HTT, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, ATN1, AR, and TBP) are the cause of several neurodegenerative diseases including Huntington’s disease (HD), six different spinocerebellar ataxias (SCAs), dentatorubral-pallidoluysian atrophy, and spinobulbar muscular atrophy. The expanded CAG repeat length in the causative gene is negatively related to the age-at-onset (AAO) of clinical symptoms. In addition to the expanded CAG repeat length in the causative gene, the normal CAG repeats in the other polyQ genes can affect the AAO, suggesting functional interactions between the polyQ genes. However, there is no detailed assessment of the relationships among polyQ genes in pathologically relevant brain regions. We used gene co-expression analysis to study the functional relationships among polyQ genes in different brain regions using the Allen Human Brain Atlas (AHBA), a spatial map of gene expression in the healthy brain. We constructed co-expression networks for seven anatomical brain structures, as well as a region showing a specific pattern of atrophy in HD patients detected by magnetic resonance imaging (MRI) of the brain. In this HD-associated region, we found that ATN1 and ATXN2 were co-expressed and shared co-expression partners which were enriched for DNA repair genes. We observed a similar co-expression pattern in the frontal lobe, parietal lobe, and striatum in which this relation was most pronounced. Given that the co-expression patterns for these anatomical structures were similar to those for the HD-associated region, our results suggest that their disruption is likely involved in HD pathology. Moreover, ATN1 and ATXN2 also shared many co-expressed genes with HTT, the causative gene of HD, across the brain. Although this triangular relationship among these three polyQ genes may also be dysregulated in other polyQ diseases, stronger co-expression patterns between ATN1 and ATXN2 observed in the HD-associated region, especially in the striatum, may be more specific to HD.Cytosine-adenine-guanine (CAG) repeat expansions in the coding regions of nine polyglutamine (polyQ) genes ( HTT, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, TBP, ATN1, and AR, respectively (Polyglutamine (polyQ) diseases are a family of nine neurodegenerative disorders caused by a cytosine-adenine-guanine (CAG) trinucleotide repeat expansion in the coding region of one of the polyQ disease-associated genes. PolyQ diseases include Huntington’s disease (HD), six spinocerebellar ataxias (SCAs) dentatorubral-pallidoluysian atrophy (DRPLA), and spinobulbar muscular atrophy (SBMA), each with its own causative gene: ectively . With thectively . The CAGectively . In HD pectively . It is tectively .HTT CAG repeat length  . In HD, t length , while it length . There it length . For exa the AAO . The obsHTT affects the brain, multiple studies used genome-wide expression analysis of post-mortem samples collected from different brain regions. By comparing the brain region-specific expression profiles of HD patients and healthy controls, the highest number of differentially expressed genes was found in the caudate nucleus and to a lesser extent in the cerebellum . We analyzed gene co-expression networks within the different brain regions to identify region-specific relations among polyQ genes and their underlying functional mechanisms.We aim to find common mechanisms through which the nine polyQ genes could interact with each other in order to understand how these interactions could affect neuropathology in polyQ disorders. Our approach consisted of interrogating healthy brain gene expression data from the Allen Human Brain Atlas (AHBA)  as its h1  if there was one probe for a gene, this probe was selected; (2) if there were two probes for a gene, we selected the probe with the highest variance (measured per brain and then averaged across the six brains); (3) if there were more than two probes for a gene, we chose the probe with the highest connectivity (measured as the sum of the Pearson correlations per brain and then averaged across the six brains). These steps resulted in 19,992 genes selected for further analysis.In our analysis, we included a brain region affected in HD patients compared to healthy controls defined according to gray matter changes in MRI scans . SeveralU-tests to identify up- and downregulated genes. Genes with a p-value lower than 0.025 in either the upper tail or lower tail in five out of six brains were considered differentially expressed.We examined gene expression differences between samples located inside and outside the HD-associated region. Differential expression was assessed for each brain separately using two one-tailed Mann–Whitney r) as a measure of co-expression between two genes. Gene pairs showing a co-expression greater than 0.5 were considered to be related as they have similar expressions across tissue-specific samples. The co-expression threshold was based on our observation of the polyQ co-expression distributions in different regions  Direct co-expression: co-expression between two polyQ genes indicates a strong interaction based on their spatial expression patterns. (2) Shared co-expression: a significant overlap of the two co-expressed gene sets of two polyQ genes suggests that these two polyQ genes are indirectly related to each other through other genes with which they interact. (3) Functional overlap: if two polyQ genes indirectly interact, the overlap between the functional terms enriched in their corresponding sets of co-expressed genes point out whether the two polyQ genes are also functionally related. We assessed the significance of shared co-expressed genes between two polyQ genes using one-tailed Fisher’s exact test. We considered a functional overlap between two polyQ genes when their respective gene sets share at least 10 enriched functional terms.2 R package. In the network, nodes represents polyQ genes (each with a unique color) as well as genes co-expressed with polyQ genes (each assigned the color of the polyQ gene with which it is co-expressed). The node color is mixed when genes are co-expressed with multiple polyQ genes. Edges are colored by the polyQ gene that they connect to. We used ‘Fruchterman-Reingold’ and ‘label adjust’ options for the layout. The circular co-expression plots for each examined region were visualized using Cytoscape  were downloaded from the HUGO Nomenclature Committee  and inclATXN2 was significantly downregulated in five out of six brains . Out of the differentially expressed genes, 711 genes showed a significant higher expression in the HD-associated region (upregulated) and 2,101 genes showed a lower expression (downregulated). The set of downregulated genes was enriched in terms related to cytoplasm, mitochondrial processes, cellular component organization, DNA damage recognition, synapses, autophagy, and metabolic processes .We examined the functional relations among the polyQ genes by analyzing their spatial co-expression using the high-resolution AHBA. Co-expression of the polyQ genes was analyzed in the healthy brain using the AHBA samples within regions associated with gray matter changes in HD patients (HD-associated region). In addition, we analyzed seven anatomical brain structures to assess whether polyQ gene co-expression patterns in the HD-associated region are reflected within these structures. All anatomical structures had a sufficient number of samples to perform co-expression analysis . ATN1 had the highest number of co-expressed genes in all examined regions except the mesencephalon and pons where only HTT had more co-expressed genes. The largest number of genes co-expressed with ATN1 was observed in the striatum , followed by the parietal lobe , and the frontal lobe . ATXN2 had the second largest number of co-expressed genes in the parietal lobe, followed by the striatum and the frontal lobe. TBP had only one gene co-expressing in the cerebellar cortex. For all sets of genes co-expressed with polyQ genes we performed functional enrichment analysis to obtain sets of functional GO terms (Benjamini-corrected p < 0.05). This was repeated for all inspected brain regions . The numbers of shared co-expressed genes for different brain regions are shown in Figure 4A, and their respective co-expressed genes in the HD-associated region are listed in Supplementary Table Figure 4B) was measured as the number of shared enriched functional terms between their corresponding sets of co-expressed genes . The same pair also shared most (490) co-expressed genes among all polyQ gene pairs. TBP and ATXN7 were not co-expressed with other genes at all. While both genes show low mean expression levels in the examined regions, the variance is similar to other polyQ genes .In the HD-associated region, ATN1 and ATXN2 in the HD-associated region was also observed in the frontal lobe (r = 0.53), parietal lobe (r = 0.52), as well as the striatum (r = 0.56) that showed the highest correlation . In the striatum, ATN1-ATXN2 was also the only co-expressed polyQ gene pair similar to the HD-associated region. ATN1 and ATXN2 had the highest correlations in the striatum and they also had the highest number of overlapping co-expressed genes and functional terms in the striatum. This indicates that the co-expression patterns between ATN1 and ATXN2 found in the HD-associated region are particularly pronounced in the striatum.Co-expression relationships of polyQ genes in the HD-associated region were also observed in other anatomical structures. The direct co-expression between HTT, ATN1, and ATXN2 across all studied regions . When considering the overlap between sets of enriched functional GO terms , the triangular relationship between these three genes is only observed in the frontal and parietal lobes of the cerebral cortex. In the striatum, we observe a very strong functional overlap between ATN1 and ATXN2. Together, these observations suggest a strong regional and functional relatedness between the three genes rather than just the measured co-expression.There is a significant overlap between sets of co-expressed genes of ATXN1 shows significant overlap of functional terms with HTT in the HD-associated region, frontal lobe, parietal lobe, and striatum. ATXN1 was also found to share functional terms with ATN1, ATXN2, and HTT, forming a clique of four polyQ genes in the parietal lobe. Finally, the co-expression patterns in the HD-associated region seemed to be dominated by the co-expression patterns within the frontal lobe, parietal lobe, and striatum, which were also the regions described as part of the HD-associated region in the imaging study .The co-expression patterns among polyQ genes within the HD-associated region overlapped with those observed within anatomical structures involved in HD neuropathology. In the striatum, the main structure affected in HD, ed in HD . The moted in HD . In a pred in HD , suggestHTT, ATN1, and ATXN2 in all examined brain regions. It would be interesting to evaluate whether this triangular relation between HTT, ATN1, and ATXN2 is dysregulated in all polyQ diseases, especially HD, DRPLA, and SCA2. In addition, we observed strong connectivity of ATXN2 and AR with HTT in the pons that has been described to undergo atrophy in SCA2  has been linked extensively to the pathogenesis of neurodegenerative diseases, including HD and other SCAs  suppressing polyQ aggregation by promoting proteasomal degradation (CHIP has also been implicated in several SCAs indicating it is directly involved in disease pathology (UB4R has a fundamental role in calcium signaling and neuronal survival and may contribute to neurodegenerative conditions (UBA1 is an important regulator of cellular protein homeostasis and contributes to the pathogenesis in SBMA and HD (ATN1 and ATXN2 is likely to be affected. The low expression of these genes in certain brain regions could indicate their increased vulnerability to dysregulations of the UPS. We suspect co-expressed polyQ genes to have a role in AAO, as they and their shared co-expression partners seem to be involved in functions that have been associated to polyQ diseases.Ubiquitin may be involved in HD pathogenesis through the relationships between her SCAs . Aggregaher SCAs . We founradation . CHIP haathology . The gennditions . This genditions . It is nnditions . UBA1 isA and HD . AltogetThere are several issues that limit a one-to-one comparison between the co-expression relationships reported in the present study and phenotype-genotype associations described previously . First, To validate our findings in other brain gene expression data we need a dataset with a spatial sampling resolution that allows co-expression analysis within substructures of the brain. Although the UK Brain Expression Consortium  sampled We showed that polyQ genes are co-expressed in the healthy brain and that their relationships are also specific to certain brain regions including a region associated with HD. Our aim was to find co-expression patterns between polyQ genes in different brain regions. The co-expression networks are likely altered in polyQ diseases due to interaction changes, especially in brain regions associated with the disease. The fact that these findings could not be validated on other expression datasets implies the importance of follow-up studies to understand more about the mechanisms behind polyQ diseases. We show that gene expression in the healthy brain may render specific regions vulnerable to expression changes based on gene co-expression networks.AK, NA, MR, and AM designed the study. BL provided funding and participated in the conceptualization of the study. JvdG provided the imaging data, which was processed by OD. AK performed the data analysis. AK, NA, WvR-M, MR, and AM interpreted the data and wrote the manuscript with input from all authors. AM and MR supervised the overall project. The final manuscript was read and approved by all authors.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.


European freshwater habitats have experienced a severe loss of plant diversity, regionally and locally, over the last century or more. One important and well-established driver of change is eutrophication, which has increased with rising population density and agricultural intensification. However, reduced disturbance of lake margins may have played an additional key role. The geographical variation in water chemistry, which has set the scene for – and interacted with – anthropogenic impact, is much less well understood. We took advantage of some recently completed regional plant distribution surveys, relying on hundreds of skilled citizen scientists, and analyzed the hydrophyte richness to environment relations in five contiguous South-Scandinavian regions. For three of the regions, we also assessed changes to the freshwater flora over the latest 50–80 years. We found a considerable variation in background total phosphorus concentrations and alkalinity, both within and between regions. The prevalence of functional groups differed between regions in accordance with the environmental conditions and the species’ tolerance to turbid waters. Similarly, the historical changes within regions followed the same trend in correspondence to the altered environmental conditions over time. Small submerged species decreased relative to tall submerged and floating-leaved species along the regional and historical eutrophication gradients. These changes were accompanied by systematically greater relative abundance of species of higher phosphorus prevalence. We conclude that species traits in close correspondence with anthropogenic impacts are the main determinants of local, regional and historical changes of species distribution and occupancy, while pure biogeography plays a minor role. Conservation measures, such as re-oligotrophication and re-established disturbance regimes through grazing and water level fluctuations, may help reduce the tall reed vegetation, restore the former richness of the freshwater flora and safeguard red-listed species, although extended time delays are anticipated in nutrient-rich regions, in which species only survive at minute abundance in isolated refugia. The species richness of freshwater vascular plants has declined, both regionally and locally, throughout Europe during the last century. The change has been driven by loss and deterioration of habitats . Many laIncreased application of fertilizers in agriculture and increased outlet of sewage from growing human populations during the 20th century led to turbid lake waters and alteration of the submerged vegetation from oligotrophic, bottom-dwelling species toward eutrophic canopy-forming or floating-leaved species tolerant of turbid waters . CessatiThough these fundamental changes of the freshwater flora have been reported from many local biotopes, the broad-scale relationships of freshwater species abundance to species traits and environmental conditions across large geographical regions have rarely been assessed. Here, we perform an analysis of richness-environment relationships in five contiguous lowland regions, two in Denmark (East-Denmark and West-Denmark) and three in Southern Sweden . The five regions share almost the same species pool of about 90 species of aquatic vascular plants, but environmental conditions follow a pronounced gradient of decreasing nutrient levels and alkalinity (Alk) along with sparser human population density, proportion of agricultural land use and proportion of calcareous and clayish soils in the order: East-Denmark, West-Denmark, Scania, Blekinge, and Småland.Historically, Denmark and Southern Sweden have had a species-rich and luxuriant flora of freshwater plants because of the shallow lowland streams and the many ponds and lakes of highly variable size and water chemistry . FreshwaHere, we report the analysis of broad-scale patterns of species distribution and abundance based on recent meticulous search for plant species, including freshwater species, by numerous skilled botanists in voluntary atlas projects examining several hundred 5 km × 5 km quadrats covering large, representative proportions of Denmark and Southern Sweden. We used this general background to perform a statistical evaluation of relative occupancy of all freshwater species. We excluded a few recent immigrant species, which may not yet have filled their potential distribution and species of uncertain taxonomic status. We first characterize the five regions with respect to human population density, land use, climate as well as Alk and total phosphorus (TP) concentrations of a large representative number of lakes (>5 ha). We then determine the occupancy of freshwater species in the five regions according to plant height, trophic preference and life-form. Finally we evaluate the historical development of the freshwater flora in East-Denmark, West-Denmark, and Scania, which have all undergone habitat loss and cultural eutrophication as well as reduction of grazing and vegetation cutting on the banks of lakes and streams during the last century.We tested three main hypotheses for the contemporary biota composition and historical change of the freshwater flora that derive from the results of recent studies of a large number of local lakes and streams . We hypo(1)Occupancy of small and oligotrophic species increases, while the occupancy of tall and eutrophic species decreases along the regional gradient of lower population density, farming intensity, and water TP concentrations.(2)Occupancy of species with life-forms tolerant of turbid water increases, while occupancy of species with life-forms intolerant of turbid waters decreases along the regional gradient of higher water TP concentrations and turbidity.(3)Occupancy of species has changed with the historical eutrophication in East-Denmark, West-Denmark, and Scania in a way resembling the pattern across the contemporary nutrient gradient between regions.1 and Sweden2. All five regions are lowland regions (maximum elevation: 131–377 m a.s.l) with a similar climate . All five regions were rich in lakes (>5 ha) (0.015–0.13 lakes km-2), and although size of the largest lakes varied , this difference is not important to the presence of submerged plants which are mostly confined to the shallow parts of small and medium-sized lakes and streams. Land use was separated into rural, agricultural, forest and nature areas. The majority of forests are used for timber production and for that reason were not included in the nature area.Geography, land use, and demographic data were taken from the national statistical bureaus of Denmark3 in the period 2012–2016. Average site-specific measurements in surface waters were used in the statistical analyses.Danish data on TP and Alk in lake waters were collected as part of the national monitoring program NOVANA, . We extrAzolla filiculoides, Callitriche obtusangula, Elodea nuttallii, and Lemna turionifera) and still have very restricted distribution ranges were excluded together with two taxa of Ranunculus penicillatus (ssp. pseudofluitans and ssp. penicillatus), which are extremely rare, difficult to identify and have probably been over-looked in some regions. Differences in level of identification between surveys were handled by lumping subspecies and a limited number of problematic species (see Supplementary Information).Species data were collected from recent regional floristic surveys in South Scandinavia . In all In all investigations, species incidence was scored as occupancy in grids of 5 km × 5 km cells, except in Scania where collection originally used a 2.5 km × 2.5 km grid size. Georeferenced primary data from Scania (obtained through GBIF) was resampled onto a 5 km × 5 km grid in a GIS environment [ArcGIS v. 10.5, with the national geodetic datum of Sweden (RT 1990) as specified by 2 grids is on the order of 35 km assuming the mean density in Denmark.Several hundred grids were meticulously examined in each of the five regions. Within the grids, all standing waters (lakes and ponds: hereafter referred to as lakes) and running waters  of different size were studied for the presence of aquatic plants. Although the exact number of freshwater localities is unknown it amounts to several thousand within each region. In Denmark, for example, there are about 100,000 lakes  and about 64,000 km of streams . Total nIn each of the five regions, occurrence of individual species was converted to a proportion by dividing by the number of investigated grid cells. These frequencies of occurrences were used in the statistical analyses.severe reduction (>50% reduction in distribution or occurrence), moderate reduction (obvious but <50% reduction in distribution or occurrences), little or no change, moderate increase (50–100%), strong increase (>100% increase in distribution or occurrence) , earlier broad-scale investigations enabled assessment of species distributional changes during the course of a 50–80 years period. Species distributions obtained in the recent Danish investigation  were comurrence) . Similar2 from the rich sediment source decreases because of longer diffusion path. Elodeid species are predominantly capable of efficient bicarbonate use to assist the diffusive entry of CO2 (Juncus bulbosus and Potamogeton gramineus) the prevalent life-form was applied. Life-form groups were included in the statistical analyses as a nominal variable to enable quantification of between group differences.To quantify the differential response by species or species groups, trait data regarding plant height, trophic prevalence and life form was compiled. Plant height was scored as maximal stem length for elodeid species  and maximum leaf length for rosette species of the isoetid life form  is shown in Supplementary Table post hoc test. To analyze how individual species traits were related to species frequency of occurrence in the investigated regions we used linear regression models with species frequency as the dependent variable and individual species traits as the explanatory variable (lm in R). Species frequency of occurrence was logit-transformed prior to analysis. As the relationship between traits and frequency of occurrence could change between regions, they were included as a covariate. Significant interactions between regions and traits, as tested by likelihood ratio tests, were interpreted as changing trait-frequency relationships. In case of a significant interaction, we made pairwise contrasts between regions to test which regions had significantly different trait-frequency relationships using the lstrends function in the lsmeans-package  was analyzed using Kruskal–Wallis test followed by Dunn’s -package .The historical changes of species occurrence were measured on an ordinal scale  and analyzed using ordinal logistic regression . SimilarFigure 1). Forests in Denmark and Southern Sweden are subject to intense forestry, but not to fertilization. These gradients of population density and farming intensity from Denmark to Småland, combined with much lower natural fertility of soils toward the East, cause lower nutrient input from domestic sewage, agricultural fields and nature areas and may account for the significant  10-fold difference in in-lake median TP concentrations across the study area . The majority of Danish lakes are eutrophic and hypertrophic (25–75 percentiles: 80–230 μg TP L-1) and few approach the oligotrophic threshold (10 μg TP L-1). Toward West-Denmark the concentration tends to decrease  and when moving to the Southern Swedish region of Scania the concentration decreases significantly . The majority of Småland lakes are nutrient poor (25–75 percentiles: 11.0–23.6 μg TP L-1) and some reach the supra-oligotrophic level (1 μg TP L-1). Blekinge resembles Småland in water phosphorus concentration  and also with respect to population density and land use.There was a ten-fold difference in population density and a five-fold difference in landscape proportion of arable land along the geographical gradient from East-Denmark via West-Denmark, Scania and Blekinge to Småland, while forest cover showed a six-fold difference in the opposite direction . Most lakes in Blekinge and Småland are softwater lakes in accordance with the dominance of sandy soils and silicious bedrocks. Alk is significantly lower compared to East-Denmark and West-Denmark , while Småland but not Blekinge is significantly different from Scania . The range of alkalinities is extensive in all five regions, however, with some softwater lakes being present in East-Denmark, West-Denmark, and Scania and some hardwater lakes in Blekinge and Småland. Medium Alk and TP are highly correlated across regions .Most lakes in East-Denmark, West-Denmark, and Scania are hardwater lakes in accordance with the dominance of calcareous soils, however, with significantly decreasing Alk from East-Denmark over West-Denmark to Scania , while Bray–Curtis similarity accounting for species abundance were markedly different between regions. East-Denmark, West-Denmark, and Scania (73–83%) formed one group and Blekinge and Småland (82%) another group of high internal similarity. Bray–Curtis similarity was low (46–57%) in the comparison of East-Denmark and West-Denmark with Blekinge and Småland (Table 2). Scania had a medium Bray–Curtis similarity (66–68%) to neighboring Blekinge and Småland.The five regions resembled each other closely in terms of species composition . There was a significant positive relationship between plant height and frequency of occurrence in the alkaline and nutrient-rich regions of East-Denmark, West-Denmark, and Scania, while the relationship was not significant in the nutrient-poor and low-alkaline regions of Blekinge and Småland (Table 3). Pairwise contrasts of the slopes of the regression line of species occurrence versus plant height tended to be steeper in East-Denmark and West-Denmark (p < 0.1) compared with lower values close to zero in Blekinge and Småland (Table 3). Thus, the mean plant height of submerged species weighted according to their occurrence decreased gradually from 98 cm in East-Denmark to 77 cm in Småland (Table 3).The relationship of species occurrence to plant height changed significantly across the five regions  in accordance with falling phosphorus concentrations in the waters . There was a significant positive relationship between plant species’ trophic preference (ICM) and occurrence in East-Denmark, West-Denmark, and Scania , while Blekinge showed no relationships and Småland showed a significant inverse relationship with species of low phosphorus preference being more abundant than species of high phosphorus preference (Table 4). Thus, the mean trophic preference of all species weighted according to their occurrence differed gradually from 6.4 in East-Denmark to only 4.3 in Småland (Table 4). Among submerged freshwater plants there was a significant positive relationship between plant height and phosphorus preference, though the correlation was weak .Occurrence of all freshwater species, including submerged and floating-leaved forms of different trophic prevalence for phosphorus, changed significantly across the five regions (Life-forms of freshwater species were divided into four categories (1–4) which are expected to represent a gradient of increasing tolerance to light attenuation in the water  and increasing preference for nutrient richness. The three submerged life-form categories have increasing plant height and trophic preference to phosphorus, and among floating-leaved forms unrooted species rely solely on nutrients from the water. Thus, mean ICM values shifted among life-forms in the order: 4.0 , 4.9 , 6.4  and 7.4 .p < 0.01), whereas a more even occupancy of life forms was observed in the two nutrient-poor, low-alkaline regions . Differences between regions in occupancy were particularly strong for isoetids, which had a 50-fold higher median occupancy in Småland (20%) than in East-Denmark  along the TP gradient . Floating-leaved species showed the opposite pattern, decreasing in median occupancy from 30 to 10% along the gradient, though the trend was not significant .In the three nutrient-rich and alkaline regions , median occupancy differed markedly from life-form 1 to 4  and the average change for all three regions was significantly correlated to the pattern observed along the spatial gradient of increasing TP concentrations from Småland to Denmark . There was a significant positive relationship  between plant height and classes of historical changes showing that smaller species declined while larger species changed less and in few cases increased . The interaction between region and plant height was not significant , indicating that the relationship between historical changes and plant height did not differ between regions.Historical changes in species abundance during the 20th century in East-Denmark, West-Denmark, and Scania were significantly correlated  but showed a highly significant positive effect of ICM on historical change .The relationship between trophic preference (ICM) and historical change was very similar with no significant interaction . Also, in all regions, small isoetids declined more than tall elodeids and floating-leaved plants . Furthermore, in Scania small elodeids also declined significantly more compared to tall elodeids and floating-leaved plants .To analyze the relationship between life-form groups and species’ historical changes, we tested if the median historical change differed between life-forms in each region. In all three regions, the median historical change differed significantly between life-form groups . Among the most common freshwater species in all regions are three floating-leaved species, which have maintained high occupancies throughout the last century .Another key result was the overall agreement between the regional difference of species occupancy hication  as a reshication  and, up hication  and greahication . The his-Denmark . Isoetid-Denmark , the Net-Denmark , emphasi-1) and decreasing Secchi transparency (5.5–1.7 m). While the original single isoetid and 11 small elodeid species disappeared, all nine tall elodeid and four rooted floating-leaved species survived  are apparently dependent on a certain level of bicarbonate to grow well  grow at bicarbonate concentrations close to zero  in photosynthesis, whereas it is unavailable to isoetids and floating-leaved plants, which – in addition to COectively . Althougll e.g., , all elo to zero . Thus, tHistorical eutrophication during the last century cannot be treated as a single mechanism, however, because it was accompanied by complex changes in disturbance regimes. Traditional agricultural practice included cattle grazing and hay making on lake shores and along streams . These pEutrophication enhances density and height of tall emergent plants , therebystance  and the similar Holocene vegetation history  are slow, and dispersal capacity is low because species only survive in low numbers in distant refugia. This is the situation for isoetids in East-Denmark, in which nutrient levels are high and isoetids are rare or have gone extinct . It is dEstablishment of a rich flora, including isoetids and short elodeids, is even possible under nutrient-rich conditions, provided that the lake shore is kept open by grazing and physical exposure removing tall amphibious and emergent species and allowing colonization of small species from neighboring sites. This is the scenario in the restored Lake Filsø established on fertile farmland in West-Denmark . Strong Plantago uniflora  can support high aquatic plant biodiversity . Growth uniflora . Locallyrawdowns . EnforceIn summary, we could predict parallel regional and historical changes in freshwater floras across nutrient gradients, through the use of species traits , which have previously been applied successfully to changes in terrestrial floras. We identified a need to include additional species traits in future fine-grained analyses in order to better account for dispersal potential, competitive ability and tolerance of physical perturbation. Recovery obtained through re-oligotrophication, grazing of banks and physical perturbation may follow a trajectory toward greater abundance of threatened species and overall higher richness, albeit probably with a prolonged time delay in relatively eutrophic regions.KS-J, LB-S, and HB developed the idea. PH, JS, and DC organized the Danish data and KS-J, PH, and LB-S extracted the remaining data. LB-S, KS-J, TN, and DC analyzed the data. LB-S performed the statistical analysis. KS-J wrote the paper with assistance from HB and LB-S. All authors commented on an earlier draft of manuscript.JS was employed by company BioPix.dk. The other 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.


Pre-operative kidney volume is an independent predictor of glomerular filtration rate in renal cell carcinoma patients. Compensatory renal growth (CRG) can ensue prior to nephrectomy in parallel to tumor growth and benign parenchyma loss. We aimed to test whether renal metabolite abundances significantly associate with CRG, suggesting a causative relationship.Tissue metabolomics data from 49 patients, with a median age of 60 years, were previously collected and the pre-operative fold-change of their contra to ipsi-lateral benign kidney volume served as a surrogate for their CRG. Contra-lateral kidney volume fold-change within a 3.3 +/- 2.1 years follow-up interval was used as a surrogate for long-term CRG. Using a multivariable statistical model, we identified metabolites whose abundances significantly associate with CRG.Our analysis found 13 metabolites in the benign  and 163 metabolites in the malignant  tissues that significantly associate with CRG. Benign/tumor fold change in metabolite abundances revealed three additional metabolites with that significantly positively associate with CRG .At the pathway level, we show that fatty-acid oxidation is highly enriched with metabolites whose benign tissue abundances strongly positively associate with CRG, both pre-operatively and long term, whereas in the tumor tissue significant enrichment of dipeptides and benzoate (positive association), glycolysis/gluconeogenesis, lysolipid and nucleotide sugar pentose (negative associations) sub-pathways, were observed.These data suggest that specific biological processes in the benign as well as in the tumor parenchyma strongly influence compensatory renal growth. Chronic kidney disease (CKD) is a recognized major public health problem, associated with substantial morbidity and mortality.Renal parenchyma volume, estimated by imaging, has been shown to correlate well with kidney function . FurtherMetabolomics, the systematic analysis of chemicals and small molecules, is a highly promising tool in kidney disease and cancer research ,8. In thTo this end, we compiled a cohort of 49 patients with a median age at nephrectomy of 60 years with CRG as well as benign and tumor tissue metabolomics data . Contra/Data collection followed patient written informed consent and Memorial Sloan Kettering Cancer Center (MSK) institutional review board approval. For the current analysis we used data collected as previously reported in our recent publication . BrieflyAbundances of metabolites were median normalized and those which fell below the limit of detection were imputed as the minimal median normalized value among the cohort of patients. However, any metabolite for which more than 75% of its measurements from the cohort of patients were below the limit of detection was eliminated from all further analyses. As a result, 736 and 724 of the 877 metabolites measured in the benign and tumor tissues, respectively, were retained.Out of a cohort of 138 patients we randomly picked 52 patients with pre- and post-operative imaging (CT scan or MRI). CT and MRI examinations were performed using the standard clinical abdomen–pelvis imaging protocols. Cross-sectional images were exported to Aquarius iNtuition Edition software . The software calculated 3D right and left renal parenchymal volume. Then, subsequent to manual rendering of the tumor area by the observer, the software automatically calculated the 3D tumor volume. Benign renal tissue was defined as the normally enhanced areas. We excluded non-parenchymal tissues, such as renal sinus fat, the collecting system, and non-enhancing cysts. Contra-lateral kidney volume (VC- Volume Contra), benign kidney volume on the tumor side (VI- Volume Ipsi), and tumor volume (V-Tumor) were calculated . We defiPatient and disease associated metadata (covariates) including patient characteristics, comorbidities, tumor pathologic and clinical stage, and nuclear grade were also obtained . All sam2 (the proportion of the variance in the dependent variable that is predictable from the independent variable) and Q2  of each model fit, although their informativeness is limited due to the high number of variables versus the low number of samples in our data [Thévenot et al. [Our aim was to find benign and tumor tissue metabolite abundances and/or patient and disease covariates which are significantly associated with CRG variation and may hence suggest a causative relationship. As this study uses retrospective data, the small sample size was pre-determined and hence the number of independent variables dramatically exceeds the number of dependent variables. As a result, a standard linear model would be inadequate to account for the collinearities in this high-dimensionality. Therefore, we sought for a multivariable regression model appropriate for this task. We chose the orthogonal partial least squares (OPLS) as it benefits from the strength of principal components analysis (PCA) regression on the one hand, and on the other hand its results are easier to interpret than those obtained by partial least squares (PLS) regression analysis . Specifiour data . Comparit et al.  S4 Fig)2 . However, after false discovery rate (FDR) correction none of these associations remained significant at a = 0.05 .To explain the variation in CRG  we combiIn the benign tissue, 13 metabolites were found to have a significant positive association with CRG , eight oWe next tested whether specific biological sub-pathways are significantly enriched with metabolites strongly associated with CRG. In the benign tissue, this revealed enrichment of positive associations of carnitine metabolism, medium-chain fatty acids (MCFAs), and fatty-acid monohydroxyl sub-pathways (FDR adjusted p < 0.05) . CarnitiIn the tumor tissue, significant enrichment of dipeptides (positive association) and benzoate, glycolysis/gluconeogenesis, lysolipid and nucleotide sugar pentose (negative associations) sub-pathways were observed (FDR adjusted p < 0.05) . DipeptiNotwithstanding, these analyses may miss metabolites whose benign versus tumor anti-correlated fold changes are significantly associated with CRG. We thus fitted the OPLS model to the benign/tumor fold change in metabolite abundances, in addition to the relevant clinical covariates , as indeFinally, we tested the effect of metabolite abundances in the benign tissue on LT-CRG  and benzoate, glycolysis/gluconeogenesis, lysolipid and nucleotide sugar pentose (negative association) sub-pathways among tumor metabolites strongly associated with CRG  supportsInterestingly, we recently reported higher levels of dipeptides to correlate with a higher cancer stage . These fOur current analysis found a negative association between the glycolysis/gluconeogenesis pathway in the tumor tissue and CRG. We recently reported an increase in abundance of metabolites of the upper glycolysis pathway alongside a decrease in the abundance of metabolites in the lower glycolysis pathway in clear cell RCC, suggesting a shift towards the pentose phosphate pathway . HoweverThat the Lysolipid pathway in the tumor tissue is negatively associated with CRG, whereas the opposite direction of association was found in the benign tissue, may further support a crosstalk between the benign and malignant tissue with respect to CRG, which is intriguing considering the importance of membrane remodeling in CRG .Finally, of all tumor metabolites significantly associated with CRG, only 3-indoxyl-sulfate was found to have a negative effect. This metabolite and p-cresol sulfate were both suggested to repress angiogenesis ,30, hencNotwithstanding our results, our study has several limitations. First, our cohort is heterogeneous including both partial and radical nephrectomy patients. However, as most of the analysis included pre-operative CRG, type of surgery that followed should not entail major bias. Second, the fact that our tissue samples were collected from only the tumor bearing kidney may mean that our results do not faithfully represent the bilateral CRG process. This limitation, however, is practically inevitable due to the impracticality of acquiring tissue from a contralateral kidney for prospective studies. Also, a similar approach is often applied in diagnostic biopsies for kidney disease. Last, our study is considerably underpowered in terms of sample size versus the number of variables, which stems from the fact that obtaining human samples is a big challenge. However, the fact that the OPLS model, which is designed to handle such designs, reported statistically significant results indicates a trend in the data and hence addition samples should not only amplify it but may also reveal additional metabolites and biological pathways that are significantly associated with CRG.Our work identifies benign parenchymal- and tumor-specific metabolites and metabolic sub-pathways strongly associated with CRG, which therefore may be driving it. This deepens our understanding of kidney pathophysiology  and provS1 FigDistribution of CRG across the cohort of 49 patients.(TIF)Click here for additional data file.S2 Fig2 and Q2 values of each model fit are also reported in panels E-H.QQ-plots of the OPLS fits and root mean squared errors (RMSE) computed for the 8-fold cross-validation OPLS fits and an OPLS fitted to the randomized data. Panels A and B and E and F describe OPLS fit to metabolite abundances in benign and tumor tissue, respectively, with pre-operative CRG as response. Panels C and G describe OPLS fit to benign/tumor metabolite abundance fold change with pre-operative CRG as response. Panels D and H describe OPLS fit to metabolite abundances in benign tissue with LT-CRG as response. P-values in panels E-H are the statistical significance of comparing each pair of corresponding cross-validation and randomized RMSEs. In addition, the R(TIF)Click here for additional data file.S3 FigCompensatory renal growth (CRG) was measured for 42 patients by two observers and the histogram of the fold change of their CRG measurements identifies two patients for which the CRG measurement of observer 1 was more than 25% than that of observer 1  and one patient for which the CRG measurement of observer 1 was less than 75% than that of observer 1 .(TIF)Click here for additional data file.S4 FigThe X-axis is the FDR adjusted p-value  based on a univariable regression and the Y-axis is the OPLS VIP score. Panels A and B describe OPLS fit to metabolite abundances in benign and tumor tissue, respectively, with pre-operative CRG as response. Panel C describes OPLS fit of benign/tumor metabolite abundance fold change with pre-operative CRG as response. Panel D describes OPLS fit of metabolite abundances in benign tissue with LT-CRG as response. Statistically significant variables are defined as those with VIP score > 1 and p’-value < 0.05 and are colored red. Variables with VIP score > 1 and p’-value > 0.05 are colored orange, and the remaining variables are colored gray.(TIF)Click here for additional data file.S1 TableMetabolite abundances in benign, tumor, and benign/tumor fold change are indicated by their compound ID . The Metabolites sheet provides the metadata for each metabolite by its compound ID, such as biochemical name and sub-pathway.(XLSX)Click here for additional data file.S2 TableIn each sheet, the univariable and OPLS analyses are provided for each variable in terms of its effect and the associated statistical significance.(XLSX)Click here for additional data file.


The number of older cancer patients is rising. Especially in older people, treatment considerations should balance the impact of disease and treatment on quality of life (QOL) and survival. How a cancer diagnosis in older people interacts with concomitant frailty to impact on QOL is largely unknown. We aimed to determine the association between frailty and QOL among community-dwelling older people aged 65 years or above with and without a cancer diagnosis cross-sectionally and at 12 months follow-up.Data were derived from the TOPICS-MDS database. Frailty was quantified by a frailty index (FI). QOL was measured with the subjective Cantril’s Self Anchoring Ladder  and the health-related EuroQol-5D  at baseline and after 12 months. To determine associations, linear mixed models were used.7493 older people  were included. Dealing with a cancer diagnosis (n = 751) was associated with worse QOL both at baseline , EQ-5D:-0.03 ) and at follow-up , EQ-5D:-0.02 ). A ten percent increase in frailty was also associated with a decrease in QOL at baseline , EQ-5D:-0.12 ) and follow-up , EQ-5D:-0.07 ). When mutually adjusting for frailty and a cancer diagnosis, associations between a cancer diagnosis and QOL only remained significant for CSAL at baseline ), whereas associations between frailty and QOL remained significant for all QOL outcomes at baseline and follow-up. No statistical interactions between cancer and frailty in their combined impact on QOL were found.Cancer diagnosis and frailty were associated with worse health-related and self-perceived QOL both at baseline and at follow-up. Differences in QOL between older people with and without a cancer diagnosis were explained to a large extent by differences in frailty levels. This stresses the importance to take into account frailty in routine oncologic care. The number of older patients qualifying for oncologic treatment is rising. Due to concomitant multi-morbidity and frailty among these patients, physicians need to deal with complex treatment decision-making processes–6. In orThe data for this study were derived from The Older Persons and informal Caregivers Survey Minimum DataSet (TOPICS-MDS) repository. This isThe following inclusion criteria were utilized to select appropriate research projects for which TOPICS-MDS data were in the repository:Community-dwelling people who were 65 years or older at baseline;The study setting was primary care or general population;Longitudinal data were available in the separate research project  at 12 months follow-up.The primary outcomes were QOL at baseline  and QOL after 12 months, respectively. Independent variables of prime interest comprised frailty and cancer diagnosis. Operational definitions for these variables and measurement can be found below. Adjustment variables were age, gender and education level.In the TOPICS-MDS baseline questionnaire for care receivers (T0), self-perceived and health-related QOL were measured by a modified Cantril’s Self Anchoring Ladder and the EuroQol-5D, respectively. Both QOL outcomes were also included in the TOPICS-MDS follow-up questionnaire for care receivers which was gathered after 12 months (T12).Modified Cantril's Self Anchoring Ladder (CSAL)CSAL is a one-dimensional index ranging from 0 (completely unsatisfied with life) to 10 (completely satisfied with life) and measures self-perceived general QOL. We usedEuroQol-5D (EQ-5D)The EQ-5D utility score measures health-related QOL. Five diTo quantify frailty, frailty indices based on the concept of deficit accumulation were used. SpecifiIn the TOPICS-MDS baseline questionnaire, individuals were asked to tick boxes which illnesses and conditions they had at the moment or had had in the past 12 months. To differentiate between people with and without cancer, we used the self-reported presence of a type of cancer . Beyond this information, no information on type or severity of cancer was available.Since QOL is associated with the education level of people, we adjusted for education level in our analyses. In the TOPICS-MDS baseline questionnaire, individuals were asked what the highest level of education was that they had completed as defined by Verhage. We clasWe used the TOPICS-MDS dataset for care receivers available in January 2017 (version TOPICS_2.0). Based on our inclusion criteria, 14 projects were appropriate to answer our research question. For these studies, intervention groups were excluded since the interventions may have influenced respondents’ frailty and/or QOL. Furthermore, respondents living in nursing homes and respondents with an unknown cancer diagnosis status at baseline were also excluded. If the respondents’ date of follow-up assessment was outside a 6 months window (3 months before or 3 months after the intended 12 months follow-up), these respondents were excluded in the analyses. Studies in which the follow-up date was exceeded in more than 60% of the respondents were excluded as a whole. In the end, 7493 respondents in 11 projects were included in our analyses .Demographic characteristics were compared between respondents with and without cancer. Student’s t-test was used for continuous data and the chi-square test was used to compare categorical data. The proportions of frailty deficits endorsed among frail respondents with cancer were compared with those among frail respondents without cancer and were considered clinically relevant if the proportions differed by 5%. To determine the association between frailty and QOL, we used linear mixed models to account for clustering within individual research projects. Independent variables included age, sex, education level, frailty, cancer diagnosis status, the interaction term frailty*cancer diagnosis and  the score on the respective QOL measure at T0. Associations with the primary determinants were examined in unadjusted models as well as models adjusted for age, sex and education level. Data were analyzed using the statistical software program SPSS version 22.In total, 7493 respondents were included in this study. Significant differences in age, frailty and outcome variables existed between included respondents and excluded respondents . Of the Analysis of the differences in deficits between frail respondents with and without cancer  showed tUnivariable analyses showed that CSAL and EQ-5D scores at baseline and follow-up, in addition to being related to frailty and cancer diagnosis, were significantly higher in respondents who were younger, and higher educated. For the EQ-5D at baseline and at follow-up, also males scored significantly higher in univariable analyses . AdjusteWhen mutually adjusting for frailty and a cancer diagnosis, parameter estimates for the strength of the associations between a cancer diagnosis and types of QOL diminished considerably and only remained significant for CSAL at baseline , p<0.05). Vice versa, the strength of the associations between frailty and QOL diminished much less and remained significant for all QOL outcomes at baseline , EQ-5D: -0.12 ) and follow-up , EQ-5D: -0.07 ). The interaction term frailty*cancer diagnosis was neither significant for QOL scores at baseline nor for QOL scores at follow-up.The purpose of this study was to examine the association between frailty and cancer in their combined impact on self-perceived and health-related QOL in community-dwelling older people aged 65 years or above. We showed that older people with cancer were more frail than people without cancer and that the type of frailty differed. Cancer negatively affected QOL of older people cross-sectionally and longitudinally, which was to a large extent associated with higher frailty levels of people with cancer. Irrespective of cancer diagnosis, frailty was independently associated with lower patient-reported QOL at baseline as well as after 12 months, both subjectively rated and concerning health-related QOL.Previous research in oncology care mainly focused on health-related QOL among patients with specific cancer types, 31. FewStrength of this study is the large sample size which resulted in outcomes with high ecological validity. In addition, we used patient-reported QOL both concerning health-related QOL and on a subjective scale in general. The cancer group was based on one question in the TOPICS-MDS questionnaire. Misclassification might have happened, however, the accuracy of a self-reported cancer diagnosis compared to registry data is quite high, 47. We Based on this study we recommend to take into account patient’s frailty beside the cancer diagnosis as part of routine oncology care to be able to accurately predict natural progression of cancer not only in terms of survival but also in terms of QOL. This enables physicians to deliver tailor-made care and pre-habilitation options for cancer treatment based on patients’ needs. Pro-active interventions aiming to optimize older patients’ frailty may be related with positive results for these patients. In addiBetween community-dwelling older people aged 65 years or above with and without a cancer diagnosis, differences in quality of life are explained to a large extent by differences in the frailty levels. Therefore, non cancer-related vulnerability should be taken into account as part of routine oncologic care. This may ultimately help to decrease the occurrence of negative outcomes and guide treatment plans on how to optimize patients’ quality of life.S1 TableCSAL: Modified Cantril's Self Anchoring Ladder, range 0 to 10, where 10 indicates the best score for present life as rated by individuals. EQ-5D: EuroQol-5D utility score, range -0.33 to 1.00 where a score below zero is indicative of a health state worse than death. TOPICS-FI38: TOPICS-MDS frailty index consisting of 38 items to quantify frailty, range 0 to 1, where participants with a score equal to or above 0.25 are considered to be frail. T0 indicates the baseline measurement, T12 indicates the measurement after 12 months.(DOC)Click here for additional data file.S2 TableaReferences are in bold, for example: mean CSAL female = 7.07, male = (7.07 + 0.02) = 7.09.Education level: education levels as defined by Verhage were cla(DOC)Click here for additional data file.


To evaluate the factors potentially associated with the decision ofadmission to the intensive care unit in Brazil. An electronic survey of Brazilian physicians working in intensive careunits. Fourteen variables that were potentially associated with the decisionof admission to the intensive care unit were rated as important (from 1 to5) by the respondents and were later grouped as "patient-related,""scarcity-related" and "administrative-related" factors. The workplace andphysician characteristics were evaluated for correlation with the factorratings. During the study period, 125 physicians completed the survey. The scores onpatient-related factors were rated higher on their potential to affectdecisions than scarcity-related or administrative-related factors, with amean ± SD of 3.42 ± 0.7, 2.75 ± 0.7 and 2.87 ±0.7, respectively (p < 0.001). The patient's underlying illness prognosiswas rated by 64.5% of the physicians as always or frequently affectingdecisions, followed by acute illness prognosis (57%), number of intensivecare unit beds available (56%) and patient's wishes (53%). After controllingfor confounders, receiving specific training on intensive care unit triagewas associated with higher ratings of the patient-related factors andscarcity-related factors, while working in a public intensive care unit (asopposed to a private intensive care unit) was associated with higher ratingsof the scarcity-related factors. Patient-related factors were more frequently rated as potentially affectingintensive care unit admission decisions than scarcity-related oradministrative-related factors. Physician and workplace characteristics wereassociated with different factor ratings. Thesedecisions may be associated with the patients' clinical characteristics,-4 but they are also influenced by non-clinical factors, such asICU bed availability.,4,5Decisions on admission to the intensive care unit (ICU) are performed routinelyworldwide.-8 Moreover, the triage processes seemto be contextually and culturally sensitive, with great variability betweendifferent countries,9,10 and, even, within countries. Specifically, in low-income and middle-incomecountries, in which resources are more scarce,-14 such triagedecisions may be more heavily influenced by non-patient related factors. However, in Brazil and otherlow-income and middle-income countries, there is paucity of data regarding theseallocations decisions.-19Despite the development of guidelines, there is no international consensus on how tomanage these triage decisions.In this study, we aimed to analyze the factors that potentially influence Brazilianphysicians' decisions of admission to the intensive care unit and to examine theassociation of specific physician and workplace characteristics with thesefactors.Hospital das Clínicas of theFaculdade de Medicina of the Universidade de SãoPaulo (USP) institutional review board ,which approved the utilization of an electronically obtained informed consent.This study was approved by the This study was an electronic, self-administered survey. An online questionnaire wasdeveloped and made available electronically .A convenience sample of Brazilian physicians with experience in critical care wasinvited to participate in the study by medical specialty e-mail groups, social medianetworking and personal contacts. Invitations were sent on three differentoccasions, within two-week intervals, initiating in October 2015.Respondents were included if they were licensed practicing physicians who werecurrently working in ICUs. Participants were excluded if consent was not obtained orif the research questionnaire was not fully completed. Participants were notrequired to be board-certified in critical care to participate in the study.The online survey included the demographic and professional characteristics of therespondents and their ICU, such as whether there was high-intensity staffing. The survey included variables related to the respondents'exposure to situations of ICU bed scarcity and triage in their practice . Other characteristics were also collected:gender, age, state and city of current practice, age, date of graduation frommedical school, fellowship in intensive care medicine , board certification in intensive care medicine, othermedical specialization, number of hours per week working in ICU, working in publicor private ICU, "closed" or "open" ICU and number of beds in the ICU. If thephysician worked in more than one ICU, it was asked that the responses reflect theICU in which the physician worked most of the time.To assess the importance of factors in the decisions of admission to the ICU, therespondents were asked to rate 14 factors as being potentially important todecision-making, using a Likert scale from 1 (never affects decisions) to 5 . Later, those factors were gathered into three groups: (1)patient-related factors ; (2) scarcity-related factors  and (3)administrative-related factors .TM  and Statistical Package forSocial Sciences (SPSS), version 21.0  were utilized as databaseand statistical software, respectively.Microsoft Excel 365t-test. Categorical variables weredescribed as a number (percentage) and were analyzed by chi-squared or Fisher'sexact test, when appropriate. The relevance of factors related to the decisionof admission to the ICU, as measured by the Likert scale, was analyzed as adichotomous variable  or as an ordinal variable. Thegrouped variables  were calculated as the overall mean of the factors comprised in eachgroup. A post hoc analysis evaluating the difference between physicians workingin public or private ICUs was performed because it was thought to be ofrelevance.Continuous data were described as the mean ± standard deviation and wereanalyzed by Student's The correlation of the respondent characteristics with the factors associatedwith the decision of admission to the ICU was evaluated by a Spearman's rankcorrelation test. All respondent characteristics that were associated with a pvalue < 0.1 were entered in a multivariable linear regression model tocontrol for confounding. Collinearity was assessed using the tolerance test andvariance inflation factor. Three different models were built, one for each groupof variables . A two-tailed p value of 0.05 was considered significant in allanalyses.In the study period, 178 physicians logged on to the questionnaire and 125 (70.2%)complete responses were collected and analyzed. The respondent characteristics aredepicted in Most respondents  were male, mean age ± SD was 37 ± 7.4years and 95 respondents (76%) were board-eligible or board-certified in criticalcare. Almost all respondents worked in high-staffed ICU, but close to half of therespondents worked in public ICU or closed ICU, as opposed to private or open ICU.Of the 48 respondents that worked in open ICU, most  worked in privateICU. Only 38 (30.4%) respondents were rarely exposed to situations of ICU bedscarcity and 52 (41.6%) were rarely involved in the ICU triage process . The majMost respondents  rated the underlying illness prognosis as always orfrequently affecting the decisions of admission to the ICU . PreviouThere were significant differences between the physicians working in public orprivate ICU in the ratings of individual factors . PhysiciOverall, patient-related factors were given higher scores (in a range from 1 to5) than scarcity-related factors and administrative-related factors, with a mean± SD of 3.42 ± 0.7, 2.75 ± 0.7 and 2.87 ± 0.7, and p< 0.001 for the comparison between patient-related factors and scarcity- oradministrative-related factors and p = 0.12 for the comparison betweenscarcity-related factors and administrative-related factors .When comparing physicians working in public or private ICU , scarcitDifferent respondent characteristics were associated with patient-relatedfactors, scarcity-related factors or administrative-related factors . After aIn this study of factors potentially affecting Brazilian physicians' decisions ofadmission to the ICU, we have found that patient-related factors were mostlyregarded as always or frequently affecting these decisions. However,scarcity-related factors, such as ICU bed availability, were also rated as highlyinfluencing these decisions. Receiving specific training in ICU triage wasassociated with higher ratings in patient-related factors, while receiving specifictraining in ICU triage and working in a public ICU (as opposed to a private ICU)were associated with higher ratings in scarcity-related factors. and in theNetherlandsdemonstrated similar results. However, one study evaluating physicians from theUnited States (US) and Israel demonstrated different patterns, with less US physicians rating ICUadmission decision details regarding patient-related factors as important.This survey demonstrated that patient-related factors were more frequently rated aspotentially affecting decisions on admission to the ICU. Other studies performed inSwitzerlandMost respondents in the survey indicated that they had experienced situations of ICUbed scarcity; however, 41% were rarely or never involved in the ICU triage process.Because the respondents who rarely experienced situations of ICU bed scarcity andwere rarely involved in the ICU triage process usually worked in private ICU, thismight reflect the different resource availabilities and ICU workflows in public andprivate ICU. This result is further supported by the fact that most respondentsindicated that private ICU were functioning as open ICUs.post hoc analysis, we found significant differences between thephysicians working in public and private ICU. Overall, scarcity-related factors weredeemed as more important by physicians working in public ICU, whileadministrative-related factors were deemed as more important by physicians workingin private ICU. Nevertheless, patient-related factors were deemed as more importantthan scarcity-related or administrative-related factors for both physicians workingin public and those in private ICU, even though there were differences in theratings of specific factors, such as the influence of acute illness prognosis andprevious performance status on the decision of admission to the ICU. It is possiblethat these rating differences reflect actual differences in practice, contributingto inequalities in the delivery of care. Whether such differences are drivenexclusively by resource availability or if other factors, such as different workflowdesigns and different financial incentives, are contributory remains elusive.In a We found that some respondent characteristics were associated with higher ratings ofpatient-related factors and scarcity-related factors as potentially affectingdecisions on ICU admission. Specific training on ICU triage was associated withhigher ratings of patient-related factors and scarcity-related factors. Although notspecified, training in ICU triage may be a modifiable factor in enhancing triageprocesses. Working in a private ICU was associated with a lower rating ofscarcity-related factors as potentially affecting decisions on ICU admission.Although this association was adjusted for respondents who were rarely exposed tosituations of ICU scarcity and were rarely involved in ICU triage, this result mostlikely represents a difference in resource availability in daily practice. Of note,although statistically significant, these associations were weak and should beinterpreted with caution. We could not calculate the exact response rate because wedid not have a specific denominator, but we had a high completion rate. Moreover,this study was based on a convenience, non-aleatory sample, which further increasesthe risk of bias. Despite this limitation, there were responses from all fiveregions and from 15 of the 27 states in Brazil and, aside from an overrepresentationof the Northeast region, the distribution of respondents followed the distributionof intensivists among the different regions of Brazil. Respondents were younger than the averageintensivist in Brazil, but they had the same gender distribution, as it has beendemonstrated that the mean age of intensivists in Brazil is 47.5 ± 9.5 and69.8% are male. However, thesedata reflect thedistribution of physicians certified in intensive care medicine, not of all thephysicians working in ICU. In our study, there was a high rate of physicians whowere board-eligible or board-certified in intensive care medicine, which probablydoes not reflect the current reality in most ICU in Brazil, in which most physiciansworking in the ICU are not intensive care specialists. Intensive care specialistswill probably have different attitudes toward ICU admission than non-specialists,which may bias our results. Nonetheless, although it is not required to be anintensive care specialist to work in an ICU, it is required that all ICU have amedical director who must be board-certified in intensive care medicine. Because ICUdirectors are usually involved in developing ICU admission policies and guidelines,in addition to helping in difficult cases, it is possible that our results aregeneralizable.This study utilized a web-based survey, which may have advantages, such as a higheraccuracy of responses and a lower risk of errors from its limited manual data entry,but it also exhibits variable responses rates. that isgovernment-led with no direct payment from users, there is also a private sectorthat covers a smaller proportion of the population. These characteristics have led to inequalitiesthat are translated into the availability of healthcare resources, includingICU. However, althoughthese characteristics are unique to Brazil, other countries face similarchallenges. Finally,this survey intended to measure the factors that potentially affect decisions onadmission to the ICU in a hypothetical manner. It is possible that actual behaviorsmay differ from what is depicted in a survey, which could also bias the results.Additionally, Brazil has a unique health system in that, although there is a UnifiedHealth SystemIn this survey of Brazilian physicians working in intensive care units,patient-related factors were more frequently rated as potentially affectingintensive care unit admission decisions than scarcity-related oradministrative-related factors, even though intensive care unit bed availability wasalso an important factor. Specific training on intensive care unit triage wasassociated with higher ratings of patient-related factors and scarcity-relatedfactors, while working in a public intensive care unit (as opposed to privateintensive care unit) was associated with higher ratings of scarcity-relatedfactors.


Of participants who completed questionnaires of feasibility (n = 24), 61.9% reported that they were satisfied using the application to monitor their food intake, and 47.7% liked getting personal information about their dietary intake from the application. However, more than 70% of participants answered that it was burdensome to use the application or that they had trouble remembering to record their food intake. The mobile application Diet-A offers the opportunity to monitor dietary intake through real-time feedback. However, use of Diet-A may not provide accurate information on the food intake of adolescents, partly because of the recording burden.Given the increasing social and economic burden of chronic disease and the need for efficient approaches to prevent and treat chronic disease, emphasis on the use of information and communication technology (ICT)-based health care has emerged. We aimed to test the feasibility of a mobile application, Diet-A, and examine whether Diet-A could be used to monitor dietary intake among adolescents. In a three-month pre–post intervention study, 9 male and 24 female high school students aged 16–18 years consented and participated in this study. Participants were instructed to record all foods and beverages consumed using voice or text mode input. Nutrient intake was measured using 24-h recalls pre- and post-intervention. We compared nutrient intake data assessed by Diet-A application with those assessed by 24-h recalls. Participants tended to underreport intakes of nutrients compared to those assessed by two 24-h recalls. There were significant decreases in sodium ( The World Health Organization (WHO) reported that deaths caused by noncommunicable chronic diseases (NCD) are expected to increase from 36 million in 2010 to 44 million in 2020 [Advancements in technology have been expected to help health professionals and health care consumers teach and adopt healthy behaviors and prevent chronic disease in a cost-effective way. Diet is a major risk factor or prevention-related factor of noncommunicable chronic diseases including cancer, cardiovascular disease and diabetes. To prevent and control a noncommunicable chronic disease, the WHO established a strategy for healthy eating and physical activity and prepared a series of action plans , suggestAssessment of dietary intake and intervention in dietary behaviors are key components in health care and public health. Accurate methods and tools to assess food and nutrient intake are essential in monitoring nutritional status. Twenty-four-hour dietary recall, dietary records, and food frequency questionnaires (FFQs) are commonly used to measure dietary intake . Twenty-2 and found that self-guided intervention with use of a mobile phone could be a potential alternative to the face-to-face treatment [Health-related behaviors, such as healthy eating and regular exercise, could lead to improvement in population health only if they are sustained. Mobile technologies can help people record their food intake and monitor their diet in real time. The ENGAGED randomized controlled trial examined the efficacy of a smartphone-supported weight loss program among adults whose BMI was between 30 and 40 kg/mreatment . In the reatment . The conAccording to research released in 2015, Korea 83%) has been ranked fourth among 56 countries in smartphone penetration, outranked only by the United Arab Emirates(UAE) (90.8%), Singapore 87.7%), and Saudi Arabia (86.1%) [.7%, and 3% has beTherefore, we developed a self-monitoring mobile dietary application, Diet-A, and tested its feasibility in a pre–post intervention study. We also aimed to examine whether the mobile application could be a useful tool for adolescents to monitor their dietary intake.We developed a dietary self-monitoring mobile application called ‘Diet-A’; its development process has been published elsewhere ,10. The 2), diabetes, hypertension, hypercholesterolemia and hypertriglyceridemia among Korean adults 30 years or older was 32.5%, 11.9%, 30.4%, 15.9% and 17.6%, respectively [To provide a short message for disease prevention, we chose four major metabolic disorders—obesity, diabetes, hypertension and dyslipidemia—which are increasing in prevalence in Korea and are the major chronic disorders regularly reported by Korea Health Statistics. The Korea Health Statistics 2013: Korean National Health and Nutrition Examination Survey (KNHANES VI-1) reported that the prevalence of obesity  was calculated as body weight (kg) divided by the square of the height (m). Twenty-four-hour recalls were conducted by 5 to 6 trained interviewers at pre-intervention. After the researcher described the functions and how to use the application, participants downloaded the application on their mobile phones. Participants were instructed to record their dietary intake using Diet-A for three months and to record all the foods, dishes, and beverages that they consumed on that day, if they started recording their diet. We also asked interim questions about good and bad aspects of Diet-A and the reasons. At post-intervention, participants completed questionnaires about the feasibility and usability of the application. The items on the feasibility questionnaire were adopted from previous feasibility studies of mobile diet applications ,16,17,18t-test. All statistical analyses in our study were performed using the SAS software package, version 9.4 , and all tests were evaluated using two-tailed tests of significance at the p < 0.05 level.The demographic characteristics between male and female participants were compared using Fisher’s exact test for categorical variables and the Wilcoxon rank-sum test for continuous variables. A paired t-test was used to compare the differences in estimated dietary intake between the two 24-h recalls and Diet-A. Nutrient intake from pre- and post-24-h recalls was compared using paired p = 0.016). The mean BMIs were similar between male and female students. Among all participants, 8 male (88.9%) and 13 female students (54.2%) used the mobile application, Diet-A, in the study period. Dietary intake was recorded by Diet-A for an average of 12.2 days (range: 1–47 days) per participant with at least 1 meal per day and 87.9% of participants recorded at least 2 meals per day during the 3-month intervention period  and calcium (p = 0.034) compared with the 24-h recall before the intervention. For other nutrients, we did not find significant differences between pre- and post-intervention. In the 24-h recall after the intervention, one male and one female student reported that they suffered from gastritis and enteritis, and one of the girls reported that she had tried a one-meal-a-day diet. When we conducted a sensitivity analysis by excluding these three participants, we did not observe significant differences in any of the nutrients between pre- and post-intervention 24-h recalls. The mean values of energy and nutrient intakes estimated by pre- and post-intervention 24-h recalls were comparable, as shown in n = 9). They reported that they were able to get information about their dietary habits and eat meals at regular times. In addition, they believed that they increased their intake of vegetables and fruits and reduced their intake of fast food such as hamburgers or fried chicken during the intervention study. The participants also reported that the main reasons why they stopped using Diet-A were the burden of recording their meals every time they ate and a lack of some foods, such as new commercial products, in the database.On the interim questionnaire for compliance with Diet-A, we asked what good and bad things they found in using the mobile application (n = 21), 61.9% reported that they were satisfied using the application to monitor their dietary intake, and 65.0% of participants who answered in item 8 reported that this application was helpful for monitoring the food consumed. Additionally, 57.1% of participants slightly agreed or totally agreed that they were able to learn about their dietary intake during the period they used a Diet-A. However, when they were asked about the difficulties of application use, 71.4% reported that it was burdensome to record their diet and sometimes they did not remember to record their diet (85.7%).The feasibility results are presented in We developed a mobile application called ‘Diet-A’ that was designed to record dietary intake and provide users with real-time feedback. Diet-A provided real-time feedback on selected nutrients relative to the Korea Dietary Reference Intakes and a short message focusing on the prevention of obesity, diabetes, hypertension and dyslipidemia.We selected total energy intake for obesity, refined carbohydrate intake for diabetes, sodium intake for hypertension and saturated fat for dyslipidemia based on the following evidence. A long-term excess of energy intake leads to obesity by altering the balance between energy intake and expenditure, and maintaining energy balance is a key strategy in the management of obesity . For preAccurate dietary assessment is essential for improving the feasibility of dietary mobile applications. Twenty-four-hour recall, food records, and food frequency questionnaires (FFQs) have been widely used for dietary assessment in epidemiology studies, but none of these methods estimate dietary intake without error ,26. TherDuring the last few years, clinical trials for managing chronic diseases using ICT have been actively conducted. Use of websites or mobile devices as a self-monitoring system resulted in greater weight loss compared to conventional care in several weight loss intervention studies. In a 12-month randomized clinical trial of 69 obese adults, the PDA group lost over 3.8 kg more than the standard group . David &Diet-A has features that help individuals improve their dietary habits, including real-time feedback on selected nutrients, disease risk, and food recommendations. Provision of real-time data allows users to read immediate feedback or to get individualized dietary advice. In our study, 65.0% of participants who answered the question reported that this application was the way they monitored their dietary intake, and 61.9% of participants reported satisfaction with realizing their own dietary intake. Although the adolescents in the study favorably answered that they were able to monitor their diet, the present study did not show improvement in their diet when we compared their diet pre- and post-intervention. We selected adolescents in this intervention study because we believed that a mobile application could be a better alternative approach to measure their diet compared to conventional methods because adolescents had more confidence in the use of mobile technology than adults ,35. HoweThe study has several limitations. First, the sample was small and unrepresentative of the general population. Second, this application did not include a function to measure the user’s physical activity; estimated energy requirements were computed as ‘low-active’, assuming the typical exercise level of Korean high school students. It may be necessary to add a motion sensor function in future studies. We chose only one nutrient per disease for a short message targeting the prevention of metabolic disorders, but given that multiple factors play roles in the development of chronic disease, further studies are needed to implement various components.Diet-A is developed to provide users with real-time feedback on their diet and assess food and nutrient intakes. Diet-A is equipped with voice recognition and photography functions for accurate recording. It focuses on four chronic diseases, namely, obesity, diabetes, hypertension, and dyslipidemia, providing users with real-time feedback. However, dietary recording remains burdensome even with the application. Overall, our study revealed some challenges with regard to the use of our mobile application for dietary assessment and self-management of dietary habit, including underestimation, promotion of motivation to use, and improvement of the food and dish database.


Union rate and BV in the low-magnitude group of RFH model were also significantly higher than those in the other groups . BV/TV in the low-magnitude group of RFH model was significantly higher than that in control-VA group (59.4±14.9% v.s. 35.8±13.5%). On the other hand, radiographic union rate (10% in centrifuge acceleration group v.s. 20% in control-CA group) and micro-CT parameters in RFH model were not significantly different between two groups in the constant acceleration studies. Radiographic images of non-union rib fractures showed cartilage at the fracture site and poor new bone formation, whereas union samples showed only new bone. In conclusion, low-magnitude vibration acceleration promoted bone formation at the trunk in both BMP-induced ectopic bone formation and rib fracture healing models. However, the micro-CT parameters were not similar between two models, which suggested that there might be difference in the mechanism of effect by vibration between two models.All living tissues and cells on Earth are subject to gravitational acceleration, but no reports have verified whether acceleration mode influences bone formation and healing. Therefore, this study was to compare the effects of two acceleration modes, vibration and constant  accelerations, on bone formation and healing in the trunk using BMP 2-induced ectopic bone formation (EBF) mouse model and a rib fracture healing (RFH) rat model. Additionally, we tried to verify the difference in mechanism of effect on bone formation by accelerations between these two models. Three groups (low- and high-magnitude vibration and control-VA groups) were evaluated in the vibration acceleration study, and two groups (centrifuge acceleration and control-CA groups) were used in the constant acceleration study. In each model, the intervention was applied for ten minutes per day from three days after surgery for eleven days (EBF model) or nine days (RFH model). All animals were sacrificed the day after the intervention ended. In the EBF model, ectopic bone was evaluated by macroscopic and histological observations, wet weight, radiography and microfocus computed tomography (micro-CT). In the RFH model, whole fracture-repaired ribs were excised with removal of soft tissue, and evaluated radiologically and histologically. Ectopic bones in the low-magnitude group (EBF model) had significantly greater wet weight and were significantly larger  than those in the other two groups, whereas the size and wet weight of ectopic bones in the centrifuge acceleration group showed no significant difference compared those in control-CA group. All ectopic bones showed calcified trabeculae and maturated bone marrow. Micro-CT showed that bone volume (BV) in the low-magnitude group of EBF model was significantly higher than those in the other two groups (3.1±1.2mm Non-union and delayed union are clinical complications of bone fractures that can impair activities of daily living and may require additional surgery. Conservative treatments using physical stimuli have been developed. Low-intensity pulsed ultrasound (LIPUS) is one of the useful and representative treatment apparatus to promote fracture healing by its mechanical stimulus, micromotion, at fracture site . In addiIn vivo studies have shown that vibration acceleration can promote bone formation in young adults and decrease bone loss in elderly individuals and postmenopausal women as well as in both small and large animals [ animals –17. Thes animals –14, 17.  animals , 19. BotOur hypothesis was that vibration acceleration has a positive effect on bone formation and healing, because the direction and amplitude changes of acceleration are important factors to generate the mechanical stimulus that promote bone formation. However, the effects of accelerations are not same between two models leading to the different results in measured parameters.All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the Physiological Society of Japan. The study was also approved by the Animal Use Committee at the Osaka University Graduate School of Medicine .in vivo experimental rodent models: BMP-induced ectopic bone formation (EBF) and rib fracture healing (RFH) models [This study evaluated the effects of acceleration modes on bone formation and fracture healing using two ) models –23. Two ®  were implanted into a dorsal subfascial pocket of 40 five-week-old male ICR mice . The skin was sutured and the animals were then maintained in cages. The 40 mice were divided randomly into five groups. After operation, we observed feeding quantity and body weight and disinfected the wounds at least once a day. A significant change body weight was not found in mice postoperatively compared to those preoperatively. Each four mice were housed in a cage (20 x 10 cm and 10 cm height). A commercial solid diet  and water were supplied ad libitum. Temperature and humidity in the animal room with 12:12 hr light:dark cycle were maintained at 20~24°C and 40~60%, respectively.As described previously , under aThe rib fracture healing model was generated in 25 four-week-old female Wistar rats  using the method previously described . Each ra™ and animal centrifuge) between two acceleration models. Therefore, we set total two different control groups, one in each acceleration model. In vibration acceleration groups, animals in low-  and high-  magnitude groups were put in a cage on a Power Plate® Pro5HP™ . Actual acceleration in each group was measured using an accelerometer  in the cage . In the EBF model, ectopic bone was dissected from the dorsal subfascial pocket and the surrounding soft tissue was carefully removed. Each ectopic bone underwent macroscopic observation, wet weight measurement, radiological assessment by radiography and microfocus computed tomography (micro-CT), and histological evaluation. In the RFH model, both whole eighth ribs were cut from the vertebra and the surrounding soft tissue was excised. Radiological and histological evaluations were performed on the rib fracture sites. Fractured ribs were examined radiologically in two dimensions with a soft X ray apparatus  after sacrifice and judged to be healed or not as described previously . The fraFormation of new bone in each BMP/atelocollagen pellet or fracture site was evaluated using a micro-CT system . Each sample was scanned at 25-μm intervals at 50 kV and 200 μA. With an angular step of 0.3°over an angular range of 180°, 600 radiographic projections were acquired. The spatial resolution was 24μm. Next, 3-D images were reconstructed with a threshold of 1,000 Hounsfield units using TRI3D-BON software . Region of interest (ROI) in EBF model was whole ectopic bone. In RFH model, ROI was the space between 1-mm proximal and distal planes from the center of the bone defect; these planes were perpendicular to the bone axis . EvaluatSamples were decalcified with ethylenediaminetetraacetic acid (pH 7.4) after 10% neutral formalin fixation. After decalcification, the samples were dehydrated in a graded ethanol series, cut along the coronal plane at the center of ectopic bones or at the midline of the defect in fracture site, and embedded in paraffin. Sections (3 μm thickness) were each mounted on individual slides, and stained with hematoxylin and eosin (H&E) or safranin-O for observation under a light and polarized light microscope .All data are expressed as mean ± standard deviation (SD). The Mann-Whitney U test was used for statistical analysis of differences between groups. A statistical difference between experimental groups was regarded as significant when the p-value was <0.05.Body weights of animals before surgery and at sacrifice did not show any significant differences between groups in each model Tables  and 2.In vibration acceleration groups, ectopic bones in the low-magnitude group were macroscopically and radiographically larger than those in the high-magnitude and control-VA groups . The meaIn constant acceleration groups, there were no parameters with significant difference in the size of ectopic bone between the control-CA and centrifuge acceleration groups macroscopically or radiographically . MoreoveHistologically, all ectopic bones in all five groups were similar to those in previous reports. The calcified trabeculae and maturated bone marrow were visible .In vibration acceleration groups, the union rate of rib fracture in the low-magnitude group was higher than that in the other two groups (60% vs. 0% in the high-magnitude group and 10% in the control-VA group) . ResultsIn constant acceleration groups, the union rates of rib fracture between the centrifuge acceleration and control-CA groups have no significant differences (10% in the centrifuge acceleration group vs. 20% in the control-CA group) . SimilarHistologically, in radiographic non-union samples, cartilage filled the defect at the fracture site and new bone formation was poor, whereas only new bone filled in the defect in radiographic union samples. The osteoid was not observed in both non-union and union samples and no clear differences in cells associated with fracture healing such as osteoblast and osteoclasts in new bone formation region were found between both samples. .in vivo bone formation in humans and animals [The positive effects of acceleration by WBV training on bone healing or density have recently been established. Vibration acceleration can also promote  animals –17. All A previous study investigating the mechanism of vibration acceleration showed that controlled dynamic loading using varying frequencies (1 to 10 Hz) produced perturbations of the intermedullary pressure in adult female rats . ConsequLow-magnitude vibration acceleration promoted bone formation in both EBF and RFH models; however, micro-CT results differed slightly between models. In the EBF model, BV was significantly higher in the low-magnitude vibration group than those in the high-magnitude group and control-VA, while BV/TV remained unchanged. This suggests that the vibration acceleration influenced the quantity of BMP-induced bone matrix formation, possibly due to the promotion of cell migration, proliferation and differentiation of newly produced bone. On the other hand, in the RFH model, BV and BV/TV in the low-magnitude vibration group were significantly higher than those in the other one or two groups. These results suggest that the newly produced bone in RFH model has smaller pores like cortical bone than that in EBF model, as the rib is healing and returning to cortical bone-like structure. The vibration acceleration may also have some influence on the bone marrow cells associated with the differentiation of newly bone at the fracture site, resulting in the higher BV/TV. BMP-induced ectopic bone formation model was one of highly-reproducible model for investigation of bone formation and easy to apply intervention, whereas the condition in case of natural fracture can be reproduced in rib fracture healing model with bone marrow cells as well as cytokine including BMP at fracture site. If the evaluations of two acceleration modes between two models would not be performed, we had not known these results associated with clues to the mechanism of the effect of vibration acceleration on bone formation as well as its effect. Therefore, we adopted two acceleration modes for two models, EBF and RFH models. However, to verify the precise mechanism in detail, further investigations are required.Previous animal experiments showed that LMHF acceleration by WBV promoted fracture healing and increased bone mineral density –8, 9, 10in vivo time-course analysis including histological evaluation as well as an in vitro study.We acknowledge that this study has some limitations. First, the vibration magnitude and frequency were limited since our study used a power plate that is a vibration exercise machine for humans. Therefore, we have to modulate vibration frequency and magnitude to determine the optimal conditions for bone healing and formation. Second, the number of rodents in this study was small and thus if we concluded no significant difference from our results, it may be necessary to have the data in large number rodents. However, some significant differences were found in low-magnitude vibration group. Therefore, we concluded that low-magnitude vibration acceleration promoted bone formation and healing. Finally, though this study showed only the positive effect of WBV on bone healing and formation in small animals, the precise mechanisms of the effects are unknown. Regarding histological analysis, further evaluation including of analysis using immunostaining should be performed, but only simple findings was described in this study. In the near future, we hope to investigate the mechanism of vibration acceleration on the bone using an Low-magnitude vibration acceleration promoted bone formation at the trunk in both BMP-induced ectopic bone formation and rib fracture healing models. However, the micro-CT parameters were not similar between two models, which suggested that there might be difference in the mechanism of effect by vibration between two models.S1 FigControl (vibration), low-magnitude and high-magnitude acceleration groups in the vibration acceleration study, and control (constant) and centrifuge acceleration groups in the constant acceleration study. Low: low-magnitude acceleration group, High: high-magnitude acceleration group, Centrifuge: centrifugal acceleration group.(TIFF)Click here for additional data file.S2 FigControl (vibration), low-magnitude and high-magnitude acceleration groups in the vibration acceleration study, and control (constant) and centrifuge acceleration groups in the constant acceleration study. Low: low-magnitude acceleration group, High: high-magnitude acceleration group, Centrifuge: centrifugal acceleration group. Union: union ribs (left column), Non-union: non-union ribs (right column). (No union ribs in high-magnitude vibration acceleration group).(TIFF)Click here for additional data file.