New Zealand (NZ) is considering transitioning from 3-yearly cervical cytology screening in women 20–69 years (current practice) to primary HPV screening. We evaluated HPV-based screening in both HPV-unvaccinated women and cohorts offered HPV vaccination in New Zealand (vaccination coverage ~50%).A complex model of HPV transmission, vaccination, cervical screening, and invasive cervical cancer was extensively validated against national population-based datasets. Sixteen potential strategies for HPV screening were considered.Most primary HPV strategies were more effective than current practice, for both unvaccinated women and cohorts offered vaccination. The optimal strategy for both groups was 5-yearly HPV screening in women aged 25–69 years with partial genotyping for HPV 16/18 and referral to colposcopy, and cytological triage of other oncogenic types. This is predicted to reduce cervical cancer incidence and mortality by a further 12–16% and to save 4–13% annually in program costs (excluding overheads). The findings are sensitive to assumptions about future adherence to initiating screening at 25 years.Primary HPV screening with partial genotyping would be more effective and less costly than the current cytology-based screening program, in both unvaccinated women and cohorts offered vaccination. These findings have been considered in a review of cervical screening in NZ. The New Zealand (NZ) National Cervical Screening Programme (NCSP) was established in 1990 . Three-dCompass (NCT02328872). A parallel pilot service evaluation project using primary HPV screening in NZ, known as ‘Compass NZ’ (ACTRN12614000714684) has also completed recruitment.Consideration of a transition to primary HPV testing for cervical screening in NZ has been prompted by the emergent international evidence on the increased effectiveness of primary HPV screening compared to cytology. Women who test negative for cervical HPV infection have a substantially lower risk of later developing high-grade cervical intraepithelial neoplasia (CIN 2/3 and CIN3+) ,5,6,7 anIn this context, in 2015 New Zealand initiated a process of policy review of the NCSP to consider a potential transition to primary HPV screening. The aims, therefore, of the current study were: (1) to construct, calibrate and validate a complex model of HPV vaccination and cervical screening in NZ; (2) to determine, in collaboration with the NZ NCSP, a set of possible strategies  suitable to the NZ context using HPV as the primary screening test; (3) to evaluate the lifetime effects and costs associated with each of these strategies, in relation to current practice for cervical screening, in both vaccinated and unvaccinated cohorts, taking into account the levels of vaccine coverage achieved in NZ; and thus (4) to identify optimal future screening approaches in NZ in both vaccinated and unvaccinated women.A dynamic model of HPV transmission and vaccination , coupled with a deterministic Markov model of the natural history of CIN and cervical screening and invasive cervical cancer survival , was used to simulate cervical disease and screening in NZ. This model platform, adaptable to different settings, has been previously used to evaluate various cervical screening and follow-up management strategies in Australia, NZ and England ,19,20,21The natural history model incorporates different disease progression and regression rates for HPV 16 infections, HPV 18 infections and other oncogenic HPV infections . The dynamic HPV transmission model, adapted from previous work, ,22,23 taCurrent practice (CP) for cervical screening within the NCSP was modelled based on existing clinical management guidelines . AdherenWe took a health services perspective, considering screening, diagnosis and treatment costs for the NZ government. All costs are presented in NZ dollars  and were inflated to 2017/18 values. The costs were obtained from the NCSP with the costs of cervical cancer treatment further calibrated to achieve an average cost of $23,116 per cancer patient as reported in 2008/09 in NZ . All cosper se but assign high amount of disutility for having abnormal screening outcomes, and Set 3 did not assign any disutility to the experience of being screened per se but assigned a small disutility for having abnormal screening outcomes.Because we have previously demonstrated in similar evaluations for Australia  and EnglFurther details of model structure, assumptions and the data sources are described in the supplementary document A total of 16 primary HPV screening strategies, determined in a series of consultations with the NCSP and its Advisory Group, were modelled see . The strIn the base case, primary HPV testing was assumed to have a test sensitivity of 96.4% and a specificity of 90.3% for CIN2+ detection in 20–69 years, consistent with the pooled absolute test sensitivity and specificity reported in a meta-analysis . CompareThe screening strategies were evaluated under two scenarios with respect to HPV vaccination. The ‘unvaccinated scenario’ assumed all women were unvaccinated, and thus this scenario tends to reflect outcomes in older cohorts. The ‘vaccinated scenario’ assumed women were offered HPV vaccination with uptake as observed in the cohort born in 1997 who were offered HPV vaccination at 12 years in 2009 through the HPV Immunisation Program  that comFor the strategies that assumed screening starts at 25 years, in the base case we modelled a ‘rapid uptake’ at 25 years, which assumed an invitation was issued and that women currently starting screening before age 25 years would all have their first screening test at 25 years under the new recommendations. The compliance to a 5-yearly interval was derived by assuming the proportion of women have early re-screening, on-time screening and late re-screening remained similar to that observed under current practice in NZ (see supplementary document The outputs of the model included cumulative lifetime risk (CLR) and age-specific rates of cervical cancer incidence and mortality, cytology test outcomes and histology test outcomes from age 10 to 84 years. Age-standardised rate (ASR) of cervical cancer incidence and mortality were calculated assuming the World Health Organisation (WHO) population.. The aveThe overall lifetime cost, LYs and QALYs associated with each strategy were calculated as the model output of cost, life-years and QALYs of the cohort accrued from 10 to 84 years and discounted at a rate of 3.5% p.a. from 20 years (the age of currently initiating screening). The total cost associated with the NCSP in 2017 was estimated assuming the 2017 NZ population . The effUnivariate sensitivity analysis was performed on a list of key model assumptions listed in The National Cervical Screening Programme Register data used to inform model parameters were de-identified. The Cancer Council NSW Human Research Ethics Committee approved the transfer of these data to the researchers and the New Zealand Ministry of Health approved their use in this modelled evaluation.The calibrated model outcomes showed close correspondence with observed age-specific rates of HPV prevalence ,25, HPV In the NZ population of 2.3 million women aged <85 years in 2017, if current practice (CP) for screening was retained, a total of 160 new cervical cancer cases, 45 cervical cancer deaths and 4,038 women detected with histologically-confirmed high-grade were estimated to occur in the unvaccinated scenario; with 92 new cancer cases, 26 cancer deaths and 2,645 cases of histologically-confirmed high-grade disease in the vaccinated scenario . The preThe relative effectiveness of each primary HPV screening strategy in relation to CP was similar in both unvaccinated and vaccinated scenarios. Twelve out of the sixteen primary HPV strategies were predicted to be associated with a decrease (of 2–20%) in cervical cancer incidence and mortality. Strategies varied in terms of their impact on the number histologically-confirmed high-grade cases, ranging from a +/-7% difference compared to CP .Partial genotyping strategies (S2 and S4 strategy groups) were associated with a 1–16% relative decrease  in cancer incidence and mortality when compared to the equivalent non-partial genotyping strategies (S1 and S3 strategy groups); HPV and cytology co-testing strategies (S3 and S4 strategy groups) were associated with a modest (<1–3%) relative decrease in both scenarios when compared to the equivalent non-co-testing strategies (S1 and S2 strategy groups).Within each strategy group, strategies assuming primary HPV screening starting at age 25 years (strategy sub-groups a and b) were predicted to be associated with a 3–15% relative decrease in cancer incidence and mortality when compared to the equivalent ‘switch-over’ strategy (strategy sub-groups c and d); strategies that refer ‘intermediate risk’ women to immediate colposcopy (strategy sub-groups b and d) were predicted to be associated with a 2–12% relative reduction in both scenarios when compared to the equivalent strategies that referring ‘intermediate risk’ women to 12 months follow-up (strategy sub-groups a and c). However, this difference was minimised in partial genotyping strategies, where the relative difference was only 2–5% (since the highest risk HPV16/18 positive women are referred directly to colposcopy in such strategies), compared to 5–12% in strategies without partial genotyping.Compared to CP, starting screening at 25 years with primary HPV testing (strategy sub-groups a and b) was predicted to be associated with a higher cancer incidence rate in 25–29 year-old women (the first 5 years after screening begins) but a significantly lower rate in 30+ years . PartialScreening according to current practice is predicted to cost $31.7M to the NZ government in 2017 (unvaccinated) and $25.9M (vaccinated) . This inAll strategies assuming primary HPV testing (S1 and S2 groups), were more effective than CP, and all except S2b were also cost-saving. All co-testing strategies (S3 and S4 groups) were both more effective and more costly than CP. Supplementary cost-effectiveness analyses were undertaken using health utilities to incorporate the effect on morbidity as well as mortality. Findings varied widely with the different utility sets. Strategy S2a was predicted as the most cost-effective (and cost-saving) strategy in both the unvaccinated and vaccinated scenarios when QALY weights set 1 and 3 were assumed; all primary HPV screening strategies were predicted to be less effective than current practice when QALY weights set 2 was assumed. Detailed outcomes of the supplementary analysis are provided in the supplementary document About 5,020 women in the unvaccinated scenario and 3,350 women in vaccinated scenario were estimated to undergo treatment for precancer under CP; most primary HPV screening strategies would result in a broadly similar number of women undergoing treatment .Under CP screening management, given the adherence assumptions used, a woman was predicted to experience 14.1 screening and/or follow-up episodes on average in her lifetime in the unvaccinated scenario and 14.0 episodes in the vaccinated scenario. This number will reduce to an average of 9.7  episodes for primary HPV screening strategies screening starting from age 25 years (sub-strategies a and b), and to an average of 11.0  episodes for the ‘switch-over’ strategies (sub-strategies c and d) . On averUnivariate sensitivity analysis was conducted for S2a and S2c in both unvaccinated and vaccinated scenarios. The predicted costs were found to be sensitive to the assumptions made for the cytology test cost, the screening initiation patterns, test characteristics of HPV test for primary screening or for triage testing on women with low-grade cytology results (which changes the predicted comparator outcomes), the cost of HPV testing, the accuracy of specific HPV partial genotyping outcomes and the aggressiveness of natural history in both scenarios. For S2a, the predicted life-years were found to be sensitive to the assumptions made for the aggressiveness of natural history , and adherence to the recommendation to initiate screening at age 25 years (i.e. for most women to initiate screening at or close to that age and not later). Increased test sensitivity for both LBC as a triage test for women with a positive HPV test result and HPV as a triage test for women with low-grade cytology outcome were found to have minimal impact on the cost and life-years estimated for S2a. The detailed outcomes of the sensitivity analysis are provided in the supplementary document We have performed a comprehensive health and economic evaluation of primary HPV testing for cervical cancer screening in New Zealand, which takes into account the impact of a national HPV vaccination program in that country. In this analysis, we found broad similarities in the relative outcomes between alternative screening strategies when considering unvaccinated women and women offered vaccination. Most  of the primary HPV screening strategies we considered were predicted to be more effective in reducing cancer incidence and mortality compared to current screening practice. Many of the primary HPV screening strategies, with the exception of co-testing, were also predicted to be less costly than current practice. In the cost-effectiveness analysis, primary screening with partial genotyping for women aged 25–69 years, with direct referral of HPV16/18 positive women to colposcopy and LBC triage for women positive for other oncogenic types with 12 month follow-up for LBC negative and low-grade results in that group (Strategy S2a) was identified as both more effective and also cost saving compared to current practice, and in comparative incremental analysis was the most cost-effective strategy in both scenarios. This strategy was predicted to be associated with a further 12–16% reduction in cervical cancer incidence and mortality and a saving of $NZ1.3 M—$3.2 M (4–12%) of the total program cost of screening compared to current practice. A variation of this strategy, where women testing positive for other (non-HPV16/18) oncogenic types but who had low-grade cytology were referred directly for colposcopy (Strategy S2b), was marginally more effective, but not cost-effective  compared to a $20,000-$50,000 per life-year saved indicative willingness-to-pay threshold in New Zealand (where the upper end reflects the approximate GDP per capita in March 2015).The three QALY weight sets assessed in the supplementary analysis of this study were obtained from three different studies ,49,50 anboth more effective and cost saving when compared to current screening practice, reducing cervical cancer incidence and mortality in New Zealand by a further 12–16% compared to current levels. A strength of the evaluation is that it used a comprehensive and calibrated model of cervical screening, based on detailed clinical management guideline recommendations, taking into account the observed screening behaviour. We conducted detailed evaluations of a wide range of primary HPV testing management options and these findings will inform policy makers of the potential benefit as well as the impact on resource utilisation associated with primary HPV testing in NZ.The current evaluation was performed on behalf of the National Cervical Screening Programme (NCSP) and overseen by the programme’s Advisory Group. This was done in the context of the recent announcement by the Australian government that it will transition to 5-yearly HPV screening in women aged 25–70 years by May 2017; the effectiveness modelling and economic evaluation to support that decision in Australia harnessed the same modelled platform and methods as for the current evaluation in New Zealand . As for A limitation of this study was that a number of influential parameters related to future screening practices, by necessity, were based on assumptions. However, these assumptions had been extensively discussed with in-country experts at the NCSP. For the primary HPV testing strategies, we assumed ~80% of women will attend for their first screening test at 25 years (based on the current uptake rate observed in women aged < = 25 years) and that the compliance rate to a 5-year screening interval recommendation will be consistent with that currently observed for the recommended 3-year interval, including limited early re-screening. After consultation, we also assumed the cost of HPV test would reduce to $35 per test when HPV test was used as the primary screening test (assuming a lower unit test cost could be negotiated with a larger volume of tests being purchased). The sensitivity analysis has found that the cost outcomes are sensitive to this assumption and thus this is a prime consideration for decision-makers in the transition to primary HPV screening. However, the optimal strategy for HPV screening with partial genotyping is still predicted to be cost saving compare to current practice when assuming HPV test cost was $40 per test in sensitivity analysis.This evaluation was conducted using assumptions that were favourable for cytology testing and conservative with respect to primary HPV testing. Specifically, the base case assumptions for HPV test characteristics were derived such that the relative test sensitivity and specificity (compared to base case cytology test characteristics) was consistent with the lower end of the 95%CI of the findings of a major meta-analysis –that is,We found that most of the primary HPV screening strategies were predicted to increase the number of women requiring access to diagnostic services, if the impact of vaccination is not considered. This finding implies that the colposcopy referral and histology evaluation rates may increase initially after a transition to primary HPV screening but are expected to drop over time as cohorts offered vaccination age and enter the new HPV screening program. However, it should be noted that the current estimates represent ‘steady state’ outcomes and transitional fluctuations in test volumes and referral rates would be expected after the transition of the screening program.We found that 5-yearly primary HPV screening would be more effective and cost-saving compared to the current 3-yearly cytology screening program in NZ, and that managing women based on HPV partial genotyping was a highly effective, and the most cost-effective, option. We also found that co-testing with cytology is not cost-effective. These findings are consistent with the results of similar modelled evaluations conducted for England and Australia ,10.It is notable that, although NZ and Australia have differing recommendations for current practice for cervical cytology screening , the optimal strategy identified in this evaluation for NZ  is very similar to the proposed pathway for primary HPV screening identified in the National Cervical Screening Program Renewal for Australia . It alsoThe assessed primary HPV screening strategies were determined in a series of consultations with the NCSP and its Advisory Group, and were modelled in the context of New Zealand, which has an effective cervical screening program that provides 3-yearly routine screening using cytology test with a high test sensitivity (~79% for CIN2+ detection) for 20–69 years. Although the optimal screening strategy identified by this study is also similar to the proposed pathway identified in the National Cervical Screening Program Renewal for Australia, some ofCompass will play a role in this process- Compass NZ (ACTRN12614000714684) has recruited five hundred 25–64 year-old women presenting for cervical screening for 5-yearly HPV screening with partial genotyping for HPV 16/18, to assess feasibility of this approach via participant acceptability, promoting GP, nurse and colposcopist education, and facilitating laboratory implementation. Since Compass NZ utilises the same pathway for primary HPV screening as that identified in this modelled analysis as the optimal approach, it represents a practical experience of primary HPV screening with this pathway before its implementation in NZ.NZ had a challenging history with respect to the original introduction and development of cervical screening ,51. TherWe predict that primary HPV screening with genotyping for HPV16/18 will be both more effective and less costly than the current program for 3-yearly cytology-based screening in NZ, in both unvaccinated women and in cohorts offered vaccination. These findings are in accordance with modelled evaluations for Australia and England and are consistent with the data from large scale international trials of primary HPV screening. Any change in national policy will involve extensive public consultation and will take into consideration broader criteria beyond cost-effectiveness.S1 Appendix(DOCX)Click here for additional data file.


Two-point linkage analyses of whole genome sequence data are a promising approach to identify rare variants that segregate with complex diseases in large pedigrees because, in theory, the causal variants have been genotyped. We used whole genome sequence data and simulated traits provided by Genetic Analysis Workshop 18 to evaluate the proportion of false-positive findings in a binary trait using classic two-point parametric linkage analysis. False-positive genome-wide significant log of odds (LOD) scores were identified in more than 80% of 50 replicates for a binary phenotype generated by dichotomizing a quantitative trait that was simulated with a polygenic component (that was not based on any of the provided whole genome sequence genotypes). In contrast, when the trait was truly nongenetic (created by randomly assigning affected-unaffected status), the number of false-positive results was well controlled. These results suggest that when using two-point linkage analyses on whole genome sequence data, one should carefully examine regions yielding significant two-point LOD scores with multipoint analysis and that a more stringent significance threshold may be needed. The development of low-cost, high-throughput sequencing technologies has reignited the interest in family-based study designs for finding causal genes in rare and common diseases. Linkage analysis was designed for the investigation of rare variants of large effect and is robust to allelic heterogeneity. Most researchers are familiar with using multipoint methods for assessing linkage, which leverage cosegregation of a marker-haplotype with the trait in a family as a proxy for the location of causal variants. Whole exome sequencing (WES) and whole genome sequencing (WGS) of several or all individuals in a pedigree has now become feasible. These data provide information about genetic variability at a very high resolution. Multipoint linkage analyses of such dense sets of markers are time-consuming, do not scale well to millions of markers, and intermarker linkage disequilibrium (LD) can cause inflated type I error rates when pedigree founders are not genotyped . A powerGenotype data for all 8,348,674 sequenced and imputed single nucleotide variants (SNVs) on the odd-numbered chromosomes were analyzed. There were 959 individuals with genotypes; 430 individuals without genotype data were added to provide complete pedigree information for linkage analysis, and one member was removed from each of two monozygotic twin pairs. There were 413 founders: 108 had genotypes and phenotypes, 9 had genotypes but no phenotypes, and 296 had neither. Because our goal was to estimate the false-positive rate under the null hypothesis of no linkage, we used two phenotypes that were simulated independently of any genotyped SNVs in the GAW18 data set. We requested the details of the simulation, and therefore knew the generating model of the simulated traits. The first analyzed phenotype was a binary trait based on the provided quantitative phenotype Q1 that was simulated by the GAW18 data providers to allow estimation of false-positives. Q1 was simulated as a normally distributed quantitative trait correlated among family members with heritability = 0.68, but was not influenced by any of the genotyped SNVs [Several pedigrees contained loops that cause problems for likelihood estimation in the linkage analysis program we used. Because our goal was to estimate false-positive rates under the null , we chose very simple strategies for breaking these loops to produce simple pedigrees. Different approaches to break loops were applied depending on their complexity. Removing the unaffected individual T2DG2501049 in pedigree 25 broke a loop caused by a woman with children from two brothers. The simple loops in pedigrees 2 and 3 were removed by duplicating an unaffected individual (T2DG0200031 and T2DG0300138) with both parents and children in the pedigree. Duplicated individuals had the same genotype and phenotype data but modified family relationships. Pedigrees 6 and 7 were excluded because their more complicated loops could not have been broken with the simple approaches used for the other pedigrees. Thus we had a set of simple pedigrees that allowed analysis of false-positive rates of two-point parametric linkage. In analyses of real data, use of a program that can analyze large pedigrees with loops intact would have optimal power.http://cran.r-project.org/web/packages/paramlink/index.html). A dominant model was chosen because no, or only a very low number of, rare allele homozygotes are observed for rare SNVs and because we were seeking to evaluate type I error rate rather than power. In a real analysis, one could either use a model from segregation analysis or could utilize dominant, recessive, and additive models with reduced penetrance [Allele frequencies of all SNVs were estimated with PLINK , using fnetrance  and adjunetrance -13. Penenetrance , with a netrance . The GWSResults are presented separately for the two different unlinked phenotypes.For the binary trait based on the provided quantitative null trait Q1, 43 of 50 replicates showed at least one GWS LOD score, with values ranging from 3.3001 to 5.685 , only two out of the 50 replicates had GWS results. In one replicate, one SNV on chromosome 1 had a combined LOD score of 3.336 that was driven by a LOD score of 3.334 in family 3. The MAF was estimated as 0.0085, and all seven affecteds in family 3 were heterozygous for the rare allele. Only three of 53 unaffecteds in this family were heterozygotes. In another replicate, 19 SNVs in a 4.6 Mb region on chromosome 3 had combined LOD scores between 3.308 and 3.751, with family 5 having the largest family-specific LOD scores, ranging from 2.128 to 2.578. These SNVs were in two blocks with nearly complete LD, and estimated MAF was 0.389 on average. Again, all affecteds, or all but one affected, were heterozygous or homozygous for the rare allele in family 5.Linkage analysis fell out of favor in the genome-wide association studies era, but the power of families can be well leveraged in sequencing studies. The advent of large-scale WES and WGS studies has rekindled interest in family-based analysis designs ,14. FamiIn this study we were interested in type 1 error rates of two-point linkage analysis of a binary trait under the null hypothesis of no linkage. GAW18 provided only one quantitative trait simulated under the null hypothesis of no linkage  so we chose to dichotomize this trait; we also simulated our own qualitative trait that had no genetic effects whatsoever. As our purpose was evaluation of false-positive rates for traits under the null, issues of power do not apply, and this approach is reasonable. However, we do not recommend dichotomization of a quantitative trait in real data because linkage analysis is generally more powerful for a quantitative trait compared to a binary dichotomization of that trait. Incorporating covariates into the linkage analysis can also improve power in a real study especially for established risk factors.Our analysis revealed very different results for the two null phenotypes. We observed a large number of false-positive results for the polygenic trait, although it was simulated without using any of the provided genotypes and was unlinked to the provided chromosomes. Most of the false-positive results observed for this trait involved SNVs with relatively common minor alleles, and only 12 of the 2398 significant SNVs across all 50 replicates had arbitrary MAF of 0.0001. Although incorrect specification of both the trait model and the marker allele frequencies can inflate type I error rates, estimation of marker allele frequencies from the data (as we did here) has been shown to control false-positive rates even when the trait model is misspecified, so model misspecification is not likely the cause of the type 1 error rate inflation observed here. Differences in pedigree structure and model misspecification also appeared to have little effect on false-positive rates of two-point LOD score linkage of complex traits in a study of a single marker with 8 equifrequent alleles . The simIn contrast, the number of false-positive results based on the classic LOD score threshold of 3.3 is well controlled if affected-unaffected status is randomly assigned , even when more than eight million SNVs are analyzed by two-point parametric linkage analysis.Both two-point and multipoint linkage analysis can detect linkage to a region containing a causal variant even if the causal variant was not genotyped. Even though we only evaluated LOD scores at θ = 0, in a real analysis, one should also obtain the LOD score at the maximum likelihood estimate of θ to detect SNVs very close to a causal variant. One must be careful interpreting a two-point analysis because the most significant two-point LOD may not indicate the actual causal variant, but simply the closest variant to the untyped causal variant as, for example, when the causal variant is not analyzed because it is in a region with low coverage or was filtered out because of bad quality. One should examine the strongest linkage region for variants dropped because of low quality and possibly resequence the most strongly linked region.This study of false-positive rates in WGS data simulated under the null hypothesis of no linkage to any chromosomes provided in the data set suggests that a higher LOD score threshold may be required when analyzing a complex trait with nonzero heritability. However, more extensive simulations are required before such new thresholds can be proposed. Reestimating allele frequencies and performing multipoint linkage in regions showing significant two-point scores may help to resolve this problem. Single significant SNVs with no supporting linkage to nearby variants may be eliminated by the multipoint analysis. This remains an area of future research.The authors declare that they have no competing interests.CLS, SS, and JEBW designed the study; CLS, SS, and CDC conducted analyses. CLS and SS drafted the manuscript. All authors read and approved the manuscript.


The functions of the different assembly stages in exocytosis and the role of Munc18-1 in SNARE assembly are not well understood. Using optical tweezers, we observed four distinct stages of assembly in SNARE N-terminal, middle, C-terminal, and linker domains . We found that SNARE layer mutations differentially affect SNARE assembly. Comparison of their effects on SNARE assembly and on exocytosis reveals that NTD and CTD are responsible for vesicle docking and fusion, respectively, whereas MD regulates SNARE assembly and fusion. Munc18-1 initiates SNARE assembly and structures t-SNARE C-terminus independent of syntaxin N-terminal regulatory domain (NRD) and stabilizes the half-zippered SNARE complex dependent upon the NRD. Our observations demonstrate distinct functions of SNARE domains whose assembly is intimately chaperoned by Munc18-1.Synaptic-DOI:http://dx.doi.org/10.7554/eLife.09580.001 Plants, animals and other eukaryotes transport many large molecules within their cells inside membrane-bound packages called vesicles. These vesicles can fuse with the membrane of a target compartment in the cell to deliver their contents inside, or fuse with the cell’s membrane to release the contents outside of the cell.Membrane fusion is carried out by a group of proteins called SNAREs. These proteins are embedded on the membranes of both the vesicle and its target, and they bind to each other to form a tight complex. This complex docks the vesicle to the target and then acts like a “zipper” to pull the two membranes close enough to fuse. The best-studied SNARE proteins act in nerve cells and fuse vesicles to the cell’s membrane in order to release molecules called neurotransmitters. This process is essential for communication between nerve cells, and relies on a protein called Munc18-1. However, it is not well understood how SNARE proteins assemble into the complex and how Munc18-1 regulates this process.Ma et al. have now used a tool called “optical tweezers” to pull an assembled SNARE complex apart in the laboratory and then observe how it folds and assembles in a step-by-step process. These experiments showed that the complex assembled in four stages and not three as has been reported in previous work. SNARE proteins are made up of four parts called domains, and Ma et al. observed that the N-terminal domains were the first to bind to each other. Next, the binding progressed to the middle domain, then to the C-terminal domain and finally to the linker domain. An intermediate, half-zippered form was also observed.Ma et al. next analysed each domain in more detail and found that the N-terminal and C-terminal domains drive the docking of vesicles to the target membrane, the middle domain is crucial for assembling the SNARE complex correctly, and all three domains regulate the fusing of the membranes. Further experiments showed that Munc18-1 promoted the assembly of new SNARE complexes and stabilized the half-zippered form, rather than stabilizing the complex after it had fully assembled. This study will provide a new tool to examine many other proteins that regulate SNARE assembly, and a basis to understand the role of SNARE proteins in brain activity.DOI:http://dx.doi.org/10.7554/eLife.09580.002 SNAREs are evolutionarily conserved molecular machines that drive fusion of transport vesicles with their target membranes, thereby transferring materials and information between different cells or cellular compartments . In the 2+ influx, the trans-SNARE complex rapidly zippers to complete its assembly and membrane fusion. However, an alternative model suggests that SNARE assembly only starts after Ca2+ triggering and completes in one step without any intermediates  of syntaxin . The NRDormation   obtained by pulling and then relaxing a single SNARE complex  showed f'Materials and methods'). In the partially-zippered state 3, the VAMP2 CTD and the t-SNARE CTD are unfolded from layer +8 to layer +3 and layer +5, respectively, whereas the remainder of the SNAREs are largely helical as in the four-helix bundle. This structure is consistent with that of a different trans-SNARE complex located on the yeast vacuole  kBT, and 22 (±3) kBT, respectively, where kB is the Boltzmann constant and T= 296 K the room temperature, with kBT=0.59 kcal/mol. Thus, the SNARE complex has total zippering energy of 68 (±4) kBT, including 8 (±2) kBT LD folding energy  and L84A (+8) at the borders of the CTD had only minor effects on SNARE assembly . In contocytosis . The synosis  that reversibly transited with the major state 11 . State 1Next, we pulled the Munc18-bound state 11 to high forces. We saw a small extension jump in a force range of 22-28 pN, showing that the Munc18-bound state 11 unfolded into the NTD-unfolded state 5 . Indeed,In conclusion, we observed that Munc18-1 interacts with the SNARE bundle in at least two modes and differentially regulates SNARE zippering: In an NRD-dependent mode, Munc18-1 stabilizes the half-zippered state, while in an NRD-independent mode, Munc18-1 structures Tc.BT for the free energy of the closed state relative to the open state, based on the measured mechanical work required to unfold the closed state. The relatively small energy difference revealed that the closed syntaxin could spontaneously open to participate in SNARE assembly. This finding is surprising, because it is generally believed that Munc18-1 tightly binds the closed syntaxin with nano-molar affinity, or −20 kBT association free energy (-1), Munc18-1 must remain attached to the syntaxin molecule during the whole syntaxin transition process  . The refe energy . Howevere energy . Given t process , suggest process . This fi process   and F77A (+6) in VAMP2 and M71A/I192A (+5) in SNAP-25B, also dramatically reduce the rate of exocytosis , characterized by the contour length i lof i-th intermediate state or transition state and its associated free energy iV at zero force. These parameters were determined by fitting the model-based calculations to the experimental measurements. To do so, we first established the experimental observables as a function of the simplified energy landscape. We calculated the total extension iX and energy iG for each state in the presence force. Both quantities constituted contributions from three components: the unfolded polypeptide, the folded portion of the protein, and the DNA handle  the effective trap stiffness of the two optical traps and R the sum of the bead radii. Given the experimental conditions and contour length of a state il, we could solve its state force iF by substituting where where Given the state energies, we computed the state populations based on the Boltzmann distribution and the transition rates in terms of Kramers’ formula. Finally, we simultaneously fit the calculated state forces, populations, transition rates, and extension changes to the corresponding measurements using the nonlinear least-squares method, yielding the best-fit parameters for the simplified energy landscape. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your submission has been favorably evaluated by Vivek Malhotra (Senior Editor), and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled "Munc18-1-regulated stage-wise SNARE assembly underlying synaptic exocytosis" for peer review at The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This paper describes a technically impressive study, in which the authors used an optical tweezers to map out the energetics and kinetics of neuronal SNARE assembly. Two technical features distinguish this work from earlier efforts. First, the authors figured out how to crosslink VAMP and syntaxin so that they could observe assembly as well as disassembly. Second, they determined assembly energetics/kinetics for a well-chosen set of VAMP  mutants. They then went on to determine assembly energetics/kinetics in the presence of Munc18. The authors demonstrate: .Major comments:Overall:(a) The paper’s structure needs to be reorganized. Some important points are treated summarily , while some irrelevant ones are emphasized.(b) Please take care not to base structural interpretations on single distance measurements. While the models for states 1-8 are undeniably useful, and while the mutagenesis results as well as previous studies provide some additional support, the models should be presented and discussed more cautiously. Even the presence or absence of, for example, SNAP-25 in each of these states does not strike the reviewers as a foregone conclusion.(c) As a specific example of interpretive ambiguity, the authors assume for the unfolding state 4 in (d) The suggested switching of Munc-18 interactions likewise appears to be arbitrary. The authors make no effort to justify that interpretation in terms of what is known about Munc18 structure and biochemistry. There are many ways in which two apparently distinct activities can be interpreted, without requiring distinct modes of interaction. The authors overstate the case by saying (rather pompously) "we discovered (sic) that Munc18-1 interacts with the SNARE bundle in at least two distinct modes".(e) The authors claim that they have "quantitatively verified the tight thermodynamic coupling between SNARE zippering and membrane fusion and revealed distinct functions of different SNARE assembly stages." They have done nothing of the kind. They have done a fine job of detecting intermediates in a useful and valuable model, in which individual SNARE complexes are pulled and relaxed, and they can (and should) be pleased with that accomplishment. But when several SNARE complexes are pulling on the same two membranes and the membranes are pulling back on them, as is the case for exocytic fusion , then the relative heights of barriers, etc., might well be different. That difference doesn't diminish the value of the numbers provided here, but it does rule out egregious overstatement. Even the interpretation in the third paragraph, subsection “Energetics and kinetics of SNARE assembly underlying exocytosis”, seems stretched – why do the phenotypes of those mutations reveal "an essential role of independent MD assembly"? Yes, messing up the MD messes up the mouse, but how does even the correspondence to the kinetic observations reported here demonstrate "independent"  assembly?Part 1:Although the authors basically performed the same experiments as in Gao et al. (2012), there seem to be two differences. The first is that unfolding in the C-terminal half of the SNARE complex, previously treated as one process, has now been resolved into CTD and MD unfolding (states 3 and 4), presumably because they did less temporal averaging of the data and plotted the extension traces over 1kHz. The second difference is that they moved the crosslinking site to layer -6. This difference likely separated NTD unfolding (state 5) from dissociation of SNAP-25 (state 6) more clearly than in Gao et al. (2012), in which the crosslinker was at layer -7. Thus, before dissociation of SNAP-25 at a higher force, they could see reversible NTD unfolding and refolding transitions.In The authors claim that "the middle domain plays a pivotal role in robust and correct stage-wise SNARE assembly, and its mutation leads to changes in SNARE assembly pathway and accuracy" and that "the MD serves as a checkpoint to further ensure proper SNARE assembly as a master switch to control CTD assembly". It is hard to understand how the reduced observation of the intermediate 3 (observed for the L60A and L63A mutants) supports these rather strong arguments. In the third paragraph of the subsection “Energetics and kinetics of SNARE assembly underlying exocytosis”, the authors allude to unpublished results, but this citation is not an appropriate way to justify the arguments. The authors should either weaken these claims or provide experimental evidence to support their arguments.Part 2:Data showing different effects of Munc18 are mixed confusingly together, making part 2 extremely hard to follow, even for a reader who knows the field. In particular, in From the extension difference between states 8 and 5 , observeOverall recommendation:1) Present the structural models for the kinetic states more cautiously and explain any alternatives.2) Support the interpretation of the role of the MD with data .3) When revising what we have called "part 2", comply with each of the three paragraphs under "part 2", above.4) Provide details (in the Methods) for the nonlinear least-square fitting that converts dwell times to deltaG (or deltaG double dagger).eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Vivek Malhotra as the Senior Editor.Thank you for submitting your work entitled "Munc18-1-regulated stage-wise SNARE assembly underlying synaptic exocytosis" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The reviewers agreed that the manuscript is greatly improved, but it remains, despite the revision, challenging and complex. In particular, two parts need attention, as follows.Essential revisions:A) The subsection entitled "Position-dependent effects of layer mutation on SNARE assembly", in which the authors discuss single-molecule mechanics of 15 SNARE complex mutants, was very difficult to follow. We propose the following changes:1) In 2) In the same figure, the current way in which FECs are marked is difficult to recognize. The colors and patterns are too similar. One way is to depict the corresponding transitions right next to individual FEC regions.3) How is "MD+NTD" different from "MD_NTD"? The reviewers could not find proper explanations neither in the main text nor figure legends.4) In b)  Essential revisions:The paper is, in some respects, two separate reports. In one, the authors characterize 15 different SNARE-complex mutants, previously studied by different methods and in different experimental setting. As the authors correctly point out, a coherent treatment by a uniform method is needed. In the other, the authors examine the effects of Munc18 on assembly and disassembly of single SNARE complexes. There are many new observations and insights, but the conclusions are less convincing than those of the first part (see below). Major comments: Overall: (a) The paper’s structure needs to be reorganized. Some important points are treated summarily , while some irrelevant ones are emphasized.We agree with the comments made by the reviewers. Following the reviewers’ suggestion, we have made a major revision of our manuscript.(b) Please take care not to base structural interpretations on single distance measurements. While the models for states 1-8 are undeniably useful, and while the mutagenesis results as well as previous studies provide some additional support, the models should be presented and discussed more cautiously. Even the presence or absence of, for example, SNAP-25 in each of these states does not strike the reviewers as a foregone conclusion.We understand the reviewers’ concern here. The wide applications of optical tweezers in protein folding studies and our research experience in this field have led our belief that optical tweezers can be used to reliably determine the conformations and energies of protein folding intermediates. We have written a separate manuscript dedicated to our methods of data analysis, which was favorably reviewed in another journal. Basically, we combine our high-resolution single-molecule measurements, the crystal structure of the fully assembled SNARE complex, including its linear topology, and results of SNARE mutations, truncations, and protein binding to model the conformations of SNARE intermediates. We have described salient features of our methods, including key mathematical formulae, in the Data Analysis part of the Materials and methods section. In addition, we have added First, we have moved our description on SNAP-25 binding earlier in the text (subsection “Four stages of SNARE assembly in four domains”).Second, we have elaborated our method of data analysis in the subsection “Energetics and kinetics of SNARE assembly”.Finally, we have added a new section “Vc peptide induces folding of t-SNARE C-terminus and attenuates SNARE zippering” to describe effects of Vc peptide on SNARE assembly. As a result, we have added a new main figure, In conclusion, these revisions, our previous results , and results obtained from complementary approaches obtained in other groups , corroborate the data analysis methods and the structural models derived by us.(c) As a specific example of interpretive ambiguity, the authors assume for the unfolding state 4 in simultaneously fit the extensions of the intermediates 2-4 to the experimental measurements to determine the borders of these states. The fitting avoids the scenario proposed by the reviewer, because shifting the border between states 4 and 5 towards the N-terminus will shorten the extension change of NTD transition.The reviewers pointed out an important feature in our data analysis. In our model, SNAREs fold and unfold in a sequential and stepwise manner, between the well-defined fully assembled state (state 1) and the fully unfolded state (state 6). The total extension change is fixed by the extension difference between the two states. We essentially performed an interpolation to locate the conformations of intermediate states based on their measured relative extensions. Thus, an increase in the extension change of one folding step must be compensated by a decrease in the extension change of another folding step. In our analysis, we first determine the t-SNARE conformation (state 5) based on its extension relative to the completely unfolded state (state 6) and the Vc-peptide-bound state  The suggested switching of Munc-18 interactions likewise appears to be arbitrary. The authors make no effort to justify that interpretation in terms of what is known about Munc18 structure and biochemistry. There are many ways in which two apparently distinct activities can be interpreted, without requiring distinct modes of interaction. The authors overstate the case by saying (rather pompously) "we discovered (sic) that Munc18-1 interacts with the SNARE bundle in at least two distinct modes".We thank reviewers for the insightful suggestion. Following the suggestion, we have tried to integrate our observations to previous findings on Munc18-1 and also compared the differences in the Discussion part. As a result, we have made major revision in the Discussion part and also the model figure . In part“In conclusion, we observed that Munc18-1 interacts with the SNARE bundle in at least two modes and differentially regulates SNARE zippering: In an NRD-dependent mode, Munc18-1 stabilizes the half-zippered state, while in an NRD-independent mode, Munc18-1 structures Tc.”(e) The authors claim that they have "quantitatively verified the tight thermodynamic coupling between SNARE zippering and membrane fusion and revealed distinct functions of different SNARE assembly stages." They have done nothing of the kind. They have done a fine job of detecting intermediates in a useful and valuable model, in which individual SNARE complexes are pulled and relaxed, and they can (and should) be pleased with that accomplishment. But when several SNARE complexes are pulling on the same two membranes and the membranes are pulling back on them, as is the case for exocytic fusion , then the relative heights of barriers, etc., might well be different. That difference doesn't diminish the value of the numbers provided here, but it does rule out egregious overstatement. Even the interpretation in the third paragraph, subsection “Energetics and kinetics of SNARE assembly underlying exocytosis”, seems stretched – why do the phenotypes of those mutations reveal "an essential role of independent MD assembly"? Yes, messing up the MD messes up the mouse, but how does even the correspondence to the kinetic observations reported here demonstrate "independent"  assembly?We are sorry for our overstatements. Fully accepting the reviewers' criticisms, we have revised the relevant sentences as “Combining our findings with previous results on exocytosis, we confirmed distinct functions of different SNARE assembly stages in synaptic exocytosis.”“SNAREs couple their exergonic folding to membrane fusion . Such a thermodynamic coupling mechanism predicts that any perturbation in SNARE assembly impacts membrane fusion. Significant progresses have been made to test the prediction in the past two decades . However, a quantitative test requires accurate and comprehensive measurements of both the energies and kinetics of SNARE assembly and the rates of membrane fusion for wild-type and various mutant SNARE complexes.”and“Other MD mutations L60A (+1) and L63A (+2) abolished the independent MD transition and thus changed the SNARE folding pathway. Yu et al. recently examined the impact of both mutations in synaptic exocytosis using cultured mouse neurons . They found that the two mutations abolished both spontaneous and evoked exocytosis, implying an important role of independent MD assembly in exocytosis. The comparison suggests that an obligate pathway of SNARE assembly may be required for membrane fusion.”In addition, we have added many phrases to emphasize numerous previous reports on electrophysiological measurements of exocytosis. For example, in the Abstract:Comparison of their effects on SNARE assembly and on exocytosis reveals that NTD and CTD are responsible for vesicle docking and fusion, respectively, whereas MD regulates SNARE assembly and fusion.”“We found that SNARE layer mutations differentially affect SNARE assembly. Finally, we recognize that multiple SNARE complexes are required for membrane fusion, as is shown in our model figure  and had Part 1: Although the authors basically performed the same experiments as in Gao et al. (2012), there seem to be two differences. The first is that unfolding in the C-terminal half of the SNARE complex, previously treated as one process, has now been resolved into CTD and MD unfolding states 3 and 4), presumably because they did less temporal averaging of the data and plotted the extension traces over 1kHz. The second difference is that they moved the crosslinking site to layer -6. This difference likely separated NTD unfolding (state 5) from dissociation of SNAP-25 (state 6) more clearly than in Gao et al. (2012), in which the crosslinker was at layer -7. Thus, before dissociation of SNAP-25 at a higher force, they could see reversible NTD unfolding and refolding transitions. In  and 4, pWe thank the reviewers for correctly summarizing our main findings presented in the manuscript.The authors claim that "the middle domain plays a pivotal role in robust and correct stage-wise SNARE assembly, and its mutation leads to changes in SNARE assembly pathway and accuracy" and that "the MD serves as a checkpoint to further ensure proper SNARE assembly as a master switch to control CTD assembly". It is hard to understand how the reduced observation of the intermediate 3 (observed for the L60A and L63A mutants) supports these rather strong arguments. In the third paragraph of the subsection “Energetics and kinetics of SNARE assembly underlying exocytosis”, the authors allude to unpublished results, but this citation is not an appropriate way to justify the arguments. The authors should either weaken these claims or provide experimental evidence to support their arguments.Following the reviewers’ suggestion, we have weakened our claim on the significance of the MD, as is described in our preceding replies. During revision of this manuscript, the unpublished work referred in our previous manuscript has been published . We have updated the citation. The major and common change caused by L60A and L63A is a combination of both CTD and MD into one transition unit, which demonstrates a change in the SNARE assembly pathway. Therefore, we suggest that a conserved SNARE folding pathway, including an independent MD folding, may be required for membrane fusion.Part 2: Data showing different effects of Munc18 are mixed confusingly together, making part 2 extremely hard to follow, even for a reader who knows the field. In particular, in We thank the reviewers for pointing out our poor figure organization. Following the reviewers’ suggestion, we have made major efforts to re-organize the figures in the whole revised manuscript. We make sure that each figure focuses on one subject matter. We believe these changes substantially improve the readability of the second part of the manuscript.From the extension difference between states 8 and 5 , observed only in the absence of NRD , the authors argue that Munc18 would bind to the C-terminal part of the t-SNARE proteins and induce a coil-to-helix transition. But then, state 4 with bound Munc18 should have a smaller extension than state 4 without Munc18. 8 and 5 , observeThe reviewers would be right if VAMP2 were free to zipper in this case. However, the average extension change upon NTD zippering on Munc18-1 bound t-SNARE complex , but with much smaller transition rate than that seen in the previous construct . As a re“The different transition kinetics seen in the two syntaxin constructs is not surprising, because the syntaxin molecule was pulled in two approximately perpendicular directions compare . It is kOverall recommendation: 1) Present the structural models for the kinetic states more cautiously and explain any alternatives.Yes, we have done so. Please see our response to the Major comments (b) and (c).2) Support the interpretation of the role of the MD with data .Yes. Please see our reply to the last comment in Part 1.3) When revising what we have called "part 2", comply with each of the three paragraphs under "part 2", above.Yes. Please see our replies to the comments in Part 2 above.4) Provide details (in the Methods) for the nonlinear least-square fitting that converts dwell times to deltaG (or deltaG double dagger).Yes, we have provided details in Methods for the nonlinear least-square fitting to derive the energies of intermediate states and transition states.Essential revisions:A) The subsection entitled "Position-dependent effects of layer mutation on SNARE assembly", in which the authors discuss single-molecule mechanics of 15 SNARE complex mutants, was very difficult to follow. We propose the following changes: 1) In Following the reviewers’ suggestion, we have explicitly shown the name of the domain to which each mutant belongs. In addition, we have color-coded the mutation names to blue, green, and red based on their associated NTD, MD, and CTD, respectively. The same color-coding is applied to mutation names in 2) In the same figure, the current way in which FECs are marked is difficult to recognize. The colors and patterns are too similar. One way is to depict the corresponding transitions right next to individual FEC regions.We have improved the labeling by adding domain names next to the corresponding transitions in FECs, as the reviewers suggested. In addition, we have simplified the labeling by only showing the abnormal transitions of the mutant SNARE complexes compared to the wild-type.3) How is "MD+NTD" different from "MD_NTD"? The reviewers could not find proper explanations neither in the main text nor figure legends.The “MD+NTD” transition means sequential MD and NTD transitions in an overlapping force range, whereas the “MD_NTD” transition indicates two-state transition of MD and NTD as a combined new domain. To address the reviewers’ concern, we have added 4) In As is described in our reply to the preceding question, we have depicted the difference between sequential transitions and cooperative transitions in b) We thank the reviewers for pointing out our confusing description regarding


This paper reveals the importance of RFID spatial and temporal data analysis in real-time status monitoring of workpieces in wisdom manufacturing workshops.Radio Frequency Identification (RFID) technology has been widely used in many fields. However, previous studies have mainly focused on product life cycle tracking, and there are few studies on real-time status monitoring of workpieces in manufacturing workshops. In this paper, a wisdom manufacturing model is introduced, a sensing-aware environment for a wisdom manufacturing workshop is constructed, and RFID event models are defined. A synthetic data cleaning method is applied to clean the raw RFID data. The Complex Event Processing (CEP) technology is adopted to monitor abnormal conditions of workpieces in real time. The RFID data cleaning method and data mining technology are examined by simulation and physical experiments. The results show that the synthetic data cleaning method preprocesses data well. The CEP based on the Rifidi The time threshold range is set with the lower threshold low-tht, and the higher threshold high-tht. Time attribute of stayed event cagvSE1, cncmSE1, cncmSE2, cncmSE3 or cagvSE2 is queried to judge whether the time attribute is within the scope of the threshold or not. If the threshold is exceeded, a stayed (machined) time anomaly occurs. For example, stayed (machined) event cncmSE1 is made up by simple events sncmAE1 and sncmLE1, the stayed (machined) time anomaly on workstation ncmN1 is monitored by the following statement:ncmN1 (time in [low-tht: high-tht]);Select time from Query the higher threshold:sncmAE1 tag from pattern [every sncm1AE = AE -> timer:interval (high-tht) and not sncmLE1 (tag.tag.ID = sncmAE1 tag.tag.ID)];Select Query the lower threshold:sncmAE1 tag from pattern [every sncmAE1 = AE -> timer: interval (low-tht) and sncmLE1 (tag.tag.ID= sncmAE1 tag.tag.ID)];Select For AGV workstations (3) Workbench BlockingwtN1 and wtN2, time thresholds of caching raw materials and products are set by wtt-th1 and wtt-th2. Time attribute of stayed event cwtSE1 or cwtSE2 is queried to judge the time attribute within the scope of the threshold or not. If the threshold is exceeded, an abnormal workbench blocking condition occurs. For example, stayed event cwtSE1 is made up of simple events swtAE1 and swtLE1, the anomaly of workbench blocking on workstation wtN1 is monitored by the following statement:swtAE1 tag from pattern [every swtAE1 = AE->timer:interval (wtt-th1) and not swtLE1 (tag.tag.ID= swtAE1.tag.tag.ID)];Select The workbench is used to cache raw materials or products in workshops. If the cache time of raw materials or products is very long, the workbench will be blocked. For workbench workstation i denotes a workpiece serial number, and j denotes a workstation serial number:Abnormal conditions of workpieces are monitored in real time through RFID data mining, which results in a real-time state matrix: If ® Edge Server; (3) abnormal condition monitoring is evaluated by physical experiments.In this section, synthetic data cleaning and data mining techniques are evaluated experimentally. Three key points are illustrated: (1) the synthetic per-tag data cleaning algorithm is evaluated under the aid of MATLAB and a physical reader; (2) the simulation experiment of abnormal condition monitoring is tested by the RifidiIn order to run experiments across a wide variety of scenarios, a physical reader and fifteen tags are set up to generate experimental data. The synthetic data cleaning algorithm is programmed in MATLAB. Two tag movement behaviors are investigated. The first is that a tag is static, which simulates how to place a tag suitably within the reader detection region, and the tag is monitored in a lower average errors per epoch. The second is that a tag is moved at a random initial velocity, which simulates dynamic environments such as tagged items on an AGV or conveyor belt, and the tag is monitored in a lower average errors per epoch at an appropriate velocity.The reader detection model is built based on the RFID tag-reader detection regions. Generally there are three distinct regions of operations of a passive RFID reader tag system: major detection region, minor detection region and outside of detection region . The majIn the major detection region, the probability of detection maintains the max value (MaxReadRate). The tag read rate then degrades gradually with increasing distance to the minor detection region. In the outside of detection region, the read rate goes down to 0%. The main difference on this detection pattern when the tags are operated in different environments lies in the percentage of the reader’s detection range corresponding to its major detection region. The experimental parameters are summarized in The evaluation metric for per-tag cleaning is average errors per epoch. The average errors per epoch is calculated as MajorPercentage parameter simulates the reliability factors that affect the tag detection rates such as tag orientation and the radio frequency interference, while varying the distance (x) parameter simulates the tag-reader signal attenuation with distance. The MajorPercentage is varied between 0 and 100%. A lower value of MajorPercentage corresponds to a more unreliable environment and higher value of MajorPercentage corresponds to a more controlled environment.Varying the MajorPercentage is varied. The results from data collected at every 1 cm within the range from 0 to 20 cm (the reader’s detection range is approximately 20 cm) are averaged. The repetitions of every data collection point are 20 times. The average errors per epoch is measured at each value for MajorPercentage between 0 and 1. At first, the average errors per epoch is tested on the conditions of different levels of reader unreliability. For the static tag tests, the tag is static, but the MajorPercentage increases, the accuracy of the two methods improves due to more reliable raw data. Especially, when the MajorPercentage is more than 0.6, the synthetic method works well. This region is adopted to monitor tagged workpieces in this paper. The difference of the synthetic method and SMURF method is evaluated by independent samples t-test ", NrwNoDepartedListener);ncmN1, a mini-type CNC milling machine is thought as the workstation ncmN2 A computer control system is used to control two CNC machines. The metal workpiece 1 and workpiece 2 with anti-metal ceramic tags are machined separately on workstation ncmN1 and ncmN2 Readers are placed on one side of every workstation to monitor the arrived and left (departed) time in real time. The real-time monitoring data is sent to the upper computer to process by the internet with routers.In order to test the effectiveness of the proposed method, a physical experimental system for workpiece monitoring in real time is established and the experiments are carried out on the test rig. The architecture of the physical experiment system is shown in The experimental parameters are summarized in idw = 300833B2DDD9014000000001) is processed on workstation ncmN1 for less than 20 s; workpiece 2 (idw=300833B2DDD9014000000002) is processed on workstation ncmN2 for more than 25 s. The physical experiment result is shown in There are two workstations in the physical experiment system, while there are nine in the simulation experiment. In the physical experiment, it is assumed that workpiece 1 (ncmN1 are shown in low-tht (20 s). As such, a machined time anomaly occurred, and a14 element in status matrix was 0. It was expressed that an abnormal condition of workpiece 1 occurred on workstation ncmN1. In high-tht (25 s). Element a25 in status matrix was 0, which expressed that an abnormal condition of workpiece 2 occurred on workstation ncmN2.The abnormal condition of workpiece 1 on workstation In this study, a RFID-based method of abnormal condition real-time monitoring of workpieces in wisdom manufacturing workshops is presented. RFID is used to detect the spatial-temporal information of workpieces at every workstation. Synthetic data cleaning and data mining based on CEP are applied to process RFID data in real time. The synthetic data cleaning method works better than SMURF in guaranteeing tag completeness and dynamics. Such real-time condition monitoring of workpieces provides a basis for proactive job shop scheduling in wisdom manufacturing.However, this study only focuses on three abnormal conditions for real-time monitoring of workpieces in the wisdom manufacturing workshops. In the future work, more abnormal conditions such as urgent workpiece arrival, incorrect workpiece quantities and misplaced parts will be monitored by searching, aggregating and matching the EPCs of the workpieces, and the abnormal results being sent to the web. At the same time, we also plan to study the Prognostics and Health Management (PHM) of processing equipment in manufacturing workshops. For example, tools can be monitored and diagnosed with different sensors such as cutting force, vibration and acoustic emission, and deep learning is adopted to complete the tool condition monitoring and prognostics.


The complete genome sequence of soybean allows an unprecedented opportunity for the discovery of the genes controlling important traits. In particular, the potential functions of regulatory genes are a priority for analysis. The basic helix-loop-helix (bHLH) family of transcription factors is known to be involved in controlling a wide range of systems critical for crop adaptation and quality, including photosynthesis, light signalling, pigment biosynthesis, and seed pod development. Using a hidden Markov model search algorithm, 319 genes with basic helix-loop-helix transcription factor domains were identified within the soybean genome sequence. These were classified with respect to their predicted DNA binding potential, intron/exon structure, and the phylogeny of the bHLH domain. Evidence is presented that the vast majority (281) of these 319 soybean bHLH genes are expressed at the mRNA level. Of these soybean bHLH genes, 67% were found to exist in two or more homeologous copies. This dataset provides a framework for future studies on bHLH gene function in soybean. The challenge for future research remains to define functions for the bHLH factors encoded in the soybean genome, which may allow greater flexibility for genetic selection of growth and environmental adaptation in this widely grown crop. Arabidopsis with severe phenotypes as a result of a lesion in a bHLH-encoding gene. Development and dehiscence of the seed and seed pod (silique) [Basic helix-loop-helix (bHLH) transcription factors belong to a large family of genes present in the shared ancestor of plants, animals, and fungi, and this family has undergone an expansion in the land plant lineage . Often rsilique) , 15, 16 silique) , 17–21 aα-helices in the bHLH domain and the other motifs outside the conserved bHLH domain are required for protein-protein interactions and function [The bHLH transcription factors have been extensively characterized at the sequence and structural level. In animals, the best-known and most thoroughly characterized bHLHs are well-known regulators and proto-oncogenes such as c-Myc, Max, and E47, where in many cases structural data on the proteins and their interaction with DNA molecules is available . Many anfunction –32. Arabidopsis) may not be the most appropriate tool to identify all the bHLH-encoding genes in a genome such as soybean. An alternative approach is to use data from the conserved HLH motif across the kingdoms of life to develop a hidden Markov model (HMM) that allows the detection of the bHLH domain across highly divergent sequences without the need for extensive sequence identity [http://pfam.sanger.ac.uk/) uses a curated alignment approach to provide constantly updated HMMs for most characterized protein families, including the HLHs. HMMs for the subsidiary motifs of the different bHLH families can be generated using protein alignments.Classification of bHLH proteins is usually based on sequence homology within the conserved bHLH domain. In recent years, a number of proteins have been identified genetically that represent novel and atypical bHLH proteins that were not previously classified by homology-based approaches, in some cases because a basic domain was lacking , 33–36. identity . This ty Arabidopsis thaliana, rice, poplar, and other plants with whole-genome sequences, but these studies have not examined the conservation and diversification of this family in the soybean [Several recent genome-wide studies have examined and classified the bHLH protein family in soybean , 39–42.  soybean –45. A st soybean . They ar Arabidopsis, and rice gene models were originally identified from annotation versions contained in the Phytozome v7.0 package. The most recent version of the soybean assembly (Assembly v.2.0 and gene models for Phytozome v.10) was compared with our data; however since stringent new rules for inclusion of genes in the newer assembly and annotation resulted in the loss of 62 conserved bHLH genes including several with confirmed expression data, the legacy annotation and assembly were retained for this analysis (http://www.phytozome.net/) [http://hmmer.janelia.org/) was applied to the predicted open reading frames (ORFs) of soybean . No e-value cutoff was initially applied in order to maximize detection of poorly annotated and orphan bHLH sequences. A cutoff was applied during manual curation (see next section). A total of 329 hits to unique predicted proteins containing putative HLH domains were initially identified. These included 31 ORFs not annotated as bHLHs by the soybean genome annotation and a small group of HLH proteins that lack the basic domain  , 47, 48.Gmax109; ) using tmain see . Data onribed in .http://dx.doi.org/10.1155/2015/603182). We then examined the revised list for sequences that were atypical or were unlikely to be true bHLHs. Several studies have used a stringent cutoff restricting the number of mismatches to the canonical bHLH motif found in mammals [e-value) and no sequences with incomplete bHLH domains or that contained stop codons were included).Sequences were manually curated to identify mispredictions of splice sites, which often led to omission of part of the bHLH domain in the genome annotation, reducing the hidden Markov model score. Sequences that included in-frame stop codons in the Williams-82 genomic sequence or were missing part of the HLH domain were removed, and these soybean gene models are listed in Additional File 2  and models generated and used for searching as described for the other motifs.Assignment of subsidiary motifs in the bHLH or HLH proteins was performed using the alignment of motifs provided in Supplemental Material by Pires and Dolan . Hidden http://meme.sdsc.edu) we searched for up to 10 new sites between 2 and 300 residues wide. Using “discriminative motif discovery” a file was supplied containing the bHLH motif plus the sequences used to create the hidden Markov models from the known bHLH secondary motifs. A single strongly significant motif, Motif 40, was detected. As above, the alignment supplied by MEME for the new motif was used to create a hidden Markov model, and this model was used to search the soybean bHLHs to determine which of them contained the motif. As before no e-value cutoff was applied, however the Family X sequences all showed e < 10−7 while the two Family IX sequences GmbHLH262 and 261 showed e-values of 0.0025 and 0.003, respectively.Using MEME ; in animals these bHLHs recognize the E-box subset CACGTG [5-E9-R13 motif and thus could be candidates for binding the G-box sequence. The conservation of these residues that are potentially involved in protein-DNA interaction within soybean is an indication that the DNA-binding function and also the recognition sequence may be conserved.The conservation of key residues within the basic region of the bHLH domain can predict both the potential to bind DNA and the preferred recognition sequence. The E residue at position 9 within the basic domain has been shown in protein crystal structures to make contact with the major groove of the DNA (reviewed in ). This pt CACGTG . This set CACGTG , 26, 27.9 residue (74 proteins) only eight have five or more basic residues within the basic region. The number of basic residues has been previously used as a criterion to classify HLH proteins as having the potential to bind DNA [9 residue is thought to be required for E-box binding, it is possible that these proteins bind DNA at a non-E-box DNA sequence, although this has not yet been demonstrated for plant bHLHs [Of the soybean bHLH proteins that lack the Ebind DNA . Since tnt bHLHs . The remnt bHLHs , 35, 57.nt bHLHs , 58, 59. Arabidopsis and rice bHLH families [Most plant bHLHs have a conserved intron structure with three introns within the bHLH domain , 39, 40.families , 39, 40. Arabidopsis bHLH sequence representing each of the major subfamilies, was generated based on the alignment of the bHLH domain  was constructed from this alignment using RAxML [ Arabidopsis, SAC51/AtbHLH142, which is involved in spermidine synthase-mediated stem elongation [ Arabidopsis families; however several ambiguous sequences were observed in the phylogeny  for new motifs, by excluding the known secondary motifs plus the bHLH domain. A single motif was detected with the consensus sequence GLCLVPVScTqqVgseNGADYWAPayggg . This sequence is strongly conserved in all the GmbHLH sequences 165–184 (Family X). In the Supplemental Material this is named Motif 40, although it follows closely the distribution of Motif 20; the NGADYWAP portion of this motif is similar to Motif 20 and it likely represents an expanded version of this motif. Much weaker similarity to Motif 40 is also observed in GmbHLH261 and GmbHLH262 of Family XI. This motif is also conserved in several Arabidopsis bHLHs in Family X.In addition to the bHLH domain analysis, any of the bHLH subfamilies can also be distinguished by the presence of one or more characteristic motifs outside the bHLH domain , 37, 55. Arabidopsis and O. sativa homologs, BLAST searches of the predicted Arabidopsis and rice proteomes were conducted with the predicted full-length coding sequences of the soybean bHLHs [ Arabidopsis proteins with >50% amino acid sequence identity, and 288 had hits with an e-value of less than 10−25 . A total of 151 soybean bHLHs shared >50% sequence identity with rice, 255 with an e-value of less than 10−25. In order to increase confidence that true orthologs were detected, a reciprocal BLAST search of the soybean genome was performed using full-length Arabidopsis and rice bHLH sequences, and the orthologs are presented in Additional File 5. Of the Arabidopsis bHLHs identified as matching soybean bHLHs, all but one of the Arabidopsis sequences are known from previous classification of bHLH proteins in Arabidopsis [ Arabidopsis protein not previously classified as a bHLH is At2g40435, a protein with homology to 5 soybean HLH proteins. The soybean proteins lack a complete basic domain, show a reasonably conserved HLH motif but have some divergence from previously described consensus sequences, and match the HLH HMM with relatively low scores. At2g40435 has strong similarity to these soybean proteins in this predicted HLH region, but HMMER does not find a match to the HLH HMM at all. Five rice proteins  were also not in previous classifications of rice bHLH proteins  [To compare the soybean bHLHs toan bHLHs . A totalbidopsis , 37, 40. File 5) , 39. AllBecause of the recent duplication of the soybean genome , many so Arabidopsis orthologs of these preferentially nodule-expressed bHLHs do not have known biological functions and do not cluster in any particular subfamily. Fifteen bHLH transcription factors are enriched in expression during soybean seed development. Among these are homologs of TARGET OF MONOPTEROS 5 and TRANSPARENT TESTA 8, both of which have known roles in Arabidopsis embryo development  [Using a survey of the publicly available transcriptome data for soybean , it was  File 6) , 62.The whole-genome duplication events in soybean that occurred 59 million years ago and 13 million years ago have clearly had a substantial impact on the size of the bHLH family in soybean when compared to other plant species , 40, 44.α-helix may also be involved in DNA contact, increasing specificity of genomic sequence targets [ Arabidopsis and soybean, although soybean contains many more bHLH-encoding genes, implying a broadly similar set of functions for each bHLH family may be conserved. All but one of the conserved motifs identified in bHLH subfamilies were found in soybean bHLH genes. A small number of these motifs are known to be involved interactions with other proteins (such as the APB and leucine zipper motifs) [Based on sequence conservation within the basic domain, it can be predicted that the majority of soybean bHLHs (77%) have the potential to bind DNA, and 58% of all soybean bHLHs are likely to recognize the core G-box sequence, of which there are over 17,000 represented in the promoters of actively transcribed genes in the soybean genome . There is experimental evidence that different bHLH proteins have a preference for certain nucleotides flanking the core G-box sequence and that residues in the second  targets , 56, 63. motifs) , 55. Arabidopsis Family X proteins. The strongly conserved NGADYWAP portion of this motif is similar to Motif 20, with which it shares very similar distribution. Motif 40 also contains the GLCL sequence contained in Motif 22. We do not believe Motif 40 is related to Motif 22 because flanking conserved residues of Motif 22 are very different to those of Motif 40, and because the distribution of Motif 40 is strongly correlated with and predictive of membership in Family X. Motif 20 also correlates with Family X but is not found in all members with strong significance, perhaps due to greater discriminative power and sensitivity for the longer motif.Using MEME a long, highly conserved motif was identified, which we term Motif 40. This sequence is strongly conserved in all Family X bHLHs in soybean. Much weaker similarity was found in two Family XI proteins. The sequence is also strongly conserved in KIDARI, ATBS1, and PRE1, and these were used in our analysis. In the case of LONESOME HIGHWAY, a clear ortholog was present in soybean  but this gene was not predicted to contain a bHLH domain using our HMM search. In the case of PAR1 and PAR2, by contrast, no homolog was apparent within the soybean bHLH proteins identified here using the HMM. While PAR1 and PAR2 have BLAST hits in soybean, they are not clearly orthologs. The BLASTP e values of the best hits are in the range 10−15 to 10−16, which represents strong similarity but is a lower level of confidence than for the orthologs of other genes such as KIDARI. This may indicate that the putative HLH domains of LONESOME HIGHWAY, PAR1, and PAR2 are divergent to an extent where structural similarity to canonical bHLHs is limited, or it could indicate that the HMM we used is not sensitive to this type of HLH domain, since it did not include representatives of this type of atypical bHLH.Recently a number of “atypical” bHLH genes have been described. The HMM method identified the bHLH domain in the closest soybean homologs of several of these genes including Arabidopsis gene  not within the set of classified bHLH proteins. Interestingly, while all five soybean proteins are predicted to contain a HLH domain using the HMM approach, the same method does not predict a bHLH domain in the highly similar At2g40435 protein. The amino acid similarity between these proteins is very strong in the predicted HLH region of the soybean proteins, and the HMM score for the HLH prediction in the soybean proteins is not strong. The putative HLH domain is located at the extreme N-terminus of the soybean proteins, which fall into bHLH subfamily III but are not predicted to bind DNA. The At2g40435 protein is thus a candidate for an atypical bHLH. The intriguingly high level of sequence conservation in these genes across Arabidopsis and soybean may point to an important, previously unknown biological function, possibly connected with the bHLH similarity.However, five soybean predicted HLH genes were identified, highly similar to anWe have identified a large number of candidate genes from a family with likely regulatory roles in many processes critical for soybean growth and genetic improvement. These results establish a foundation for future functional genomics studies to target bHLH-controlled processes for modification and improvement in soybean.As Supplementary Material we have included the following. Addititional File 1: an Excel spreadsheet file that includes names and expression data for all of the soybean bHLH-encoding gene models.Additional File 2: A list of soybean gene models excluded from the analysis due to an incomplete bHLH domain or predicted in-frame stop codon.Additional File 3: A full-text alignment of all 319 soybean bHLH domains in pdf format.Additional File 4: a detailed phylogenetic tree of the soybean bHLH genes including full names and predicted family membership.Additional File 5: An Excel spreadsheet showing the presumptive rice and Arabidopsis orthologs of the soybean bHLH genes.Additional File 6: An Excel spreadsheet containing a list of bHLHs enriched for expression in seed or nodules.Additional File 7: The alignment of 319 soybean bHLH genes in PHYLIP format for computer analysis.Additional File 8: A LOGO illustration of the putative Motif 40.


Plasmodium falciparum is an abundant, intrinsically disordered protein that is GPI-anchored to the surface of the invasive blood stage of the malaria parasite. Recombinant MSP2 has been trialled as a component of a malaria vaccine, and is one of several disordered proteins that are candidates for inclusion in vaccines for malaria and other diseases. Nonetheless, little is known about the implications of protein disorder for the development of an effective antibody response. We have therefore undertaken a detailed analysis of the conformational dynamics of the two allelic forms of MSP2 (3D7 and FC27) using NMR spectroscopy. Chemical shifts and NMR relaxation data indicate that conformational and dynamic properties of the N- and C-terminal conserved regions in the two forms of MSP2 are essentially identical, but significant variation exists between and within the central variable regions. We observe a strong relationship between the conformational dynamics and the antigenicity of MSP2, as assessed with antisera to recombinant MSP2. Regions of increased conformational order in MSP2, including those in the conserved regions, are more strongly antigenic, while the most flexible regions are minimally antigenic. This suggests that modifications that increase conformational order may offer a means to tune the antigenicity of MSP2 and other disordered antigens, with implications for vaccine design.Merozoite surface protein 2 (MSP2) of  Recent decades have seen an increasing recognition that many proteins naturally lack a defined folded state, and that their function depends instead on conformational disorder ,2. Such Plasmodium falciparum merozoite, the form of the parasite that invades red blood cells during the blood-stage of infection, which is responsible for symptomatic and severe malaria. Although the specific function of MSP2 has not been defined, it appears to play an essential role in blood-stage replication; it is retained on the merozoite surface during invasion and then degraded soon after invasion is complete . Th. ThJ(ω) ion data .1H]-15N NOE is generally negative. These results reflect conformational dynamics dominated by local processes on timescales shorter than 1 ns. In contrast, the relaxation of a conventionally structured protein is dominated by overall rotational diffusion , with relatively small contributions from faster local processes. For FC27 MSP2 we observe relaxation properties that are in excellent agreement with those we reported previously under more acidic conditions -15N NOE values greater than 0.6 [per se [Previous studies of structured antigens have established that increased epitope flexibility tends to increase antigenicity ,66, in cthan 0.6 , reflect [per se , whereasStaphylococcus aureus fibronectin binding protein [The consistency of the antigenic profiles we have measured here between animals and with other previous studies in mice and in humans, strongly suggests that these profiles are determined by the intrinsic immunogenicity of the recombinant MSP2 antigen. As such, the correlation we observe between conformational restriction and antigenicity probably reflects a tendency for more flexible regions of MSP2 to be less immunogenic. The mechanistic basis underlying this tendency is currently unclear, though several possible explanations are worthy of consideration. It has been suggested that the unusual residue composition of disordered and repetitive antigens may give rise to extensively cross-reactive responses, which fail to mature into high-affinity and specific antibodies . Alterna protein . This ef protein ,72. In tLittle is known about which MSP2 epitopes contribute to a protective immune response. Vaccine-derived protection mediated by MSP2 appears to be strain specific ,73, suggThe correlation established here begs the question of causation: is it possible to modulate the immunogenicity or antigenicity of a disordered antigen by altering its flexibility? Antigen flexibility could be modulated by directly modifying the antigen by addition of bulky residues or disulfide bonds at sites flanking a target epitope. Alternatively, simply changing the formulation of the antigen may have the desired effect. For example, the N-terminal region of MSP2 can be conformationally stabilised by interactions with lipid membranes, in a way that may better reflect the conformation of MSP2 on the merozoite surface . These p