SD/-Trp-His-Leu medium in the primary screening were isolated and retransformed into the CG1945 cells containing the corresponding SH2 bait plasmids. The improved LacZ assay was then performed. Clones that were positive for all of the reporter assays and confirmed by three independent improved LacZ tests were selected for specific interactions The GAL4 BD-SH2 fusion bait plasmids were transformed into the yeast strain CG1945 using the lithium acetate protocol. The transformants were grown on SD/-Trp plates and spread on SD/-Trp-His plates for self-activation estimation. Those transformants without background growth or growth inhibited by 3-amino-1, 2, 4-triazole were selected for the subsequent screening. Approximately 3.01×10HEK293T cells 50 values were calculated by regression analyses using SPSS 17.0.HEK293T cells were washed 5 times with ice-cold PBS buffer and scraped off the plates into cell lysis buffer , 0.15 M sodium chloride, 0.001 M EDTA, 0.5% sodium deoxycholate, 0.1% SDS and 0.001 M PMSF). Lysates were incubated on ice for 5 minutes, vortexed 2 times for 30 seconds each and centrifuged at 12,000 rpm for 15 minutes at 4°C. After centrifuging twice, the supernatant was incubated with anti-Flag antibody  for 6–8 h at 4°C. Protein G agarose (40 µL) was added into 1 mL of the above mixture and coupled to the anti-Flag antibody for 3 h at 4°C. The immunoprecipitates were washed 2–3 times with PBS buffer (PH 7.4), and the sediments were diluted with 5×SDS electrophoresis buffer and heated at 97°C for 10 minutes. The proteins were separated by SDS-PAGE and transferred to PVDF membranes . The membranes were incubated with anti-GFP antibody  and goat anti-rabbit secondary antibody  to identify target proteins. For the competition analyses, the immunoprecipitation complexes were mixed with anti-Flag antibody and different concentrations of the synthesized phosphorylated ligands or the nonphosphorylated ligands. The proteins were separated by SDS-PAGE, and the SH2 domain of Grb7 protein was analyzed using anti-GFP antibody. Western blot analysis was carried out using the Image J In BRET, the Renilla luciferase (mRLUC) is fused to the candidate protein, and the green fluorescent protein mutant (EYFP) is fused to the target protein. Interactions between the two fused proteins bring the luciferase and the green fluorescent protein into close proximity, which promotes resonance energy transfer from mRLUC to EYFP The structure of the nonphosphorylated ligand 41-A (GIPTQPTTSSEPSPPSNPP WDP) was first built by MOE. This sequence had no homology sequence with known structure in the Protein Date Bank (PDB) database and no predicted secondary structure, such as α helix or β strand element. Therefore, a linear, extended conformation was set as the primary conformation of this ligand. Then, the energy of this ligand was minimized in an AMBER99 force field to build its final conformation for docking. Then the structure of the human Grb7 SH2 domain complex with a ten amino acid-peptide was downloaded from the PDB database (ID: 2l4k). The structure of the SH2 domain was extracted for docking. Thus, a docking method was used to predict the structure of the domain-ligand complex.3 cells per well at 18 h after transfection. 20 µL of MTS was added into 100 µL of cell culture medium, and the mixtures were incubated at 37°C for 1 h before measurement. The absorbance at 492 nm of transfected cells and control DMEM medium was measured separately at 18 h, 24 h, 36 h, 48 h and 60 h after transfection on Synergy4 Multi-Mode Microplates Readers . The absorbance was adjusted by differences in the absorbance between cell culture medium and control medium without seeded cells. The data were analyzed by double-factor variance analysis using SPSS 17.0.The MTS cell proliferation assay was performed to analyze the proliferation of SK-BR-3 breast cancer cells  according to the manufacturer's instructions . SK-BR-3 cells at 70%–80% confluency were transiently transfected respectively with plasmids of 41-mRLUC-3×Flag or mRLUC-3×Flag and Mega Tran 1.0 . Beginning from 12 h after transfection, 400 µg/mL G418  was used to select for the transfected cells Table S1Amino acid sequences of nine nonphosphorylated peptides.(DOC)Click here for additional data file.


Pandemic (H1N1) 2009 influenza A virus was detected in Norwegian pigs in October 2009. Until then, Norway was regarded free of swine influenza. Intensified screening revealed 91 positive herds within three months. The virus was rapidly transmitted to the susceptible population, including closed breeding herds with high biosecurity. Humans were important for the introduction as well as spread of the virus to pigs. Mild or no clinical signs were observed in infected pigs. Surveillance of SIV in 2010 revealed that 41% of all the Norwegian pig herds had antibodies to pandemic (H1N1) 2009 virus. Furthermore, this surveillance indicated that pigs born in positive herds after the active phase did not seroconvert, suggesting no ongoing infection in the herds. However, results from surveillance in 2011 show a continuing spread of the infection in many herds, either caused by new introduction or by virus circulation since 2009. Swine influenza A viruses (SIVs) are enzootic in most European pig populations . The SIV The clinical picture of pandemic (H1N1) 2009 infections in experimentally and naturally infected pigs was described as mild respiratory illness, increased temperature, and decreased appetite , 11, 12.Mycoplasma hyopneumoniae [ The pig production in Norway consists of approximately 2500 herds, mainly small family farms, with about 1.5 million slaughtered pigs in 2010. National surveillance and control program shows that the pig population is free for Aujeszky's disease (AD), transmissible gastroenteritis (TGE), porcine respiratory corona virus (PRCV), porcine reproductive and respiratory syndrome (PRRS), and eumoniae , 18. Theeumoniae . The sur This paper consists of studies that describe the initial spread of pandemic (H1N1) 2009 virus amongst Norwegian pig herds, the control measures initiated, and the infection status 20 months after the introduction. The paper also discusses the manifestation of the infection in the Norwegian pigs given that they had no prior immunity to any influenza A viruses and are free from most other viral respiratory diseases.Following the primary outbreak in October 2009, an intensified surveillance was immediately initiated to investigate the extent of SIV infection in the pig population in Norway, referred to as study 1 in the results section. The study targeted the following categories of herds: (i) herds where pigs with influenza-like signs were observed, (ii) herds where the pigs had been exposed to humans with verified pandemic (H1N1) 2009 virus infection or influenza-like illness (ILI), and (iii) herds with a history of contact with or in close proximity to infected herds through transfer of animals, usage of common equipment, for example, transport vehicles to slaughterhouse, and visitations from farmers of infected farms. In herds suspected to have an active infection, nasal swabs were collected from 20 pigs at different ages, with priority of sampling pigs with clinical symptoms. In herds without clinical signs, blood samples were collected from at least 20 pigs (sows or slaughtered pigs) for serological testing. Calculation of sample size was done assuming 100% test sensitivity and a within-herd prevalence >15%. The sample size implies that the risk of having 20 nonreactors from a positive farm is less than 4%. During the follow-up study, there was a special focus on the prevalence in nucleus and multiplier herds, hereafter referred to as study 2. The nucleus herds are closed herds meaning that they have no contact with other herds except via semen from the Norsvin AI station. The multiplying herds are either closed or obtain replacement sows from a single nucleus herd. They can sell sows to one or several conventional herds. The biosecurity level is high in both types of herds. Serological surveillance was carried out to investigate the possible spread of pandemic (H1N1) 2009 to these herds in the early phase of the outbreak. Between 1st of October 2009 and 31st of January 2010, blood samples were collected from 20 slaughtered pigs from 112 nucleus and multiplier herds. A third study was initiated to follow the course of the pandemic (H1N1) 2009 infection. From the herds that tested positive for pandemic (H1N1) 2009 in 2009, 38 herds consisting of 13 nucleus herds, 20 multiplier herds, and five conventional integrated herds were selected for the study. From each herd, blood samples from 20 slaughtered pigs were tested for antibodies against pandemic (H1N1) 2009. Blood samples were taken on the condition that 6 to 12 months had elapsed since the herd first tested positive and that the pigs tested were born at least 2 months after that time. An additional testing was performed in 9 herds about 18 months after the pandemic (H1N1) 2009 introduction. Study 4 consists of data from the national surveillance programme for SI in Norwegian pigs in 2009 and 2010. The program includes all nucleus and multiplier breeding herds and the nucleus unit of all sow pools. In addition, a random selection of conventional pig herds, 300 integrated and piglet-producing herds and 60 fattening herds, was selected from a population of approximately 2500 herds. Ten pigs in each herd were tested. With the exception of pigs from finishing herds, which are sampled at the abattoirs, the blood samples are collected from sows in the herds.Clinical and epidemiological information recorded by The Food Safety Authority was available from 43 pig herds suspected to be infected. The information was collected, while the herd was being sampled during the early phase of the outbreak between 10th of October 2009 and 31st of December 2009. The selection of herds was based on the same criteria as described previously. The size of these herds ranged in numbers from 5 to 2000 pigs (average 550 pigs). The investigation used a structured questionnaire consisting of questions on epidemiological information including clinical picture within the herd, likely source of infection, contact with people diagnosed with pandemic influenza or had ILI symptoms and being in contact with pigs while sick, movement of animals in or out of the farm, and handling and biosecurity measures.Initially, serum samples were analyzed for specific antibodies against A/Sw/Belgium/1/98(H1N1) and A/Sw/Flanders/1/98(H3N2) using hemagglutination-inhibition (HI) assays according to the method described in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals . FollowiμL of the sample material using the automatic extraction instrument NucliSens easyMag  according to the manufacturer's instruction. Detection of influenza A virus was performed as described by the World Health Organization, The WHO Collaborating Centre for influenza at CDC Atlanta [ Nasal swabs , individual or in pools of two, were placed in 1 mL of transport medium (EMEM 2% IBFS/Tris) before they were shipped to the laboratory. Total RNA was extracted from 200  Atlanta . AmplifiμL extracted RNA was amplified using 46 primer pairs and a one-step RT-PCR Kit . The resulting overlapping amplicons, carrying primer-derived M13 terminal sequences, were sequenced with M13 primers using the ABI BigDye Terminator v1.1 Cycle Sequencing Kit and the ABI 3130 Genetic Analyzer . Sequences were assembled using Sequencher v.4.9  and compared to related H1N1 virus sequences using BioEdit software [ Full genome sequencing of isolate A/sw/Norway/02_11342/2009(H1N1) was carried out through a modified version of the WHO sequencing primers and protocol provided by the WHO Collaborating Centre for influenza in CDC, Atlanta, USA (28 April 2009) . Brieflysoftware .Descriptive statistics and calculation of true population prevalence using confidence intervals were used. To measure the association between county prevalence and the pig density by county, we used simple linear regression or scatter plot analysis performed in Stata10.After the introduction of pandemic (H1N1) 2009, the intensified surveillance (study 1) shows that 54 of the 114 herds tested were positive for pandemic (H1N1) 2009 virus by RT-PCR, and 55 of the 140 herds tested by serology were positive for antibodies against pandemic (H1N1) 2009. Altogether 91 herds tested positive from 10th of October to 31st of December 2009. In the 55 seropositive herds, 59% (range 5% to 100%) of the individual samples from each herd were positive. From the 54 RT-PCR-positive herds, an average of 65% (range 5% to 100%) of the individual samples from each herd were positive. Geographically, the infected herds were found throughout Norway . In 2010In study 2, a total of 2349 blood samples from 112 nucleus and multiplier herds were tested for antibodies against influenza A virus. Testing of the nucleus herds was completed by the 18th of December 2009, and the multiplier herds were finished on the 31st of January 2010. The surveillance shows that 27.1% of the nucleus herds and 34.4% of the multiplier herds were seropositive for pandemic (H1N1) 2009. Between six and 12 months after a herd was seropositive for antibodies against pandemic (H1N1) 2009 virus, blood samples were collected from slaughtered pigs born at least two months after the initial detection (study 3). In total, 1127 pigs from 38 herds were tested. Three of the 38 herds tested positive for antibodies against pandemic (H1N1) 2009 virus. The seropositive herds were multiplier herds located in the South West region where pig farming density is highest . Pigs frr = 0.013, p = 0.023) with the pig density by county . In 60% (21/35) of these infected herds, there was evidence to suggest that humans were the most likely source of infections. In these herds, the farmer, farm worker, or the farmer's family members were diagnosed with pandemic (H1N1) 2009 (33% or 7/21) or had ILI just before or during the outbreak (66% or 14/21). Twenty percent (7/35) reported contact with another infected farm through humans or movement of infected animals as source of infection. One farmer of a positive herd had bought pigs from a positive herd and also had one farm worker with ILI during this period. Forty-nine percent (17/35) had reported that clinical signs were observed in the pigs. The most common clinical signs reported were coughing, fever, and loss of appetite, lasting for less than two weeks. No mortality was reported. Nineteen percent (8/43) of the herds from which epidemiological information was gathered tested negative although 3 of these herds had histories of contact with humans with ILI.Full genome sequence analysis was carried out on nasal swab RNA from an infected pig in the index herd, as well as from the virus isolated from a sick staff member at the same farm. The sequences of these two full viral genomes were virtually identical (99.9985% identity) (Acc. no JQ253790-JQ253797). Moreover, the HA sequence showed at least 99.8% identity to other pandemic (H1N1) 2009 circulating in humans in Norway at the same time. The complete sequence further indicated that the virus had not acquired known resistance mutations in the neuraminidase gene.Pandemic (H1N1) 2009 virus was first detected in Norwegian pigs on the 10th of October 2009 [ The first confirmed human case of pandemic (H1N1) 2009 virus in Norway was in May 2009 . It was  The national surveillance program for SIV in Norway shows that the first herd positive for antibodies against pandemic (H1N1) 2009 virus was sampled on the 30th of September 2009 . As show Norway has previously been free of SIV as documented by a national surveillance program for swine . AlthougConsistent with other studies , there w Herds positive for pandemic (H1N1) 2009 virus in 2009 were tested again in 2010. The results show that there was no seroconversion in pigs born at least two months after the virus was first detected, indicating that there was no ongoing infection in the herds. A possible explanation may be that the Norwegian pig herds are small, and many herds practice batch farrowing of three, five and a half, or seven weeks intervals, with strict sectioning of the herds with all-in all-out system. However, results from the surveillance in 2011 show that some pigs born in 2010/2011 are seropositive, indicating an ongoing spread of the infection. It is not clear to what extent this is caused by new introduction from humans or continued virus circulation between susceptible pigs since 2009. Continuing surveillance is important to show whether the virus has become enzootic in the Norwegian pig population. There have been several reports of reassortant virus that have genes from pandemic (H1N1) 2009 virus and other SIV subtypes –33. As lPandemic (H1N1) 2009 virus was introduced to the Norwegian pig population in September/October 2009. The virus spread rapidly to a large proportion of all categories of herds all over the country, including closed, high-biosecurity nucleus herds. Humans seem to have been important for the introduction and spread of the infection. The clinical impact was mild, and in many infected herds, no clinical signs were observed. The ongoing national surveillance program will reveal whether the virus will become enzootic in the Norwegian pig population.


HAMP domain is a ubiquitous module of bacterial and archaeal two-component signaling systems. Considerable progress has been made recently in studies of its structure and conformational changes. However, the mechanism of signal transduction through the HAMP domain is not clear. It remains a question whether all the HAMPs have the same mechanism of action and what are the differences between the domains from different protein families. Here, we present the results of unbiased molecular dynamics simulations of the HAMP domain from the archaeal phototaxis signal transducer NpHtrII. Two distinct conformational states of the HAMP domain are observed, that differ in relative position of the helices AS1 and AS2. The longitudinal shift is roughly equal to a half of an α-helix turn, although sometimes it reaches one full turn. The states are closely related to the position of bulky hydrophobic aminoacids at the HAMP domain core. The observed features are in good agreement with recent experimental results and allow us to propose that the states detected in the simulations are the resting state and the signaling state of the NpHtrII HAMP domain. To the best of our knowledge, this is the first observation of the same HAMP domain in different conformations. The simulations also underline the difference between AMBER ff99-SB-ILDN and CHARMM22-CMAP forcefields, as the former favors the resting state and the latter favors the signaling state. Escherichia coli), meanwhile others are regulated by chemo- and photoreceptors (the CheA kinases that are widespread among bacteria and archaea).Many microorganisms live in highly variable environments, and as a consequence they have developed sophisticated signaling systems. Many schemes of signal transduction rely on a phosphotransfer reaction and employ two main components: a histidine kinase, whose activity depends on the signal, and a response regulator protein histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins and phosphatases  or this is a result of a forcefield bias. To that end, we have performed additional simulations #3 and #4, with the structures from the simulation #1 taken as starting poses . In the The eigenvector corresponding to PC1 characterizes the structural details of the transition between the two discovered states. To determine it in the best way possible we have applied the analysis to the concatenated trajectory that includes all the simulations. The same PC1 was used to obtain all the results presented here –7. CompaIt could be useful to estimate the free energy difference and the value of the energy barrier between the two states. However, the results obtained with the AMBER and CHARMM forcefields differ not only in the magnitude of the energy difference but also in its sign. Inclusion of the adjacent NpHtrII domains into the model might also change the energetics of the transition from one state to another. Thus, we do not assess the aforementioned energetic properties as the results of such assessment may be misleading. The presented simulations may serve as an example that, despite considerable progress in forcefield development After establishment of the two states of the NpHtrII HAMP domain we proceed to the structural analysis. The most notable conformational rearrangement associated with the first principal component is the relative longitudinal displacement of the HAMP domain helices AS1 and AS2 ,  or  conformation , as both numbers can be determined for each trajectory snapshot. The analysis shows that the correspondence is clear and unambiguous . We use The most significant difference between the resting and the signaling states of the NpHtrII HAMP1 domain is the longitudinal shift of the helices AS1 and AS1’ relative to the helices AS2 and AS2’  upon theInterestingly, the residue F124 resides in the same coiled coil layer as the residue A291 of the Af1503 HAMP domain. Mutations of A291 to bulkier hydrophobic aminoacids render the domain, which is natively dysfunctional, able to conduct the signal in Taz (Tar-HAMP-EnvZ) chimeras Here, we have presented the results of unbiased molecular dynamics simulations of the NpHtrII HAMP1 domain. The domain adopts two conformations that share many features with the two HAMP domain states observed experimentally. To the best of our knowledge, this is the first example where the atomic structures of two different conformations of the same HAMP domain are presented.Although the simulations of the HAMP domain without the flanking modules may be misleading Natronomonas pharaonis HtrII (residues 85–133). The initial model was obtained by automated homology modeling procedure by SWISS-MODEL server In all the simulations we have studied the first HAMP domain of Natronomonas pharaonis lives in. The box size was chosen so that the minimal distance between the atoms of periodic images of the protein was 1.6 nm. The resulting system was minimized with a steepest descent algorithm using a tolerance of 1,000 kJ mol−1 nm−1 and a step size of 0.01 nm. After that, the solvent was equilibrated for 10 ps at a constant volume (NVT ensemble) and then for 10 ps at a constant pressure (NPT ensemble). The protein atoms were harmonically restrained during the solvent equilibration. The starting frames for the simulations #3 and #4 were chosen randomly from those of the simulation #1 with a needed HAMP domain state and are shown in −5 bar−1.Molecular dynamics simulations were conducted using the GROMACS software version 4.5.3 To restrain alpha-helicity, harmonic potentials were applied to the φ and ψ dihedral backbone angles. The average values of −63.8° and −41.1° α, O) of the HAMP domain alpha-helices. Atoms of the linker were not included in the analysis as the linker was very flexible and adopted variable conformations that correlated weakly with the state of the alpha-helices. The principal component 1 determined for the concatenated trajectory that includes all the simulations was used throughout the analysis. Comparison of the principal components determined for individual simulations with those determined for the concatenated trajectory is presented in Principal components analysis (PCA) Figure S1Comparison of the principal components obtained from the individual simulations with those obtained from the concatenated trajectory. The plots show the dot products of the corresponding principal components, with the black color corresponding to 1 and the white color corresponding to 0. The first 20 components were taken for each trajectory. The eigenvectors were normalized to 1 prior to multiplication. The first principal component (PC1), obtained from the concatenated trajectory, is present in both CHARMM simulations (A and C), absent from the AMBER simulation that starts from the homology model (B), but present in the AMBER simulation that starts from the signaling state, obtained previously in the CHARMM simulation. Roughly diagonal structure is observed in all plots. The principal components other than the first one are not very well conserved in different trajectories.(EPS)Click here for additional data file.Movie S1Transformation of the HAMP domain structure corresponding to the first principal component of the PCA analysis. The structure is colored blue in the resting-like states and red in the signaling-like states.(MP4)Click here for additional data file.Movie S2Example of the signaling-to-resting state transition from the molecular dynamics simulations. Presented is a part of the trajectory 3 from the simulation 4 . The arrows highlight the motions of the helices AS1 and AS1’. The trajectory is smoothened with a window of 11 ns in order to remove fast motions. Projection of the structure on the first principal component is also shown.(MP4)Click here for additional data file.