Trypanosoma brucei (Tb)Arl13, the sole Arl13b homolog in this evolutionarily divergent, protozoan parasite. TbArl13 has conserved flagellar functions and exhibits catalytic activity towards two different TbArl3 homologs. However, TbArl13 is distinctly associated with the axoneme through a dimerization/docking (D/D) domain. Replacing the D/D domain with a sequence encoding a flagellar membrane protein created a viable alternative to the wild-type TbArl13 in our RNA interference (RNAi)-based rescue assay. Therefore, flagellar enrichment is crucial for TbArl13, but mechanisms to achieve this could be flexible. Our findings thus extend the understanding of the roles of Arl13b and Arl13b–Arl3 pathway in a divergent flagellate of medical importance.The small GTPase Arl13b is one of the most conserved and ancient ciliary proteins. In human and animals, Arl13b is primarily associated with the ciliary membrane, where it acts as a guanine-nucleotide-exchange factor (GEF) for Arl3 and is implicated in a variety of ciliary and cellular functions. We have identified and characterized This article has an associated First Person interview with the first author of the paper. Highlighted Article: All roads lead to cilia – how the essential flagellar enrichment of Arl13 is achieved in trypanosome cells using a fundamentally different strategy compared with that of animal cells. Flagella/cilia provide critical motility and sensory functions in eukaryotic cells from protists to mammals alike. Their roles in human physiology and diseases (ciliopathies) have spurred renewed interest in this ancient organelle . The cilMutations in Arl13b are associated with Joubert syndrome (a ciliopathy), and Arl13b-null animals display a wide range of ciliary defects that compromise ciliogenesis, cilia length extension, ciliary motility and sonic hedgehog signaling . ConcomiTrypanosoma brucei, the single homolog of Arl13b (TbArl13) lacks both the palmitoylation motif and the C-terminus tail – both of which are essential for Arl13b ciliary targeting in higher eukaryotes. In light of recent evidence that Arl13b may also have roles outside of the cilia . To further analyze Tb927.10.5230, we performed phylogenetic reconstruction, sampling all predicted trypanosome Arf/Arl GTPases and major Arf/Arl family members from representative eukaryotic model systems . The resulting phylogenetic tree confidently placed Tb927.10.5230 in the same clade with all other known Arl13/Arl13a/Arl13b proteins included in the dataset . Because the duplication of Arl13 to Arl13a and Arl13b is likely a vertebrate invention  show that TbArl13 possesses a highly conserved GTPase domain, with characteristic absence of the ‘Switch 2’ catalytic glutamine residue (DxxGQ) that is highly conserved in other small GTPases (Chlamydomonas reinhardtii (Cr)Arl13b . TbArl13 also lacks a long C-terminal tail – another characteristic, although less-conserved feature of Arl13b . Taken together, our bioinformatic analyses show that TbArl13 has highly conserved catalytic domains (a GTPase domain and the CC motif) but is divergent in elements that have been implicated in targeting and/or regulatory functions.The most unusual feature of TbArl13 is the presence of an N-terminal extension, which is predicted to be a ‘dimerization/docking domain of the PKA regulatory subunit’ . D/D is a class of protein targeting domains best characterized in PKA, but they have also been found in non-PKA proteins . The D/DT. brucei cells (the proliferative form found in tse-tse fly midgut) by tagging one endogenous TbArl13 allele with a yellow fluorescent protein (YFP) reporter followed with a triple HA tag . When using a protocol that efficiently extracts membrane-associated proteins such as the dually lipidated calflagin Tb24 .We first investigated the intracellular localization of TbArl13 in procyclic form (PCF) e HA tag B. Highlye HA tag C. In alle HA tag B–G. Somegin Tb24 , we consThere are two major cytoskeletal structures in the trypanosome flagellum: the axoneme, which is nucleated from the basal bodies, and the paraflagellar rod (PFR), which only assembles after the flagellum exits the flagellar pocket and at a distance to the basal bodies ; Fig. 1GFig. S5A). These results demonstrate that the D/D domain fully accounts for the flagellum targeting and axonemal association of TbAr113.The D/D domain was first characterized and best studied in the regulatory subunits of PKA . The subSince the D/D domain is also known to mediate PKA regulatory subunit dimerization , we askeFig. S5B) and reduced TbArl13–mNeonGreen in the cytoskeletal fraction  using a cumate (Cmt)-inducible system  in cellsfraction F in cellfraction D. Similafraction . FurtherNotably, overexpression of the TbArl13 D/D domain is not well tolerated by the cells as high expression levels could not be maintained for more than 2 days (data not shown). The dominant-negative effect of D/D overexpression in BSF cells will be discussed below, where it is more pronounced.Fig. S6A). At 72 h after tetracycline washout, TbArl13 expression was not detectable . Unlike control cells that each contain single or duplicated flagella that tightly adhere to the cell body via the flagellum attachment zone (FAZ), TbArl13-iKO cells displayed severe flagellar defects (The cellular functions of TbArl13 were investigated by Arl13 inducible knockout (iKO) in PCF cells. Both endogenous TbArl13 alleles were replaced with drug resistance genes, and ectopic TbArl13–YFP expression was achieved through a tetracycline (Tet) inducible cassette stably incorporated into the genome  E. For flFig. S6C), generating ‘monster cells’ with multiple flagella and nuclei, indicative of cytokinesis failure  TbArl13 mutants were stably transfected into BSF cells with stable integration of Tet-inducible TbArl13-RNAi A,B.As shown in Fig. S6D). Strikingly, Tb24CDiR–YFP was able to support continued cell proliferation in the absence of endogenous TbArl13  by swapping the D/D domain of TbArl13 with the entire sequence of the well-established flagellar membrane-associated protein calflagin Tb24 . As expe TbArl13 C,E. ThesT. brucei.Interestingly, we found that DDiR–YFP expression alone had an inhibitory effect on cell proliferation before expression was lost and growth recovered F. CombinT. brucei.To investigate the interaction network of TbArl13, we performed proximity-dependent biotin identification (BioID) of potential TbArl13-interacting partners . Specific protein hits were ranked based on their relative protein content . NotablyT. brucei . The localization of all three Arl3 homologs were first screened by overexpressing YFP-tagged recombinant proteins. TbArl3A–YFP was enriched at the nucleus and along the flagellum, as well as forming a punctum at the base of the flagellum . TbArl3C–YFP was highly concentrated at the base of the flagellum, with a weaker presence along the flagellum. TbArl3B–YFP, however, appeared to be completely excluded from the flagellum, and adopted a reticulated pattern that closely resembled the PCF mitochondrion . Consistent with the BioID results, only TbArl3A–VC and TbArl3C–VC exhibited positive interaction with TbArl13–VN, allowing recombined Venus fluorescence to be detected both at the base and along the flagellum .Bi-fluorescence complementation BiFC;  was empllagellum B. Additi lysates C. ReciprFig. S7E,F). Interestingly, TbArl3C seems to have a much higher intrinsic nucleotide exchange rate compared to TbArl3A, further suggesting that the two Arl3 homologs may be regulated and/or function differentially. Bacterially produced GST–TbArl3B was not soluble in our hands. Considering that TbArl3B did not interact with TbArl13 in BiFC and in vivo pulldown assays, it is unlikely that it is an interacting partner of TbArl13 and was thus not included in the GEF activity assays.Arl13b has been shown to be a GEF for Arl3. To investigate whether both TbArl3A and TbArl3C could be regulated by TbArl13 in the same fashion, we performed GEF assays using fluorescent, mant-labeled nucleotides . His–TbATo investigate whether TbArl3A and TbArl3C have flagellar functions, we generated classic GTP-locked mutants by altering the catalytic glutamine residues at Switch 2 region to a leucine residue. Inducible expression of TbArl3A-Q70L–BB2 and TbArl3C-Q77L–BB2 both drastically inhibited flagellum biogenesis, with both flagellum and FAZ markers reduced into stubs or dots in most cells at 20 h post induction A,C. In sT. brucei. Multiple lines of evidence support that TbArl13 is a bona fide Arl13b ortholog. At the sequence level, the catalytic domain is highly conserved among TbArl13 and other Arl13b orthologs. At the cellular level, TbArl13 localizes to the flagellum and is critical for flagellar biogenesis and functions. At the molecular mechanism level, TbArl13 catalyzes guanine nucleotide exchange in trypanosome Arl3 homologs. Trypanosomes (euglenozoan) are one of the most divergent groups of extant eukaryotes  suggest that they are found in a wide range of non-PKA proteins including some flagellar proteins . Thus, DTaking advantage of this simple D/D-mediated flagellar-targeting mechanism, we explored the relationship between the localization and functions of TbArl13. The chimeric Tb24CDiR–YFP, which could enrich on the flagellum membrane but did not form a gradient distribution along the flagellum, could functionally replace WT TbArl13. By contrast, CDiR–YFP, which could enter the flagellum but showed no enrichment compared to the cytoplasm, could not rescue TbArl13-RNAi cells. These results suggest that neither the axoneme association nor the gradient-like distribution is essential for TbArl13 function. Instead, flagellar enrichment of the TbArl13 catalytic domain, via the D/D domain or Tb24, is required A–C. The The dominant-negative effects of expressing just the D/D domain could be due to its activity as an orthosteric inhibitor, as these domain truncations can compete with WT TbArl13 for docking partners on the axoneme D–F. SincTbArl13 likely dimerizes due to the D/D domain. Interestingly, it was reported that human Arl13b (which does not possess a D/D domain) could also self-associate through the ‘N-terminal domain’ . A laterFig. S1B), it is uncertain whether TbArl3B is a true Arl3 ortholog. Our results suggest a possible expansion and/or specialization of Arl3 functions in T. brucei. Considering the well-established role of Arl3 as a cargo displacement factor, an intriguing scenario is that the differential localization of the two TbArl3 proteins allows for different cargos to be released at different flagellar locations. Mammalian cells have only one Arl3, which is found in multiple structures including the centrioles , the cilia and the Golgi  (https://github.com/PatrickKueck/AliCUT) to remove the positions that were not unambiguously aligned. Both Maximum Likelihood (ML) and Bayesian phylogenetic inferences were conducted on the resulting alignment. Model testing was performed with ProtTest 3 (v. 3.4.2)  (Putative Arf/Arl GTPases were retrieved by searching ARF domain (Domain ID: IPR024156) in the Tritryp Database (pdb.org) . Large ppdb.org) . Phylogepdb.org) , based opdb.org) . Alignmepdb.org)  and ALIC. 3.4.2) . The bes. 3.4.2) , where t. 3.4.2) . Two par(v. 1.6) .https://www.ebi.ac.uk/interpro/). The COILS server was used for coiled-coil prediction . Multiple sequence alignments were visualized using Sequence Manipulation Suite . The D/Dediction . Palmitoediction .2. Cell transfections were carried out based on published protocols   at 28°C. All BSF cells were maintained in HMI-9 medium containing 10% heat-inactivated fetal bovine serum (Hyclone) at 37°C with 5% COrotocols . Trypano YTat1.1  cells st YTat1.1  were use13 cells . For BSFression) . A PCR-bression) .Table S1). Point mutations were introduced into WT TbArl13 or TbArl3 genes by site-directed mutagenesis using inverse PCR (Plasmids used in this study are listed in the supplementary information (erse PCR . For TbAE. coli. Antiserum was further affinity purified on PVDF membrane strips loaded with GST–TbArl13 (Table S2).Rabbit anti-TbArl13 polyclonal antiserum was generated using purified His–TbArl13 expressed in –TbArl13 . Other aUnless otherwise stated, PCF and BSF cells were fixed with 4% paraformaldehyde before or after extraction with 0.25% NP-40 or Triton X-100. The fixed cells or cytoskeleton samples were blocked with 3% BSA and then incubated with primary and secondary antibodies. Images were captured either on a Zeiss Axio Observer Z1 fluorescence microscope with a CoolSNAP HQ2 CCD camera (Photometrics) or on an Olympus FLUOVIEW FV3000 confocal microscope.For TEM, PCF and BSF cells were fixed and resin-embedded based on established protocols . Ultra-tFig. S5B. The anterior tip of the axonemes were determined using either the beginning of mCherry or mNeonGreen signals, whichever was stronger. A correlation coefficient was calculated using Graphpad Prism 6.All images were adjusted and edited for presentation using ImageJ. For all images subject to measurement and comparison, both acquisition and image display parameters were kept constant. For flagellar fluorescence intensity quantification, axonemes were traced using freehand tool in ImageJ (line width=3 pixels). The average intensity values along the first 2 µm of the anterior axonemes were used for analysis in VN and VC fragments were cloned from pBiFC-VN155 (I152L) . Protein identification threshold was set at 1% false discovery rate (FDR) based on reversed protein sequences. ‘Protein content’ was calculated using the emPAI method (T. brucei flagella (PCF cells stably transfected with pDEX-FLiR-MycBirA were induced with 10 µg/ml cumate for 24 h. These cells and WT PCF cells (negative control) were then cultured in the presence of 50 µM biotin for an additional 24 h. The procedures for streptavidin-based pulldown of biotinylated proteins following a published protocol . To maxiI method , which eflagella  using thin vitro pulldowns of purified His–TbArl13 and GST–TbArl3A/C beads were carried out through standard protocols  following the manufacturer's protocol.Reciprocal rotocols  and were2. Excess nucleotides were removed by desalting columns . 0.5 μM mant-GDP-labeled GST–Arl3A or GST–Arl3C were incubated with different concentrations of His–TbArl13 and unlabeled GTP (20-fold excess) at 20°C. The GTP-binding assay was performed using the RhoGEF exchange assay kit  based on the manufacturer's instructions. 4 μM purified GST–Arl3A or GST–Arl3C were incubated with or without 1.5 μM His–TbArl13 in the presence of 1.5 μM mant-GTP at 20°C. All fluorescence data was recorded with a Tecan Infinite M200 plate reader. Both GEF and GTP-binding assay results were normalized to the initial fluorescence reading. All experiments were repeated six times. Kobserved (Kobs) was obtained by fitting the data as a one phase-exponential decay using Graphpad Prism 7.Guanine nucleotide exchange assays were conducted based on established protocols . Briefly


The antibody seroprevalence of young stock can be a useful indicator of recent or current infection in a herd. We examine the factors that contribute to the assessment of herd exposure to disease, via spot testing for antibody, using bovine viral diarrhoea virus (BVDv) as an example. A statistical distribution of seroprevalences for BVDv in beef herds identified three groups of herds: low, intermediate and high within-herd BVDv antibody seroprevalence. We tested two assumptions –the intermediate seroprevalence group of herds is assumed to be negative for BVDv at the herd level and alternatively if this group is assumed to be positive. We found that: The herd-level sensitivity and specificity are sensitive to the assumption regarding the herds with intermediate seroprevalence. If an appropriate cut-point is chosen, reducing the sample size from ten to five does not produce a large drop in herd-level test performance. Increasing the cut-point may be valuable at the outset of an eradication programme. Increasing the sample size and decreasing the cut-point is advantageous towards the end of an eradication programme, to minimise the risk of positive herds being misclassified. The framework presented here illustrates how seroprevalence screening may be understood and assessed. This principle has been employed in the screening of herds for infectious diseases, such as bovine viral diarrhoea virus (BVDv) in cattle.For most infectious agents, antibodies measured in animals by an ELISA test provide information about the recent or historic exposure of those animals to the infectious agent. The prevalence of animals with high antibody levels  on the basis of the detection of antibody-positive animals, a subsequent and more extensive search for antigen-positive  animals within the herd typically will be conducted. A particular aim of such subsequent screening is the identification of persistently infected (“PI”) animals, which are epidemiologically important for the continued spread of disease7.BVDv is the infectious agent of a serious cattle disease of economic importance8. The variation in seroprevalence amongst herds that are truly BVDv-positive as well as among those that are truly BVDv-negative (i.e. not infected) will impact the results of a screening test. Among the other factors that will affect the performance of a screening test are the following:The sensitivity and specificity of the antibody test at the level of the individual animal;The number of animals to be tested or sampled; andThe use of an appropriate cut-point or threshold for the number of test-positive animals in the sample at which the herd (or flock) is deemed positive.Using the presence of antibody-positive animals to identify infected herds is dependent upon several factors. Levels of within-herd antibody seroprevalence have been found to vary across populations of herdsTogether, these factors contribute to the specificity and sensitivity of the screening test at the level of the whole herd.i.e., the number of seropositive animals which must be detected in the sample in order for the herd to be deemed positive. For example, the first mandatory testing stage of the Scottish eradication scheme9 required five or ten young animals to be tested per management group depending on the age of the young stock with an implicit cut-point of one positive animal albeit with scope for reinterpretation by the veterinarian in the case of low numbers of animals with low or inconclusive antibody levels10.Screening programmes for BVDv which utilise testing for antibodies in individual sera from young stock vary in the number of animals they require to be tested and in the cut-point, Thus the detection of even a single antibody seropositive animal in the sample was sufficient for the herd to be deemed positive for BVDv. In the eradication programme proposed for the Netherlands an initial screening of five young stock leads to additional testing if two or more tested animals are found to be antibody positive .5. However, there is also evidence of a middle group or class of herds with intermediate seroprevalence  and the latter stages of an eradication scheme . We also consider the importance of the middle group and how it may affect the interpretation of the results of screening for antibodies in young stock.As noted above, the within-herd BVDv antibody seroprevalence varies among herds. The statistical distribution of seroprevalence amongst young stock is generally U-shaped with a large proportion of herds with zero or close to zero seroprevalence and a large proportion with close to 100% seroprevalenceTreatment of herds with intermediate levels of antibody seroprevalence as either BVDv negative or positive;Testing of five or ten animals; andUse of a cut-point or threshold  of different numbers of animals.Results of test performance at the herd-level are presented for the following combination of scenarios Fig. :TreatmenFrom results illustrated in Fig. Table 13. Possibly of greater interest to the policy maker is the probability that any herd randomly selected from a population of herds is truly positive and tests negative . Here we use the term “overall probability of a false negative” to distinguish this value from the false negative rate. The overall probability of a false negative is simply the true herd-level prevalence multiplied by the false negative rate.The “false negative rate” is the probability that a truly positive herd tests negative The overall probability of a false negative naturally increases linearly with herd-level prevalence Figs  and 4. IWe illustrate the consequences of our assumption for the status of the intermediate seroprevalence group for a scenario provided within the Dutch eradication scheme in which the number of animals tested is five and the cut-point is two Table . This deIn Table In Figs We do not present figures for the median overall probability of false negatives when herds with intermediate within-herd seroprevalence are treated as negative because in that situation the median rate of false negatives is zero for both a cut-point of one or two positive animals.For most infectious agents, antibodies measured in animals by an ELISA test provide information about the recent or historic exposure of those animals to the infectious agent. The prevalence of animals with high antibody levels  may thus be used to identify herds that have been or are being exposed to high levels of the infectious agent, provided that estimates of seroprevalence can be interpreted as a herd-level result. In this study we employ the example of BVDv to demonstrate how the herd-level sensitivity and specificity might be estimated on the basis of an interpretation of seroprevalence data collected from young stock in Scottish beef suckler herds.14 found that the within-herd antibody seroprevalence from herds in which an antigen-positive animal was identified was 87% whilst it was only 43% in herds in which no antigen-positive animal was found. Therefore the within-herd prevalence of antibody-positive animals may be effectively used as a proxy or indicator for the presence or absence of antigen-positive animals.A natural gold standard for defining a herd as BVDv positive is evidence of circulating virus in the herd and therefore evidence of the presence of at least one persistently or transiently infected animal in the herd, which might be detected on the basis of screening animals for virus or for BVDv antigens. Such screening would require a great deal of testing , and the consequences of a false negative result could be substantial. Alternatively, however, the epidemiology of BVD virus is such that the presence of BVD virus in the herd is amenable to screening by testing for antibodies in animals of any age, and particularly in young stock. A study17 although it has been suggested that as few as three young stock might need testing17 especially if the antibody test is done in tandem with antigen testing18. A BVDv eradication programme now being initiated in the Netherlands involves initial antibody spot-testing of five animals, with a cut-point for the number of antibody-positive animals of two. One clear benefit of choosing a cut-point of two (as opposed to one) is that it reduces the risk of false positives and thus reduces the risk of unnecessary and expensive secondary testing of the whole herd. Our work informs decisions regarding sample numbers and threshold values under different conditions.When a number of individual animals are tested for antibody in order to determine a binary  herd status, there are two important decisions to be made. The first is the number of animals to be tested and the second is the cut-point for the number of positive animals at or above which the herd is deemed positive. Commonly the number of animals to be tested in BVDv eradication programmes is 5–10 young stock19. In our analysis the optimum appears to be two positive animals out of ten, if the intermediate seroprevalence group is considered positive and a cut-point of around seven positive animals if the intermediate seroprevalence group is considered to be negative  to have a sensitivity and specificity of 100% and 98.2% respectively in comparison to a virus neutralisation test. In our simulations we have used the sensitivity and specificity from the statistical distributions reported in the study8 on which our simulations are based. The sensitivity  is 96.3%  and the specificity is 98.8% .Our approach to estimating the Our study is based on the categorisation of individual antibody scores into a binary positive or negative result by comparing the antibody scores with a cut-off. This is the conventional way of interpreting individual antibody results and is both convenient and easily interpreted. However, when we scale up to testing at the group level it is possible that the loss of information that is involved in categorising a continuous antibody score into a binary positive or negative is an important loss of information. For example, it is possible that having several calves with an antibody score just below the cut-off is a better predictor of a positive herd than having only one calf just above the cut-off. Current eradication schemes tend not to be set up to report individual antibody scores, and it is not known how frequently such a situation might occur. To take into account the individual antibody scores would require further investigation, ideally using data pertaining to individual antibody scores from sampled calves from herds with and without PI animals. The interpretation would require a multivariate analysis to optimally interpret several antibody scores without recourse to the binary categorisation. Such an approach might improve the herd-level test performance but might be unattractive to veterinary practitioners and to producers because it would involve complex and non-transparent calculations.8. The Scottish study found three classes, characterised by low, intermediate and high seroprevalence. Our analyses are based on the assumption that those groups represent different epidemiological herd statuses. The analyses are made complex by the fact that the epidemiological status of the group of herds characterised by intermediate levels of antibody seroprevalence is uncertain. Ideally we would have data from the same herds on both the within-herd seroprevalence for young stock and whether or not an infected animal exists in the herd. With such data we would be able to confidently describe the status of herds in the group with intermediate seroprevalence, but in the absence of such data we can consider the two extremes – i.e. that all herds in this group are either negative or positive. It is probable that this group of herds with intermediate seroprevalence consist of a mixture of truly positive and truly negative herds, in which case the correct interpretation would lie between the two extremes presented here.The extent to which antibody seroprevalence can be used to determine herd-level status of infection is dependent on the level of separation of the frequency distribution of seroprevalence values in negative herds compared to the distribution in positive herds see Fig. . The gre12. On the other hand at least one study exists in which there was no obvious middle component in the distribution of within-herd seroprevalence5.It is worth considering the wider evidence regarding the existence of a group of herds with intermediate seroprevalence levels. Other studies have presented data suggesting the possible existence of such a groupExistence of residual maternal antibody derived from dams in young stock that have not, in fact, been exposed to the virus.The young stock have been vaccinated.The young stock were exposed to low levels of virus  leading to a low antibody response.Fomite contamination – leading to low antibody response and hence low prevalence.Exposure to a particular strain of virus that causes low antibody response.Heterogeneity in ages of the sampling group – if there are some new recruits to the group post-exposure they will be antibody negative and will “dilute” the group and hence reduce the prevalence.There are various possible causes of intermediate seroprevalence in some herds:In our analyses we have combined the intermediate seroprevalence group with either the low seroprevalence (negative) group or the high seroprevalence (positive) group. In doing so, we have effectively created a mixture of two, rather than three, groups. The resulting mixture depends upon the relative proportional contribution each group gives and we have, implicitly, used the relative proportions outlined in the original description of the three groups Fig. . It is lThe large differences in herd-level test characteristics such as sensitivity, specificity, false negative and false positive rates, depending on whether the intermediate seroprevalence group is assumed to be negative or positive Figs  and 2 hiIt is also worth noting that whatever the true status of intermediate seroprevalence group, the assumption that it represents truly positive herds results in estimates of the herd-level sensitivity which are lower than, or equal to, the actual herd-level sensitivity: this then represents a “worse case” estimate of herd-level sensitivity. It is therefore a “conservative” estimate for herd-level sensitivity. If this group were composed either partly or wholly of truly negative herds, then the actual herd-level sensitivity would be higher than predicted on the basis of the assumption that they are all truly positive. The opposite relationship can be seen for herd-level specificity: assuming that the intermediate seroprevalence group is positive results in estimates for this parameter which might be higher than the actual value. Whether it is preferable to under-estimate herd-level sensitivity or specificity of a test may be dependent on other conditions, as discussed below. Which of these test characteristics (sensitivity or specificity) is more important may determine which assumption, regarding the intermediate prevalence group, is the more “conservative” or risk-averse at a particular time.6. Therefore it is important that the test sensitivity is particularly high during the final stages of eradication even if this comes with the increased risk of false positives. To achieve a meaningful and maximum sensitivity indicates a cut-point of just one antibody positive animal. Thus the overall probability of a false negative is reduced if the number of animals tested is increased to ten and the cut-point is held at one  that some positive herds may be incorrectly classified as negative. This is because as the population approaches total susceptibility, the epidemiological, economic and political consequences of reintroduction become substantialone Figs , 3. The one Figs  will reqIn conclusion, we find that:The true status of the intermediate seroprevalence group is crucial, and analyses of herd-level sensitivity and specificity are highly dependent on the assumed status of this group. Misunderstanding the true status of this group will lead to imperfect design of a screening scheme and in particular poor selection of cut-points;Reducing the number of young stock screened for BVDv antibody from ten to five need not result in a substantial drop in herd-level sensitivity and specificity if the appropriate cut-point is selected;Increasing the cut-point to a value greater than one for the number of antibody seropositive animals before treating a herd as positive may be useful at the outset of an eradication scheme; The benefit of doing so is to increase the specificity, and reduce the cost of unnecessary secondary whole herd screening that is associated with a false positive.Increasing the number of animals tested, reducing the cut-point to one, or adopting both actions in the latter stages of an eradication scheme is warranted in order to maximise the herd-level sensitivity and thus reduce the number of false negative herds to as great an extent possible.We believe this paper provides a useful example of a framework for understanding the complexity of measuring herd exposure to an infectious agent using a spot-test approach. It enables us to consider how the ‘test’ should evolve over the course of an eradication scheme.et al.8 identified and described three statistically distinct groups or classes of herds on the basis of their within-herd seroprevalence levels: these groups are characterised by low, intermediate and high antibody seroprevalences. Brülisauer et al.8 described these groups statistically thus making them amenable to simulation . We explore here the consequences of both the extreme scenarios that this intermediate group is fully negative or fully positive whilst recognising that it could be a mixture of both negative and positive herds.We hypothesised that each group reflects an epidemiologically important class of herd with the group characterised by low seroprevalences reflecting negative status herds and the group with high seroprevalences reflecting positive status herds. The status of herds with intermediate seroprevalences remains unclear and has several possible explanationsWe then envisaged the relatively high herd-level prevalence which might be expected at the outset of an eradication scheme and compared it with the relatively low prevalence anticipated in the latter stages. We sought to demonstrate the varying rate of false negatives, false positives and predictive values dependent on the prevalence as well as the screening design.The simulation was carried out using the following procedure which is also illustrated in a flow diagram Fig. .Figure 68. We then simulated the true antibody seroprevalence in young stock within each herd , based on the range of seroprevalence values reported for the class of each herd. All simulation parameter values were based on published estimates, as presented in Table We constructed a population of 2000 simulated herds , which were distributed among the three classes of herds described above . The proportion of herds in each class was simulated based on published dataFrom each simulated herd, a sample of a fixed number of animals was drawn (the exact sample size varied depending on the scenario: see below for more detail). No assumption for the total number of young stock in the herd was made as this was not necessary – it was merely assumed that each herd had sufficient young stock to fulfil the desired sample. The true status (antibody-positive or antibody-negative) of each animal in the sample was determined based on a random binomial distribution defined by the overall proportion of antibody-positive animals in the herd. The test status of each animal in the sample was then determined on the basis of its true status and the (imperfect) test performance parameters  drawn from the triangular distributions described in Table We then envisaged a cut-point for the number of test-positive animals in the sample at, or above which, the sampled herd would be deemed to be positive. If the number of test positives was below that cut-point then the herd was interpreted as being negative.Therefore, we had 2000 herds each of which was from one of the three classes of herd . The low-seroprevalence class of herd was described as negative, and the high-seroprevalence as positive. The intermediate seroprevalence group of herd was treated as either negative or positive depending on which scenario we selected. This status was, in effect, our (simulated) “true” status for the herd. For each herd we also had the number of individual test-positive animals, and therefore when compared to a chosen cut-point the herd was designated “test positive” or “test negative”. As the test is not perfect, some herds may be truly negative but test-positive and vice versa. By summing over all herds we then calculated the proportion of “true” positive herds that tested positive and similarly for negative herds to estimate the herd-level sensitivity and specificity of the screening regimen.Different sample sizes (the number of young stock tested);Different cut-points; andDifferent assumptions about the group of herds characterised by intermediate seroprevalence.Different scenarios were examined to explore the consequences of important contributing factors on the predicted herd-level specificity and sensitivity:23. The low BVDv herd-level prevalence scenario was hypothesised at 0.5% based on work by Løken and Nyberg (2013)24 in which after 7 years of eradication the prevalence had dropped to 0.5%.The resulting simulated herd-level sensitivity and specificity estimates were used to estimate rates of false negatives and false positives and to estimate positive and negative predictive values under two different scenarios: (a) high BVDv prevalence as typical of the early stages for an eradication scheme and (b) low BVDv prevalence as typical of an “end-game” scenario towards the latter stages of an eradication scheme. The high BVDv herd-level prevalence was set at 60%variation or uncertainty, we ran the model 2000 times (to provide a 95% precision of ± 0.5% on a proportion of 1%), thus giving a range of values for herd-level sensitivity and specificity.Each run of the model provided a single estimate of the herd-level sensitivity and specificity, based on 2000 herds. In order to estimate the 8.The data used in this study were all generated through simulation based on the statistical distributions described in a publicly available paperSupplementary Information


In this case, an isolated persistent left superior vena cava (PLSVC) was present. A PLSVC is a remnant of the left cardinal vein which is normally obliterated during early embryological development, leaving the ligament of Marshall . FailureA PLSVC usually coexists with a right-sided superior vena cava (RSVC) or, less frequently, with a persistent bridging vein. In our case, however, a very rare isolated PLSVC was present, in which regression or obliteration of the RSVC occurs. Though rare, PLSVC is the most common venous anomaly, with a prevalence ranging from 0.3 to 0.5% in the general population and up to 10% in patients with congenital heart disease . An isolIn patients with an isolated PLSVC, especially right ventricular lead placement can be technically demanding due to the acute angle between the coronary sinus ostium and the tricuspid valve. In our case, we used a pigtail-shaped stylet to loop the lead in the right atrium and enter the right ventricle.During our procedure, it became clear that advancement of the leads through the RSVC was impossible, so we decided to cross over to the PLSVC. Eventually, a right-sided dual-chamber pacemaker was successfully implanted. Left-sided device implantation through a PLSVC may be complex, but also remains a feasible approach. Long-term results of pacemaker implantation in patients with an isolated PLSVC are good . In conc


Schizosaccharomyces pombe at 2.1 Å resolution. A structural comparison between two yeast L-aspartate aminotransferases revealed conserved enzymatic mechanism mediated by the open–closed conformational change. Compared with higher eukaryotic species, L-aspartate aminotransferases showed distinguishable inter-subunit interaction between the N-terminal arm and a large domain of the opposite subunit. Interestingly, structural homology search showed varied conformation of the N-terminal arm among 71 structures of the family. Therefore, we classified pyridoxal 5ʹ-phosphate-dependent enzymes into eight subclasses based on the structural feature of N-terminal arms. In addition, structure and sequence comparisons showed strong relationships among the eight subclasses. Our results may provide insights into structure-based evolutionary aspects of pyridoxal 5ʹ-phosphate-dependent enzymes.L-aspartate aminotransferase is a pyridoxal 5ʹ-phosphate-dependent transaminase that catalyzes reversible transfer of an α-amino group from aspartate to α-ketoglutarate or from glutamate to oxaloacetate. L-aspartate aminotransferase not only mediates amino acid and carbohydrate metabolism but also regulates the cellular level of amino acids by catalyzing amino acid degradation and biosynthesis. To expand our structural information, we determined the crystal structure of L-aspartate aminotransferase from  L-aspartate aminotransferase AST, EC 2.6.1.1) is not only a key metabolic enzyme that links amino acid metabolism to carbohydrate metabolism through reversible transamination reaction but also an enzyme that regulates the cellular level of amino acid by catalyzing amino acid degradation and biosynthesis [.6.1.1 isSaccharomyces pombe shares relatively high sequence identity with those of chicken cytosolic (46%), Saccharomyces cerevisiae (49%), and Escherichia coli (37%). On the other hand, S. pombe AST shares relatively low sequence identity with those of Pyrococcus horikoshii (16%), Thermus thermophilus (17%), and Thermotoga maritima (15%). The structure of cytosolic or mitochondrial AST from chicken [Saccharomyces cerevisiae [Escherichia coli [AST enzyme is highly conserved across species, owing to its essential role in varied metabolic pathways. ASTs are found from bacterial to eukaryotic species, with sequence identity ranging from 20%–80% . AST froytosolic 6%, Sacchia coli 3%. On theevisiae 4%, and Eshia coli , 11 havehia coli –14. Altharitima 1%. The sthia coli , 13.Among three families  of PLP-dependent enzymes, the aminotransferases were classified as an α-family according to the characteristics of their catalytic reaction and sequence similarity . Thus, GAST generally forms a homodimer consisting of two active sites in the vicinity of subunit interfaces; these active sites bind to its cofactor PLP and substrate independently. Each subunit is composed of three parts: large domain, small domain, and N-terminal arm. The active site is situated in the cavity formed by two large domains and one small domain. Conformational transition of the small domain was observed in accordance with the binding of dicarboxylic inhibitors , 12, 19.Schizosaccharomyces pombe. Based on the structural analysis of SpAST and other homologues, distinguishable inter-subunit interaction was observed between the N-terminal arm and the large domain of the subunit opposite to the N-terminal arm. In addition, the conformation of the N-terminal arm of PLP-dependent enzymes can be categorized into eight types via structural homology search. This study may provide insights into structural and evolutionary relationships for a broad spectrum of ASTs.In this study, to suggest a broader view of the three-dimensional structure and show the structural features of PLP-family proteins, we determined the crystal structure of AST from Schizosaccharomyces pombe L-aspartate aminotransferase  was amplified from its genomic DNA and cloned into pET26b. An accidental point mutation D153E was found in the recombinant DNA. However, the mutation was unlikely affect the enzymatic function due to its location as far from the active site or dimeric interface. The plasmid was transformed into BL21 (DE3) star, and cells were grown in LB medium. The recombinant SpAST protein was induced by 0.3 mM isopropyl-β-D-thiogalactopyranoside at A600 of 0.6, and cells were further incubated at 20°C for 18 h. Cells were then collected and lysed by sonication; cell debris was then precipitated by centrifugation. The supernatant was incubated with Ni+-NTA resin for 90 min, and the C-terminal His6-tagged SpAST protein was eluted by 250 mM imidazole after being washed with 5 mM imidazole. Samples were further purified using Hiprep 16/60 sephacryl S300 column , which was equilibrated with 20 mM Tris pH 7.5, 150 mM NaCl, and 2 mM DTT. Proteins were concentrated to 24 mg/mL and stored at −80°C until further use.The gene encoding SpAST protein was performed by employing hanging drop vapor diffusion method at 20°C. The crystal was observed at a concentration of 24 mg/mL protein in 20% (w/v) polyethyleneglycol 8000 and 0.1 M sodium HEPES (pH 8.5). Crystals were then cryoprotected using a crystallization solution containing 21% (v/v) glycerol and flash frozen in liquid nitrogen. The crystal was diffracted and collected at 100 K at 0.9795 Å on beamline 5C at the Pohang Accelerator Laboratory , using a Pilatus-3 6M detector (Switzerland) [1 space group.Crystallization of Saccharomyces cerevisiae AST  as a search model [The structure was determined by molecular replacement method using AutoMR software of the PHENIX software package with AST from ch model , 23. Thech model  and thench model . The modch model , using XSpAST from DALI server [The homologous proteins were searched by aligning their structures to that of I server . The subI server  and MegaI server  softwareSpAST forms a very stable homodimer with an unusually extended interface  has a central beta sheet, which encompasses 47 residues across seven strands surrounded by alpha helices, thereby resembling Rossmann fold. The small domain (residues 16–47 and 323–409) contains two antiparallel beta strands and is surrounded by five helices. Two domains are linked with each other through a long alpha helix α12. Fifteen amino acids present in the N-terminal extended from α1 of the small domain and formed a loop. This loop wraps across the subunit and interacts with the loop α9–β6, helix α10, and loop β7–α10 of the large domain.The structure of nterface . The solThe subunits of the homodimer are tightly associated mainly with their large domains through the salt bridges and hydrogen bonds present between side chains as well as between the main chain and the side chain. The water molecules also significantly contribute to inter-subunit interaction. Also, many hydrophobic residues are aligned at the interface contributing hydrophobic interaction between two molecules of dimer. Among 85 residues involved in the dimeric interaction, 7 residues formed salt bridges, 28 residues formed hydrogen bond, and 40 residues contributed hydrophobic interactions. Two tyrosine residues in both of the large domains, Tyr298 in helix α11, forms a face-to-face pi interaction with each other within a distance of 4 Å, contributing to stable dimerization of the dimer. Tyr70 and Tyr260 of both the large domains are also in close distance within 3.6 Å with a nearly perpendicular shape . This alUnexpectedly, two additional electron densities were found at the active site and the surface that could be fitted to phosphate and glycerol molecules . NotablySpAST is composed of Tyr70, Ser107, Gly108, Thr109, and Arg263 residues which recognize the phosphate group of PLP; Asn191, Asp219, Tyr222, and Lys255 for interacting with the pyridine ring of PLP; Asn191, Arg289, and Arg383 are involved in identifying the substrate (SpAST were compared with either S. cerevisiae (ScAST) or chicken cytosolic (GgAST) enzyme, most of the side chains, except those of Lys255 and Arg289, were found to have similar conformations. The residues corresponding to Lys258 and Arg292 of S. cerevisiae and chicken are shown to interact with their cofactor PLP and the inhibitor maleate, respectively , which spans both large and small domains for the comparison. The helix α12 of SpAST was tilted 11.2° away from central core of the dimer , α1 makes two salt bridges with loop α5-β2 and α10 in the same subunit. The region of N-terminal arm forms helical structure and contact large domain of the partner subunit [Most of the aminotransferases including  binding . The N-t subunit . These fThe two antiparallel β-sheets, instead of helix α1, were observed over the active site in subclass II enzymes . AlthougBacillus subtilis (PDB code 4MGR) was classified as subclass III and showed the most striking feature among the subclasses. The subclass III showed that the N-terminal region was further extended toward the large domain of the opposite subunit and formed a winged-helix DNA-binding domain (WHD)  from in (WHD) . As a reT. denticola  structure, the Lys8 of N-terminal arm makes hydrogen bond and salt bridge of the main chain of Glu32 on loop α1-α2, and side chain of Glu381 in helix α15, respectively [The subclass IV showed domain folds similar to subclass I, except for helix α1 and N-terminal arm. The helix α1 rotated almost perpendicular to that of subclass I, and the N-terminal arm followed a completely different path . NotablyPseudomonas putida , even helix α1 is missing and replaced by short loop, leaving the cavity of active site widely open. But this is not a common feature of subclass V.The structures belonging to subclass V contained relatively short N-terminal arm followed by helix α1, which could not contribute to dimerization interface . ExceptiBurkholderia pseudomallei . The helix α1 was extended by three turns and reached to the large domain of opposite subunit  showed no rigid body rotation on the small domain upon substrate binding, but clearly showed subunit rotation between monomers in homodimer [Methanocaldococcus jannaschii (PDB code 3F9T) of subclass VII also contained this loop, which indicates that two subclasses are closely related.The structures belonged to both subclasses VII and VIII shared similar structural feature on its N-terminal arm, forming a helical bundle with the helices in large domain of the opposite subunit . Tryptopomodimer . The majThe N-terminal arms of subclass III and VIII form extra fold which might be involved in further oligomerization as seen in the structure of gabR of subclass III (PDB code 4MGR), in agreement with the report of Lindqvist and co-workers . InteresSpAST. The phylogenetic tree data were generated using Maximum-Likelihood method Click here for additional data file.


This regulation process is highly sensitive to force conditions and protein-glycan interactions as the process occurs in flowing blood. There are two sites on A2 domain (N1515 and N1574) bearing various N-linked glycan structures. In this study, we used molecular dynamics (MD) simulation to study the force-induced unfolding of A2 domain with and without a single N-linked glycan type on each site. The sequential pullout of β-strands was used to represent a characteristic unfolding sequence of A2. This unfolding sequence varied due to protein-glycan interactions. The force-extension and total energy-extension profiles also show differences in magnitude but similar characteristic shapes between the systems with and without glycans. Systems with N-linked glycans encountered higher energy barriers for full unfolding and even for unfolding up to the point of ADAMTS13 cleavage site exposure. Interestingly, there is not much difference observed for A2 domain structure itself with and without glycans from standard MD simulations, suggesting roles of N-glycans in A2 unfolding through long-ranged protein-glycan interactions. At its matured form, a monomeric vWF has a total of 2,050 residues, and its 13 domains are connected in the following sequence: D′-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CT/CK 8, collagen type VI9, and heparin10. The A3 domain contains the binding sites for collagen at the damaged vessel spot. Nonetheless, it has been recently noted that the A1 domain could be shielded from binding to platelets due to its N- and C- terminal flanking regions and the adjacent A2 domain12. The A2 domain can be sufficiently extended or even unfolded under increased hydrodynamic loading, which may permit the A1 domain to interact with platelets. The A2 domain also contains a proteolytic site for a plasma protease  to regulate the size of vWF multimers13. Various asparagine (Asn) residues have been found to be highly glycosylated, and the number of N-linked glycans carried by typical sized vWF multimer is reported to be more than 300. For the A2 domain only, several glycoforms are determined on two specific sites, N1515 and N157414.von Willebrand Factor (vWF) is a blood glycoprotein that binds to platelets and collagen in the process of hemostasis. vWF is known to be synthesized and secreted by vascular endothelial cells and megakaryocytes/CK Fig. 3. A dime15 that are observed on two separate scales: whole polymer elongation and individual domain unfolding. For the former, Schneider et al. and Ouyang et al. independently showed that vWF multimers adopt a globular conformation in quiescent and low extension flow conditions. When extension rate exceeds a threshold value , vWF multimers transit to an elongated conformation17. For the individual domain unfolding behavior, A2 domain unfolding at high extension rate is preceded by domain elongation at intermediate extension rate. Experimental studies also demonstrated that ultralarge vWF (ULVWF) multimers, secreted from blood before released from Weibel-Palade bodies in endothelial cells18, were easier and more efficient to bind to GP1b than plasma vWF19. This is because, for a given extension rate, ULVWF multimers exhibit more elongation, exposing more sites for binding, than typical vWF in plasma. In healthy conditions, ADAMTS13 would cleave the peptide bond between Tyr1605 and Met1606 in A2 domains in the ULVWF multimers20; this prevents thrombosis and maintains a proper size range for functional vWF in blood. However, the cleavage site is located in the central part of the A2 domain and can be exposed only when the A2 domain is unfolded22. In disease conditions, this size regulation process could be disrupted. For example, clots formed due to aggregation of ULVWF multimers are reported to be one of the causes of thrombotic thrombocytopenic purpura (TTP)23. A significant number of Type 2 von Willebrand disease (vWD) is caused by mutations of the A2 domain, which may have some relationship with easy exposure of the cleavage site and thus its cleavage, resulting in a deficiency in making sufficiently long vWF to initiate clotting and stop bleeding25.Interestingly, a distinction of blood clotting stages has been characterized when vWF exhibits different types of molecular behaviors26. To mimic the flow induced unfolding process, external forces can be applied to the terminals of an A2 domain to move them apart. This can be done in experiments by optical tweezer and atomic force microscopy techniques or in atomic scale simulations. Chen et al. performed molecular dynamics (MD) simulations based on a published homology model of the A2 domain28, which gave first insight into force induced A2 domain unfolding sequence. Interlandi et al. later also performed force-induced unfolding simulations based on a crystal structure29. They concluded that the mutations related to certain vWDs  made the unfolding of the A2 domain easier than wild-type (WT) under similar external forces. This is indeed consistent with some vWDs related to those mutations whose certain hydrophobic residues were changed into charged side chains. McKinnon et al. reported that N-linked glycan itself could interact with ADAMTS13, preventing the A2 domain from being cleaved30. This work gave first insight into the N-linked glycans’ influence on ADAMTS13 proteolysis process of A2 domain.Experiments and simulations have been employed to elucidate the mechanical properties of vWF A2 domain unfolding and associated ADAMTS13 cleavage. Near to the wound site on a vessel wall, the extension rate increases dramatically, exerting large extension forces on vWF multimers. These extension forces would likely to invoke some A2 domains into unfolded states, so that the platelet/collagen binding and ADAMTS13 regulation process can be accelerated2+ can protect A2 domain cleavage by ADAMTS1333. An experimental study showed that the N-linked glycan on N1574 also stabilized the A2 domain against ADAMTS13 proteolysis34. In this work, Lynch et al. have performed experiments with WT A2 and truncated glycan structures for thermal-induced unfolding and the ADAMTS13 proteolysis. They concluded that glycans could essentially prohibit ADAMTS13 binding, suggesting that N-linked glycans can protect the already unfolded A2 domains from being cleaved by ADAMTS13. This result gives more insightful evidence about roles of glycan N1574 in modulating ADAMTS13 proteolysis process. In blood, as described previously, due to the high shearing and extension at the wound spot, the A2 domain undergoes unfolding by flow-induced forces much more than by thermal fluctuation. Thus it is still unclear whether and how N-linked glycans influence the force-induced unfolding process of the A2 domain.Recent biochemical and biophysical studies elucidated that the vicinal disulfide bonds in the C-terminal of A2 (C1669-C1670) with Ca14. Moreover, there are also researches showing that vWF proteolysis by ADAMTS13 is not a fast on/off process35, so that the simulations in this study were performed until the A2 domain was fully extended36 to provide insight into its size regulation process. To further investigate the influence of glycans on protein structure, standard  MD simulations of A2 domain with and without glycans (each with two replicas) were also performed.In this study, we performed MD simulations aiming to elucidate the influence of N-linked glycans on force-induced A2 domain unfolding process by comparing unfolding sequence, unfolding force, as well as energy with and without N-linked glycans. The N-linked glycans in this study were chosen to be the glycoforms found in human bloodAs shown in Fig. DEE). There are six representative SASA states during the unfolding process, and a clear jump in SASA can be seen between two adjacent states, representing an unfolding event of one of the β-strands. These states represent: (A) no β-strand unfolding, (B) β6 unfolding, (C) β5 unfolding, (D) β4 unfolding, (E) β3/β1 unfolding, and (F) fully unfolded A2, respectively. More specifically, state A represents the state where each β-strand is in a folded state, and only α6 is pulled out from the main body of the A2 domain. Thus, the sudden SASA jump between states A and B represents β6 unfolding. The same trend goes as each β-strand unfolds. And, as shown by Chen et al.27, pullouts of β5 or β4 is a sliding pathway which shows a clearer SASA jump. On the other hand, β1 unfolding is more like an unzipping pathway, going with a gradual SASA increase shown between states D and E. Clearly, the unfolding of either β6, β5, or β4 among all four systems happens almost at the same DEE, showing overall significant overlaps in the different pulling schemes. However, for the transition from state D to E +G Fig.  also cleC+G Fig. . This inDEE are shown in Fig. 37, or at most 0.1 mm/s38).The long-ranged protein-glycan interactions not only influence the unfolding sequence, but also increase the instantaneous forces and energy for A2 domain unfolding. The instantaneous force profiles as a function of Force fluctuations can be seen before β6 unfolds due to pre-adjustments of protein conformations during early pulling stages. In fact, the largest differences in the force profiles for pulling Nter versus Cter occur during the first few unfolding events . This is because β6 (close to the Cter) is always the first β-strand that is pulled out regardless of the Nter or Cter pulling. For the Cter pulling, residues close to the Cter are pulled out from the rest of the A2 domain that remains relatively stationary. For the Nter pulling, instead of pulling out the residues close to Nter, the bulk of the A2 domain is re-oriented and moves with the Nter group. Thus, in addition to the forces/energies required to pull out residues close to the Cter, additional forces/energies are required to move the bulk of the A2 domain with the Nter group. So that, the external forces can be transmitted to the structure near the Cter, overcoming the energy barrier to break the H-bonds for β6 unfolding . Moreover, Fig. In order to investigate whether such pre-adjustments have influences on the overall unfolding evens, we performed another independent simulation for each pulling Cter/Nter strategy up to β6 or β5 unfolding , the difference between force profiles with glycans versus without glycans starts to increase . 2), which is slightly over 2% of that in 2)). Clearly, the viscous force contribution to the force difference in Fig. To examine the influence of the viscous force (due to the bulky glycans) on the force difference between systems les Fig.  become pTo investigate the reason why systems 34. Here, we present a mechanistic study focusing on the influence of N-linked glycans on the force-induced unfolding of the A2 domain. We have performed MD simulations of force-induced unfolding process with the crystal structure of vWF A2 domain. Glycans were built based on two natural glycoforms14. Force-induced pulling was achieved with strategies of pulling through different terminals. Comparing among four independent pulling simulations, the glycans show some influences on the unfolding sequence and required forces/energy. Due to the long-ranged protein-glycan interactions on certain residues, β3 was pulled out earlier than β1 in some glycan systems while the opposite sequence happened in the systems without glycans. In addition, the systems with glycans showed about 25% more energy for full extension and 10% more until exposure of ADAMTS13 cleavage site than the systems without glycans. By examining the H-bond count from standard  simulations and calculating the interaction patterns among pulling simulations, it is clear that glycans make force-induced unfolding harder not by changing the protein structure in folded states, but through protein-glycan interactions in unfolded states. As mentioned in Methods, the glycan sequence used in this work contains the shared structure occurred in most glycoforms at each site. Thus, our results suggest that interactions between human plasma vWF and glycans are prevalent and influence the unfolding of A2 domain. Glycoforms may vary among individuals with vWDs, which has not been fully understood yet. Nonetheless, regardless of higher levels of brunches or truncated glycan structures, our work provides insight into a general role of glycans in A2 domain unfolding. We hope that our results may contribute to some investigations for glycan-related vWDs.It has been shown that N-linked glycans of vWF A2 domain can prohibit its binding with ADAMTS13 and thus proteolysis by ADAMTS1339. We used the CHARMM36m force field for protein40 and the CHARMM36 force field for carbohydrates43. A crystal structure for vWF A2 domain (PDB ID: 3GXB) was taken from Protein Data Bank44. We used a TIP3P water model45 and counter ions of Na+ and Cl− with the concentration of 0.15 M were added to neutralize the system. All simulation systems and parameters were set up using CHARMM-GUI47. Visualization and analysis were done by VMD48.In this work, all MD simulations were performed with NAMDFor A2 domain pulling simulations, the protein structure was initially placed in a rectangular water box with a size of 800 Å by 80 Å by 80 Å. The pulling forces were applied to the center of mass (COM) of each terminal residue . We performed four simulations: pulling N1495 (by fixing C1672) or C1672 (by fixing N1495) with or without glycans at N1515 and N1574. For convenience, pulling N1495 is called “Nter” pulling and pulling C1672 “Cter” pulling. In addition, the following symbols are used for the pulling systems: 2 and its moving speed to be 5 Å/ns. As a previous work showed, the fully extended length of a single A2 domain is 570 Å22, thus all simulations stopped when the terminal to terminal  distance reached this value, which is approximately 114 ns. The human vWF A2 domain has six β-strands (β1–β6) connected by loops with various lengths49, and these strands were used as references for A2 domain unfolding events. In this work, the sequences of glycan N1515 and glycan N1574  to model both N-glycan structures50; Glycan Reader & Modeler uses the PDB glycan structures from GFDB (http://www.glycanstructure.org/fragment-db)51. Figure The spring constant for pulling was set to be 5 kcal/mol/Å574 Fig.  were cho52 and the electrostatic interactions were calculated by particle-mesh Ewald method53 with a mesh size of ~1 Å for fast Fourier transformation and sixth order B-spline interpolation. SHAKE algorithm was used to constrain bond lengths involving hydrogen atoms54 and the simulation time-step was 2 fs. We first relaxed the system in a canonical ensemble (NVT) with T = 300 K with harmonic restraints to all solute atoms. The constant temperature was controlled by Langevin dynamics with the damping frequency of 50 fs−1. 100–120 ps isothermal isobaric ensemble (NPT) was then applied to adjust the solvent density. To control the constant pressure, Langevin piston method was used. A dihedral restraint force constant was set to be 1 kcal/(mol·rad2) in order to keep the carbohydate chair conformation during these equilibration steps. To perform the force-induced unfolding simulation of A2 domain , COLVARS method was used55 and the COMs of two terminal residues were calculated firstly as the external forces’ initial positions. The effective spring potential  acting on a pulling terminal residue was calculated using the following equation:k is the spring constant, v is the moving speed of the spring potential, The van der Waals interactions were smoothly switched off over 10–12 Å by a force-based switching functionSupplementary InformationMovie S1


With the rapid development of nano-preparation processes, nanocrystalline materials have been widely developed in the fields of mechanics, electricity, optics, and thermal physics. Compared to the case of coarse-grained or amorphous materials, plastic deformation in nanomaterials is limited by the reduction in feature size, so that they generally have high strength, but the toughness is relatively high. The “reciprocal relationship” between the strength and toughness of nanomaterials limits the large-scale application and development of nanomaterials. Therefore, the maintenance of high toughness while improving the strength of nanomaterials is an urgent problem to be solved. So far, although the relevant mechanism affecting the deformation of nanocrystalline materials has made a big breakthrough, it is still not very clear. Therefore, this paper introduces the basic deformation type, mechanism, and model of single crystals, polycrystalline materials, and thin films, and aims to provide literature support for future research. Yσ is the yield strength, σ0 is the lattice resistance, k is the material correlation coefficient, and d is the average grain size. This is an approximation. The more general formula is to use a power expression with exponent n, where 0.3 ≤ n ≤ 0.7. However, the Hall–Petch equation is strictly valid for polycrystalline materials with grain size larger than 1 μm, whereas for nanocrystals there are some significant deviations. When the metal grain size is reduced to the nanometer scale (d < 100 nm), the strength of the material will be significantly improved . S. S29]. Sc, crystal dislocation occurs  is related to the stress required to move the dislocations on the slip plane. Schmid’s law states that when the shear stress acting along the slip direction on the slip plane reaches a critical value τn occurs b 30]:. Where n is the normal vector to the slip plane and s is the slip direction. When λ = φ = 45° (soft orientation), τ = σ/2, the slip system possess the largest tensile stress; when λ = 90° or φ = 90° (hard orientation), τ = 0. Crystal orientation can make the movement of dislocations easy or difficult. Under identical conditions, more the slip systems in the crystal, more will be the spatial orientations that can be assumed during the slip process, which is beneficial for slip and, therefore, leads to high plasticity.In this formula, τ3+ and 500 mol ppm Ni2+ impurities. In a pure crystal containing a small amount of Fe2+, two different strain-hardening regions were identified with a CRSS of 0.73 ± 0.07 kg/mm2. Moreover, even if the concentration of Fe3+ is very low, precipitation-hardening can be observed during aging. The effect of heat treatment on Ni+2-doped crystals is very small. Studies have shown that Fe3+ has a much greater effect on MgO hardening than Ni2+. The common face-centered cubic (FCC) crystal has a slip direction of <1 1 0> and a slip plane of {1 1 1}. Therefore, the face-centered cubic crystal has 12 slip systems. The slip direction of the body-centered cubic (BCC) crystal is <1 1 1> and the slip surfaces that may appear are {1 1 0}, {1 1 2}, {1 2 3}. Thus, there are 48 potential slip systems for the BCC crystal. Although there are more slip systems in the BCC crystal than in the FCC crystal, the slip systems of BCC cannot appear simultaneously. Therefore, the plasticity of the BCC crystal is not as good as that of the FCC crystal. The slip direction of the hexagonal close-packed (HCP) crystal is <1, 1, −2, 0>; the slip surface is {0, 0, 0, 1}, the prism surface is {1, 0, −1, 0} and the pyramidal surface is {1, 0, −1, 1}. There are three slip systems of HCP. Owing to the small number of slip systems of HCP, its plasticity is generally not as good as FCC and BCC ,32. TablIn addition to impurities, many factors affect the CRSS of materials. Spitzig et al.  studied c-axis stretching, there will be tension twinning or stretching twins. It has a significant contribution to structural evolution and a substantial effect on plastic flow during large deformations. Rawat et al. . I. I29]. It et al.  used then et al.  studied n et al. , impuritn et al. ,42, or on et al. ,44 can hThe plastic deformation of crystals is a complicated process and it is of significance to study the deformation mechanism. Yoshinaga et al.  studied In general, low stacking faults can catalyze cracking metals and alloys with high twinning activity, whereas high stacking faults can catalyze slippage of cracked metals and alloys. Kim et al.  revealedThe plastic deformation of each of the crystal grains in a polycrystal is substantially similar to that of a single crystal, and the polymorphous plastic shape is still slip-like. Kocks et al.  comparedThe mechanical properties of polycrystalline materials can be adjusted by varying the degree of nucleation, propagation, and interaction of dislocations and shear bands. As metals and alloys are mostly polymorphic, the interaction between defects and grain boundaries is particularly significant. The grain boundaries accord higher strength and hardness to polycrystals than that of single crystals. The finer the crystal grains in polycrystals, the larger the ratio of grain boundary regions and the strength and hardness of metals and alloys. In bulk polycrystalline materials, the grain boundary acts as a barrier to dislocation motion, expressed by the classical Hall–Petch relationship, which describes the increase in the yield strength of polycrystalline metals as the grain size decreases. The particle size can reach 40 nm ,51. HoweThe above-mentioned deformation of the polycrystal has two characteristics: (1) the deformation of the crystal is affected by the grain boundary and the crystal grains having different orientations; (2) the plastic deformation of any one of the grains is not in an independent, free deformation state, and the surrounding grains are required to simultaneously adapt to the deformation to maintain the bonding between the grains and the continuity of the entire object. Therefore, establishing a reasonable model to describe the plastic deformation of polycrystals has always been a hot topic for researchers. In the polycrystalline plastic model, the material is considered to be a composite of many preferred orientation distributions . The plastic behavior of the metal polycrystal depends on the conversion of the microscopic physical quantity to the macroscopic physical quantity as well as on the appropriate treatment of the grain boundary effect. To describe a polycrystal, many homogenization models have been proposed based on the single-crystal plasticity theory. The most commonly used are the Sachs model , the TayIn the 1920s, Sachs first proposed the model of polycrystal plastic deformation. This model assumes that the deformation of each grain in the polycrystal is free, that is, the stress states of the polycrystal are continuous and the same as the stress states imposed by the outside world. However, in the actual situation, the same stress everywhere will lead to a discontinuity in the strain distribution, that is, the strain will not be coordinated and will thus leads to cracks . TherefoIn the 1930s, Taylor  proposedBoth the Sachs model and the Taylor model ignore the actual heterogeneity that occurs during polymorphic deformation, i.e., they do not consider intergranular interactions between the grains. Takajo et al.  used theEvidently, the above-mentioned various polycrystalline plastic models have their respective scopes of application, and they still cannot completely simulate and explain the deformation behavior and texture evolution of all grains under different loading deformation modes. At present, there are several noteworthy problems in the various models. First, certain aspects of the crystal’s constitutive relationship are not clear enough, such as work-hardening relationship, strain rate sensitivity, temperature influence, slip mechanism, and selection criteria of twinning system. The second is the interaction between grains, such as the role of grain size, stress balance between grains, and strain coordination. Therefore, further in-depth study of the crystal plastic constitutive model of the material is still needed.φ model for the viscoplastic large deformation of polycrystalline materials based on a new nonlinear intermediate method. The method was applied to predict the stress–strain response and texture evolution of face-centered cubic metals under tensile, compressive, and plane strain compression. In addition, the effects of weight parameters on sliding activity, stress–strain response, and texture transitions such as copper and brass textures were also analyzed  to form a cover layer having a certain thickness (less than 10 microns) and strengthening, protection, or special function. According to its mechanical properties, a thin film can be categorized as a brittle or ductile film. There are many factors affecting the mechanical properties of the film material. The elastic modulus and stress–strain relationship of the film affect the stress change of the film material after deformation, and the strength factor determines whether the film is damaged during use. The mechanical properties of the film material directly determine the range and lifetime of the thin-film material.In the past ten years, nano-films have played a significant role in the development of nano/microelectronic devices owing to their high strength, wear resistance, and good resistance to radiation ,72,73. TIn the grinding process of thin-film materials, it is necessary to simultaneously remove thin layers with very different properties. Under the action of mechanical load, burrs on the soft layer, debris on the brittle layer, and interfacial delamination will inevitably occur. This surface damage and structural failure can have a significant negative impact on the connection between the two thin-film modules, ultimately affecting the performance of the device . A varieIn the past two decades, the structural failure of thin-film systems has been extensively studied. Delamination at interfaces is a common form of failure in the processing of thin-film structures ,84. StudLu et al.  depositeFlexible electronic devices typically deposit a thin metal film on a polymer substrate. However, delamination of a conductive metal film  from the substrate may result in the loss of the electrical function. To improve the bonding strength between the metal film and the substrate, different brittle intermediate layers such as Cr, Ti, or Ta ,89,90 maIn recent years, nanomaterials have been used in increasing number of fields. Therefore, research on the preparation process and deformation mechanism of nanomaterials has received extensive attention. The irregular arrangement of atoms on the grain boundaries makes the energy stored inside the material high. Therefore, the grain boundaries become ready sources of defects. On one hand, under the energy drive, the grain size will gradually grow to reduce the area of the grain boundary; on the other hand, the size, distribution, and deformation mode of the grain are closely related to the material properties, service life, and different grains sizes. However, when studying the plastic deformation of materials, the classical model does not take into account the changes in the internal microstructure of the material and the unevenness of the deformation. The deformation phenomenon cannot be well explained. Therefore, studying the deformation mechanism of metal materials is of great significance for better study of the mechanical properties of metal materials. With the diversification of the mechanical properties of materials, existing material models have been unable to provide a reasonable description. The FEM is an effective calculation method for calculating the plastic deformation of materials. It can reflect the anisotropic properties of materials well and has important guiding significance for studying the deformation unevenness of materials and the evolution of texture. With the continuous development of characterization methods and calculation methods, the understanding of the interaction of grain boundary/twist boundary, dislocation, crack, mechanical twinning and shear band in nanostructured metal materials will continue to deepen. The law of strength and toughness of metal materials can be theoretically revealed, which provides a theoretical basis for the preparation of nanomaterials and their engineering applications.


Authors of a recent paper demonstrate that, like ERMES (ER-mitochondria encounter structure) in fungal cells, PDZD8 (PDZ domain containing 8) tethers mitochondria to the ER in mammalian cells. However, identifying PDZD8 as a “functional ortholog” of yeast Mmm1  is at odds with the phylogenetic data. PDZD8 and Mmm1 are paralogs, not orthologs, which affects the interpretation of the data with respect to the evolution of ER-mitochondria tethering. Our phylogenetic analyses show that PDZD8 co-occurs with ERMES components in lineages closely related to animals solidifying its identity as a paralog of Mmm1. Additionally, we identify two related paralogs, one specific to flagellated fungi, and one present only in unicellular relatives of animals. These results point to a complex evolutionary history of ER-mitochondria tethering involving multiple gene gains and losses in the lineage leading to animals and fungi. We do not dispute the authors’ interesting results with respect to PDZD8 function in mammalian cells. However, claims made by the authors regarding the evolutionary relationship of this gene with fungal homologs represents a misuse of the term ortholog, where the term homolog or, more appropriately, paralog is correct. This misuse has consequences for interpreting data in the paper, including in the domain-swapping experiments conducted as part of the complementation assay. The misclassification affects how their data should be interpreted and results in confused explanations of how ER-mitochondria contact sites evolved in animals and fungi. Approaching the data with correct terminology alleviates these problems and illuminates interesting possibilities about trait evolution in fungi and animals.Hirabayashi2). However, orthologs can diverge and perform different functions in different lineages . An example of heterofunctional orthologs is animal Miro , which, in yeast, is called Gem1 (GTPase EF-hand protein of Mitochondria) and has been suggested to interact with ERMES (ER-mitochondria encounter structure) in fungi4. Miro is important for microtubule-dependent mitochondrial motility in animals . However, although a Miro ortholog is present in all eukaryotic lineages, it does not function in microtubule-dependent mitochondrial motility in yeast,Neurospora,Arabidopsis, orDictyostelium7. Thus, functionality alone does not imply orthology.Orthology and paralogy are special cases of homology . Ortholoet al.1 swap the SMP domain from PDZD8 into the Mmm1 protein inSaccharomyces cerevisiae thereby rescuing the defects imparted by loss of Mmm1. But, they also successfully rescue Mmm1 defects by swapping the SMP domain from its paralog,S. cerevisiae Mdm12 , into theS. cerevisiae Mmm1 protein. The domain swapping experiments should not be interpreted to mean that the proteins (or even the domains) carry out the same function. Instead, these experiments suggest that the paralogous SMP domains from PDZD8, Mmm1, and Mdm12 are biochemically isofunctional in this specific scenario . However, it must be stressed that this does not mean that the proteins themselves are isofunctional. The fact that mammalian PDZD8 and similar proteins from other animals have long C-terminal extensions containing accessory domains (e.g. a PDZ domain and cysteine-rich C1 domains), while Mmm1 and Mdm12 do not, suggests these proteins have different or additional mechanisms of function. Thus, we cannot say that the full proteins are isofunctional homologs and especially not isofunctional orthologs.Any SMP domain is homologous to any other SMP domain because they have shared ancestry, but only those arising via speciation are orthologs. To demonstrate that PDZD8 and Mmm1  are “functional orthologs”, Hirabayashi11; but it has also been demonstrated that PDZD8 is not an ortholog of ERMES components . This can be seen very clearly in the SMP-domain proteins ofCapsaspora owczarzaki, which include orthologs of all the ER-mitochondrial contact site SMP proteins . This organism is closely related to animals12, but still retains both a complete ERMES complex and PDZD8 and represents a future model for investigating their differential functions. Interpreting the work of Hirabayashiet al.1 from a comparative perspective demonstrates that mitochondria-ER tethering is a function that is conserved deep within this gene family predating the duplication that gave rise to PDZD8 and Mmm1, and indeed, all known ERMES paralogs. This is important because it allows us to phrase the next questions, ‘how have different paralogs expanded, or changed, in function? ’ and ‘why have some evolutionary lineages maintained multiple paralogs while others have tolerated loss?’. When uninformative terms like ‘functional ortholog’ are used, especially in exceptional papers like Hirabayashiet al.1, biologists interested in explaining functional differences between organisms are being misled. We stress that only comparative evolutionary methods can identify the starting data for phrasing the above questions and functional data cannot be used when inferring orthology or paralogy. Once questions of orthology and paralogy have been resolved, questions of functional conservation and divergence can be addressed at the bench.The fact that PDZD8 is a homolog of SMP domain-containing proteins like Mmm1 has been demonstrated previously14. The sequences used to generatehttps://github.com/mbzdlb/PDZD8.All sequences were downloaded from publicly available databases (e.g. Joint Genome Institute or NCBI) or obtained from published transcriptomic and genomic data et al. comment on a recent seminal study by Hirabayashiet al., which describes the biochemical characterization of the SMP domain-containing protein PDZD8, as an ER-mitochondria tether important for the regulation of Ca2+ dynamics in mammalian neurons. Although the functional data presented by Hirabayashiet al. is sound and of great interest, their interpretation of the homologous relationship between PDZD8 and the yeast SMP domain-containing protein Mmm1 is incorrect. Mmm1 is part of the ERMES complex, which tethers the ER to mitochondria in yeast and is important for the exchange of phospholipids between these organelles. This complex contains two further proteins of the SMP family, Mdm12 and Mdm34. Wideman et al.1 and we2 have previously shown that some eukaryotes contain all three of these ERMES proteins as well as PDZD8, indicating that they are paralogs. Hirabayashiet al., however, misinterpret PDZD8 to be a functional ortholog of yeast Mmm1, and substantiate this relationship experimentally by substituting the SMP domain of yeast Mmm1 with that of PDZD8 to rescue the phenotypic defects caused by the deletion of Mmm1. Here, Widemanet al. present convincing data to show that PDZD8 is a paralog of Mmm1 and not its ortholog. The arguments presented by Widemanet al. are sound and will help to clear the confusion regarding the evolutionary relationship between these proteins.In this correspondence, Wideman Minor comment: The human PDZD8 protein contains a C1 domain as well as a coiled-coil segment at its C-terminal end. Do other orthologs also contain these domains? If so, the domain composition of PDZD8 shown in Fig. 2 should be updated to show this. Also, HHpred searches suggest that the PDZ domain in human PDZD8 is inserted into the C2 domain; is this the case for other orthologs too?I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In this correspondence, Wideman and colleagues show that PDZD8  is not an ortholog of the ERMES subunit Mmm1p. This conclusion by Wideman et al. is well founded, and the underlying argument is well explained in the text here.1). There, the SMP domain from mammalian PDZD8 substituted for the function of  the Mmm1p SMP domain in yeast, which was used to support the view that “The SMP domain of PDZD8 is functionally orthologous to the SMP domain found in yeast Mmm1.” However, a definition of orthologue that I accept is “Homologous sequences are orthologous if they are descended from the same ancestral sequence separated by a speciation event” [https://en.wikipedia.org/wiki/Homology_(biology)]. By that definition, I agree with Wideman et al. that bioinformatic studies indicate PDZD8 and all 3 ERMES subunits with SMP domains, including Mmm1p, are paralogs, not orthologs. The same point of non-orthology has been said before in Wideman et al., 2015, but was then missed in the first paper that focussed on the function of PDZD8  can be substituted by the other’s SMP domain. There are several points where the current article might be marginally improved by revision:2. Improve text and diagram describing domains within PDZD8: apart from the SMP and PDZ domains, PDZD8 is described as containing a C1 domain e.g. NCBI human PDZD8 (1154aa): 841-883. Here that domain is missing from the diagram. In addition, a C2 domain is shown in the diagram in an incorrect way, because the C2 domain sequence in PDZD8 is split. ~65 aa lies between the SMP and PDZ domains (320-372), then the rest (695-800) is before the C1 domain. See Figure 1B in Wong et al. 2017 The text would benefit from a brief analysis of likely branching of the SMP protein family, based on supplementary data from the 2015 paper, possibly plus extra work to place the different Mmm1 groupings and the two new designations (X and Y). The  text should explcitily state that the Mmm1s in C. owczarzaki and other holozoa are variant, rooting onto the main tree separately from fungal Mmm1s, despite the shared presence of the 2 accompanying motifs. This raises another point. The diagram adds to cladistic analysis of SMP domain sequences alone by looking for accompanying motifs. How can using accompanying motifs be justified, when accompanying domains are excluded from the orthology/paralogy argument? As SMP sequences can be inherited separately from other elements, I do not fully grasp that C. owczarzaki-1867 is included in Mmm1, while C. owczarzaki-5382 is excluded from Pdz8.Fungi” refers to each of the three times it appears in the diagram. Is it always the same set of fungal sequences or a different group in each case? How many?Say what “Say that the figure is not the same as appears in ref 11 or its supplementary data, but based on the same data set. Say that Tcbs have been excludedFor “closely related protists” (line 3) say if every protist has been included or a selection has been made (and how)Identify falgellated fungi (compared to non-flagellated)Add description of one/two asterisks to key, not at bottom of long legend.   Understanding the figure might be improved by more information in the legend: Data set on Git Hub: I note that some sequences are incomplete. This should be made explicit.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.