Higher incidences in enteric infectious diseases during summer have been reported. Here, we assessed whether the presence of seasonality in intestinal ESBL-Escherichia coli/Klebsiella pneumoniae (ESBL-E/K) carriage in the general Dutch population exists. From 2014 to 2017, the faecal carriage of ESBL-E/K in healthy individuals was determined in three cross-sectional studies in the Netherlands, including 5985 subjects. Results were pooled to identify seasonal trends in prevalence (by month of sampling). Multivariate logistic regression analysis was used to calculate pooled odds ratios and 95% confidence intervals. Results were adjusted for age, sex, antibiotic use and travel. Overall prevalence of ESBL-E/K carriage was 4.3% (n = 260 ESBL-E/K-positive), with differences between months ranging from 2.6% to 7.4%. Compared to January, the monthly prevalence of ESBL-E carriage was highest in August  and September . The observed monthly differences in ESBL-E/K carriage rates suggest that there is seasonal variation in exposure to ESBL-E/K other than due to travelling and antibiotic use. This should be taken into account in designing future ESBL-E prevalence studies in temperate regions.Infections due to extended-spectrum  Infectiibiotics . Besidesibiotics . The preibiotics –6.Escherichia coli), seasonal variation is ubiquitous in temperate regions, with increased human disease incidence during warmer months [E. coli is observed [E. coli survival in water and soil, which is probably related to the increased competition for nutrients following the increased activity of environmental bacteria at higher temperatures [With enteric zoonotic diseases  [Seasonality in healthcare-associated infections, mainly bloodstream infections (BSIs), caused by Gram-negative bacteria including observed . More rer months , 12. Howerature) .et al. on the faecal carriage of ESBL-E in a limited number of patients attending an emergency room of two hospitals in Barcelona and persons involved in food-borne outbreaks showed that there was a temporal distribution in ESBL-E carriage with a decrease in the prevalence in May and an increase in July [et al. assessed the incidence of carriage (including both colonised persons and persons with infections) of ESBL-producing E. coli and K. pneumoniae (ESBL-E/K) in hospitalised patients in two German university hospitals by using routine laboratory surveillance data. They found an increasing trend in the incidence of ESBL-E/K carriage during summer compared to winter [So far, seasonal variation in asymptomatic ESBL-E carriage in the general population has not been investigated. Two studies investigated the seasonal trends of ESBL-carriage in patients. One study by Mesa  in July . Howevero winter .It is important to know whether seasonality (or monthly variations) is present in the asymptomatic carriage of ESBL-E in order to design future prevalence studies more appropriately by decreasing the chance of finding factors associated with ESBL-E carriage which is due to a difference in exposure (i.e. moment of sampling). The aim of the present study is to assess whether there is a seasonal difference in the carriage of ESBL-E/K in the general Dutch population. The outcomes of this pooled analysis may also help to better understand the variations in ESBL-E/K carriage rates.E. coli and K. pneumoniae was determined in three different large cross-sectional studies in the Netherlands in the selections of predominantly healthy individuals in the population at large: the Livestock Farming and Neighbouring Residents' Health Study (VGO) [Enterobacter cloacae complex were excluded from our analyses. Data on antibiotic use and travel were collected via self-reported questionnaires in all three studies, except for antibiotic use in the VGO study. In the VGO study, antibiotic use was derived from general practitioner (GP) electronic medical records and data were only included when the GP registered prescriptions for ≥46 weeks during the calendar year and if the patient was registered at the particular GP for at least three-quarters of the year [From 2014 to 2017, the faecal carriage of ESBL-producing dy (VGO) , the ESBdy (VGO)  and a stdy (VGO) . The VGOdy (VGO) . In the dy (VGO) , 6, 16. the year . Data onχ2 test. A P-value <0.05 was considered statistically significant. Univariate and multiple logistic regression analysis was performed and pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. All variables available were included into the multivariate analysis since these were either general characteristics , known risk factors for ESBL-E/K carriage (antibiotic use during the last 6 months and travel during the last 6–12 months) or our variable of interest (month of sampling). To correct for multiple testing, the Benjamini–Hochberg procedure with a 10% false discovery rate was used. Furthermore, a supplementary analysis was executed to assess if the pooled logistic regression analysis was justified compared to a multilevel logistic regression model that corrects for between-study variation (by using study as a random effect). All analyses were performed using SAS (version 9.4).Results from the three studies were pooled to study the presence of seasonal trends in prevalence throughout the year (by month of sampling). The trend in monthly prevalence was tested using the Mantel–Haenszel E. coli/K. pneumoniae) from isolates taken between 2014 and 2016. ESBL production was estimated by ESBL confirmatory tests, or, when no data on confirmatory tests were available, by resistance against cefotaxime/ceftriaxone and/or ceftazidime.Furthermore, we also checked data from the Dutch national AMR surveillance system, based on routine data from medical microbiological laboratories , to inveχ2P-value of 0.036). In univariate analysis, monthly differences in the prevalence of ESBL-E/K carriage were observed in our data, with the highest prevalence in August and September compared to January (n = 6395 participants of whom 291 (4.8%) were the carrier of ESBL-E/K)), results were very similar with a little increase in the ORs for August and September (data not shown). The random-effect estimates for the study were not statistically significant in the supplementary analysis, indicating that there was no significant variation between the studies. The ORs from the multilevel model were very similar to the ORs from the pooled logistic regression analysis.A total of 8636 faecal samples of an equal amount of persons had been analysed in the three studies, and 5983 (69.3%) fulfilled the criteria to be included in this pooled analysis with a median age of 58 years (interquartile range 47–66) . Of thos January . In addiχ2P-values were non-significant, both overall and when a distinction was made between hospital-acquired (blood sample taken >2 days after admission) and community-acquired BSIs (data not shown).Finally, no clear seasonal trend could be observed in the monthly ESBL/E-K BSI prevalence data from the national antimicrobial resistance surveillance system: all Mantel–Haenszel In this pooled analysis with the data of three consecutive years (February 2014–April 2017) from 5983 individuals, ESBL-E/K carriage rates in the general population varied over the months of the year. Adjustment for age, sex and known risk factors for ESBL-E/K carriage (travel and antibiotic use) negligibly influenced the results from the univariate analysis and other factors seem to play a more important role to the exposure. We hypothesise that the higher ESBL-E/K carriage rate in August and September is caused by an increased chance of exposure to and/or acquisition of ESBL-E/K, potentially in combination with other factors including human behaviour/social and environmental factors.E. coli has been reported in temperate regions [E. coli and ESBL-E/K, while for non-ESBL-producing K. pneumoniae, conflicting results were found between different studies with regard to seasonal variations [et al. on community-acquired bacteraemia caused by E. coli in Denmark observed peak dates for different subgroups in August and September [As described above, increased incidence of enteric zoonotic diseases during warmer months including Salmonellosis, Campylobacteriosis and infections with Shiga toxin-producing  regions . This seriations , 15. Recriations , 12, sugriations , 15. Inteptember , which aE. coli and K. pneumoniae during summer in humans have been suggested. However, the causal mechanisms driving this are generally unknown and different processes seem to counteract each other, like increased animal shedding vs. decreased survival outside the host with increasing temperatures. In addition, it is very likely that it is an interplay between environmental and human behavioural/social factors. The abundance and diversity of genes encoding ESBLs differ significantly between open community and livestock and their products, indicating that animal production and foodborne transmission have a lesser contribution to ESBL carriage in humans than previously assumed in the Netherlands [et al. found that fluoroquinolones and β-lactams in general showed the strongest associations between temperature and resistance, which supports the mechanism-specific impacts of temperature on resistance [et al. showed in a population with a minimal environmental and genetic variation that despite overall gut microbiome stability within individuals over time, there are consistent and significant population-wide shifts in microbiome composition across seasons. In general, a lower gut microbiome diversity was observed during summer than during winter [Several explanations for increased rates of infections and ESBL carriage of herlands , and humherlands –20. Therherlands . Neverthherlands , potentiherlands . Howeverherlands . Furtherherlands . This inherlands . Moreovesistance . Finallyg winter . This seThe current analysis was performed by pooling data from three different cross-sectional studies in the Netherlands using univariate and multiple logistic regression analysis. All participants were from the general population, aged ≥18 years and similar laboratory techniques to identify ESBL-E/K carriers were performed. The supplementary analysis with random effects for the study did not alter our results. It was therefore decided to keep the analysis as simple as possible.A limitation of the study is that we only had data available of reported travel during the last 6–12 months (depending on the study), and we did not know the exact timing of the reported travel relative to the faecal sampling. Therefore, we might not have been able to adjust our analyses completely for travel. A second limitation is that, although data were available for three consecutive years, the number of samples for some months in this period was too low to perform a longitudinal analysis check whether the effect observed in August and September occurred yearly.E. coli and K. pneumoniae BSIs. A limitation of this analysis was, however, that the distinction between hospital-acquired and community-acquired infection based on the surveillance data might have been biased as the date of hospitalisation was missing in a substantial part of the infections. Furthermore, we could not include incidence data on ESBL-E/K BSIs because no monthly denominator is available in the surveillance system on routine diagnostics.Finally, the analysis of the national surveillance data showed no clear seasonal trend in the monthly proportion of BSIs caused by ESBL-E/K out of all In conclusion, differences in monthly ESBL-E/K carriage rates in the general community were found in the Netherlands, which may indicate that the factors during warmer seasons could influence the probability of an individual to become colonised. However, no seasonal trend in the proportion of ESBL-E/K BSIs was seen in the national surveillance data, indicating that the increased ESBL-E/K carriage rate did not result in an increase in community-onset invasive infections. In addition to adjusting for known risk factors, we advise to consider seasonal fluctuations in carriage rates when designing future ESBL prevalence studies in the general community and among risk groups  in temporal regions, as these might partly adjust for human behavioural/social and environmental factors that affect ESBL-E/K carriage in the community. We therefore recommend to sample participants within the same time window (i.e. during the same season but preferably (the same fraction) year-round) and not consecutively.


Background: Whole-genome studies of vine cultivars have brought novel knowledge about the diversity, geographical relatedness, historical origin and dissemination, phenotype associations and genetic markers. Method: We applied SOM (self-organizing maps) portrayal, a neural network-based machine learning method, to re-analyze the genome-wide Single Nucleotide Polymorphism (SNP) data of nearly eight hundred grapevine cultivars. The method generates genome-specific data landscapes. Their topology reflects the geographical distribution of cultivars, indicates paths of cultivar dissemination in history and genome-phenotype associations about grape utilization. Results: The landscape of vine genomes resembles the geographic map of the Mediterranean world, reflecting two major dissemination paths from South Caucasus along a northern route via Balkan towards Western Europe and along a southern route via Palestine and Maghreb towards Iberian Peninsula. The Mediterranean and Black Sea, as well as the Pyrenees, constitute barriers for genetic exchange. On the coarsest level of stratification, cultivars divide into three major groups: Western Europe and Italian grapes, Iberian grapes and vine cultivars from Near East and Maghreb regions. Genetic landmarks were associated with agronomic traits, referring to their utilization as table and wine grapes. Pseudotime analysis describes the dissemination of grapevines in an East to West direction in different waves of cultivation. Conclusion: In analogy to the tasks of the wine waiter in gastronomy, the sommelier, our ‘SOMmelier’-approach supports understanding the diversity of grapevine genomes in the context of their geographic and historical background, using SOM portrayal. It offers an option to supplement vine cultivar passports by genome fingerprint portraits. Grapevine (Vitis Vinifera) is one of the oldest of the cultivated plants for which living progenitors still exist. The broad geographic area of the wild grapes and a bewildering number of different forms expanding from Europe to Asia and Caucasus continue to intrigued researchers, with a crucial question about the origin and domestication of the grape. Archaeological and historical studies suggested that the cultivation of the domesticated grape (V. vinifera L. subsp. sativa) started about 10,000 to 8000 years ago, from its putative wild ancestor (V. vinifera L. subsp. sylvestris), and the primary center of domestication was located between the Near East [http://www.oiv.int/). Different authors evidenced the presence of secondary domestication centers, where spontaneous hybridizations among wild plants and cultivated forms or targeted selection, created the pattern of the modern Western European cultivars [Grapes are not only tasty, but also one of the most economically and culturally important crops. They are used for both winemaking and fresh (‘table’) consumption. Grape is unique, not only because it is a major global perennial crop, but also because of its historical and cultural connections with the development of humans. According to the OIV  the surface area of the world vineyard is estimated at 7.4 million hectares, with a production of 76 million tons of fresh grapes and 260 million hectoliters of wine  and wild (V. Vinifera L. subsp. sylvestris) grapes suggest at minimum two separate domestication events, one derived from the Transcaucasian wild grape and another one in Western Europe, where cultivars experienced introgression from local wild grape [In the last years, whole-genome studies of vine cultivars using genotyping and sequencing technologies have added novel knowledge about the diversity, geographical relatedness, historical origin, phenotype associations, genetic markers and distribution paths of the vine ,9,10,11.ld grape ,7. Assocld grape . The furHere, we apply SOM (self-organizing maps) portrayal , a neuraStrengths of the applied method are dimension reduction and visualization capabilities . ParticuThis study aims at applying SOM-portrayal to the genetics of vine, in order to generate SNP-landscape of grape genotypes, to relate its topology to the geographical distribution, to possible paths of cultivar distribution, to selected genetic markers, and link them to grape utilization. In gastronomy, the sommelier is the wine ‘waiter’ (or ‘waitress’), who recommends combining wine with food based on traditional knowledge about the character of wine varieties, their taste and geographic origin, and thus to support the decision-making of guests for a successful dinner. In analogy, our ‘SOMmelier’-approach aims at supporting understanding genetic diversity and relatedness of grapevine genomes in the context of their geographic and historical background by application of SOM portrayal.Grape cultivars data: grapevine genetic SNP- and phenotype data were taken from [https://urgi.versailles.inra.fr/Species/Vitis/Data-Sequences/Genotyping-data repository.ken from . The datHuman population data: For a comparison of genomic relatedness between worldwide geographic strata, we analyzed genome-wide microarray SNP data (Illumina 650Y arrays) from the Human Genome Diversity Project (HGDP) , as descOur analysis concept is based on the following main ingredients: (i) transformation of SNP data into excess minor allele frequencies (eMAF), which increases sensitivity for subtle differences between the genomes of the vine accessions; (ii) dimension reduction of the more than 10,000 SNP data into a matrix of 2500 so-called ‘meta-SNPs’, using unsupervised clustering and their visualization in terms of individual accession portraits using SOM machine learning. (iii) further reduction of dimension by segmentation of the SNP-portraits into about one-to-two dozen so-called spot clusters of correlated SNPs, which serve as fingerprint features of the vine genomes; (iv) diversity analysis of accessions using different approaches . Each of the approaches used enables discovering different aspects of the mutual relatedness between the cultivars. (v) So-called pseudotime analysis as a special type of diversity analysis to analyze genetic similarities in terms of multibranched trees with possible impact for temporal diversification of vine cultivars; (vi) phenotype associations to map information of vine utilization on the SNP landscapes.In order to further process the data, we coded the genotype of each SNP using a trinary code, with ‘0′ for homozygous major alleles (AA), ‘1′ for heterozygous alleles (Aa and aA), and ‘2′ for homozygous minor alleles (aa).ij = Nij/N , where Nij is the number of SNPs with the respective allele in the data set. The minor allele frequency of a SNP is MAF = 2 paa + 1 (paA + pAa) with paa < pAA. With the SNP-code introduced above one finds for its mean value averaged over all cases, <SNP-code> = pAA * 0 + (pAa + paA) * 1 + paa * 2 = 2 MAF, i.e., the mean SNP-code of a SNP equals twice its MAF. For further data processing, we define a SNP-score by centralizing the SNP-code of each SNP with respect to its mean value, SNP-score = SNP-code – 2 MAF. One obtains SNP-score = −2 MAF for the major allelic SNPs, SNP-score = 1 – 2 MAF for heterozygous SNPs and SNP-score = 2 (1 – MAF) for the minor allelic SNPs.First, let us consider one SNP in the data set of N cases, e.g., of N =783 cultivar samples. The fractions of the three genotypes of this SNP are defined as pSecond, let us consider a group of n correlated SNPs in the data set, and calculate the mean of their SNP-score in one selected sample. In the next subsection, we show that such a group of SNPs is given, e.g., by a meta-SNP, or a ‘spot-module’ as obtained below in SOM-analysis. Analogous consideration as above deliver the result for the group-averaged SNP-score, <SNP-score> = 2 (MAF - <MAF>) where <…> here denotes group averaging referring to one selected sample. Hence, the group-averaged SNP-score estimates the deviation of the mean MAF of the considered SNPs, in a certain sample from their mean in the considered population. Accordingly, it can be understood as an excess MAF-value (eMAF), where positive SNP-scores mean higher frequencies than in the population while negative values refer to reduced frequencies. The centralization of each feature (here the SNP-code of each SNP) with respect to its mean value averaged over all samples is applied as a standard preprocessing step in SOM analysis. Features space of centralized values is more sensitive to subtle differences between samples than the feature space of non-centralized values.Euclidean distance between the SNP- profiles as a similarity measure. It ensures that SNPs with similar profiles cluster together in the same or in closely located meta-SNPs. Each meta-SNP profile can be interpreted as the mean profile averaged over all SNP profiles of the respective meta-SNP cluster. For each cultivar accession, the meta-SNP score obtained provides the excess MAF . It is defined as the difference between the mean minor allele frequency (MAF of meta-SNP) of all SNPs collected in this meta-SNP in this particular sample, minus the mean MAF of these SNPs averaged over all samples. The eMAF values of each cultivar accession are visualized by arranging them into a two-dimensional M = 50 × 50 grid, and by using a red to blue color-code for maximum to minimum eMAF-values in each of the grid images. These images ‘portray’ the genetic landscape of each accession studied in units of the eMAF SNP-score. We used SOM as implemented in the “oposSOM” R package [SOM-portrayal of SNP data was performed as described previously . In shor package . Cultiva package  for deta package  to extraCategorical phenotypes such as the utilization of grapes for table or wine usage were associated with the SOM SNP-landscape by performing ANOVA  of SNP-metagenes of the sub-collection of cultivars of a certain phenotype  and coloring the meta-SNP pixels in the SOM according to the obtained p-value (in units of -log(p)), from red (high) to blue (low). Phenotype correlation maps were obtained by calculating the point biserial correlation between the eMAF profile of each metagene and the respective phenotype profile, which is given, e.g., for ‘table vine utilization’ by categorical values ‘0′ (no table usage) and ‘1′ (table usage). The metagenes of the map were then colored between red (maximum correlation) and blue (minimum correlation). Point serial correlation de facto provides difference portraits between cultivars of the respective phenotype and all others, e.g., between the mean portrait of cultivars of wine utilization, minus the mean portraits of cultivars of non-wine utilization.Accession diversity analysis was performed based on meta-SNPs using principal component analysis (PCA), similarity net and minimum spanning tree plots, based on correlation metrics between the individual SOM-portraits as implemented in oposSOM . t-SNE  of correlated meta-SNPs in the chosen cultivar . AccordiThe inspection of the individual accession portraits  reveals The spot-profiles show the mean values of the eMAF-scores averaged over all meta-SNPs included in the respective spot among the cultivars and, particularly, their region-specific alterations C. LargesNext, we analyzed the distribution of selected SNPs across the map. Laucou et al.  identifiThe chromosome-wise distribution of SNPs shows local enrichment in most cases . Overall, we find an association between vine utilization and specific areas of the SOM, which reflects an East-to-West gradient with preferential table usage in the East and wine usage in the West in agreement with . Note thTo increase geographic resolution, we calculated country-wise SNP-landscapes using ANOVA -portrayal A. The ANIn order to evaluate the relatedness between the individual SNP-portraits of cultivars, we calculated similarity nets. They are based on Pearson′s-correlation coefficients between the individual SOM-portraits and arrange them in a way that highly correlated cultivars were connected by lines and locate in the close mutual distance B, left. In the next step, we analyzed SNP-portraits and their diversity on the level of individual cultivars. An inspection of their gallery  indicateTo further evaluate the relatedness between the cultivars, we calculated a minimum spanning tree (MST), which, in contrast to the correlation net discussed above, connects only most similar cultivars and, in consequence, generates ‘paths’ of closest similarities C. The MSNote, that the structure of the vine-MST is completely different from the MST of human genomes of worldwide populations taken from the human genome project. This MST of human genomes possesses an almost linear backbone, with only a few side branches. It reflects the ‘out of Africa’ basic migration history of modern humans, which initially proceeded mostly along one major path. Hence, MST presentation seems to reflect the mechanisms and paths of genome dissemination. In summary, diversity analysis on single cultivar level reveals a marked intermixing between the different geographic regions where, however, cultivars from the South-Caucasus and Near East from the trunk for diverging branches of vines are typical for IBER (and ITAP), WCEU, MFEA and BALK (and RUUK) regions, respectively.For a more detailed study of possible paths of the distribution of grapevine, we applied so-called ‘pseudotime’ (PT) analysis. It arranges cultivars according to their mutual genetic similarity along possible multi-branched paths, using a diffusion-law based algorithm . The obtThe consecutive alterations in feature space observed were supported by PT-profiles of selected spots B. The eMThe consideration of grape utilization along the different PT-paths clearly reveals the enrichment of table vines in branches 0 and 4, while wine cultivars strongly enrich in the branches 1–3 at later PTs C, middleIn summary, multibranched PT-analysis resolves the ‘transverse’ distribution of cultivars among geographic regions also in ‘longitudinal’ PT-dimension, by sorting them according to mutual similarities of their genomic landscapes. We find the accumulation of grapes used for wine production at later PTs especially in Europe, which suggests the diversification of wine cultivars at later times compared with table grapes.We applied SOM artificial neural network-based machine learning to genome-wide SNP data of 783 grapevine cultivars, in order to visualize and to analyze the landscape of grapevine genomes, in terms of topological features such as ‘mountains’ and ‘valleys’, referring to positive and negative eMAF values, respectively .We complemented this genetic feature (SNP-) landscape by grapevine accession-similarity plots. Each of them visualizes different aspects of the mutual relatedness of cultivars. Principal component plots distribute accessions in (eMAF) distance-scale. It results in local overcrowding of cultivars with similar genomes, e.g., from Near East, Caucasus and Middle Asia while Western European and Iberian cultivars separate into virtually different data clouds of large variability between sample genomes. PCA requires separate projection-plots to cover the essential properties of similarity space. The (correlation) similarity net projects multidimensional sample space into two dimensions. It reveals major routes of grape distribution around the Mediterranean Sea and the genetic barrier between Iberian and Western European cultivars, evident also in the feature landscape. In contrast, the minimum spanning tree (MST) generates path-like structures. For worldwide human genomes, the MST reflects roughly the ‘out-of-Africa’ migration history, in terms of a virtually linear path starting from Sub-Saharan Africa and ending in East Asia, with side branches towards Europe, America and Oceania. Interestingly, the grapevine MST splits into four mean branches, enriching West European, Iberian, Balkan and Middle East grapes, and with cultivars from South Caucasus and Fertile Crescent  at their central crossroad. A detailed comparison of individual (ANOVA-) portraits further supports this result, using the feature landscapes in a country-wise resolution.Overall, our genomic landscape and the different sample similarity plots are consistent with the historical knowledge and previous genetic findings of grapevine domestication and distribution ,10. AccoV. vinifera ssp. sylvestris), particularly in Western Europe [Hence, grape growing and winemaking in classical time disseminated from ‘Iberia-to-Iberia’, starting in or near the ancient kingdom Iberia in Middle Georgia in South Caucasus in the East and ending at the Iberian Peninsula in the west. The combined action of selection, breeding, admixture and migration have shaped the cultivated grape diversity. Substantial genetic diversity has been maintained, subsequent to domestication derived from Transcaucasian Wild Grape, possibly due to several events of introgression from local wild vine Silvestris-varieties, Wild grapes  analysis. This method has been originally developed for describing cell differentiation using single-cell transcriptomic data ,32, and Our ‘SOMmelier’ approach visualizes essential aspects of grapevine genomes related to geographic distribution, paths of dissemination and vine utilization. ‘SOMmelier’ portrays vine genomes in terms of individualized images. They are intuitive, meaning that they don’t need specialized genetic knowledge for interpretation. Together with vine genome landscapes described here, we propose to use individual genomic portraits as an option to supplement vine cultivar passports as fingerprint characteristics of their genomes. Such fingerprint portraits consider virtually the whole diversity of the vine. Note also that we here discuss accession portraits mainly on the level of geographic regions as a sort of worked example to support the interpretation of genomes of individual accession portraits. Our study also demonstrates that bioinformatics methods proven before in analytic tasks on different omics realms, mostly transcriptomics, but also epigenomics, proteomics and human genomics, provide reasonable results if applied to vine cultivars as an example of plant genomes. Our approach thus extends the methods toolbox for plant genetics by providing novel approaches which complement established ones. Their pros and cons should be evaluated in future applications. The ‘SOMelier ‘method opens the opportunity to process larger genotype data, obtained by, e.g., whole genome sequencing and/or increased number of cultivars included.


Advanced microanalytical techniques such as high-resolution transmission electron microscopy (HRTEM), atom probe tomography (APT), and synchrotron-based scanning transmission X-ray microscopy (STXM) enable one to characterize the structure and chemical and isotopic compositions of natural materials down towards the atomic scale. Dual focused ion beam-scanning electron microscopy (FIB-SEM) is a powerful tool for site-specific sample preparation and subsequent analysis by TEM, APT, and STXM to the highest energy and spatial resolutions. FIB-SEM also works as a stand-alone technique for three-dimensional (3D) tomography. In this review, we will outline the principles and challenges when using FIB-SEM for the advanced characterization of natural materials in the Earth and Planetary Sciences. More specifically, we aim to highlight the state-of-the-art applications of FIB-SEM using examples including (a) traditional FIB ultrathin sample preparation of small particles in the study of space weathering of lunar soil grains, (b) migration of Pb isotopes in zircons by FIB-based APT, (c) coordinated synchrotron-based STXM characterization of extraterrestrial organic material in carbonaceous chondrite, and finally (d) FIB-based 3D tomography of oil shale pores by slice and view methods. Dual beam FIB-SEM is a powerful analytical platform, the scope of which, for technological development and adaptation, is vast and exciting in the field of Earth and Planetary Sciences. For example, dual beam FIB-SEM will be a vital technique for the characterization of fine-grained asteroid and lunar samples returned to the Earth in the near future. Most physical, chemical, and biological processes on Earth involve the interaction of naturally occurring materials at the macroscopic, submicron to nanoscopic scale. Beyond the Earth, unique astrophysical processes are recorded in planetary materials by, for instance, high pressure impact-related minerals  and highIn recent decades, advances in microbeam analytical technology, such as micro-Raman spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), atom-probe tomography (APT), and scanning transmission X-ray microscopy (STXM), enable one to accurately determine the morphology, crystal structure, and elemental, organic functional chemical and isotopic compositions of natural and synthetic materials at the micro-, nano-, or even the atomic scale –5. HowevThe concept of the focused ion beam technique using a liquid Ga ion source was originally developed by Seliger and Fleming in 1974 , 9. One Since then, FIB-SEM has become a diverse multifunctional tool for revealing structural, elemental, and isotopic chemical information in natural materials down to the nanometer scale. However, for their effective characterization, analytical methods require adaptation when using the dual beam FIB-SEM system. This review addresses the advanced use of FIB-SEM for a range of application in the natural sciences: from TEM analysis of small lunar soil grains, APT analysis of Pb isotopes in Earth-based zircons, and 3D slice and view of Earth-based shale kerogen to the characterization of extraterrestrial organics by coordinated synchrotron-based STXM-TEM. In addition, we address the key adaptations required in the FIB-SEM analytical methodologies for these different applications in Earth and Planetary Sciences. Finally, we assess the future development of the dual beam FIB-SEM technique.+ beam is extracted from the ion gun, it is accelerated with the voltage of 1~30 kV and then passes through the condenser and objective lenses. When the incident beam scans the sample surface, various ion beam-material interactions occur, the products of which can be collected and analyzed by multiple detectors.A schematic of the FIB-SEM system is illustrated in + ions and the target material can offer imaging, milling, and deposition. These are the generic processes performed in the operation of the dual FIB-SEM system. Specific adaptations to the FIB sample preparation method however need to be tailored for the preparation of samples specific to particular analytical techniques (The interaction between Gachniques .+ beam can produce high-resolution secondary electron (SE) images with enhanced contrast of a sample surface, as illustrated in + ion than an electron means that much fewer Ga+ ions are backscattered, therefore producing more SEs than that from the electron beam with similar beam size and currents, producing a higher signal/noise ratio in the images. Additionally, secondary ions (SI) can also be collected at each point of a raster for producing ion images for chemical or isotopic analysis, examples being secondary ion mass spectroscopy (SIMS) [Similar to a typical SEM, the incident Gay (SIMS) –19.2 for SiO2 [Samples can be microscopically removed  by ion-atom collisions . A surfafor SiO2 .+ ions are inevitably included [2, and others can be deposited using the GIS system.Unlike milling, the FIB-SEM system can offer precise, localized deposition of materials by using a gas injection system (GIS). In this process, the metal-containing organic compound is heated to a gas that flows out of a narrow tube above the sample surface. When ions or electrons scan a selected region, the metallic components of the precursor will be deposited on the surface of substrate . To depoincluded . Pt, C, Energy-dispersive X-ray spectroscopy (EDS) is the most wildly used method for measuring major elements (0.1 at %) in a sample, and its elemental detection range is 4Be~92 U . Unlike It is well known that TEM has the capability to image the structure and chemistry of materials at the nanometer scale. Spherical aberration (Cs) correction techniques can extend TEM imaging to the sub-Angstrom scale. The high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) and electron energy loss spectroscopy with the energy resolution better than 10 meV have been used in condensed matter physics and material science . For Ear+ milling method is effective for material sciences. However, one disadvantage of this technique is that artifacts may be introduced during mechanical thinning. Another disadvantage is that the milling region cannot be exactly controlled due to the broad ion beam. These problems have been addressed by FIB-SEM in the past two decades. The FIB system makes it easy to extract a microsized foil from the desired site on the sample surface. The foil is then attached to the grid and thinned down to the desired thickness (<100 nm).Prior to the use of FIB-SEM, TEM foils of terrestrial rocks and extraterrestrial materials were prepared using ultramicrotome and argon ion milling. The ultramicrotome is widely used in the preparation of biological samples. Examples of its application are for the characterization of acid mine drainage sediments  to interThe FIB and TEM techniques has been a routine analytical technique for terrestrial samples, such as ore minerals, natural diamonds, high-pressure experiment products, and microfossils –39. Ciobx) with npFe by-products of space weathering were observed in an Mg-Fe silicate fragment, as shown in x and npFe probably formed directly in micrometeorite shock-induced melt, instead of solar-wind generated vapor deposit or irradiated rim. This new observation will shed light on space weathering processes on the Moon and airless celestial bodies.The FIB-SEM is also readily used for the preparation of TEM samples in the study of extraterrestrial materials, such as presolar grains, high pressure mineral phases, and other meteorite components , 46–48. + beam [+ beam can also reduce the amorphous layer thickness. Therefore, it is necessary to design an appropriate sample preparation method according to the characteristics of different geological samples . For ultrathinning of submicron particles on a surface, initial E-beam capping rather than the Ga+ beam eliminates any possible sputtering of small particles on a surface [It should be noted that the high-energy ion beam will damage the sample surface, forming an amorphous layer. Previous studies have shown that accelerating voltage is a major factor affecting the thickness of amorphous layer , 55. Tak+ beam . Surface surface . Additio surface , 57.Atom probe tomography is a kind of field ion microscopy (FIM). A pulsed voltage is applied to the needle-shaped tip sample, breaking atomic bonds at the surface. The evaporated ions are projected onto a position-sensitive detector by a strong electrostatic field. The measurement of the ion flight times, from laser pulse to detector impact, allows the ion identities to be determined by time-of-flight mass spectrometry . The cheWhen applied to geological samples, which are commonly insulating, APT is usually operated in a “pulsed laser” mode that promotes field evaporation , 63.207Pb/206Pb ratio, though the average bulk value of 207Pb/206Pb is in good agreement with SIMS data at the micron scale [Zircon is one of the best minerals for U-Pb dating due to its high physical and chemical stability. The oxygen isotopic composition of ancient zircons is useful for inferring the formation time of the hydrosphere and the habitability of the early Earth . Howeveron scale , 64. Theon scale , 59. In on scale , 65, 66.E/∆E: ~3000) enables a stack of X-ray images to be taken at different energies around the X-ray Absorption Near Edge Fine Structure (XANES) of an element of interest. Elemental, chemical, mineralogical, and perhaps more uniquely organic functional chemical distributions can be obtained across the X-ray image [Synchrotron-based Scanning Transmission X-ray Microscopy (STXM) takes images of X-ray transparent samples via the raster of a stage behind a fixed highly focused X-ray beam down to the nanometer scale . The useay image , 69. XANay image . Here, way image  for theiay image .Organic matter is found in a wide range of planetary materials such as chondrites, interplanetary dust particles (IDPs), Martian meteorites, micrometeorites, and comets. The morphology, molecular composition, and distribution of OM can better constrain its hydrothermal evolution in the early solar system , 74.  Fi+ beam can be used for serial milling of the sample, and at the same time, a sequence of SEM cross-sectional images can be obtained. Then, segmentation and 3D visualization of the sample can be rendered using commercial software  [The microscopic features of rocks in 2D can be well analyzed using scanning electron microscopy. However, 3D is crucial for understanding the true distribution of phases in samples. In the dual beam system, the Ga, Avizo) . Such 3D, Avizo) , 79, 80.μCT), SEM, MAPS mineralogy, and FIB-SEM technology [Taking oil shale as an example, characterization and modeling of the pores' shape and connectivity provide essential information on the permeability and gas accumulation space –83. Unlichnology . FIB-SEMchnology , 87–90. chnology .A typical 3D structure of shale is shown in μm3. Due to the limited analytical scales, 3D reconstruction results are sometimes not fully representative of the actual complex and highly heterogeneous structure of rocks. It is therefore necessary to combine X-ray computed tomography and SEM mosaic images for statistical analysis from micron to nanometer scale [The volume of FIB 3D tomography is usually about ~1000 er scale , 92. The+ ions on various materials can alter their properties. It is therefore vital to prepare samples using methods minimizing the alteration of the material to be characterized. This is arguably even more important for labile, soft, and low Z number materials such as organic material that are susceptible to the remobilization of their functional groups by Ga+ interactions. Here, we summarize variations on the conditions and give specific suggestions required for the different applications using FIB for either TEM, APT, 3D tomography, or organic-based STXM analyses  cross-section milling in FIB-SEM 3D tomography often has the “curtain” effect, which affects the imaging resolution and causes reconstruction artifacts [+ beam and advances in multiple beam techniques have shown great potential to address these issues. In addition, the application of the FIB-SEM system will be further extended with the development of new technologies.As discussed above, although the FIB-SEM dual-beam system has been essential and widely used on natural materials, there are still some technical limitations: (1) high-energy Gartifacts . ImproveHigher ion beam currents for improved signal/noise and higher spatial resolutions are common goals in microscopy techniques. As mentioned above, most FIB-SEM ion beam systems use a liquid Ga ion source. The ion beam current of commercial FIB system is less than 100 nA, and the best resolution is about 2.5 nm at 30 kV, as shown in + plasma FIB microscopy (PFIB) has a slightly lower resolution, but its beam current is about 20 times as large as the Ga ion beam. Higher beam current means higher sputtering rates and larger analytical volumes at the same time. This PFIB system has been employed in semiconductor industry for milling materials at hundreds of micron length scale. In addition, previous studies have shown that the depth of damage caused by the Xe+ plasma FIB is 20~40% less than that measured from a Ga+ FIB-prepared specimen [Xespecimen , 105. ThCompared with using FIB-SEM as a stand-alone instrument, synergistic techniques show unique advantages that enable us to acquire more information from one sample and unravel the complex natural processes. This has been used to reveal shock-induced trace element segregation , microbeAdditionally, with the use of the sample preparation technique of FIB-SEM system, mass spectroscopy techniques such as thermal ionization mass spectrometry (TIMS) and quadrupole inductively coupled plasma mass spectrometry (Q-ICP-MS) could offer some level of spatial resolution that was not typically available . In the FIB-SEM is a powerful tool for site-specific sample preparation of TEM ultrathin foils and APT needle-shaped samples of natural and synthetic materials and also can work as a stand-alone technique for three-dimensional (3D) tomography. This technique has been widely used to study the microstructure, chemical and isotopic compositions of terrestrial rocks, extraterrestrial materials, paleontology, and marine sciences from the micron scale down to the single-atom resolution, through combining with multiple detectors and other in situ analytical instruments, including TEM, APT, and synchrotron-based STXM as examplesFIB methodologies need to be tailored for different purposes, whether it is small particle analysis, atomic probe tomography, synchrotron-based scanning transmission X-ray microscopy, or 3D FIB-SEM tomographyMultiple ion beam microscopy and some new technologies that have emerged recently are expected to be used to study the Earth and extraterrestrial materials for better revealing complex natural processes


Background: EEG-driven research is paramount in cognitive-neuropsychological studies, as it provides a non-invasive window to the underlying neural mechanisms of cognition and behavior. A myriad collection of software and hardware frameworks has been developed to alleviate some of the technical barriers involved in EEG-driven research. Methods: we propose an integrated development environment which encompasses the entire technical “data-collection pipeline” of cognitive-neuropsychological research, including experiment design, data acquisition, data exploration and analysis in a state-of-the-art user interface. Our framework is based on a unique integration between a python-based web framework, time-oriented databases and object-based data schemes. Results: we demonstrated our framework with the recording and analysis of an n-Back task completed by 15 elderly (ages 50 to 80) participants. This case study demonstrates the highly utilized nature of our integrated framework with a challenging target population. Furthermore, our results may provide new insights into the correlation between brain activity and working memory performance in elderly people, who are prone to experience accelerated decline in executive prefrontal cortex functioning. Conclusion: our framework extends the range of EEG-driven experimental methods for assessing cognition available for cognitive-neuroscientists, allowing them to concentrate on the creative part of their work instead of technical aspects. We propose an integrated development environment which encompasses the entire technical "data-collection pipeline" of cognitive-neuropsychological research, including experiment design, data acquisition, data exploration and analysis in a state-of-the-art user interface. In human subjects testing, our framework extends the range of EEG-driven experimental methods for assessing cognition available for cognitiveneuroscientists, allowing them to concentrate on the creative part of their work instead of technical aspects. I.Electroencephalogram (EEG) recordings of superimposed neuronal electrical activity can be used to provide insights into the cooperative behavior of neurons, which underlie cognition EEG is widely utilized across cognitive-neuropsychological research, where a participant is often asked to complete computerized interactive stimuli-driven tasks , while his brain activity and task-related responses are recorded. These neuropsychological tasks are usually programmed in a dedicated software suite, such as the commercialized E-Prime Over the past few decades EEG technology has evolved from addressing clinicians needs with cumbersome setups which require long assembly time, hair removal and a wired computer, to small, easily assembled, wireless systems, which can be commercialized for meditation or gaming Here, we describe a new Integrated Development Environment (IDE), which facilitates task creation, EEG recording, data synchronization and data analysis. Our IDE is based on a Graphical User Interface (GUI) and does not require programming expertise , group information , task definition data (stimuli parameters) and task results . Here we used two type of databases: SQLite Our framework utilized the ’Cortex API’ III.One of the most accessible EEG lines of products is commercialized by Emotive. The Emotive EPOC was shown to have better accuracy then the common alternatives for control task IV.Our framework currently supports programming of three main types of neuropsychological tasks: N-back task (i.e. executive attention brain states), open vs close eyes task , and affective/emotional processing task (negative versus positive affect brain states) using the IAPS , groups of participants and tasks. All can be saved or loaded from external files. Once a task is authored and chosen, the system searches for available headsets (via Bluetooth). When a headset is chosen, the EEG contact wizard is loaded, where the contact quality of the electrodes (2D / 3D) is monitored via a standard color scheme. In this wizard, band power from each electrode is recorded and displayed. Finally, a participant (or a group of participants) is chosen and the task is executed. Once data is acquired, data cleaned and analyzed to generate the EEG mean values represented in the results of different frequency bands. Data can be visualized with our data exploration wizard, in which each patient’s behavioral data can be viewed with his EEG data, clicks and colored indication lines for clicked correctly, incorrectly, and avoided. Finally, results can be exported to Excel. Few screen shots are shown in VI.A typical workflow with our system initiates with designing the neuropsychological task using our task authoring tool. Once the task is specified, the researcher defines the human subjects, individually, or from a previously defined group of subjects. Experiment starts with setting up the EEG equipment, connecting it wirelessly to the framework and then ensuring good electrode conductance using our EEG contact wizard. Once contact level is sufficient, the researcher chooses the desired task and execute it. The system stores the recorded data (EEG and responses (e.g. key presses)). The researcher can aggregate results from different groups, end explore the data using our tasks visualization tool. Finally, data and statistical analysis can be downloaded and explored using our data analysis tool. Full details are given in the supplemental manual.We exemplified our framework by conducting a 2-Back task (recognizing target from two stimulus-displays ago, for further details see VII.One of the prominent commercial solution for conducting cognitive-neuropsychological research is E-Prime. While proved useful, E-Prime cost money and more importantly is closed for modifications and application-specific tailoring. Open-source software packages extended the available frameworks for cognitive-neuropsychological research with the development of PsyToolkit, which was recently shown to have comparable performance Current available integrated EEG software framework include the OpenBMI Our framework integrates the entire pipeline of cognitive-neuropsychological research, providing state-of-the-art graphical user interface for experiment design, data acquisition, data exploration and analysis. Thus, extending the range of packages available for cognitive neuroscientists, will allow researchers to concentrate on the creative part of their work instead of on time-consuming technical aspects that delay preparation and execution of EEG-driven clinical studies assessing human behavior and cognition.


Objective: Constipation is one of the most frequent non-motor symptoms (NMS) in Parkinson's disease (PD), causing great disturbance to patients. The present study investigated the prevalence and the clinical features of constipation in patients with PD and explored the difference between prodromal and clinical constipation of PD.Methods: A total of 186 patients with PD were recruited into this study. Subjective constipation was defined by ROME III criteria. Demographic and PD-related clinical information of the participants were collected. The PD patients were objectively assessed by a spectrum of rating scales of motor symptoms, non-motor symptoms, and quality of life.Results: In total, 51.61% (96/186) of PD patients suffer from constipation. Compared with patients without constipation, the patients with constipation were prone to have restless leg syndrome, depression, and anxiety and have higher scores of the non-motor symptoms scale. Among patients with constipation, 21.88% (21/96) patients had constipation in prodromal stage. Compared with patients with constipation in clinical stage, patients with prodromal constipation had a lower age of constipation onset , longer timespan from constipation onset to motor symptom onset . Patients with prodromal constipation were predominantly tremor onset  and usually had a better quality of life . Depression was the only risk factor of constipation in PD patients. Body mass index, depression, and anxiety were factors that affected the life quality in patients with constipation.Conclusions: Our results supported the high incidence of constipation in patients with PD and that, in some patients, constipation occurred before the onset of motor symptoms. The specific clinical characteristics of patients with constipation and with prodromal constipation help to make early diagnosis, to discover the relationship between constipation and PD, and to further explore the pathogenesis of this degenerative disease. Parkinson's disease (PD) is a common neurodegenerative disorder characterized by motor  and non-motor symptoms  . ConstipAlthough progressive dopaminergic denervation is the cardinal pathology in substantia nigra pars compacta (SNpc) of patients with PD, other systems, such as the serotonergic system, are affected as well. Over the last decade, studies have suggested that a progressive and non-linear loss of serotonergic terminals takes place in PD and that serotonergic dysfunction in PD is associated with the development of motor and non-motor symptoms –7.via the vagus to the lower brainstem and the dopaminergic nigrostriatal system , were recorded for all participants. General clinical information, including levodopa equivalent daily dose , age ofConstipation was defined through the ROME III functional constipation criteria . The ROMThe severity of PD was assessed by Hoehn–Yahr (H&Y) stage and the Unified Parkinson's disease rating scale (UPDRS). The motor symptoms of PD patients were evaluated by UPDRS III . Onset oEach participant also completed the Pittsburgh Sleep Quality Index (PSQI), Epworth Sleepiness Scale (ESS), Rapid Eye Movement Sleep Behavior Disorder Screening Questionnaire (RBD-SQ), restless leg syndrome (RLS) diagnosis, Hamilton's Depression Scale (HAMD), Hamilton's Anxiety Scale (HAMA), Mini-mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), and Parkinson's Disease Non-motor Symptoms Scale (PD-NMSS). The PSQI is a self-report questionnaire that assesses nighttime sleep over a 1-month time period, with a global score ranging from 0 to 21. Higher scores represent poorer subjective sleep quality . The ESSt-tests were used to analyze the measurement data of two groups with a normal distribution. Non-normal distribution data were analyzed using non-parametric tests (Mann–Whitney test). The enumeration data were analyzed using χ2 test. We used logistic regression analysis to assess the correlations between the different factors and constipation in PD patients. Linear regression analysis was performed to figure out the risk factors of PAC-QoL. P < 0.05 was considered as statistically significant.The measurement data are expressed as mean ± SD (standard deviation), and the enumeration data are shown as numbers (rate). Two independent-sample A total of 186 idiopathic PD patients were ultimately included in this study. There were 95 male patients (55.60%) and 91 female patients (44.40%). The mean age was 67.53 ± 8.52 years old (range: 42–87 years), with a mean disease duration of 68.11 ± 69.59 months (range: 3–420 months).In 186 PD patients, 96 cases (51.61%) were with constipation (Cons-PD), and 90 cases (48.39%) were with no constipation (nCons-PD). Among the 96 PD patients with constipation, 21 cases (21.88%) experienced constipation at prodromal stage or before the onset of motor symptoms , and 75 cases (78.12%) experienced constipation at clinical stage or after the onset of motor symptoms .P = 0.043). The prevalence of RLS in the Cons-PD group was significantly higher than in the nCons-PD group (P = 0.043), but there were no significant difference in other sleep disorder evaluations, including PQSI score, ESS scores, and RBD. The scores of HAMA and HAMD were significantly higher in the Cons-PD group than in the nCons-PD group . The score of NMSS in the Cons-PD group was also significantly higher than in the nCons-PD group.The demographic variables and the clinical features of Cons-PD and nCons-PD groups are compared in 2 = 4.091, P < 0.001). The timespan from constipation onset to motor symptom onset in Cons-prodromal-PD was longer than in Cons-clinical-PD . Cons-prodromal-PD was predominantly tremor onset, while Cons-clinical-PD was non-tremor onset . There were no differences between the two groups in ESS, RBD, PQSI, RLS, MMSE, and MoCA. The HAMA and HAMD scores, which manifested a significant difference between the Cons-PD and the nCons-PD groups, were not different between Cons-prodromal-PD and Cons-clinical-PD. There was no difference in NMSS between the two groups. PAC-QoL in the Cons-prodromal-PD group was lower than in the Cons-clinical-PD group .The demographic variables and the clinical features of Cons-prodromal-PD and Cons-clinical-PD groups are compared in P < 0.01).Logistic regression analysis was performed to figure out the risk factors of PD-C in P < 0.01).Linear regression analysis was performed to figure out the risk factors of PAC-QoL in The clinical symptoms of PD are divided into motor and non-motor symptoms. Among non-motor symptoms, constipation is unanimously regarded as one of the most relevant and difficult to treat. In this study, 51.61% PD patients had constipation, in accordance with previous studies reporting 50 to 80% of constipation in PD patients  but muchWe did not find differences between Cons-PD and nCons-PD in demographic data and lifestyle, including sex, education, exposure to insecticides/herbicides, drinking Chinese liquor, and smoking. Although it has been reported that women are more prone to experience chronic constipation in general subjects  and thatWe further found that Cons-PD patients tended to have a statistically high percentage of tremor onset phenotype, and they were more prone to be accompanied by sleep disorders (RLS in particular), anxiety, depression, as well as more other non-motor symptoms. There was no significant difference in the severity of motor symptom, disease duration, age of motor symptom onset, onset location, and clinical phenotype between these two groups.Although we did not complete a statistical analysis of Medopa dosage alone, this study showed no difference between LEDs in Cons-PD and nCons-PD groups, being consistent with Frazzitta et al.  but contWe retrospectively surveyed the timespan between onset of constipation and onset of motor symptoms to investigate the clinical features between PD patients with prodromal stage constipation and clinical stage constipation. Our data showed that there were 21.88% of PD patients having constipation before the onset of motor symptoms, that is, the prodromal stage. The result was similar to but slightly lower than those of previous studies, which reported the prevalence of constipation before the onset of motor symptoms as ranging from 28.19 to 87% . CompareCorrelation analysis showed that HAMD was positively associated with constipation, suggesting that HAMD might be a risk factor for constipation in PD patients. Considering the small sample size of patients with prodromal constipation, we did not analyze the risk factors of prodromal-stage-constipation PD patients.We spotlighted in this study that depression was an independent risk factor of constipation, and PD patients with prodromal stage constipation were predominantly tremor onset. Since the underlying clinical implications and mechanisms are unclear, studying and discovering the underlying mechanisms becomes interesting and challenging.in vivo in patients with PD was significantly reduced , but we now know that 5-HT is vastly secreted in the bowel and acquired by platelets, becoming circulating or enteric, making the content of 5-HT in CNS negligible. Much attention was focused on the role of brain 5-HT in the past, but the function of peripheral or intestinal 5-HT has been increasingly concerned and understood. The American Gastroenterological Association classifies primary constipation into three groups on the basis of colonic transit time and anorectal function: normal-transit constipation, slow-transit constipation, and outlet dysfunction. Studies have shown that the colonic transit time in PD patients is twice that of the control group, and researchers considered that constipation in PD patients is related to the decrease of colonic transit function . The perOur study has some limitations. First, it is a retrospective clinical study in which the information of chronology of motor and non-motor symptoms were collected through patients' recall. Although most clinical studies have adopted this method of information collection, recall bias might partially affect the accuracy of our results. Secondly, the lack of a healthy control group may exist when asking about non-motor symptoms. Thirdly, RLS is usually diagnosed by sleep or neurology experts with face-to-face interview. Using the questionnaire for RLS diagnosis may have included RLS mimics and potentially resulted in a high prevalence. Fourthly, no bowel transit time or stool examinations were performed when evaluating constipation, which might lead to a lack of objectivity and quantification of the results. Finally, since it is a single-center retrospective study, the results still requires to be testified by more studies, including multicenter clinical studies.With limited literature search capabilities, we have not found out reports on the relationship between the raphe serotonergic system and constipation symptom in patients with PD and between the raphe serotonergic system and enteric serotonergic neurons in constipation patients with PD. In the current study, we demonstrated that depression was an independent risk factor of constipation, and PD patients with prodromal stage constipation were predominantly tremor onset. The differences in the results of various studies are related to various factors such as race, population, sample size, diagnostic definition, etc. This requires researchers to further expand the sample size and unify various concepts and evaluation standards in a future study. On the basis of the present study, we are interested in and will investigate the possible relationship between disruption of serotonergic neurons, either in raphe or in gut, and constipation in patients with PD in future studies.All datasets generated for this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by the Ethics Committee of Xuzhou Clinical Hospital of Xuzhou Medical University approved this study. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.QX and GD designed the study. CG, LB, LZ, LiX, SM, and DY collected the data. LiuX did the statistical analysis. QX and GD analyzed the data and drafted the manuscript. CX participated to draft and revise the manuscript. All authors wrote and revised the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Supplemental Digital Content is available in the text Controversies emerge over routine performances of whole-body computed tomography (WBCT) in patients with blunt polytrauma. The existing randomized and non-randomized evidence is inconclusive, and during observations of non-trauma, incidental findings, detected by WBCT, have left uncertainty regarding their consequences and optimal management. Additionally, previous meta-analyses have failed to address the limitations of primary studies and issues associated with incidental findings. Therefore, we planned a new systematic review to address these points.We will search the PubMed, EMBASE, and Cochrane Central databases from inception to December 31, 2020, with no language restriction and perform full-text evaluation of potentially relevant articles. We will include prospective and retrospective studies with a single-gate design that assessed diagnostic accuracy and/or yield of WBCT to detect traumatic injuries, and studies that assessed incidental findings detected by WBCT. Additionally, we will include randomized controlled trials and non-randomized comparative studies that assessed the effectiveness of WBCT against conventional care, including selective computed tomography (CT). Studies of patients of all ages with blunt traumatic injuries, assessed at an emergency department, will be included. Two reviewers will extract data and rate the study validity via standard quality assessment tools. The primary outcome of interest will be reduction in mortality. Our secondary outcomes will include diagnostic accuracy and yield, detection of incidental findings and clinical outcomes associated with these detections, and improvement in other non-mortality clinical outcomes. We will qualitatively assess study, patient, and intervention characteristics and clinical outcomes. If appropriate, we will perform random-effects model meta-analyses to obtain summary estimates. Finally, we will assess the certainty of evidence by the grading the quality of evidence and strength of recommendations.Ethics approval is not applicable, as this is a secondary analysis of publicly available data. The review results will be submitted for publication in peer-reviewed journals.CRD42020187852. Although the incidence rates of penetrating injury vary by geographic region, injuries attributed to blunt trauma are the most common worldwide, and are typically caused by traffic collisions and falls with approximately 1.2 million and 0.7 million deaths, respectively.Injury is a major cause of health loss with approximately 4.5 million deaths globally in 2017. and recently, as proposed in the new Berlin definition, using ≥2 injuries with an Abbreviated Injury Scale (AIS) ≥3 and at least one additional clinical variable that reflects pathological conditions. Since blunt injuries may not necessarily be externally visible, accurate and timely diagnosis of specific injuries plays a critical part in the management of blunt multiple trauma. Advanced Trauma Life Support (ATLS) recommends a standardized, structured approach to trauma patients. This approach comprises the primary survey of life-threatening injuries coupled with implementing simultaneous resuscitation based on the Airway (assessment and protection), Breathing (and ventilation assessment), Circulation (assessment), Disability (assessment), and Exposure  (ABCDE) approach and the secondary survey by systematically assessing the injured body parts and their specific treatments. In the primary survey, x-ray examination of the chest and pelvis using a portable device, and bedside ultrasound examination of the perihepatic space, perisplenic space, pericardium, and pelvis, also known as focused assessment with sonography for trauma (FAST), are recommended as the standard adjunct diagnostic imaging. In the secondary survey, computed tomography (CT) is the mainstay to identify specific injuries.Multiple trauma, also referred to as major trauma or polytrauma, the term applied to patients with multiple traumatic injuries with high mortality rates, is conventionally defined by an Injury Severity Score (ISS) ≥16, With installation of a modern, high-quality, high-speed CT scanner system, adjacent to or in the emergency department (ED), precise images, rapidly obtained through upfront pan-scanning, could theoretically provide a better evaluation of the injured sites and can lead to better management to life-threatening injuries in a timely manner even in hemodynamically unstable trauma patients with specific indications. Examples of the expected benefits include timely provision of surgical interventions, such as decompressive surgery for severe brain injuries with intracranial hypertension, and surgical interventions and/or interventional radiology (IVR) for bronchial, lung, and aortic injuries or hemothorax and hemoperitoneum.Advances in medical imaging technology have introduced multi-detector CT in clinical practice. With its rapid scanning and reconstruction speed, coupled with high quality non-invasive imaging, several routines including upfront pan-scanning protocols, whole-body CT (WBCT) in early trauma evaluation were proposed. subsequent observational studies–18 and meta-analyses–22 consistently reported that WBCT-based management strategies were associated with better clinical outcomes, including mortality, than conventional management. Consequently, WBCT has gained rapid and wide adoption in clinical practice.Since the first observation of the usefulness of upfront, routine use of WBCT in comparison with conventional management, based on selective CT, was reported in 2007, Although several intermediate outcomes, such as the time required to diagnose traumatic injuries, were improved, the effect of WBCT analyzed based on the intention-to-treat (ITT) analysis —the effect of assignment to the WBCT group—failed to show a mortality benefit. As expected, 46% of the patients in the conventional group ultimately underwent a WBCT after the primary survey; non-blinded treating personnel sequentially performed CT scans of all segmental body regions. Post-randomization confounding is a well-known consequence in pragmatic RCTs such as REACT-2 and needs special care However, the ITT effect may not always represent the ultimate target in RCTs; the per-protocol effect, an alternative to the ITT effect, which also accounts for adherence to the assigned interventions, may be of more relevance to patients and their clinicians. Nevertheless, a most recent meta-analysis that included REACT-2 performed naive synthesis methods, not rigorously addressing these methodological heterogeneities.However, uncertainties exist regarding the comparative effectiveness of WBCT-based management vs conventional care because the promising comparative data from the observational studies were not validated in a Dutch non-blinded randomized controlled trial (RCT), REACT-2, enrolling approximately 1100 patients with trauma of varied severity, including 36% non-multiple trauma patients. Clinical consequences of these findings, however, are uncertain and their optimal managements have not yet been established. For instance, a recent observation of a regional trauma registry in the USA involving 957 patients reported non-trauma abnormal findings in 40%. Nearly 90% of the CT-detected incidental findings were nontrivial and deemed to require additional medical interventions of various invasiveness, raising concerns at both individual and societal levels. Again, previous systematic reviews did not address the issues stemming from incidental findings.In addition to increased radiation exposure, WBCT necessarily detects incidental findings other than traumatic injuries, which include both benign and neoplastic lesions as well as vascular abnormalities, such as enlarged aneurysms.Given that it has been over 4 years since the most recent meta-analysis report was published, and that there has been an absence of new, rigorously conducted systematic reviews that addressed the aforementioned limitations, we planned to conduct a new systematic review and meta-analysis. Our objectives are 3 fold: (a) an update of the comparative effectiveness of WBCT-based management compared with conventional imaging-based management, (b) qualitative and quantitative syntheses of the evidence on clinical outcomes associated with incidental findings of WBCT, and (c) comprehensive evidence review of predictors that identify in which subgroups WBCT is more useful or harmful.2 Based on the established framework for assessing levels of clinical effectiveness of diagnostic tests and the analytic framework shown in Figure This systematic review and meta-analysis protocol follows the preferred reporting items for the systematic review and meta-analysis protocols 2015 statement (PRISMA-P) and in the event of protocol amendments, the date of each amendment will be accompanied by a description of the change and the rationale.KQ 1 (diagnostic accuracy):What is the diagnostic accuracy of WBCT to detect traumas?a.All patients with blunt traumatic injuries (regardless of severity)b.Patients with blunt multiple trauma onlyKQ 2 (diagnostic impact):What is the proportion of patients for whom WBCT is useful in diagnosing traumatic injuries in comparison with conventional imaging-based assessment?a.All patients with blunt traumatic injuries (regardless of severity)b.Patients with blunt multiple trauma onlyKQ 3 (management decision impact):How often does WBCT change diagnostic or therapeutic strategies in comparison with conventional imaging-based assessment?a.Use of therapeutic interventionsb.Use of supportive interventionsc.Use of diagnostic interventionsKQ 4 :What is the comparative effectiveness between WBCT-based management strategies and conventional strategies?a.Mortalityb.Time spent for specific therapeutic or supportive interventions, and ICU or hospital stayc.Time spent before ED disposition decisionKQ 5 (effect modifiers):What factors modify the effectiveness measures listed under the KQ 4?KQ 6 :In what percentage of trauma patients does WBCT detect incidental findings?a.Malignancyb.Major findings c.Moderate findings (that may cause morbidity)KQ 7 :What are the consequences related to incidental findings detected by WBCT?a.Additional management related to incidental findingsb.Subsequent clinical outcomes related to incidental findings2.1http://links.lww.com/MD/F533. The electronic search results will be imported into EndNote X9  and duplicate results will be removed. As additional searches, we will peruse the reference lists of previously reported systematic reviews and meta-analyses. No language restrictions will be set.We will search the PubMed, EMBASE, and Cochrane Central Register of Controlled Trials (CENTRAL) databases from the inception of data collection through December 31, 2020, using free text terms, such as “whole body CT” and “multiple trauma” and their synonyms. The complete search strategy and full list of databases are available in the supplementary file, 2.2,32 We will include any prospective or retrospective cohort or cross-sectional studies with a single-gate design that included ≥30 patients of all ages with blunt trauma and assessed the outcomes relevant to KQs 1, 2, 3, 5, 6, and 7. Regarding KQs 3 and 4, both RCTs and non-randomized studies of interventions (NRSIs)—cohort studies, case-control studies, controlled before-and-after studies, interrupted-time-series studies, and “quasi-” randomized studies—of any size that assessed the comparative effectiveness of WBCT in comparison with conventional management involving selective CT, will be eligible. We will exclude conference abstracts, primary studies with the outcome data unextractable from the publication, and studies based on mathematical modeling approaches, such as decision model or cost-effectiveness studies. To deal with across-study variations and insufficient reporting in the inclusion criteria of trauma patients , we will exclude studies that explicitly included >5% of patients with trauma other than blunt trauma, or studies including >10% of patients with unspecified trauma unless subgroup data on patients with blunt trauma exclusively are extractable.Table Two independent reviewers will double-screen abstracts using Abstrackr, a web-based software for citation screening . We will then peruse all potentially eligible full-text articles that ≥1 reviewers screen in for eligibility. All non-English publications will be translated into English before full-text assessment. Any discrepant results will be resolved by consensus. We will employ adjudication by a third reviewer in case of unresolved discrepancies.2.3Data will be extracted by 2 reviewers. One primary reviewer will extract descriptive data, and ≥1 other reviewer will verify all extracted data. Two independent reviewers will double-extract any numerical data on the outcomes of interest. Disagreements will be resolved by consensus, and a third reviewer will adjudicate any unresolved discrepancies. We will contact the study authors for any missing or unresolved numerical data by email. We will send 2 additional email correspondences if no response is received by 2 weeks after the previous correspondence attempt.KQs), characteristics of WBCT , characteristics of alternative testing strategies , characteristics of reference standards (for KQs 1 and 2), and definitions of reported clinical outcomes  as descriptive data.We will extract study and patient characteristics , study location , study period (enrollment year), study design, enrollment methods (consecutive or not), number of centers, clinical setting, and inclusion and exclusion criteria. or the new Berlin definition criteria for polytrauma is satisfied . We will also record data on mortality prediction models such as the trauma and injury severity score (TRISS) or its variants, if any, were reported.Patient characteristics will include the number of patients, average age (range), male sex (%), average vital sign parameters (ranges), category of injury  (%), mechanisms of injury, average ISS (range), injured anatomical parts per 5 ISS-based separate body regions  (%), and their respective average AIS (range) and injuries categorized as severe trauma as defined as AIS ≥3 (%), and patients categorized as having multiple trauma (%). Items to extract data for the mechanisms of injury will include road traffic injuries (%), falls (%), and self- or inter-personal violence (%). The average and range values to be extracted for vital sign parameters will include respiratory rate (RR) (per minutes), pulse (beats per minutes), systolic blood pressure (SBP) (mmHg), and Glasgow Coma Scale (GCS) score (points), or the Revised Trauma Score in aggregate (points). We will consider a patient with multiple trauma if an ISS is ≥16,vol).Test characteristics of WBCT will include location of CT scanner, timing of CT scanning, anatomical parts and specifications of CT scanning, use of contrast materials, and interpreter of CT results and experience. Items to extract data for the CT specifications include field of view, slice thickness and interval, X-ray tube voltage (kV), automatic tube current modulation reported as mAs product, number of detectors, pitch factors including gantry rotation speed and beam collimation, image reconstruction algorithms, and volume of CT dose index , routine indication of CT for specific body regions, if any, body regions for which CT was ultimately performed (%), WBCT performed after primary work-up (%), and other specific post-primary work-up imaging tests. Items to extract data for the CT specifications are the same as those described above.2.4KQ 4a). Here, mortality outcomes include 24-h mortality, 30-d mortality, in-hospital mortality, and overall mortality. For all subquestions under KQ 4, we will consider the effect of “assignment” to WBCT-based management  at baseline  to be the main analysis; the effect of “starting and adhering” to the WBCT-based management   will be regarded as the sensitivity analysis. We will also assess the effect of WBCT regardless of whether it is performed upfront or as the subsequent-line imaging survey .Our primary outcome of interest will be reduction in mortality from all causes by upfront use of WBCT in comparison with conventional management (KQs 4b-c) including time spent before ED disposition for multiple phases of ED stay including time from ED admission to CT, time from ED admission to diagnosis, time from ED admission to specific therapies , time from admission to discharge from ED, time spent for specific therapeutic or supportive interventions, and time spent for ICU or hospital stay by WBCT in comparison with conventional management as the secondary outcomes. Specific clinical characteristics that are associated with better mortality or other short-term clinical outcomes using WBCT  will be assessed simultaneously in this context as a secondary outcome (KQ 5).We will assess short-term non-mortality clinical outcomes (KQ 6). These outcomes will include detection of incidental cancers, aneurysms, and other incidental findings. Two other clinical outcomes related to these incidental findings—additional management required for the incidental findings and their subsequent outcomes—will also be assessed as the secondary outcomes (KQ 7). Additional management will include specific diagnostic and/or therapeutic interventions deemed necessary (KQ 7a-1) and actually performed (KQ 7a-2) for the incidental findings. The subsequent clinical outcomes will include any benefits (KQ 7b-1) and harms (KQ 7b-2) associated with the detection of incidental findings .We will also assess clinical outcomes stemming from incidental findings other than trauma, such as cancer or aneurysm, as secondary outcomes (KQ 1), change in the diagnosis of traumatic injuries between WBCT in comparison with conventional imaging-based assessment (KQ 2), management decision impact of WBCT, such as additional use of other diagnostic tests or supportive  or therapeutic interventions  ordered based on the WBCT results in comparison with conventional imaging-based assessment (KQ 3).Additional immediate outcomes, also assessed as secondary outcomes, include sensitivity and specificity of WBCT to detect traumatic injuries (2.5KQ 1). We will define sensitivity as TP/(TP+FN) and specificity as TN/(FP+TN), where TP indicates true-positive (positive WBCT and reference standard tests), FP indicates false-positive (WBCT positive and reference standard test negative), FN indicates false-negative (WBCT negative and reference standard test positive), and TN indicates true-negative (WBCT and reference standard test negative) results from the 2 × 2 contingency table including cross-classified count data according to whether WBCT and the reference standard test are positive or negative.Sensitivity and specificity will be used as the metric of diagnostic accuracy .For sensitivity and specificity, we will focus on (a) injuries that occur in 5 specific ISS-based body regions  separately and (b) 20 operationally defined individual injuries as proposed by Stengel et al. separately ,KQ 2) and again consider an individual patient as the unit of analysis. For diagnostic before-after studies, we will define absolute change in the diagnosis as the number of injured patients , detected by WBCT, subtracted by the number of injured patients detected by a conventional diagnostic strategy , which is divided by the total number of all patients tested with WBCT.We will use the percentage of absolute change in the diagnosis of traumatic injuries in each of the 5 ISS-based body regions or 20 individual injuries between WBCT and conventional imaging-based assessment as the measure of diagnostic impact (KQ 3) and incidental findings (KQ 7a), we will calculate the absolute change or relative risk of additional diagnostic or therapeutic interventions. The unit of analysis will be an individual patient. For diagnostic before-after studies, we will calculate the number of patients for whom specific diagnostic or therapeutic interventions were added or avoided according to WBCT in comparison with conventional imaging-based assessment, divided by the total number of patients tested with WBCT as the absolute change. For RCTs and NRSIs , we will use the relative risk (RR) in a specific intervention between the 2 groups.As the measures of management decision impact outcomes, specifically related to traumatic injuries (KQ 4) and incidental findings (KQ 7b), we will assess the association of use of upfront WBCT  with binary, event-type count data  by using RR or hazard ratio (HR) as the measures of outcomes. For continuous outcomes, such as length of hospital stay and other duration outcomes, we will use the difference in time spent between groups.For patient-relevant clinical outcomes specifically related to traumatic injuries (KQ 6), we will calculate the percentage of incidental findings as the outcome metric. Our main target condition of interest was incidental findings that were verified as malignancy only. We will also consider composite target conditions—major incidental findings  alone, and major and moderate findings combined  according to the commonly adopted three-grade and four-grade classification systems for incidental CT findings.For incidental findings (2.6KQ 1) and diagnostic impact outcomes (KQ 2), we will accept any reference standard tests adopted in eligible studies. However, we will prefer clinical verification including repeat and/or additional imaging, surgical or radiological interventions, and clinical follow-up and autopsy, if performed, as the appropriate clinical reference standard over WBCT (performed as the index test) only. Likewise, we will accept any methods to verify event-type and duration-type clinical outcomes for KQs 4–6; however, we will record whether a study prospectively identified the events or durations through research-oriented, study-specific methods (preferred), or relied on data, routinely collected for purposes other than research such as data from disease or trauma registries, or medical charts.For sensitivity and specificity (2.7KQ 1), 2 reviewers will independently assess the patient selection, index test, reference standard, and their flow and timing, based on the revised Quality Assessment of Diagnostic Accuracy Studies instrument tool (QUADAS-2). We will assess 4 domains of study validity: participant selection, index test, reference standard test, and flow and timing thereof. A tailored version of the tool with details of the signaling questions, operational definitions, and rules for combining the answers to produce a domain-level rating are included in the supplementary file, http://links.lww.com/MD/F533.To assess the risk of bias and concerns regarding the applicability of studies of diagnostic accuracy (KQ 2), we will assess study quality by using the original version of the Quality Assessment of Diagnostic Accuracy Studies instrument tool (QUADAS) tool with modifications proposed by Meads et al. As recommended, we will apply the adapted QUADAS tool by considering conventional imaging-based assessment  and WBCT as the  index and reference standard tests, respectively.For diagnostic before-after studies that assessed diagnostic impact , we will use established quality assessment tools. Our main interest is the ITT effect of upfront use of WBCT  on mortality. Regarding NRSIs, we will use the Risk Of Bias In Non-randomized Studies of Interventions (ROBINS-I tool). We will assess 7 domains of study validity, i.e., confounding, participant selection, classification of interventions, deviations from intended interventions, missing data, measurement of outcomes, and selective reporting. RCTs, we will use the revised tool to assess risk of bias in randomized trials (RoB 2 tool). We will assess 5 domains of trial validity, i.e., randomization processes, deviations from intended interventions, missing outcome data, measurement of outcomes, and selective reporting. We will prespecify age, GCS, SBP, RR, and ISS, and the TRISS and its variants as important mortality predictors and prediction models to consider, respectively.To assess patient management and clinical outcomes (2.8KQ 1), we will first calculate sensitivity and specificity for each study with their corresponding 95% confidence intervals (CIs). Then, we will assess between-study heterogeneity visually by plotting sensitivity and specificity separately in forest plots and in the receiver operating characteristic (ROC) space. When studies are less likely to have different explicit thresholds for diagnosing injury findings, we will obtain summary estimates of sensitivity and specificity with their corresponding 95% credible intervals (CrIs) and confidence regions for summary sensitivity and specificity by using bivariate random-effects meta-analysis with the exact binomial likelihood when ≥3 studies are available.,47 When diagnostic criteria are evidently inconsistent across studies, we will construct hierarchical summary ROC curves (HSROCs).To quantitatively synthesize sensitivity and specificity  even when a series of clinical tests are performed to verify a disease condition .tau, and I2 statistics and their corresponding 95% CrIs. An I2 > 50% will indicate intermediate heterogeneity, while an I2 > 70% will indicate high heterogeneity. To additionally explore statistical heterogeneity in studies of patient management and patient-relevant clinical outcomes , we will identify influential and/or outlier studies by constructing the Graphical Display of Study Heterogeneity (GOSH) plots if data are amenable to this analysis.For other (non-accuracy) outcomes, we will assess between-study heterogeneity visually by constructing forest plots and quantitatively by estimating the between-study SD parameter, KQ 4, 5, and 7b), we will perform a standard Bayesian hierarchical random-effects meta-analysis. For count data from RCTs, we will perform a random-effects meta-analysis using the binomial likelihood with logit or complementary log-log link in a generalized linear modelling framework. If already-estimated relative measures are the only extractable formats for RCTs, and for all other designs of comparative studies , we will use the log-transformed estimates and their variances as “plug-in” estimates and perform a random-effects meta-analysis using the standard approximate normal-normal model. Regarding prior distributions on the tau, we will use the context-specific informative prior distributions as appropriate. We will prefer data adjusted for cofounding variables over unadjusted data. For difference in continuous outcomes, such as length of hospital stay, we will perform a random-effects meta-analysis of the standardized mean difference. Again, we will prefer adjusted data over unadjusted data.For calculating summary RR or HR estimates, based on event-type data , such as proportions of detection of incidental findings, we will perform a random-effects meta-analysis of proportions using the binomial likelihood and logit link . We will use a non-informative prior distribution on the tau to cover the possibility of very large across-study statistical heterogeneity.For summary estimates of the proportion measures in non-comparative studies (2.9KQ (other than KQ 1), we will perform the funnel-plot asymmetry test if at least 10 studies are included. For studies assessing diagnostic accuracy (KQ 1), we will not assess funnel-plot asymmetry because the required tests are still less well-established and not recommended for studies of diagnostic accuracy.For studies relevant to each  Preplanned candidate factors will include design , number of centers (single- vs multi-center), study location, CT protocol, cause of trauma , and severity of included patients . For the univariable meta-regression of diagnostic accuracy, we will add a covariate in the bivariate model to simultaneously explain the statistical heterogeneity in both (logit-transformed) sensitivity and specificity.We will perform subgroup analyses and univariable random-effects meta-regressions, if appropriate.KQ 4 and 7b), if feasible, we will apply the Bayesian approach proposed by Turner et al to fully account for uncertainty derived from missing data. Otherwise, we will impute missing data based on a series of conventional methods proposed by Akl et al. For missing continuous data, we will impute missing data as proposed by Ebrahim et al.For missing binary outcome data in RCTs , we will create a standard summary of findings (SoF) tables and assess the overall quality of evidence according to the recommendations.–67 For diagnostic accuracy (KQ 1), SoF tables and the overall quality of evidence will be constructed following the approaches specifically designed for test accuracy.,69Finally, we will assess the certainty of evidence using the Grading of Recommendations Assessment, Development, and Evaluation approach.2.10www.openbugs.net) from within Stata V.16.1/SE  via a suite of “wbs” commands. All tests will be two-sided, and statistical significance will be defined as a P value <.05.All statistical analyses will be performed in the Bayesian framework using OpenBUGS V.3.2.3 (members of OpenBUGS Project Management Group; see 2.11Ethics review will not be necessary as this is a systematic review of already published data. The review findings will be disseminated through publications in peer-reviewed journals, and presentations at conferences.3 Given the immediate cost of additional tests and clinical follow-up for the incidental findings, effectiveness and harms of WBCT-based management vs. alternatives, conventional management should be comprehensively assessed as the whole management. With up-to-date systematic review methodologies, and if feasible, new meta-analytic results, we hope to clarify the actionable evidence that supports the evidence-based trauma care using upfront WBCT.Upfront use of WBCT is an alternative mode of trauma screening strategy that is becoming increasingly available. WBCT may become more accessible in the near future as more institutions install a modern, high-quality and high-speed CT scanner system, adjacent to or in the ED, which may ultimately increase the number of trauma patients screened with routine, upfront WBCT. The theoretical advantages of WBCT-based management over selective CT-based management have only been shown for intermediate outcomes, such as time required to diagnose traumas and have yet to be convincingly demonstrated for morbidity and mortality, and more importantly, for patient-relevant clinical outcomes in RCTs. Evidence is lacking on the benefits and harms stemming from incidental findings detected by WBCT. In theory, by expanding the scan targets from selected body parts to the whole body, pan-scanning could detect more injured lesions at the cost of more incidental findings, in addition to the already demonstrated increased overall radiation exposure and cost.3.1This will be the first systematic review and meta-analysis to comprehensively assess the existing evidence on diagnosis and subsequent clinical outcomes—including incidental findings—stemming from routine whole-body computed tomography for blunt polytrauma, using the established frameworks to assess diagnostic technology and its health outcomes.Comprehensive literature search and up-to-date systematic review methodologies will be used to clarify the strengths and limitations of the currently existing evidence.Meta-analyses might not be feasible if appropriately analyzed comparative data were sparse.KN, MI, and TT originated the idea; KN and TT performed literature searches and extracted data for protocol writing; KN and TT drafted the initial version of the protocol; all authors suggested amendments and approved the final version of the protocol; all authors made substantial contributions to the intellectual content of the paper and gave final approval for the final version of the manuscript. KN and TT are guarantors of the research.Conceptualization: Kyohei Nagasawa, Mitsunaga Iwata, Teruhiko Terasawa.Data curation: Kyohei Nagasawa, Teruhiko Terasawa.Formal analysis: Teruhiko Terasawa.Funding acquisition: Teruhiko Terasawa.Investigation: Kyohei Nagasawa, Teruhiko Terasawa.Methodology: Kyohei Nagasawa, Mitsunaga Iwata, Takashi Nihashi, Teruhiko Terasawa.Project administration: Mitsunaga Iwata, Takashi Nihashi, Teruhiko Terasawa.Software: Teruhiko Terasawa.Supervision: Mitsunaga Iwata, Takashi Nihashi, Teruhiko Terasawa.Validation: Kyohei Nagasawa, Mitsunaga Iwata, Takashi Nihashi, Teruhiko Terasawa.Writing – original draft: Kyohei Nagasawa, Teruhiko Terasawa.Writing – review & editing: Kyohei Nagasawa, Mitsunaga Iwata, Takashi Nihashi, Teruhiko Terasawa.