Nutrient balances computed as isometric log-ratios (ilr) provide a comprehensive understanding of nutrient relationships in the diagnostic leaf at high yield level by combining raw concentration data. Although the most appropriate method for multivariate analysis of compositional balances may be less efficient due to non-normal data distribution and limited nutrient mobility in the plant, robustness of the nutrient balance approach could be improved using Box-Cox exponents assigned to raw foliar concentrations. Our objective was to evaluate the accuracy of nutrient balances to diagnose fertigated “Prata” orchards. The dataset comprised 609 observations on fruit yields and leaf tissue compositions collected from 2010 to 2016 in Ceará state, N-E Brazil. Raw nutrient concentration ranges were ineffective as diagnostic tool due to considerable overlapping of concentration ranges for low- and high-yielding subpopulations at cutoff yield of 40 Mg ha−1. Nutrient concentrations were combined into isometric log-ratios (ilr) and normalized by Box-Cox corrections between 0 and 1 which may also account for restricted nutrient transfer from leaf to fruit. Despite reduced ilr skewness, Box-Cox coefficients did not improve model robustness measured as the accuracy of the Cate-Nelson partition between yield and the multivariate distance across ilr values. Sensitivity was 94%, indicating that low yields are attributable primarily to nutrient imbalance. There were 148 false-positive specimens  likely due to suboptimal nutrition, contamination, or luxury consumption. The profitability of “Prata” orchards could be enhanced by rebalancing nutrients using ilr standards with no need for Box-Cox correction.The “Cavendish” and “Prata” subgroups represent respectively 47% and 24% of the world banana production. Compared to world average progressing from 10.6 to 20.6 t ha The current cultivars of bananas originate from the hybridization of diploid subspecies of Musa acuminata Colla (A genome) and Musa balbisiana Colla (B genome), and they exhibit various levels of ploidy and genomic constitution, such as diploid ; triploid ; and tetraploid 2. The main banana cultivars in Brazil have genomes AA, AAA, AAB, ABB, and AAAB2. Brazil produces 6.9 × 106 tons of banana fruits annually on 480 × 103 hectares3. Although banana production in Brazil is supported by strong domestic demand representing 98% of total production4, the average yield of 14.5 tons ha−1 year−1 remains lower than the world average5 of 20.6 tons ha−1 year−1.The “Cavendish” and “Prata” subgroups represent 47% and 24% of the world banana production, respectively6. Low fruit yields are generally attributed to nutrient mismanagement and water shortage10. The K is generally the main limiting nutrient and interacts with N, Ca and Mg11. Because K is also of public health concern due to too low daily intake15, it is applied in relatively large amounts to boost banana yield and quality, potentially leading to luxury consumption. “Prata” is the dominant banana subgroup in N-E Brazil and is fertigated. Fertilization represents 16–22% of production costs compared to 14–27% for irrigation16.Banana nutrient requirements depend on yield potential, plant density, soil fertility, and root development19. S, Cu, Zn, Mn and Fe have variable mobility, and Ca and B are relatively immobile21. The indirect relationship between relatively phloem-immobile leaf nutrients and fruit yield depends on soil test and water supply regulating xylem transport to the fruit22. As source of variability and heteroscedasticity nutrient mobility from leaf to fruit could be constrained by Box-Cox exponents23 between zero (immobile nutrients unrelated to yield) and 1 (mobile nutrients related to yield).Nutrient acquisition by the banana fruit relies on plant’s ability to translocate nutrients. N, P, K, Mg, Cu, and Zn have the greatest potential for nutrient translocation from leaf to fruit11. There are several methods to interpret the results of tissue analysis. The most common method is the critical concentration range diagnosing nutrients separately24, assuming that all other factors are close to their optima25, an over-optimistic assumption. Because components of a system are intrinsically interactive and multivariate26, nutrients can be combined into balances using isometric log-ratios (ilr)27. The ilr transformation is the most suitable method to run multivariate analyses on compositional data28 such as leaf composition31. However, the ilr transformation may not return normality or homoscedasticity.Banana diagnostic leaf tissue is collected close to blooming stageilr data distribution and model accuracy. Exponents may be varied between 0 and 1, zero not contributing to ilr (30. Accuracy is computed as (TN+TP)/(TN+FN+TP+FP). However, nutrient excess at high yield level (FP specimens) is diagnosed as sub-optimal concentration, luxury consumption of contamination. Accuracy defined in terms of diagnostic power could thus be computed as (TN+TP+FP)/(TN+FN+TP+FP). The conventional ilr transformation is robust if Box-Cox coefficients of one across concentrations return greatest model accuracy and diagnostic power after varying coefficients between zero and one.Box-Cox coefficients assigned to raw concentration data could improve Our objective was to measure the robustness, accuracy and diagnostic power of nutrient balance designs for fertigated “Prata” banana. We hypothesized that high yields of fertigated “Prata” are reached within narrow foliar nutrient combinations into balances after optimizing Box-Cox coefficients assigned to raw concentration data.−1) and dry (18.35 ± 3.77 Mg ha−1) seasons. Fruit yields were normally distributed in both seasons and varied between 9 and 53 tons ha−1 year−1. The critical Mahalanobis distance across the whole dataset was 3.9 at a cutoff yield of 40 Mg fruit ha−1  used to compute the Mahalanobis distance are presented as . Very low-yielding crops in Group 4 soils had several major limitations, such as shallow (<25 cm) solum and high salinity. Soil bulk density, pH, and electrical conductivity did not limit yield in Ceará33, leaving solum thickness as the probable yield-limiting factor. Quartzispamments require major investments in fertilization and irrigation due to low nutrient and water reserves in Ceará soils (Group 3).Although nutrient imbalance appeared to be the main yield-limiting factor in Ceará state, there were 24 FN specimens in which yields were limited by other factors. There are several classes of soil that enable the soil to sustain banana production relative to soil properties34, such as those typical of Missão Velha. Mg did not appear to be yield-limiting at Missão Velha despite high K levels that are potentially antagonistic to Mg18. However, Ca, B, and S may accumulate as a result of local soil properties, such as high pH and organic matter content35. The S level likely reflected water quality due to the presence of gypsum layers in the sedimentary basin at Missão Velha36. The relative shortage of Mn can be generally attributed to high soil pH37. Banana plants are highly responsive to added Mn38, but foliar Mn diagnostic standards are elusive9. Although often shown to be in relative shortage in banana orchards39, Zn levels were adequate or in apparent excess at Missão Velha. The [B | Ca] balance among TP specimens was often high, which indicated potential Ca excess or B shortage.Levels of banana leaf nutrients such as Ca, Mg, and Mn were relatively stable at daytime temperatures between 17 and 30 °C40. Exponential increase from 0.6 to 3.1 g Na kg−1 was observed in the shoots of banana seedlings33, well below the Na level of 25 g kg−1 in leaf margins suffering from necrosis40. Any beneficial role of Na could occur below around 1 g Na kg−1, as is the case in Missão Velha. Foliar Al concentration41 normally ranges between 0.050 and 0.400 g Al kg−1. Foliar Al may accumulate in high-pH soils due to the attack of soil-borne pathogens on banana roots42. Roots react by exuding oxalic, malic, and fumaric acids43, which may chelate Al ions45. As a result, tissue Al varied widely between orchards but did not reach toxic levels.The status of Na and Al in banana leaf has been little documented. Accumulation of Na in banana seedlings may reduce biomass production−1. For AAB banana a cutoff yield of 30 tons ha−1 was suggested in Minas Gerais, Brazil46, while for AAA banana a cutoff yield close to 40 tons ha−1 was stipulated in São Paulo state47. In Ceará state, commercial yields of AAB “Prata” are generally classified as low (<30 tons ha−1), medium (30–35 tons ha−1), or high (≥35 tons ha−1). In the Ceará dataset, 106 orchards produced <30 tons ha−1, 137 orchards produced 30–35 tons ha−1, 317 orchards produced 35–45 tons ha−1, and 49 orchards produced >45 tons ha−1. Furthermore, 199 orchards (33%) produced ≥40 tons ha−1. Obviously, there is a large potential to increase yield in Ceará orchards by rebalancing nutrients.The cutoff yield to establish nutrient standards was 40 tons ha31, ternary diagrams and multivariate analyses allow the entire nutrient status to be captured49. Additionally, criticism of the misuse of multivariate analysis to analyze compositional data is reported in the literature26. Log-ratio transformations address resonance in the compositional space , subcompositional incoherence, and the intrinsically non-normal distribution in the constrained compositional space compared to the unconstrained real space required to conduct statistical analyses. The clr transformation50 is the most common log-ratio used to diagnose nutrient limitations in crops. To facilitate interpretation of the diagnosis from the Mahalanobis distance, clr indices can be ordered in ascending order from the most negative (relative shortage) to the most positive (relative excess), as in DRIS51.Whereas nutrients diagnosed separately may return low diagnostic performance53. Low accuracy was due to the high proportion of FP specimens (24%), indicating suboptimal fertilization, luxury consumption of nutrients, or leaf contamination. Fertigation appeared to be adequately run in 12% of orchards (TN+FN). Pests or unfavorable soil factors may have limited the yield of FN specimens. The fertigation regime should be rebalanced in 88% of orchards (FP+TP). Thus, nutrient mismanagement appeared to be the main yield-limiting factor at Missão Velha. Regional nutrient standards appeared to be more reliable than so-called “universal” nutrient ratio standards35.The diagnostic accuracy of 72% reported here was lower than the generally obtained value of 80%, whereas the critical Mahalanobis distance of 3.9 was close to the range of 4.1–5.9 that has been reported for other cropsilr and clr made possible performing nutrient standards. The Cate–Nelson partitioning about cutoff yield of 40 tons ha−1 and the multivariate distance across ilr values returned a critical Mahalanobis distance of 3.9, below which nutrient balances were adequate. Although appealing to improve data distribution and constrain nutrient mobility in plants, the Box-Cox coefficients assigned to raw nutrient concentrations failed to improve model accuracy.Nutrient imbalance limited yield in Ceará’s “Prata” banana orchards. Nutrient concentration ranges did not discriminate between low- and high-yielding crops but combining nutrients into clr indices should be further validated by using nutrient tests for specimens showing the Mahalanobis distance above the critical value. While 4% of orchards (FN specimens), solum thickness or other factors limited fruit yield, 96% of orchards were classified as nutritionally balanced (TN specimens) or imbalanced (FP and TP specimens). The most commonly deficient nutrient was boron, and the most commonly excessive was nitrogen. Although fertigation can enhance banana yield, the dosage of nutrients requires adjustment based on reliable diagnostic tools. Banana is the fruit crop showing yet the highest proportion of false positive specimens and the smallest number of false negative specimens. Hence nutrient imbalance is a crucial problem that could be addressed using tools of compositional data analysis.The order of nutrient limitation to yield shown by 54. Maximum and minimum temperature ranges were 31–35 °C and 19–21 °C, respectively. For comparison, optimal mean temperatures for banana crops are as follows: 22 °C for floral initiation, 31 °C for leaf growth and development, and 28 °C (range: 15–35 °C) for high commercial yields32. Total rainfall averaged 1022.6 mm, compared to 1200–1800 mm as an effective precipitation regime for banana production32.Data were collected from 2010 to 2016 in 6- to 18-year-old banana stands at Missão Velha, Ceará state, Brazil . Climate in this part of Brazil is semi-arid tropical (Aw in the Köppen-Geiger classification), with a dry winter season and rainfall concentrated in the summer season. The warmest months in the area extend from September to December (Supplementary Table 55 or Quartzipsamment56. Soil properties in the top layer (0–20 cm) are presented as . Plants were sprinkler-irrigated at a crop evapotranspiration (ETc) rate proportional to potential evapotranspiration ET0 57. To sustain high yield and quality, the banana clump was restricted to a few fruiting plants by yearly pruning. Banana yields could be affected not only by nutrient imbalance but also by diseases and pests, including nematodes58. The most frequent pest is the banana weevil (Cosmopolites sordidus), and the most frequent disease is yellow Sigatoka (Mycosphaerella musicola).Commercial orchards averaging 3 ha in size were surveyed for yield and foliar composition. Plant density averaged 1332 plants ha−1 year−1, as follows: 10% during the first 3 months, 75% between the fourth month and blooming (seventh to ninth month), and 15% up to harvest. Depending on the soil test, potassium is applied as potassium chloride (60% of K2O) every 3–15 days at total rate of 1300 to 1700 kg K2O ha−1 year−1 as follows: 0% during the first 3 months, 90% from the fourth month to blooming, and 10% toward harvest. Phosphorus is applied as reactive natural phosphate (27% of P2O5) at planting and via fertigation as monoammonium phosphate (48% of P2O5) at total rate of 160 kg P2O5 ha−1 year−1. Calcium is supplied to maintain the soil K: Ca: Mg molar ratio between 0.5: 3.5: 1.0 and 0.3: 2.0: 1.0. Manganese, zinc, and boron are applied at rates of 60 kg manganese sulfate (26% Mn) ha−1 year−1, 96 kg zinc sulfate (20% Zn) ha−1 year−1, and 20–30 kg boric acid (17% B) ha−1 year−1, respectively.In fertigated orchards, nitrogen is generally supplied as urea (45% N) every 3 to 15 days up to 440–600 kg N ha59. Four samples made of 10 subsamples were composited in each orchard. Samples were oven-dried at 72 °C for 48–96 hours and ground to less than 1 mm. N was determined by the micro-Kjeldahl method. After sample digestion in a mixture of nitric and perchloric acids60, the elements Ca, Mg, Fe Zn, Cu, Al and Mn were quantified by atomic absorption spectrophotometry, P by colorimetry, S by turbidimetry, and K and Na by emission flame photometry61. B was quantified by colorimetry62.The dataset comprised 609 observations of cv. ‘Prata’, AAB “Prata” subgroup. The period between budding and harvest varied from 8 to 12 months. Yield data were reported for the dry and wet seasons. In July and December of each year, the third most fully expanded leaf of banana plants was collected at blooming stage twice a year during the wet and dry seasons, and 10-cm-wide pieces were cut from inner halves on both sides of the midrib and at the midpoint of the laminailr, as follows64:nj+ and nj− are numbers of components at the numerator and denominator, respectively, ilr is designated as [components at denominator|components at numerator] because the log-ratio becomes more negative as values at the denominator increase. More negative numbers are located on the left-hand side of the array, as in algebra. The ilr values were used to compute the Mahalanobis distance from a reference subpopulation65. Box-Cox coefficients varying between 0 and 1 are assigned to raw concentrations at numerator and denominator, hence transforming ilr values to reach additivity, normality or homoscedasticity and possibly controlling nutrient mobility, a coefficient near zero making the nutrient immobile and a coefficient close to one making the nutrient mobile.The tissue compositional simplex comprised N, P, K, Ca, Mg, S, Cu, Zn, Mn, Fe, B, Na, and Al, as well as a filling value computed as the difference between the measurement unit and the sum of nutrients. The orthonormal balance was computed as the 21. K and Mg are antagonistic to each other22. N and P reflect protein synthesis and energy transport, respectively66. Among nutrients of limited mobility, S is involved in protein synthesis, whereas Cu, Zn, and Mn are involved in metabolism22 and fungicide formulations. Fe and Mn are involved in soil genesis55. Sodium (Na) acts as a functional element as osmoticium for cell enlargement and an accompanying cation for long-distance transport67. Plant tolerance to Al toxicity depends on Al interactions with other minerals, such as B, P, Ca, and Mg41.The balance design was elaborated following a sequential binary partition  and validation (2015–2016) datasets that were merged in case of similar accuracy. The Cate–Nelson procedure partitions data into true-negative (TN), false-negative (FN), true-positive (TP), and false-positive (FP) quadrants from the relationship between crop yield and Mahalanobis distance65. Specificity is computed as TN/(TN+FP), sensitivity as TP/(TP+FN), accuracy as (TN+TP)(TN+FN+TP+FP), negative predictive value as TN/(TN+FN), and positive predictive value as TP/(TP+FP). The centered log-ratio (clr) values of the TN subpopulation were compared with published data after adjusting the geometric mean for the number of components (Table Computations were performed by using the R statistical package version 3.4.1ibration 010–2014 SUPPLEMENTARY MATERIAL


The microbial community of the built environment (BE) can impact the lives of people and has been studied for a variety of indoor, outdoor, underground, and extreme locations. Thus far, these microorganisms have mainly been investigated by culture-based methods or amplicon sequencing. However, both methods have limitations, complicating multi-study comparisons and limiting the knowledge gained regarding in-situ microbial lifestyles. A greater understanding of BE microorganisms can be achieved through basic information derived from the complete genome. Here, we investigate the level of diversity and genomic features  from complete genomes of bacteria commonly identified in the BE, providing a first step towards understanding these bacterial lifestyles.Here, we selected bacterial genera commonly identified in the BE (or “Common BE genomes”) and compared them against other prokaryotic genera (“Other genomes”). The “Common BE genomes” were identified in various climates and in indoor, outdoor, underground, or extreme built environments. The diversity level of the 16S rRNA varied greatly between genera. The genome size, GC content and GC skew strength of the “Common BE genomes” were statistically larger than those of the “Other genomes” but were not practically significant. In contrast, the strength of selected codon usage bias  was statistically higher with a large effect size in the “Common BE genomes” compared to the “Other genomes.”Of the four genomic features tested, the S value could play a more important role in understanding the lifestyles of bacteria living in the BE. This parameter could be indicative of bacterial growth rates, gene expression, and other factors, potentially affected by BE growth conditions . However, further experimental evidence, species-level BE studies, and classification by BE location is needed to define the relationship between genomic features and the lifestyles of BE bacteria more robustly.The online version of this article (10.1186/s12864-018-5389-z) contains supplementary material, which is available to authorized users. The microbial community of the built environment (BE) is an important player in human-microbe interactions. As such, in order to build urban environments that benefit human well-being, it is necessary to study the relationship between the BE and microbial communities. As of 2016, about 54% of the world’s population is living in urban areas , suggest2 concentration, surface material) but were not found to play a significant role in the microbial community composition  to 7.0 Mb [Mycobacterium smegmatis strain MC2 155 (NC_008596)] with a median of 4.5 Mb  compared to other “Common BE genera” (27.4–73.0%) ; Additional file M. leprae Br4923 (NC_011896); Additional file Mycobacterium ranged from 0.36–1.30, suggesting that either the growth rate of different Mycobacterium species present in the BE varies drastically or that some Mycobacterium species have more “volatile” codons, as discussed below. For example, M. tuberculosis and M. leprae have S values in the lower range (0.36–0.45) and also have slow generation times of ~ 1 and 14 d, respectively  to ccupants . The GC cumented . The GCSectively , 98, 99.of 4–5 h .To further understand the 28 “Common BE genera,” we analyzed four genomic features: genome size, GC content, GC skew, and codon bias. While our study based itself on the results of previous studies to retrieve the “Common BE genera,” we aimed to demonstrate the potential of using genomic features to provide insight into microbial lifestyles and to describe the trends found in the “Common BE genera” . The “CoThis initial analysis could help begin to surmise certain lifestyles of the bacteria commonly found in the BE. For example, the S value has implications on the growth rates of bacteria  found inMycobacterium tuberculosis, one of the “Common BE genera” and pathogen with S values (0.41–0.45) below the “Common BE” and “Other” genome medians  to surmise the lifestyle of bacteria. The “Common BE genera” selected in this study have only been commonly identified by culture-based and amplicon-based sequencing studies, which have limitations as described in the Introduction. Although the “Common BE genera” have been detected in multiple BE studies (≥ 6), these bacteria may not be active in the BE. Moreover, although this study is based on completed genomes from the NCBI RefSeq database, the genomes could have been derived from environments not related to the BE. Thus, the conclusions derived from this study serve as a hypothesis for the potential lifestyles of commonly identified BE bacterial genera. Further studies are needed to accurately determine the typical BE genera and the association of BE genera with BE selection pressures.Proteobacteria, Firmicutes, and Actinobacteria), and also by randomly selecting one representative for species that have multiple strains sequenced. Of the four genomic features , only the S value showed consistent results and tended to be higher in the “Common BE genera” compared to the “Others.” This indicates that the selected codon usage bias tends to be stronger in the “Common BE genera” than in the “Other genera,” regardless of the datasets used, and that our results were less affected by biases in the available sequenced genomes. We also tested different numbers of publications  to select for BE genera. The corresponding numbers of the selected “Common BE genomes” were 1208, 1029, 922, 825, 739, and 717. Even when genera observed in at least 1 out of 54 publications were defined as the “Common BE genera,” the median S value for the “Common BE genomes” (1.14) was higher than that for the “Other genomes” (0.35) with a large effect size (Cliff’s delta of 0.548), and the Wilcoxon rank sum test returning significant result with q-value of 2.59e-126. This is consistent with the results obtained by larger numbers of publications (n > 1) to define the “Common BE genera.” Thus, selected codon usage bias tends to be larger in the “Common BE genomes” than in the “Other genomes,” regardless of the genome data set used and criteria to define BE genera.It is important to note that the results remained similar when different data sets were compared  , although there are ongoing “efforts to expand the bacterial and archaeal reference genomes…to maximize sequence coverage of phylogenetic space” . HoweverTwenty-eight bacterial genera were selected to represent the bacteria commonly identified in the BE. Although geographical location, temperature, and humidity are important factors in shaping the BE microbial composition, many of the “Common BE genera” were identified around the world. All the genera have also been observed in the human microbiome. Here, we used genomic features to demonstrate the potential of understanding the lifestyle of bacteria from the genome. Together, the genome size, GC content, and GC skew for the “Common BE genomes” showed trends similar to (were not strongly deviated from) those for the entire data set of completed prokaryotic genomes analyzed obtained from the NCBI database. On the other hand, the strength of selected codon usage bias  for the “Common BE genomes” tended to be significantly higher than that of the “Other genomes.” As such, the S value could be indicative of bacterial growth rates, gene expression, and other evolutionary processes that may play a role in the bacteria present in the BE. Further insights could be gained through more BE studies analyzing locations with fewer publications , identifying microbial communities at the species-level, and by minimizing cross-study biases.n ≥ 6 publications) and had at least one genome sequenced in the National Center for Biotechnology Information  RefSeq database [n = 28 genera). These were denoted as “Common BE genomes” or “Common BE genera” while the bacterial genera not selected were denoted as “Other genomes” or “Other genera.” Based on this criterion, 28 genera were retained  , and complete sequences of prokaryotic chromosomes (GenBank format [Firmicutes (370), Proteobacteria (222), Actinobacteria (123), and Bacteroidetes (2). The 1863 “Other genomes” belonged to 644 genera from 36 phyla, including Proteobacteria (875), Firmicutes (192), Actinobacteria (115), and Chlamydiae (110). The “Common BE genomes” and “Other genomes” were linked to the 18 environmental categories in MetaMetaDB: Aquatic, Biofilm, Compost, Food, Freshwater, Hot_springs, Human, Human_gut, Human_lung, Human_nasal_pharyngeal, Human_oral, Human_skin, Marine, Rhizosphere, Rock, Root, Sediment, and Soil. Complete listings of the genomes used in this study, along with the genomic features, are shown in Additional file RefSeq chromosome sequence accessions with the NC_ prefix were obtained from the NCBI prokaryotic genome list (k format ) were dobacteria 23, and Bhttps://cran.r-project.org/web/packages/ape) [https://www.arb-silva.de/projects/living-tree/) [To measure the genetic diversity among taxa within a genus, the mean distance (Dmean) between all pairs of bacteria was calculated . The genges/ape) . We usedg-tree/) . LTP datg-tree/) .The total number of nucleotides (A + T + G + C) was calculated from the whole nucleotide sequence of each chromosome.The relative frequency (percentage) of guanine and cytosine (G + C)/(A + T + G + C) was calculated from the whole nucleotide sequence of each chromosome.The asymmetry in nucleotide composition between leading and lagging strands of DNA replication is represented by GC skew (C-G)/(C + G). The strength of GC skew was measured by the GC skew index or GCSI  with a wAs a measure of translationally selected codon usage bias, the S value was calculated for each chromosome, as described in Sharp and co-workers  and VieiWe performed several statistical analyses to compare the values of the genomic features  between two groups of genomes: e.g., “Common BE genomes” versus “Other genomes”; and MetaMetaDB environment-associated “Common BE genomes”  versus other “Common BE genomes” .p-value obtained by the statistical test was adjusted for multiple comparisons by controlling for the false discovery rate (FDR) [We performed the Wilcoxon rank sum test  as a non-parametric statistical hypothesis test to compare the values between two groups . The p-vte (FDR) . An FDR We calculated Cliff’s delta statistic as a non-parametric effect size to estimate the degree of overlap between two distributions . A Cliffhttp://www.g-language.org) [https://www.R-project.org/) [Genome sequence analyses  were performed using the G-language Genome Analysis Environment version 1.9.1 (age.org) . Statistct.org/) .Additional file 1:Table S1. Selection of bacterial genera commonly identified in the built environment. Bacterial genera identified in 54 publications were compiled (see Table S2) and commonly identified genera were selected. All bacterial genera identified in more than about 10% of the publications (n ≥ 6 publications) with at least one complete reference genome on the NCBI RefSeq database were used in this study (n = 28 genera). Table S2. Metadata for each reference. 54 publications were compiled, including metadata for location, sub-locations, bacterial genera identified, sample type, climate (Table S4 and S5), temperature (°C), and humidity (%). If temperature or humidity was not described by the publication, the average over a certain period of time (either the timeframe stated in the publication or the publication year) was obtained from online sources. Table S3. Publication count for each “Common BE Bacterial Genus” by macro-Level BE location. Macro-level BE Locations included indoor, outdoor, underground, and extreme. Further division by type of sample is also depicted, including surface (S), air (A), water (W). Darker orange color indicates more references identified the genera in the macro BE location and sample type while lighter orange color indicates fewer references. The total number of references for each location and genera are also shown. Table S4. Köppen climate classification. Köppen climate classification was used to identify the climate for each publication’s study location. Only the climate assignment between 1981 and 2010 was used for this study. Abbreviation descriptions, latitude, and longitude values are listed. Table S5. Publication count for each “Common BE Bacterial Genus” by climate. The climate was identified for each publication’s study location based on the closest Köppen latitude and longitude values and correlated with the Köppen ID (see Table S4 for Köppen assignment). For publications describing general locations , a central location in the region was chosen for latitude and longitude. Publications without location specifics were not included, and publications in space were separated out to “Space” category. Darker orange color indicates more references identified the genera in the macro BE location and sample type while lighter orange color indicates fewer references. The total number of references for each location and genera are also shown. Table S6. MetaMetaDB environmental category assignment for each “Common BE Bacterial Genus.” MetaMetaDB is a database to search for the possible habitats a microorganism could live in and was made by collecting 16S rRNA sequences. Environmental categories for each “Common BE bacterial genus” were based on the identity threshold of 97%, corresponding to the species taxonomic level. Every species for each “Common BE genus” is listed with the corresponding environmental category, where “Y” indicates that the species has been previously identified in the category and “N” indicates the species has not been identified in the category. “Hits” indicates the number of 16S rRNA sequences used by the database. Table S7. Mean distance (Dmean) between all pairs of bacterial species for each “Common BE Bacterial Genus.” The Dmean was used to describe the genetic diversity among species within a genus. The genetic distance between a pair of bacteria was calculated with the K80 model using the ‘dist.dna’ function of the ‘ape’ package of R (https://cran.r-project.org/web/packages/ape). We used a nucleotide sequence alignment of the 16S rRNA genes in ‘The All-Species Living Tree’ Project (https://www.arb-silva.de/projects/living-tree/). LTP datasets based on SILVA release 128 were downloaded from Archive (https://www.arb-silva.de/no_cache/download/archive/living_tree/LTP_release_128/). Bacterial genera for which 3 or more taxa (N > 2) were available at LTP_release_128 were included in the 16S rRNA diversity analysis. Table S8. Genome information. Genome features reported include size (Mb), GC content (%), GCSI (GC skew index), and S value (strength of selected codon usage). A genus was deemed BE if observed in at least 6 publications out of 54. The column “BE” shows the number of references that identified the genera. Table S9. Robustness of the study. The genome data set used in this study was tested over two levels: 1) different subsets of bacteria  and also randomly selecting one representative for species that have multiple strains sequenced, and 2) testing different numbers of publications  to select for BE genera. Table S10. Genomic feature statistical analysis for each MetaMetaDB selected environmental category. Each genomic feature per MetaMetaDB environmental category was analyzed to determine statistical significance between the “Common BE genomes” associated with an environment and the “Common BE genomes” not associated. Significance is indicated by q-value < 0.05 and large effect size by Cliff’s delta |d| > 0.474. (XLSX 3660 kb)Additional file 2:Figure S1. Map of publications used in this study. The 54 publications used in this study are mapped by the closest Köppen latitude and longitude values in order to assign Köppen climate IDs by color (Table S4) . The size of the circle indicates the number of common BE bacterial genera (n = 28) identified in the publication. Publications not plotted on the map are those from the International Space Station. Figure S2. Descriptive statistics of diversity indices . Plots of diversity levels between taxa within each genus based on 16S rRNA gene sequences, with scatter plots below the diagonal, histograms on the diagonal, and the Pearson correlation coefficient (Corr) above the diagonal. The diversity levels for each genus were represented by three indices: the number of taxa (N), mean distance (Dmean) between all pairs of taxa, and phylogenetic diversity (PD). Figure S3. GC skew plots for Clostridium perfringens strain 13 (A) and Methylobacterium sp. 4–46 (B). G-language Genome Analysis Environment version 1.9.1 (http://www.g-language.org) was used to generate the GC skew plot. Figure S4. Genome size (Mb) distribution among MetaMetaDB selected environmental categories. A boxplot showing the distribution of genome sizes within each “Common BE genus” associated with an environment (purple) compared to the “Common BE genera” not associated (red). Figure S5. GC content (%) distribution among MetaMetaDB selected environmental categories. A boxplot showing the distribution of GC content within each “Common BE genus” associated with an environment (purple) compared to the “Common BE genera” not associated (red). Figure S6. GCSI distribution among MetaMetaDB selected environmental categories. A boxplot showing the distribution of GCSI within each “Common BE genus” associated with an environment (purple) compared to the “Common BE genera” not associated (red). Figure S7. S value distribution among MetaMetaDB selected environmental categories. A boxplot showing the distribution of S value within each “Common BE genus” associated with an environment (purple) compared to the “Common BE genera” not associated (red). Figure S8. GC skew plots for Mycobacterium avium subsp. paratuberculosis K-10 (A) and Mycobacterium leprae Br4923 (B). G-language Genome Analysis Environment version 1.9.1 (http://www.g-language.org) was used to generate the GC skew plot. (PDF 7950 kb)


Caenorhabditis elegans and parasitic nematodes. The current study in C. elegans expands previous work in several ways. Detecting anthelmintic bioactivity in drugs, compounds or natural products requires robust, sustained pharyngeal pumping under baseline conditions. We generated concentration-response curves for stimulating pumping by perfusing 8-channel microfluidic devices (chips) with the neuromodulator serotonin, or with E. coli bacteria (C. elegans’ food in the laboratory). Worm orientation in the chip (head-first vs. tail-first) affected the response to E. coli but not to serotonin. Using a panel of anthelmintics—ivermectin, levamisole and piperazine—targeting different ICs/NTRs, we determined the effects of concentration and treatment duration on EPG activity, and successfully distinguished control (N2) and drug-resistant worms . EPG recordings detected anthelmintic activity of drugs that target ICs/NTRs located in the pharynx as well as at extra-pharyngeal sites. A bus-8 mutant with enhanced permeability was more sensitive than controls to drug treatment. These results provide a useful framework for investigators who would like to more easily incorporate electrophysiology as a routine component of their anthelmintic research workflow.Many anthelmintic drugs used to treat parasitic nematode infections target proteins that regulate electrical activity of neurons and muscles: ion channels (ICs) and neurotransmitter receptors (NTRs). Perturbation of IC/NTR function disrupts worm behavior and can lead to paralysis, starvation, immune attack and expulsion. Limitations of current anthelmintics include a limited spectrum of activity across species and the threat of drug resistance, highlighting the need for new drugs for human and veterinary medicine. Although ICs/NTRs are valuable anthelmintic targets, electrophysiological recordings are not commonly included in drug development pipelines. We designed a medium-throughput platform for recording electropharyngeograms (EPGs)—the electrical signals emitted by muscles and neurons of the pharynx during pharyngeal pumping (feeding)—in   •Microfluidic electropharyngeogram (EPG) recordings reveal pharyngeal pumping.•E. coli or serotonin evoke sustained pumping in C. elegans in microfluidic chips.•Anthelmintic drugs inhibit pumping in a concentration- and time-dependent manner.•EPG recordings distinguish susceptible from drug-resistant worms.•The 8-channel EPG platform is a useful tool for anthelmintic research. PovertyMany anthelmintic drugs target ion channels (ICs) and neurotransmitter receptors (NTRs), which generate the electrical activity of neurons and muscles . PerturbC. elegans and parasitic nematodes  into the mouth and intestinal tract. In contrast, some species have minimal pharyngeal musculature and may feed by other means . Likewise, the electrode configuration used to noninvasively record EPGs from intact worms is analogous to that used for a human electrocardiogram  to pull food  to further validate the use of EPG recordings and automated data analysis by characterizing the effects of three anthelmintic drugs that act on ICs/NTRs to disrupt electrical signaling. The results provide a useful framework for investigators who would like to incorporate electrophysiology into their anthelmintic research workflow.The physiology and life cycle of ic model . Importaris suum . Progresnd goats , and othnd goats . The pre22.1C. elegans strains from the Caenorhabditis Genetics Center  were grown at room temperature using standard methods on Nematode Growth Medium (NGM) agar plates seeded with the OP50 strain of E. coli. Control worms (reference strain) were Bristol N2; other strains were DA1316 ; VC2937 ; CB407 ; and CB6147 . Synchronous cultures of worms were obtained by bleaching adults to obtain cohorts of eggs  stocks (1M) were prepared in dH20 and stored at 4 °C until use. The pH of PPZ solutions was adjusted to 7.0 using HEPES buffer in M9 and titrating with 12 N HCl. Working solutions of all anthelmintic drugs were prepared from stocks and used on the same day. In some initial experiments, 0.005% Fast Green (Fisher F-99) was added to solutions to confirm uninterrupted perfusate flow . The stock OP50 suspension in M9 was stored at 4 °C, used for no more than 5 d, and O.D.600 was determined before each use. As needed, new aliquots were collected from the original LB culture flask, which was kept at 4 °C and discarded after 30 d. In some experiments , Micro1401-3, Cambridge Electronic Design (CED), Cambridge, UK] and Spike2 software  at 2.5 KHz per channel.EPGs were recorded as in bus-8 worms, which have disrupted epidermis and altered surface properties  and loaded as above. Chips pre-treated with Pluronic F-127 were used for both bus-8 and N2 control worms.operties , tended operties  to reduc2.6Rseal; see Section We found no significant power in EPG signals related to pharyngeal pumping at frequencies above 100 Hz. Therefore, except when measuring high-frequency voltage noise associate with the seal resistance , to give a normalized measure of EPG amplitude. The normalized signal was passed to separate detectors for E spikes and R spikes. Each of the two detectors consisted of a linear bandpass filter followed by a detector of peaks exceeding a threshold, and had 3 adjustable parameters: low frequency cut-off  as the basis for the optimization procedure. Unpaired E spikes and R spikes were counted as “orphans.” Informally stated, the optimization procedure sought to adjust the thresholds and filter frequencies such that detected E spikes were almost always followed by detected R spikes, with few orphans and a narrow distribution of pump durations. We used an entropy measure of distribution width,The fitness function to be maximized wast = −12 to −2 min, with t = 0 min being the onset of a test solution). Overall, 74 of 2134 worms (4.5%) analyzed for Some worms were excluded from analysis based upon the inability to detect pumps reliably, or a low baseline pump frequency  adjusted to give similar peak-to-peak amplitudes during the baseline period. Transient electrical artifacts or occasional large pump signals were masked so that traces could be shown with similar gains.Because of potential variation in n = number of worms in the ensemble) and plotted as mean (line) ± 1 S.E.M. (shading). Baseline pump frequency was obtained by measuring each worm's mean pump frequency for a 10-min interval (t = −12 min to −2 min), then averaging across worms. “Normalized” pump frequency was calculated by dividing each worm's smoothed frequency vs. time curve by its mean pump frequency during the baseline period, then averaging the normalized pump frequency data across worms.Plots of pump frequency vs. time were constructed by counting pumps in 1-s time bins and smoothing the values using a Gaussian weighted sliding window (S.D. = 30 s). The smoothed frequency vs. time curves were averaged across all worms in each experimental group , as described previously  of pumps that occurred following drug onset, with CFsolution . This cueviously .50 values from Hill plots were compared using a likelihood ratio test  for pump frequency and amplitude in 10 mM 5HT were 3.6 min and 26.9 min, respectively, an approximately 6-fold difference. We discuss potential causes of the 5HT-stimulated increase in pump amplitude below.In previous studies  we perfu0 mM 5HT  to induct = 15–60 min). By our definition, an IPI includes the time interval between the E to R spike in a pump  plus the subsequent interval from the R spike to the next E spike. This definition of IPI is synonymous with the “period” of the pumping rhythm. One advantage of this definition compared to others  was calculated to be 1.15 mM. In IPI histograms from 5HT concentrations <10 mM 5HT, the mode remained stable at ∼4.5 Hz while the right shoulder increased as concentration decreased (data not shown). Thus, as 5HT concentration falls, the fundamental pumping rhythm of ∼4.5 Hz continues while gaps become more frequent and of longer duration, to produce a lower mean pump frequency.mM e.g., , which wC. elegans cuticle is relatively impermeable to solutes and the extent to which particular drugs enter the body across the cuticle vs. by ingestion is generally unknown .For experiments in which we stimulated pumping by perfusing chips with 5HT (above) or OP50  is related to the muscle's force of contraction .Variations in R effects . In cont3.3C. elegans’ usual laboratory food source, E. coli strain OP50. OP50 was suspended in M9 at a range of concentrations measured by optical density at 600 nm (O.D.600)  sat horizontally on the syringe pump for 30 min . We also confirmed that bacteria did not settle out of suspension during experiments, which could reduce the concentration that worms experience. This was done by measuring the O.D.600 of the perfusate before loading it on the syringe pump and after it had passed through the recording channels and accumulated in waste reservoirs. The mean O.D.600 of perfusate from the reservoirs was 98% of its initial value and the values did not differ significantly . We conclude that bacteria remained in suspension during experiments, at their pre-perfusion concentration, for at least 120 min.Although 5HT is a convenient stimulator of robust, sustained pharyngeal pumping, its use is sometimes undesirable: e.g., when investigating the activation of feeding behavior by natural stimuli. 600 = 4) in M9. In all experiments, worms were food-deprived for 2 h prior to the switch to OP50, which reduces pumping to low levels and enhances the response when food is again provided  appeared saturating in their ability to stimulate pumping. For these four groups, there were no significant differences between mean pump frequencies during the rapid responses (t = 2–7 min), nor during the sustained responses (t = 30–60 min) . Similarly, 600 = 3 and above, measured in a microfluidic device. Accordingly, values from these four groups (O.D.600 = 2–5) were combined; the mean pump frequency during the rapid and sustained responses to OP50 differed significantly . The underlying mechanism(s) of the biphasic behavioral response to OP50 is unknown. One possibility is sensory adaptation in olfactory neurons that detect the sudden presence of food: e.g., the C. elegans neuron AWA, which is sensitive to food-related odors and responds to odor in an initial transient that decays to a plateau  were 3.28 ± 0.14 Hz for the rapid, and 2.54 ± 0.13 Hz for the sustained, responses , whereas maximum pump frequencies in 5HT were 4.52 ± 0.08 Hz, and 4.59 ± 0.09 Hz, for 10 and 20 mM 5HT, respectively . This result contrasts markedly with the IPI histogram for 10 mM 5HT , worms pump in bouts separated by gaps. We suggest that the temporal patterning of pumping in EPG chips stimulated by OP50, or moderate concentrations of 5HT , may better resemble C. elegans behavior in natural environments than the clock-like pumping stimulated by 10 mM 5HT.0 mM 5HT D, in whi0 mM 5HT , Fig. 3B0 mM 5HT , Fig. 3Et = 30–60 min after perfusate switch) in worms exposed to low or high concentrations of OP50 . For the high concentration, worm orientation had no significant effect on pump frequency. In contrast, in the low OP50 concentration, tail-first worms (whose heads were directed into the perfusate flow) responded with significantly higher pump frequency than did head-first worms .When loading EPG chips, worms orient more or less randomly into head-first or tail-first orientation in worm traps (data not shown). In the 8-channel chip , worms cA. ceylanicum and A. suum stimulated to pump in EPG chips by blood serum or 5HT have baseline pump frequencies of ∼1 Hz and pump inhibition by IVM is readily detected at concentrations spanning several orders of magnitude  pumping in pendence , Fig. 3.3.4For subsequent experiments, all EPG recordings were performed in 10 mM 5HT in M9 (henceforth termed “M9-5HT”), to induce robust, sustained pumping suitable for detecting inhibitory effects of applied drugs . Worms wC. elegans and other nematodes . The IVM receptor, GluCl, mediates synaptic inhibition from motoneuron M3 onto pharyngeal muscles; these synapses are active between the E and R spikes to help terminate each pump . As a Glematodes . We compt to IVM . Lockeryavr-14; avr-15; glc-1 worm continued pumping long after the N2 worm had stopped . The cause of this difference is unknown, but mutations in many genes can alter worms’ pump basal frequencies; a search of the CGC website using the term “pumping” in the “description” field yields more than 40 genotypes with slow, fast or irregular pumping phenotypes. To compare strains with dissimilar baseline pump frequencies, we normalized pump frequency to its mean value during a 10 min interval (t = −12 to −2 min) during the baseline period  maintained a steady frequency for the 60 min recording period whereas switching to IVM caused concentration-dependent inhibition of pumping in both strains. The superimposed, normalized data in avr-14; avr-15; glc-1 worms were right-shifted (indicating drug resistance) compared to N2s for the four IVM concentrations tested.t = 0 min) and the end of the experiment (t = 60 min), with CF50 being the time at which 50% of the pumps had occurred  rose approximately linearly, indicating a steady rate of pumping. Unexpectedly, unlike other CF50 comparisons in this study . As seen in avr-14; avr-15; glc-1 controls was slightly right-shifted compared to N2s, due to a small increase in pump frequency in avr-14; avr-15; glc-1 worms relative to N2 worms during the final ∼15 min of the EPG recording  of pumps that occurred between drug onset , in addition to or instead of CF50; e.g., anthelmintic drug efficacy studies often compare multiple EC (effective concentration) values .Distinguishing drug-sensitive and drug-resistant worms is important for basic research . In these experiments, bus-8 worms could potentially have shown increased baseline pump frequency due to enhanced 5HT entry across the cuticle  whereas the CF50 value in 3 μM IVM was significantly reduced in bus-8 compared to N2 worms (P < 0.002). These results are consistent with the bus-8 mutation increasing worms’ sensitivity to 3 μM IVM, as assessed by inhibition of pharyngeal pumping. A similar result was obtained when 1 μM IVM was tested (data not shown).After switching to 3 μM IVM, pump frequency decreased in both strains, but more rapidly in C. elegans mutants with enhanced permeability for drug and toxicology testing. When comparing N2s with a bus-17 allele in a motility assay, 50 values for two nicotinic compounds were reduced 5- to 8-fold in the permeability mutant; the bus-17 mutant also revealed paralytic effects of two other compounds that were ineffective in N2s. bus-8 to be no more sensitive than N2s in lethality and growth inhibition assays. C. elegans permeability mutants for use in toxicology testing. All mutants showed decreased fitness (based on development rate and brood size), with a bus-5 mutant providing the best combination of sensitivity and fitness in their protocol. In the current experiments, the modest increase in sensitivity to IVM in bus-8 worms  in ematodes . Activats assays . Signifiharynges . The absunc-38 worms. In control experiments  than the rather stereotyped responses to IVM . lev-8, a LEV-resistant mutant with a defective L-AChR subunit. Control N2 and unc-38 worms maintained steady pumping during the post-switch period . The EC50 value for rapid inhibition in N2s differed significantly from those of the other three categories of inhibition: N2 sustained, unc-38 rapid and unc-38 sustained [all P < 2 × 10−5; all EC50 comparisons by a Likelihood Ratio test . Thus, unc-38 worms, and (2) by t = 45–60 min post-switch, the reduction in pump frequency was similar in the susceptible and resistant strains. Thus, LEV resistance was manifested only during an initial phase of pump inhibition.The difference in pump inhibition kinetics in N2 and tio test  with Bonunc-38 worms , and that the expulsion rate in 10 mM LEV was greater than for all other concentrations (P < 0.03). Importantly, unc-38 worms were significantly less likely to be expelled than N2s; e.g., only 10.7% of unc-38 worms in 10 mM LEV were expelled compared to 43.6% of N2s (P < 0.01). This phenomenon was not observed with other anthelmintics; e.g., in the highest IVM concentration tested (10 μM IVM) only 1.9% (1 of 52) of N2 worms were expelled. In the highest PPZ concentration tested (100 mM), 18.9% (7 of 37) of N2 worms were expelled compared to 4.7% (1 of 21) of controls, but this difference did not reach significance (P = 0.24). Thus, only LEV increased the proportion of worms that slipped through the worm trap; the possible cause of this difference is discussed below.These experiments revealed an unexpected aspect of LEV action. The dimensions of channel features in the EPG chip are optimized to ensure that Day-1 adult N2s lodge in the worm trap and rarely slip through. However, a relatively high proportion of LEV-treated worms were expelled through the worm trap into waste reservoirs see B. Table seal on EPG amplitude. As worms slipped farther into the trap and became tightly compressed, the amplitude of their EPG signals increased dramatically; e.g., in the example shown in seal increases as a worm is propelled deeper into the trap, which might explain the increase in pump amplitude over time in 5HT. To test this hypothesis, we measured the RMS amplitude of high-frequency noise in EPG recordings during LEV experiments to provide a proxy of Rseal (see Section n = 5 worms from 3 chips) was 6.5 ± 0.21 μV during the baseline period (“PRE”), 13.9 ± 5.1 μV during the time of maximum EPG amplitude (“MAX”), and 6.04 ± 0.21 μV after worms were expelled (“POST”). PRE and POST noise values did not differ significantly  whereas the MAX noise value differed significantly from both PRE and POST values (P < 0.01). These data provide strong evidence that worm position in the worm trap alters Rseal and, artifactually, EPG amplitude. As discussed above , a worm's signal had to be recorded for 60 min post-switch; any worms expelled during this period were excluded from analysis. If paralyzed worms were the strongest responders to LEV, the concentration-response analysis of pump frequency and other parameters , Fig. 7 3.7C. elegans derives from unc-49, which is alternatively spliced into multiple subunits to form a heteromeric receptor . GABAergic motoneurons inhibit body wall and head muscles, and excite enteric muscles, but there is no GABAergic innervation or GABA-Rs in the pharynx . As an areceptor . We compunc-49 worms before and after a switch from M9-5HT to M9-5HT (control experiments), which had no apparent effect on EPG activity. unc-49 worms after switching to 100 mM PPZ; pump frequency in the N2 worm decreased, in part from more frequent gaps, whereas pumping continued steadily in the unc-49 worm, consistent with a PPZ-resistant phenotype. Before proceeding further, it is necessary to discuss the use of unusually high concentrations of PPZ to obtain responses within the 60-min post-switch period. In addition to potential contributions of cuticle impermeability, PDMS drug absorption and strong pumping stimulation by 10 mM 5HT, other studies show that PPZ has intrinsically low anthelmintic activity against C. elegans. Haemonchus contortus, expressed in frog oocytes, require PPZ concentrations up to 100 mM for full activation, consistent with weak agonist activity even in the absence of a cuticular barrier . Normalized data in unc-49 worms, notably in the 40 and 100 mM PPZ groups. This effect was confirmed in CF plots  or 10 mM PPZ (P = 0.51) groups, but did so in the 40 mM (P < 0.002) and 100 mM (P < 0.01) groups. These data demonstrate a PPZ-resistant phenotype of unc-49, using microfluidic EPG recordings. Much less is known about anthelmintic resistance in unc-49 mutants compared to mutations in GluCl or L-AChR genes. In a motility assay, unc-49 worms were equally affected by PPZ.CF plots E; CF50 vunc-49 showing decreased sensitivity to the drug. PPZ, like LEV, targets receptors located outside of the pharynx, respectively activating inhibitory GABA-Rs and excitatory L-AChRs on body wall muscles. Similarly to IVM, which also causes flaccid paralysis, PPZ did not cause a significant increase in the percentage of worms expelled through the worm trap  inhibited pumping in a concentration-dependent manner, with 4C. elegans in microfluidic EPG chips. Sections E. coli bacteria. We chose to use 10 mM 5HT as the pumping stimulus against which to test inhibitory effects of anthelmintic drugs (Sections Many anthelmintic drugs act on ICs/NTRs, which control the electrical activity of neurons and muscles, and these proteins remain valuable targets for anthelmintic drug development. To facilitate the use of electrophysiological recordings in anthelmintic research, we developed a microfluidic EPG platform that collects continuous EPG recordings from 8 worms simultaneously while perfusing test solutions . In the Sections  but the Sections .C. elegans in studies of aging, toxicology and other stressors (e.g., C. elegans.The second objective was to further validate the use of EPG recordings and automated data analysis by characterizing the effects on pharyngeal pumping of three representative anthelmintic drugs that act on ICs/NTRs: specifically, we examined the effects of IVM on GluCl, LEV on L-AChRs and PPZ on GABA-Rs Sections . The latrs e.g., . For allC. elegans (Continuous EPG recordings taken before and during drug treatment provide exceptional statistical power. A typical control experiment in 10 mM 5HT, with a worm pumping at ∼4.5 Hz for 90 min, yields data on > 24,000 individual pumps; in the 8-channel EPG platform, this represents almost 200,000 pumps acquired over 90 min. A typical sample size of 20 worms in an experimental group yields almost 4 million pumps for automated analysis. Furthermore, because EPGs are recorded both before and after drug exposure in each worm, within-subjects statistical tests can be employed. The parameters analyzed in this study—pump frequency, pump duration and IPI—are only a subset of potentially informative features that can be mined from EPG recordings. The phenotypes produced by anthelmintic drugs are not especially subtle, but the ease of rapidly collecting large EPG data sets for statistical analysis facilitates identification of subtle pumping phenotypes that would be missed by traditional methods such as visually counting pumps. The throughput of the current 8-channel EPG platform could be increased by adding more recording modules per chip or running multiple chips in parallel. Importantly, the 8-channel EPG platform is compatible with a diverse range of nematode species:  elegans , other f elegans  and host elegans .C. elegans body wall muscles (The microfluidic device used here is one of many that have recently become available for studies of anthelmintics and drug resistance. The devices fall into two main classes. The first class comprises devices for characterizing locomotion, including both crawling and swimming. This is done in straight channels or arena-shaped chambers in response to anthelmintics such as LEV . In an e muscles  has not,C. elegans can help with this. The higher throughput and non-specificity of locomotion assays can be advantageous for primary screens, whereas lower-throughput secondary screening using electrical recordings can suggest specific ICs or NTRs that may underlie a drug effect. For example, in a screen of the NIH Clinical Collection library for anthelmintic candidates, primary screening was accomplished using development, lethality and locomotion assays, followed by EPG analysis for more detailed characterization (There are advantages and disadvantages to each microfluidic platform. Locomotion, like electrical recordings, provides a readout of neuromuscular activity and worm health. Locomotion can generally be measured at higher throughput than for existing electrical recording devices, and a large literature on anthelmintic effects on worm movement in non-microfluidic conditions is available for comparison. A disadvantage of locomotion assays are that they are agnostic in suggesting specific targets or mechanisms of action of candidate drugs, although mutant analysis in rization . Ultimatrization .For microfluidic EPG recordings, a microfluidic chip closely related to ours, called the NeuroChip, was introduced in 2013 . Both syIn summary, the results obtained in the current study should provide a useful framework for investigators who would like to adopt electrophysiology into their research workflow for anthelmintic drug screening, drug resistance, basic research on nematode feeding behavior, and other aspects of nematode biology.The authors declare the following potential competing financial interests: JCW, KJR, SRL and WMR own equity in NemaMetrix, Inc., which holds the sole commercial license for the patented microfluidic EPG device reported here.JCW and KJR designed the experiments and KJR performed them. WMR and JCW performed data and statistical analysis. SRL designed the microfluidic chip. JCW drafted the manuscript. All authors participated in editing the manuscript.


Influenza vaccination is effective to prevent influenza infection. However, findings about association between socioeconomic status and influenza vaccination coverage are controversial. Online survey was conducted among 4995 participants between 20 and 69 years of age throughout Japan, January 2017. We asked about history of receiving vaccination in previous year and socioeconomic status, with their reasons for having vaccination or not. Age stratified multivariable logistic regression model was conducted to estimate the odds ratio (ORs) and 95% confidence intervals (CIs) of receiving vaccine for each educational level and income. Sex, self-reported health status, marital status and income were included as covariates. The rate for receiving influenza vaccine among ≤64 year-olds and ≥65 year-olds was 32.9% and 35.4%, respectively. Among younger adults, vaccination varied by each education: junior high school, 23.6%; senior high school, 27.2%; college, 32.6%; university, 36.2%; and graduate school, 39.8%. Compared to junior high school, those from graduate school tended to be more vaccinated . On the contrary, those aged above ≥65 years old received vaccination with no significant differences across education. Likewise, among respondents aged ≤64 year-olds, income was significantly associated with influenza vaccination. Despite being “Managed by school or company” (32.5%), having “No particular reason” was the frequent reason for both receiving influenza vaccination (23.8%) or not (34.3%). Adults with higher educational level were significantly more likely to receive vaccination. Subsidizing influenza vaccination may reduce inequality in receiving vaccination for adults. Strengthening vaccination through various approaches is necessary, such as managing by school or company. The global annual influenza-associated respiratory death is estimated to be 291,243 to 645,832 . IncreasDuring pandemics, particularly young children, older population and in general people who are already ill are vulnerable. Yet, younger adults are effected the most . AccordiInfluenza vaccination is an important matter for Japan due to rapidly increasing aging population compare to other countries . In JapaThis study aimed to investigate the association between individual's educational attainment, income as proxies for socioeconomic status (SES) and influenza vaccine reception. In addition, this study searched whether the association of SES differed by age because of the Preventive Vaccination Law in Japan and searched underlying reasons that influenced individuals' behavior of receiving vaccination or not.22.1This was a web-based cross-sectional study in Japan. The survey with self-administered questionnaire was conducted by Macromill Inc. in January 2017. This global research company has access to approximately 3 million registered individuals. They are able to specify the surveyed population by age and sex. According to our target population, 20–69 year olds, the company have distributed the survey through all prefectures of Japan. Those who agreed to answer the questionnaire participated in the survey to receive point-based incentives that can be converted into cash afterwards. Our survey ended until even number of responses for each sex and age-groups were acquired. Five participants had missing value for educational attainment, thus the study included 4995 respondents.2.2Have you received influenza vaccination in the past year?’, ‘Received vaccination’ and ‘Did not receive’ as response choices.The uptake of influenza vaccine was dependent variable and data on receiving vaccination was collected through following questions: ‘2.3As independent variables, educational attainment and income were used as proxy measures of socioeconomic status. Educational attainment was divided in five categories: ‘Junior high school’, ‘Senior high school’, ‘Vocational and Technical College’, ‘University’ and ‘Graduate school’. Annual household income was categorized into four: lowest (<3 million JPY); 2nd (3–4.9 million JPY); 3rd (5–7.9 million JPY); top quartile (≥8 million JPY).2.4Sex, self-reported current health status, and marital status were adjusted as covariates. Marital status was categorized as married, divorced or lost and single.2.51) Economic reason, 2) Scariness toward injection, 3) No time to visit hospital, 4) Fear of side effects, 5) No experience of influenza, 6) Feelings of vaccine to have no effectiveness, 7) No necessity because children had grown up already, 8) No elders living with, 9) No particular reason, 10) Others. On the other hand, those who received the influenza vaccine were asked about motivating factors and stimulus: 1) Managed by school or company, 2) Advice from medical person, 3) Advice from family member, 4) Feelings of vaccine being effective, 5) Experience of severe influenza, 6) Living with children, 7) Living with elders, 8) Living with a student at present preparing for entrance exam, 9) No particular reason, and 10) Others.To examine individuals' behavioral reason toward influenza vaccination, multiple choice question was prepared, some of which were used in previous studies . ParticiIn order to analyze whether the reasons toward influenza vaccination differed between educational attainments, we combined participants according to the years of educational attainment.2.6To demonstrate the association between influenza vaccine coverage and SES variables, multivariate logistic regression was done with an adjustment of sex, self-reported current health, and marital status as covariates. The main analysis was stratified by age groups, 20–64 years and ≥65 years based on two reasons. First, according to WHO guideline, 65 years and over are recommended to take influenza vaccine . Second,2.7We considered each participant's response to the questionnaire as their consent to participate in the survey. The study protocol was reviewed and approved by the Ethics Committee of Tohoku University Graduate School of Dentistry.3A total of 4995 participants participated in the survey. Numbers of males and females were approximately equally recruited, despite the number of respondents aged 65 years old or above were relatively small compared to participants of 20–64 years old. With respect to influenza vaccination, 32.9% of younger adults and 35.4% of elders had received influenza vaccination . A slighAmong 20–64 years old participants 1489 (32.9%) were vaccinated and the influenza vaccine coverage rate increased according to levels of educational attainment: junior high school, 23.6%; senior high school, 27.2%; vocational and technical college, 32.6%; university, 36.2%; and graduate school, 39.8%. Also, influenza vaccination coverage increased by each income level: <3million, 27.3%; 3–4.9 million, 30.5%; 5–7.9 million, 34.9% and >8 million, 40.0%.p value = 0.027) compared with junior high school. On the other hand, no significant associations were observed among participants of age ≥ 65 years.Results from univariate analysis among ≤64-year-old shows that influenza vaccine coverage was significantly associated with SES. Especially, for participants who graduated from graduate school, the odds-ratio was OR 2.14, 95% CI 1.24 to 3.68 compared with individuals graduated from junior high school . After aP ≤ 0.001). There was no statistical significance between annual household income and influenza vaccination among participants of age ≥ 65 years.Likewise, among ≤64 year-old respondents' annual household income was significantly associated with influenza vaccination. Particularly, participants with >8million annual household income, the odds-ratio was OR 2.05, 95% CI 1.45 to 2.91 compared to lowest quantile. After adjusting the covariates, respondents with annual household income from top quantile were at significantly higher odds of influenza vaccination, compared to those with lowest quantile , followed by “Feelings of vaccine to have no effectiveness” (19.3%), “No experience of influenza” (19.3%), “Economic reason” (15.2%), “No time to visit hospital” (13.1%), “Scariness toward injection” (10.7%), “Fear of side effects” (10.7%), “Others” (3.4%), “No necessity because children had grown up already” (2.1%) and “No elders living with” (1.7%). Among younger adults, when we stratified by educational attainment on the reasons of not having received influenza vaccine, “No experience of influenza” (P = 0.049), “Economic reason” (P = 0.001) and “No time to visit hospital” (P < 0.001) had statistical difference between educational attainment.Managed by school or company” (32.5%), followed by: “No particular reason” (23.8%), “Advice from family member” (15.4%), “Feelings of vaccine being effective” (13.4%), “Living with children” (8.9%), “Advice from medical person” (8.0%), “Experience of severe influenza” (7.4%), “Living with elders” (4.8%), “Others” (3.5%) and “Living with a student at present preparing for entrance exam” (2.2%). Stratification on the reason for receiving influenza vaccine showed that among younger adults, “Managed by school or company” (P = 0.006), “No particular reason” (P = 0.008) and “Advice from family member” (P = 0.002) had statistical difference among educational attainment.In contrast, in 4This study showed that 20–64 year-old participants with higher educational attainment and higher annual household income had significantly higher reception of influenza vaccination compared to those with lower educational attainment and lower annual income. On the contrary, no socioeconomic inequalities were observed among those ≥65 years who were covered by the Preventive Vaccination Law in Japan. Therefore, subsidies for receiving influenza vaccination by the municipalities had the potential to reduce social inequalities in vaccination coverage. To the best of our knowledge, previous studies only investigated for inequalities on pandemic mortality  and rareIn Japan, nationwide influenza vaccination program with subsidy was launched for people only aged ≥65 since 2001/2002 season . HoweverBased on our result, older adults have lower influenza vaccination and this value is much lower than WHO global targets for vaccination coverage rates. It may be due to many municipalities provide subsidies only partially and does not have a recommended coverage goal. On the contrary, in the US, Medicare Part B covers the entire cost of the influenza vaccination for elders , and theNo particular reason” was the most frequent response among participants who had not received vaccination. On the contrary, those being “Managed by school or company” had higher vaccine uptake among participants receiving influenza vaccine. Among those being 20–64 years of age, “Managed by school or company” significantly differed between the educational attainment followed by “No particular reason” and “Advice from family member”. These points should be considered in vaccination reception because influenza vaccination coverage in Japan is still low.“Among those who received influenza vaccination, mostly managed by their company or school and received the vaccine for no particular reason was observed. By contrast, the participants who did not receive influenza vaccine had no particular reason on first of all reasons. The difference between previous studies and our results may elucidate that public concerns about influenza vaccination changed over time . Among 2Managed by school or company” was associated with higher influenza vaccination reception. We consider that spreading knowledge through education and management may have potential on increasing the vaccine coverage. For respondents aged ≥65 year-old, there was no association between education and influenza vaccine coverage. The major reason for not receiving vaccination was “No particular reason.”Moreover, there was a difference in the reasons of receiving vaccination between the age groups. Based on our result, 20 to 64-year-old individual with higher educational attainment and being “4.1This study has several limitations. First, the research was an internet self-reported survey and has a probability of recall bias. Second, the proportions of older individuals were relatively small compared to participants of 20–64 years. Highly educated older respondents may have accessed the survey and the possibility of selection bias leading to overestimation of receiving vaccination cannot be denied. Third, regional information is one of the important variables in SES. Although the study obtained information of all the Japanese 47 prefectures, information on whether the residual area was urban or rural could not be further obtained. Fourth, the precise targeted population could not be obtained by the internet research company, making it difficult for us to calculate the response rate.Previous studies have only conducted individually on health care workers , school 5Younger participants with higher educational attainment and higher household income had significantly higher reception of influenza vaccination. Although older adults aged ≥65 years have possible subsidies for influenza vaccination, the vaccination coverage did not differ between those aged <65 years and ≥65 years.The authors declare that there is no conflict of interest and no financial disclosures.


Brassica oleracea var. capitata) requires a long-term low-temperature exposure for floral induction, causing a delay in the breeding cycle. The objective of this study is to develop a method to induce flowering in cabbage without low-temperature treatment, using a grafting method. We conducted grafting experiments using two flower-induced Chinese kale cultivars  and seven radish cultivars/accessions as rootstocks and investigated the flowering response of grafted cabbage scions without low-temperature treatment. “Watanabe-seiko No.1” cabbage, when grafted onto the two Chinese kale cultivars, did not formed flower buds. Flowering was successfully induced in “Watanabe-seiko No.1” by grafting onto three out of the seven tested radish cultivars, and in “Kinkei No.201” and “Red cabbage” by grafting onto one tested radish cultivar. In “Watanabe-seiko No.1,” the earliest flower bud appearance was observed at 29 days after grafting. Seeds were also obtained from the three cabbage cultivars that flowered by grafting. Gene expression analysis of “Watanabe-seiko No.1” cabbage scions which formed flower buds by grafting, revealed high expression of the homolog of the floral integrator, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (BoSOC1), at the time of flower bud appearance. However, in the same leaf samples, we observed low expression of two homologs of florigen, FLOWERING LOCUS T (BoFT.C2 and BoFT.C6). In addition, two homologs of the floral repressor FLOWERING LOCUS C (BoFLC3 and BoFLC4), which are known to be down-regulated before flower bud differentiation in the vernalization pathway, were highly expressed, indicating that grafting onto radish induces cabbage flowering independently of the vernalization pathway. The expression level of the radish FT homolog (RsFT) in “Rat’s tail-G2,” which had highly induced flowering in the grafted cabbage scion, was higher than in the other radish cultivars. However, although “Rat’s tail-CH” effectively induced flowering in the cabbage scion, the expression of RsFT was low in this cultivar. In this study, floral induction of non-vernalized cabbage cannot be explained by the expression levels of RsFT in rootstock plants, alone. The flowering of non-vernalized cabbage would be induced by transmissible agents from rootstocks and not by the expression of cabbage FT, BoFT, from the scion itself.Cabbage ( Brassica oleracea var. capitata), the delay in the breeding cycle due to the vernalization requirement is particularly remarkable, because cabbage is a plant-vernalization-type plant, which becomes sensitive to low temperatures only after developing to a certain size by expanding many true leaves, and also requires long-term low-temperature exposure for flowering. Furthermore, cabbage is a polycarpic plant, which has ability to turn back to vegetative growth after flowering; thus, reversion from reproductive growth to vegetative growth tends to occur when vernalization is insufficient  rootstock. To our knowledge, this is the only report where cabbage has successfully flowered without low-temperature treatment. Interestingly, in this study by If we can develop a method enabling floral induction in cabbage without treatment at low temperatures, we can shorten the breeding cycle of cabbage and remove regional limitations for cabbage breeding and seeds production. Grafting is a known method for floral induction under non-inductive conditions in several kinds of plants . Kagawa Arabidopsis thaliana, which belongs to the same plant family as cabbage, Brassicaceae. The floral repressor gene FLC, which acts as an integrator of the vernalization pathway, encodes a MADS-box protein that represses the expression of the floral promoter gene FT and floral integrator gene SOC1 by directly binding to their first intron and promoter, respectively  is one of the FLC homologs in B. oleracea, and was shown to be strongly associated with the vernalization requirement by QTL analysis in broccoli  (B. oleracea var. capitata) (B. oleracea var. botrytis L.)  have been reported  , cabbageapitata) , and cauytis L.) . Meanwhiironment . FT homothaliana , and tworeported . SOC1 hothaliana . In radi pathway ,b. In se pathway .B. oleracea var. alboglabra) was investigated under controlled environmental conditions. We also discuss candidate genes important for the non-vernalization flowering of cabbage and the mechanism behind the differences in floral induction ability among rootstock plant cultivars.The final objective of this study is to develop a non-vernalization flowering method for cabbages, using a grafting method that enables a shortening of the breeding cycle and allows for seed production regardless of location or season. In this report, in order to identify rootstock plants suitable for our non-vernalization flowering method in cabbages, the flowering response of cabbages grafted onto radishes and Chinese kale , under long-day  conditions. Japanese commercial cabbage cultivar, “Watanabe-seiko No.1” , “Kinkei No.201” , and “Red cabbage”  were used as scion plants for grafting experiment. It was previously reported that “Watanabe-seiko No.1” requires vernalization at 4–9°C for 6–7 weeks to induce flower bud differentiation (R. sativus L. var. longipinnatus) “Osaka-shijunichi” , “Wakayama” (Noguchi Seed), “Shinshu-jidaikon” , and “Hayabutori-shogoin” ; and three accessions of radish for seed-pod vegetables (R. sativus L. var. caudatus) “Rat’s tail-CH” , “Rat’s tail-G2” , and “Rat’s tail-G4”  were used as rootstocks  was injected into the bag, with the opening tied. The polyethylene bags were removed 12–24 h after pollination.Cabbage scions that flowered after grafting were self-pollinated by COreatment . CabbageLn) and the length (Ll) and the width (Lw) of the biggest leaf of the rootstocks were measured immediately after grafting. Then, the total leaf area of the rootstock (La) was estimated using a preliminary calculated regression equation /(the number of the successful grafts) × 100.The number of leaves on the rootstock plants (-2 s-1 PPFD in long-day (16L/8D) conditions. After low-temperature treatment, the seedlings were returned to the growth room and continued to grow for 2 weeks. The eighth expanded leaf from the top was collected at the beginning and the end of the 8 weeks low-temperature treatment and at 2 weeks after returning to the growth room. All leaf samples were collected at 1 h before the end of the light period. Sampled leaves were immediately frozen with liquid nitrogen and stored at -80°C until RNA extraction.For gene expression analysis of flowering-related genes in the grafted rootstocks and scions, leaves were sampled as follows. The tip of third expanded leaf from the top of “Watanabe-seiko No.1” scions, which had flowered following grafting onto “Rat’s tail-CH” and “Rat’s tail-G2,” were sampled twice: at 7 DAG and within 1 week from flower bud appearance. As a control, the tip of third leaf from the top of “Watanabe-seiko No.1” scions grafted onto “Watanabe-seiko No.1” (NVstock) were sampled at 7 and 60 DAG. For the rootstock analysis, the tip of healthy and non-yellowing fresh leaves at the lowest position were sampled at 30 DAG. For comparison with “Watanabe-seiko No.1,” which had flowered by grafting, expression analysis of flowering-related genes was also performed on “Watanabe-seiko No.1,” which had been subjected to low-temperature treatment. The seedlings of “Watanabe-seiko No.1” were grown in the growth room for 10 weeks after sowing. Then, the seedlings were vernalized for 8 weeks in the growth cabinet, at a temperature of 6 ± 1°C, under LED light (660 nm:450 nm = 8:2) with an output of 90 μmol m® , following which 1 μL of 10-fold diluted reverse transcription (RT) product with pure water was used as a template for real-time RT-PCR. Real-time RT-PCR was performed using the THUNDERBIRD® SYBR® qPCR Mix  according to the manufacturer’s instructions using the LightCycler® 480 system . The real-time RT-PCR cycling was performed as follows: 95°C for 5 min, followed by 40 cycles at 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s. Single-target product amplification was evaluated using a melting curve. All real-time RT-PCR primers used in this study are listed in Supplementary Table 1 and the report of FLC (BoFLC3 and BoFLC4) and FT (BoFT.C2 and BoFT.C6), particular caution was used to design primers for the different sequences between the two homologous genes. For the expression analysis of cabbage scion and vernalized “Watanabe-seiko No.1,” primer sets BoFLC3, BoFLC4, BoFT.C2, BoFT.C6, BoSOC1, and BoActin were used. For the expression analysis of “Watanabe-seiko No.1” and Chinese kale rootstocks, BoFT.C6 and BoActin were used, whereas RsFT and RsActin were used for radish rootstocks.Total RNA was extracted from leaves using Sepasol RNA I Super G , purified using a high-salt solution for precipitation , and reverse transcribed with ReverTra AceFT homolog mRNA. RT products of total RNA extracted from the leaves of the rootstock plants were used as templates. The CDS was determined by direct sequencing of the amplification products using Sanger sequencing. The sequence data were deposited in DDBJ with accession numbers LC414370-LC414380.The PCR was performed using a primer set (Supplementary Table 2) with the largest leaf area observed in “Rat’s tail-CH” (528.5 ± 227.3 cm2)  was 287.2 ± 67.8 cm2. The scion and the rootstock were successfully connected at 1–2 weeks after grafting, and the percentage for grafting success was approximately 70–80%, without large differences observed between rootstock cultivars  Table . Flower When “Watanabe-seiko No.1” was grafted onto non-vernalized “Watanabe-seiko No.1,” Chinese kales and some of the radish cultivars, the scions continued vegetative growth and did not form flower buds at 60 DAG Table 1–C2. On 2 treatment and obtained matured seeds roughly 80 days after pollination , at both 7 DAG and flower bud appearance  in the NCBI reference sequence database of B. oleracea and R. sativus using BLAST program2. As a result, we could find three and two genes without insertion or deletion within any of the exons in B. oleracea and R. sativus, respectively  and R. sativus (Rs1.0 reference Annotation Release 100) and got the same result. Among those genes, two for B. oleracea and one for R. sativus were identical to the FT homologs reported in previous studies . Both of the other two proteins  belonged to TSF clade by phylogenetic analysis , and its expression level was significantly higher than all other cultivars. Conversely, the expression levels of FT homolog in “Rat’s tail-G2” , “Rat’s tail-G4,” and “Rat’s tail-CH” were the lowest among radish cultivars.There were differences among the expression levels of G Figure . In the FT homolog CDS revealed several amino acid sequence polymorphisms within Chinese kale cultivars and between Chinese kale and radish . In addition, in the two cultivars of Chinese kale that did not induce flower bud differentiation, the expression level of FT homolog was approximately 30 times lower than “Watanabe-seiko No.1” that had been vernalized for 8 weeks , which induced the highest percentage of cabbage scions to flower, was significantly higher than all other rootstocks , considering the relatively small difference in the total leaf area between radish cultivars , a closely related species of cabbage, the silencing of FLC homologs (BnFLC) by RNA interference using a viral vector enabled flowering without cold treatment , which originally distribute in tropical areas. This suggests that plants originating in tropical areas that do not require vernalization for floral induction may have the ability to strongly induce flowering by grafting, possibly because they maintain high expression of transmissible floral stimulating agents, like FT, under warm conditions. Although cabbage scions could produce normal seeds without low-temperature treatment after grafting onto a “Rat’s tail-CH” rootstock (Figures Alternatively, grafting is a simple method that in this study successfully induced flowering in cabbage without low-temperature treatment, in accordance with the previous report . Seeds w Figures , there mFT homolog in the rootstock plant appears related to cabbage scion floral induction. Further investigation will make it possible to identify the factors within the rootstock that contribute to flowering in the cabbage scion. This information will be useful for screening and breeding rootstocks that strongly induce flowering of the scion by grafting and thus promoting the practical use of this graft-inducing flowering method not only for cabbage but also other Brassicaceae crops.In this study, we successfully induced flowering and seed set of several cabbage cultivars without low-temperature treatment by grafting onto radish rootstocks under a controlled environment. Our results demonstrate the usefulness of this graft-induced flowering method for shortening the cabbage breeding cycle and its potential seed production in tropical areas. Interestingly, there was a significant difference in the flowering response of the cabbage scion depending on which radish cultivars were used as rootstocks. The expression level of the KM contributed to data collection, data analysis, and writing of the manuscript. YK contributed to data analysis, data interpretation, drafting the manuscript, and final approval of the version. MH contributed to the conception and design of the work, data analysis, data interpretation, writing the manuscript, and final approval of the version.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.