Atherosclerosis (AS) is the primary cause of cardiovascular disease and the incidence is extremely common; however, there are currently few drugs that can effectively treat AS. Although oridonin has been widely used to treat inflammation and cancer for numerous years, to the best of our knowledge, its protective effect against AS has not been reported. Therefore, the present study aimed to investigate whether oridonin attenuated AS.2 LSS for 30 min and subsequently treated with oridonin or transfected with a small interfering RNA (siRNA) targeting nuclear factor erythroid 2-related factor 2 (NRF2). Reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH) and glutathione disulfide (GSSG) in EA.hy926 cells were analyzed to determine the level of oxidative stress. The nitric oxide (NO) levels and mRNA expression levels of endothelial NO synthase (eNOS), endothelin-1 (ET-1) and prostaglandin synthase (PGIS) in EA.hy926 cells were analyzed to determine EC dysfunction. Furthermore, the mRNA and protein expression levels of NRF2 were analyzed using reverse transcription-quantitative PCR and western blot. In addition, zebrafish were fed with a high-cholesterol diet to establish a zebrafish AS model, which was used to observe lipid accumulation and inflammation under a fluorescence microscope.By using text mining, chemometric and chemogenomic methods, oridonin was predicted to be a beneficial agent for the treatment of AS. A parallel flow chamber was used to establish a low shear stress (LSS)-induced endothelial cell (EC) dysfunction model. Briefly, ECs were exposed to 3 dyn/cmWe found LSS led to oxidative stress and EC dysfunction; this was primarily indicated through the significantly decreased SOD and GSH content, the significantly increased MDA, GSSG and ROS content, the upregulated mRNA expression levels of ET-1, and the downregulated NO levels and mRNA expression levels of eNOS and PGIS in ECs. Notably, oridonin could improve LSS-induced oxidative stress and EC dysfunction, and the effects of oridonin were reversed by the transfection with NRF2 siRNA. Oridonin also attenuated lipid accumulation and neutrophil recruitment at the LSS regions in the zebrafish AS model.In conclusion, the results of the present study suggested that oridonin may ameliorate LSS-induced EC dysfunction and oxidative stress by activating NRF2, thereby attenuating AS.The online version contains supplementary material available at 10.1186/s12906-022-03658-2. Atherosclerosis (AS) is characterized by endothelial dysfunction, inflammation, progressive lipid deposition and vessel stiffness, which is accompanied by potential complications such as myocardial infarction or stroke , 2. PrevRecently, network pharmacology has been proposed as a novel discipline to investigate the complex interactions of compounds and biology. The potential targets of compounds are identified on a proteome-wide scale with computational ligand-based target prediction methods . MultiplRabdosia rubescens, was proven to possess antitumor activity, and it has been used to treat liver, esophageal and pancreatic cancer in the clinic [Oridonin, the major active ingredient of the traditional Chinese medicinal herb e clinic –11. Preve clinic , 13. HowAlthough AS has numerous systemic risk factors, such as hypertension, obesity and smoking, it preferentially develops near branches and bends in blood vessels, where the ECs are exposed to oscillatory or low shear stress (LSS) . Since tThe current study used both a LSS-induced EC dysfunction model and a high-cholesterol diet (HCD)-induced zebrafish AS model to verify the target, which was identified previously. The findings revealed that oridonin ameliorated LSS-induced EC dysfunction by activating NRF2, thereby attenuating AS.Oridonin (NSC-250682) was purchased from Selleck Chemicals. MTT reagent (11465007001) was purchased from Sigma-Aldrich; Merck KGaA. The other chemicals and reagents were of analytical grade.https://tcmspw.com/tcmsp.php), a database comprised of systems pharmacology data for drug discovery related to herbal medicines [http://sea.bkslab.org) was used to predict potential targets of oridonin. Finally, the potential target proteins identified were further subjected to The Pharmacogenomics Knowledge Base (http://pharmgkb.org), and Comparative Toxicogenomics Database (http://ctdbase.org) to remove noise and errors. In addition, this enabled an accurate view of oridonin targets for cardiovascular diseases, including ADCY1, ADCY2, NRF2 and NROB1  for 24 h. The cell viability was determined using an MTT assay, with ≥3 independent experiments performed in triplicate.The HUVEC cell line, EA.hy926, was obtained from the American Type Culture Collection. EA.hy926 cells were cultured in DMEM  supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, and maintained at 37 °C in a 5% COUpon EA.hy926 cells reaching the logarithmic growth phase, the cells were seeded onto a glass slide (30 × 50 mm) and treated with 0.1% DMSO, 100 μg/ml oridonin or 100 μg/ml oridonin combined with the transfection of 0.1 μmol/l small interfering RNA (siRNA) targeting NRF2  for 24 h. Following treatment for 24 h, the LSS test was performed. Briefly, a parallel flow chamber , which consists of two stainless steel plates and a silicone gasket, was used in the present study. The glass slide with confluent cells was placed on the lower plate of the chamber and then subjected to LSS induced by continuous fluid flow. SS values were modulated by the flow through the chamber.The NO levels, and ET-1, endothelial NO synthase (eNOS) and PGIS mRNA expression levels in EA.hy926 cells were analyzed to determine EC dysfunction. Following the LSS experiment, EA.hy926 cells were incubated with 50 μM NO-specific fluorescent dye, 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein diacetate . Following the incubation, the EA.hy926 cells were washed twice with PBS and then visualized and analyzed via fluorescence microscopy. The fluorescence intensity was semi-quantified from ≥3 random fields of view per slide from three different slides.The mRNA expression levels of ET-1, eNOS and PGIS in EA.hy926 cells were analyzed using reverse transcription-quantitative PCR (RT-qPCR). Briefly, following the LSS experiment, total RNA was extracted from the cells using TRIzol® reagent , according to the manufacturer’s protocol. Total RNA was reverse transcribed into cDNA using the PrimeScript™ RT Master mix  , according to the manufacturer’s protocol. The resultant cDNA was used as a template for qPCR analysis in the Thermal Cycler Dice® Real-Time system . The primers used for the qPCR were designed by Primer3 software and are listed in Table Following the LSS experiment, EA.hy926 cells were collected to measure secreted superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, glutathione (GSH) content and glutathione disulfide (GSSG) content using ELISAs , according to the manufacturers’ protocols.Following the LSS experiment, EA.hy926 cells were also incubated with the ROS-specific fluorescent dye dihydroethidium . Following the incubation, the EA.hy926 cells were washed in PBS twice and then visualized and analyzed via fluorescence microscopy. The fluorescence intensity was semi-quantified from ≥3 random fields of view per slide from three different slides.Following the LSS experiment, EA.hy926 cells were harvested for Western blot analysis. Briefly, the proteins were extracted using RIPA buffer . According to the manufacturer’s protocol, protein concentrations were determined by the BCA protein assay kit , and equal amounts of proteins were resolved on the 10% SDS-PAGE gel followed by transferring to the PVDF membranes. After blocked with 5% skim milk for 90 min at room temperature, the blots were incubated at 4 °C overnight with primary antibodies against NRF2 , HMOX1 , eNOS , SOD2 , and Beta Actin  as needed. After that, the membranes were further probed by the HRP-conjugated secondary antibodies. Blotted protein bands were visualized with enhanced chemiluminescence detection reagents (Thermo Fisher Scientific). Relative changes in protein expression were estimated from the mean pixel density using Image J, normalized to Beta Actin.Tg(fli1:EGFP) zebrafish larvae, constitutively expressing GFP in the ECs, were fed for 10 days with the HCD enriched with 4% cholesterol and supplemented with 10 μg/g fluorescent cholesteryl ester analog. A large amount of red fluorescent lipid accumulation in the zebrafish green blood vessels could be observed under a fluorescence microscope.Zebrafish lines used in this research were purchased from China Zebrafish Resource Center . All studies involving zebrafish manipulations were approved by the institutional animal use and care committee of Nanjing Drum Tower Hospital. All animal experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. About the euthanasia of zebrafish, we put zebrafish larvae in a 2 g/L tricaine solution for about 1 minute for euthanasia, meanwhile we use the lost of righting reflex, opercular movements and the silence of heart beating to confirm death. The accumulation of lipids in zebrafish blood vessels was detected to reflect early atherosclerotic plaque formation. As determined from a previous study , zebrafiTg(fli1:EGFP) zebrafish larvae were randomly divided into five groups: i) Control group; ii) AS group; iii) 1 mg/l oridonin group; iv) 50 mg/l oridonin group; and v) 100 mg/l oridonin group. In the control group, the larvae were fed with 10 μg/g fluorescent cholesteryl ester analog for 10 days. In the AS group, the larvae were fed for 10 days with the HCD enriched with 4% cholesterol, and supplemented with 10 μg/g fluorescent cholesteryl ester analog. In the various concentrations of oridonin  treatment groups, the larvae were fed for 10 days with the HCD enriched with 4% cholesterol, and supplemented with 10 μg/g fluorescent cholesteryl ester analog. At the same time, the larvae were treated with various concentrations of oridonin . Images of the caudal vasculature in the live larvae revealed that the vasculature of the control and HCD larvae were stained diffusely red, with bright fluorescent lipid deposits in the blood vessel wall visible in the HCD larvae. A study previously reported that these accumulated lipids are similar to the plaques observed during early AS [The 5 dpf early AS .Tg(mpx:EGFP) zebrafish was subsequently observed through specifically labeling the neutrophils with GFP. In the control group, 5 dpf Tg(mpx:EGFP) zebrafish larvae were fed with conventional feed for 10 days. In the AS group, 5 dpf Tg(mpx:EGFP) zebrafish larvae were fed for 10 days with a HCD enriched with 4% cholesterol. In the various concentrations of oridonin  treatment groups, 5dpf Tg(mpx:EGFP) zebrafish larvae were fed for 10 days with the HCD enriched with 4% cholesterol, and treated with various concentrations of oridonin . The quantity and recruitment of neutrophils was observed under a fluorescence microscope to determine the levels of inflammation in each group of zebrafish.The presence of inflammation in The 5 dpf wild-type AB-line zebrafish larvae were randomly divided into five groups as said above. In the control group, zebrafish larvae were fed with normal basal feed (which did not contain 4% cholesterol) for 10 days. In the AS group, zebrafish larvae were fed a HCD enriched with 4% cholesterol for 10 days. In the various concentrations of oridonin  treatment groups, zebrafish larvae were fed for 10 days with an HCD enriched with 4% cholesterol and treated with various concentrations of oridonin . After 10 days of feeding and 24 h of fasting, Nile Red staining was used to detect the lipid levels in each group of zebrafish. The stock solution (1.25 mg/ml) of Nile Red  was prepared in acetone and stored in the dark at − 20 °C. For the staining of the zebrafish, the stock solution was diluted to 50 ng/ml in egg water and incubated for 15 min at 28 °C in the dark. The fishes were washed with distilled water 3 times and anesthetized with a few drops of tricaine solution . The zebrafish were subsequently mounted in 4% methylcellulose and the extent of Nile Red staining was imaged using an Olympus SZX16 microscope (Olympus Corporation), which was used for yellow fluorescent imaging.The 5 dpf wild-type AB-line zebrafish larvae were randomly divided into five groups as said above. The description of each group of zebrafish is consistent with that described in the section describing the detection of the lipid levels in the zebrafish AS model. After 10 days of feeding and 24 h of fasting, dichloro-dihydro-fluorescein diacetate was used to detect the levels of ROS in each group of zebrafish.http://www.njjcbio.com/), according to the manufacturer’s protocols. The results of the aforementioned assays were quantified using a multifunctional microplate reader.The 5 dpf wild-type AB-line zebrafish larvae were randomly divided into five groups as said above. The description of each group of zebrafish is consistent with that described in the section describing the detection of the lipid levels in the zebrafish AS model. After 10 days of feeding and 24 h of fasting, 5 larvae from each larva in each group were randomly selected and sacrificed as one sample, and six samples were prepared for testing each index. Triglyceride (TG) levels, total cholesterol (TC) levels, SOD activity and MDA levels were measured using commercial assay kits  and data are presented as the mean ± standard deviation. The normal distribution was checked with the Shapiro-Wilk test. Statistical differences between groups were determined using an ANOVA with Bonferroni adjustment for multiple comparisons. Through combining text mining and chemogenomic prediction methods, 4 potential protein targets for oridonin were obtained Table . It was EA.hy926 cells were treated with various concentrations of oridonin  for 24 h, and the cell viability was determined using an MTT assay at 24 h. As shown in Fig. 2 for 30 min. A siRNA sequence targeting NRF2 was used to silence its expression, which was subsequently verified by RT-qPCR and western blot  zebrafish fed the HCD  zebrafish fed the HCD, while the treatment with 50 or 100 μg/ml oridonin appeared to decrease the HCD-induced increase in neutrophils , NO and ET-1, are synthesized and released by ECs. PGI2 and NO are effective vasodilators and are produced by PGIS and eNOS, respectively, while ET-1 is a potent vasoconstrictor [LSS has been reported to serve an important role in the occurrence of EC dysfunction and AS . ECs, thstrictor . In the Tg(mpx:EGFP) zebrafish indicate that HCD will increase the level of inflammation in zebrafish, and oridonin can effectively inhibit the increase in inflammation caused by HCD.LSS was previously found to increase oxidative stress levels in ECs . The finNRF2 is an important mechanosensitive transcription factor that regulates the expression of a group of genes responsible for inflammation and oxidative stress. Thus, it is considered to have a close relationship with EC dysfunction and AS. In the high/laminar SS regions of the vasculature, NRF2 was discovered to be activated, where it participated in the adaptative mechanism of the ECs to oxidative and inflammatory stress . In the To investigate whether oridonin treatment attenuated AS, zebrafish were fed a HCD to establish a zebrafish AS model. The zebrafish is a unique vertebrate model that combines the advantageous characteristics of invertebrate models  with a high degree of evolutionary conservation with mammals. Thus, zebrafish are invaluable, not only for studying vertebrate development and physiology but also for modeling human diseases . ZebrafiIt is generally considered that hyperlipidemia, inflammation and oxidative stress lead to the initiation and development of AS –31. In tThere are many limitations in this study, we revealed the potential of oridonin in the treatment of atherosclerosis, and briefly studied the role of NRF2 in the treatment of atherosclerosis. The mechanism of our research is not deep enough and we will conduct more research in subsequent experiments to specify how dose oridonin overcome shear stress-induced endothelial dysfunction and oxidative stress. A fluorescence quantification by flow cytometry will be a good supplementary result to show the changes between intracellular NO activity and we will supplement it in later experiments. Most importantly, we only found that oridonin can affect the expression of NRF2, but whether NRF2 is the target of oridonin needs more research in the future.In conclusion, the findings of the present study revealed that oridonin exerted effects in LSS-induced EC dysfunction and HCD-induced hyperlipidemia, inflammation and oxidative stress, and inhibited plaque formation in AS as observed from a sequence of testing in-silico target identification, human umbilical culture, and a zebrafish model. Therefore, in the future, oridonin may be considered as a potential therapeutic option for AS.Additional file 1: Figure S1. Effects of NRF2 siRNA on NRF2 mRNA without any other treatment. ##P < 0.01 when compared with si-NC, NRF2, nuclear factor erythroid 2-related factor 2, siRNA, small interfering RNA.Additional file 2: Figure S2. Original images of western blot in Fig. Additional file 3: Figure S3. Original images of western blot in Fig. Additional file 4: Figure S4. Original images of western blot in Fig.


FAN1, MSH3, BDNF, COMT and MAPT in predicting cognitive decline and brain atrophy. We found that carrying the 3a allele in the MSH3 gene had a positive effect on global cognitive function and brain atrophy in multiple cortical regions, such that 3a allele carriers had a slower rate of cognitive decline and atrophy compared with non-carriers, in agreement with its role in somatic instability. No other genetic predictor had a significant effect on cognitive function and the effect of MSH3 was independent of intellectual enrichment. Intellectual enrichment also had a positive effect on cognitive function; participants with higher intellectual enrichment, i.e. those who were better educated, had higher verbal intelligence and performed an occupation that was intellectually engaging, had better cognitive function overall, in agreement with previous studies in Huntington’s disease and other dementias. We also found that intellectual enrichment interacted with the BDNF gene, such that the positive effect of intellectual enrichment was greater in Met66 allele carriers than non-carriers. A similar relationship was also identified for changes in whole brain and caudate volume; the positive effect of intellectual enrichment was greater for Met66 allele carriers, rather than for non-carriers. In summary, our study provides additional evidence for the beneficial role of intellectual enrichment and carrying the 3a allele in MSH3 in cognitive function in Huntington’s disease and their effect on brain structure.An important step towards the development of treatments for cognitive impairment in ageing and neurodegenerative diseases is to identify genetic and environmental modifiers of cognitive function and understand the mechanism by which they exert an effect. In Huntington’s disease, the most common autosomal dominant dementia, a small number of studies have identified intellectual enrichment, i.e. a cognitively stimulating lifestyle and genetic polymorphisms as potential modifiers of cognitive function. The aim of our study was to further investigate the relationship and interaction between genetic factors and intellectual enrichment on cognitive function and brain atrophy in Huntington’s disease. For this purpose, we analysed data from Track-HD, a multi-centre longitudinal study in Huntington’s disease gene carriers and focused on the role of intellectual enrichment (estimated at baseline) and the genes  et al. showed that cognitive function in Huntington’s disease is affected by an interplay between genetic and environmental factors. 3a allele carriers had slower cognitive decline and cortical atrophy. In addition, high intellectual enrichment predicted slower cognitive decline and counteracted the detrimental effect of the Met66 allele in brain-derived neurotrophic factor.Papoutsi https://doi.org/10.1093/braincomms/fcac308) for a scientific commentary on this article.See Hannan (  It is characterized by a triad of symptoms, motor, psychiatric and cognitive. All Huntington’s disease gene carriers will eventually develop dementia,,1 but there is substantial variability in its onset and severity, which cannot be explained fully by CAG repeat length and age. Cognitive impairment is present in Huntington’s disease gene carriers many years before predicted disease onset and in the absence of motor symptoms.2 However, research on the genetic and environmental factors that contribute to this variability in cognitive impairment in Huntington’s disease is still limited.Huntington’s disease is a genetic, neurodegenerative disorder caused by an abnormal coronary artery angiography (CAG) repeat expansion in the Huntingtin ,14 catechol-O-methyl transferase (COMT),15 MutS homologue 3 (MSH3)16 and microtubule-associated protein tau (MAPT),17 predict cognitive function. Two of these studies have also provided preliminary evidence that intellectual enrichment is associated with less striatal atrophy in humans,13 suggesting that it supports greater brain maintenance.In Huntington’s disease, a small number of studies have so far examined the role of genetic polymorphisms and lifestyle factors on individual differences in cognitive function. More specifically, environmental enrichment,18–21 that measured changes in behaviour and brain structure over 3 years in individuals with the Huntington’s disease gene mutation in pre-manifest (maximum 15 years from predicted onset) and early stages of the disease. We quantified lifetime intellectual enrichment using level of education, pre-morbid intelligence quotient (IQ) and occupational cognitive demands12 measured at baseline. In terms of genetic polymorphisms, we selected common polymorphisms that have been previously associated with cognitive function in Huntington’s disease .The aim of our work was to provide evidence regarding the effects of intellectual enrichment and genetic factors on cognitive function and brain structure in Huntington’s disease. For this purpose, we retrospectively analysed data from a multi-centre, longitudinal study, Track-HD,18–21 In summary, 243 Huntington’s disease gene carriers (both manifest and pre-manifest) and 123 matched controls were recruited across four sites . The participants were predominantly Caucasian (97.5%), which limits our ability to test differential effects across different populations. However, it is important to note that Huntington’s disease is predominantly found in individuals with European ancestry.22 Local ethics committees approved the study at each site and all participants provided written informed consent according to the Declaration of Helsinki.Track-HD is a multi-centre, 4-year observational study in Huntington’s disease gene carriers and matched controls. A full description of the Track-HD study has been previously reported.n = 229), irrespective of disease diagnosis, were used for the analyses. Data from the matched control group were only used to create standardized scores of cognitive performance in the gene-carrier group , number correct in 45 s from the stroop word reading test (a measure of psychomotor speed), number correct adjusted for guessing (>0 means better than chance) for the five items condition from the spot the change task (a measure of working memory), number correct for negative emotions from the emotion recognition task  and variability in the inter-tap interval in a paced tapping task at 3 Hz . These tasks were included in all four visits, in addition to the circle tracing task. However, that task was excluded from the composite score because of large practice effects that persisted across all visits.23 The remaining five measures were then used to create a composite score of global cognitive function.To quantify cognitive function and change over 3 years, we created a composite score from the available cognitive measures. This composite score represents global cognitive function and is composed of the following measures from the Track-HD cognitive batteryZ-scores using the mean and standard deviation (SD) of the control group at baseline and then summed. In the paced tapping task, the reciprocal of the variance in the inter-tap interval was used, such that a higher value indicated better performance (i.e. lower variability), consistent with all other measures. Therefore, higher values in the composite score indicate better performance. When computing the composite score, if one or two measures were missing, they were replaced by the mean Z-score of the existing measures. This was the case for 21 participants in Visit 1, 7 participants in Visit 2, 3 participants in Visit 3 and 16 participants in Visit 4, respectively. If a participant had less than three out of five measures available for a visit, then we did not compute a composite score for that visit and therefore, this participant visit was not included in the analyses with cognitive function as a variable.To calculate the composite score, raw values were transformed to 24 and genotypes were extracted using PLINK software.25 Because the main outcome measure was cognitive function, we focused on single-nucleotide polymorphisms (SNPs) that had been previously associated with cognitive and psychomotor function in Huntington’s disease. Based on previous literature, we, therefore, selected the following SNPs: rs4680 on Chromosome 2215 in COMT, which relates to dopamine metabolism, rs9468 on Chromosome 1717 in MAPT, which relates to tau protein production, rs2140734 on Chromosome 15 near FAN1,26 which is involved in DNA repair and a polymorphic repeat expansion in Exon 1 on Chromosome 5 of MSH3,28 which is involved in DNA mismatch repair. In addition, we tested another polymorphism, rs6265  on Chromosome 11 in BDNF, which encodes the Val66Met polymorphism and regulates BDNF expression. Although the role of the Val66Met polymorphism in Huntington’s disease remains unknown, it has been consistently associated with cognitive function in ageing and dementia29–31 and BNDF expression may be affected by Huntington’s disease pathology.33 It also interacts with intellectual enrichment factors to predict cognitive function34: it was therefore relevant to our research question.Track-HD subjects were genotyped using Illumina Omni2.5v1.1 arraysTREM2 (rs75932628) has a role in cognitive function in Huntington’s disease.35 However, the minor allele frequency is very low in the population (0.005% in Europeans in 1000 genomes project data Phase 3) and therefore, we did not include it in our analyses.A previous study showed that the gene et al.17MSH3 predictor was coded for the presence of the three-repeat allele (3a).27 The rs2140734 (FAN1) predictor was coded for the presence of the minor allele G. It is also important to highlight that this SNP is in complete linkage disequilibrium with minor allele C in rs3512. The latter has been more widely examined in other studies and shown to be associated with age of onset and disease progression in Huntington’s disease.36 The MAPT predictor distinguished between H1 haplotype homozygotes and H2 carriers. Carrying the minor allele C in rs9468 tags for the H2 haplotype, whereas carrying the allele T tags for the H1 haplotype.37 The COMT (rs4680) predictor variable distinguished Met158 homozygotes from carriers of the Val158 allele.15 The BDNF (rs6265) predictor variable distinguished carriers from non-carriers of the detrimental allele, Met66.All genetic predictors were coded as having a binary, dominant effect, similar to the approach of Vuono BDNF, because there was an insufficient number of cases . Occupational cognitive requirements39 were estimated from the main profession recorded for each participant  = age × (CAG—35.5)].18–21 Measures of white matter integrity using diffusion weighted imaging were only introduced at Visit 4 in Track-HD; therefore, we only focused on GM volume in our study. Whole-brain T1-weighted 3D magnetization-prepared rapid acquisition with gradient echo images were acquired at 3T at all four visits . Caudate volume at baseline and longitudinal change was measured using medical image display and analysis software (MIDAS)’ semi-automated segmentation and the boundary shift integral, respectively.43 Total GM volume at baseline was measured using statistical parametric mapping 12 (SPM12). Longitudinal change was measured using a non-linear fluid registration method in MIDAS, which produced whole-brain voxel compression maps measuring change from baseline.44 Voxel compression maps were then convolved with SPM-derived GM maps to generate a change in total GM over time. The measures of caudate volume and total GM volume used in all the analyses were transformed to per cent of total intracranial volume (TIV), in order to adjust for differences in brain size. TIV was measured at baseline using MIDAS.Pathology at baseline and rate of change were quantified using structural MRI measures of caudate volume and total grey matter (GM) volume, which are robust and well-defined markers of brain atrophy in Huntington’s disease.45 The GM probability maps at baseline and voxel compression maps of change from baseline were normalized to a group template space using diffeomorphic anatomical registration through exponentiated lie algebra. Normalized images were then smoothed using an 8 mm full-width at half maximum Gaussian kernel. Full details of the MRI methods used have been published previously.46In addition to caudate and total GM volume, we also performed exploratory whole-brain analyses using voxel-based morphometry (VBM).http://www.r-project.org/) and the packages lmertest (version 3.1-1) and lme4 (version 1.1-21).Statistical analyses were performed using R version 3.6.3 . Models with cognitive function as an outcome also included the use of antipsychotic medication as a covariate see . Time wa centred .P-values and applying Bonferroni correction to control Type I error rate when multiple measures were used to test a hypothesis.Our hypotheses tested whether our variables of interest significantly predicted cognitive function or brain volume at baseline and changed over time. We used likelihood ratio tests to assess the covariate-adjusted significance of predictors on the outcome variables. We also tested for the significance of the interaction of genetic predictors with intellectual enrichment in the same way. In all the analyses, we visually inspected model residual distributions to assess plausible normality. No outliers were identified. Significance was established using two-sided P < 0.001 uncorrected at voxel level and P < 0.05 family-wise error corrected at cluster level.For VBM analyses, a binary GM mask was created using the mean normalized images from all Huntington’s disease gene carriers. This was used in all analyses. Statistical maps were thresholded at two-tailed info@chdifoundation.org.Track-HD data are available upon request after appropriate data use agreements are signed by the study funder, the CHDI Foundation. Please direct inquiries to 13 Furthermore, among pre-manifest gene carriers who are closer to predicted disease onset, those with high intellectual enrichment have a slower rate of cognitive decline than those with low intellectual enrichment.12Previous research showed that participants with early stage Huntington’s disease who had higher intellectual enrichment had better cognitive performance than those with lower intellectual enrichment.P < 0.001; t(229) = 6.744, P < 0.001.In our cohort of participants with pre-manifest and early stage Huntington’s disease, intellectual enrichment predicted mean global cognitive function and there was also a significant three-way interaction between intellectual enrichment, DBS and time  on global cognitive function (both t(224) = 2.043, P = 0.042). In contrast, an individual with 407.4 DBS (1SD above mean) and 1SD above mean intellectual enrichment had faster cognitive decline than an individual with the same DBS but 1SD below mean intellectual enrichment [slope estimate (95% CI) = −0.741  and −0.413 ] for high and low intellectual enrichment, respectively; contrast estimate (SE) high versus low = −0.327 (0.110), t(247) = −2.971, P = 0.003). To aid with interpretation of this finding, we repeated the same analysis, replacing DBS with group as an ordered factor, coding for manifest and pre-manifest individuals. There was a significant main effect of intellectual enrichment; however, the group by visit by intellectual enrichment interaction was not significant in this case  at baseline and the presence of depressive symptoms at baseline with their interactions with visit as confounds. Adding these factors did not alter the results, both the main effect of intellectual enrichment and the three-way interaction between intellectual enrichment, DBS and visit remained significant .pbon = 0.024; t(215) = 2.534, P = 0.012). However, there was no difference in participants with high DBS (for 407.4 DBS contrast estimate (SE) for high versus low intellectual enrichment was −0.0141 (0.135), t(215) = −1.044, P = 0.298). As previously, we repeated the same analysis, replacing DBS with the group as an ordered factor, coding for manifest versus pre-manifest individuals. There was no significant group by intellectual enrichment interaction on caudate volume at baseline  = −0.329  and −0.118 ] for non-carriers and carriers, respectively; contrast estimate (SE) non-carriers versus carriers = −0.21 (0.081), t(213) = −2.587, P = 0.010; MSH3, FAN1, COMT and MAPT. There was no change in the results  = −0.181  and −0.122 ] for non-carriers and carriers, respectively; contrast estimate (SE) for non-carriers versus carriers = −0.0586 (0.0168), t(196) = −3.486, P < 0.001).We next examined the relationship between MSH3 on GM volume across the whole brain. There was a significant effect of MSH3 on GM volume at baseline in the right middle temporal gyrus, the right inferior occipital gyrus and the left post-central gyrus  had higher volume and a slower rate of GM atrophy compared with non-carriers. Our results therefore suggest that carrying the 3a allele in MSH3 supported preserved cognitive function and was associated with a slower rate of neurodegeneration.Exploratory VBM analyses examined the main effect of al gyrus . There wrecuneus . Lastly,al gyrus . The eff8 To test this hypothesis in our study, we examined the interaction between all five genetic polymorphisms and intellectual enrichment on cognitive function and decline. BDNF was the only gene that significantly interacted with intellectual enrichment to predict global cognitive function at baseline  and high intellectual enrichment (1SD above mean) were: estimates (95% CI) = −6.31  and −2.57 , respectively; contrast estimate (SE) for high versus low intellectual enrichment = 3.74 (0.598), t(216) = 6.249, P < 0.001. Similarly, for Met66 allele non-carriers, with low (1SD below mean) and high intellectual enrichment (1SD above mean) estimate (95% CI) = −5.08  and −3.40 , respectively; contrast estimate (SE) for high versus low intellectual enrichment = 1.68 (0.483), t(214) = 3.486, P < 0.001. The difference between high and low intellectual enrichment was greater for Met66 allele carriers than for non-carriers, suggesting that the Met66 allele moderates the effect of intellectual enrichment on cognitive function [contrast estimate (SE) = −2.06 (0.768), t(215) = −2.678, P = 0.008; Previous research in ageing and dementia has shown that environmental factors, including intellectual enrichment, interact with genetic polymorphisms in order to predict cognitive function.BDNF interacts with intellectual enrichment to impact cognitive function, we examined the interaction between BDNF and intellectual enrichment on brain volume. The effect of the interaction of BDNF with intellectual enrichment was significant but small on both caudate and total GM volume rate of change  = 1.900, P = 0.059. Met66 allele non-carriers with high (1SD above mean) and low intellectual enrichment (1SD below mean) estimate (95% CI) = −0.0103  and −0.0094 , respectively; contrast estimate (SE) = −0.0010 (0.0007), t(205) = −1.480, P = 0.140. The difference between high and low intellectual enrichment was positive for Met66 allele carriers, but negative for non-carriers, suggesting that the Met66 allele moderates the effect of intellectual enrichment on caudate atrophy rate [contrast estimate (SE) = −0.0026 (0.0011), t(221) = −2.406, P = 0.017; To understand the mechanism by which t(214) = 2.658, P = 0.008. Met66 allele non-carriers with high (1SD above mean) and low intellectual enrichment (1SD below mean) estimate (95% CI) = −0.179  and −0.132 , respectively; contrast estimate (SE) = −0.0469 (0.0218), t(192) = −2.154, P = 0.0324. The difference between high and low intellectual enrichment was positive for Met66 allele carriers but negative for non-carriers, suggesting that the Met66 allele moderates the effect of intellectual enrichment on total GM atrophy rate [contrast estimate (SE) = −0.124 (0.0362), t(207) = −3.421, P = 0.008; This was similar to total GM volume. Met66 allele carriers with high (1SD above mean) and low intellectual enrichment (1SD below mean) estimate (95% CI) = −0.129  and −0.206 , respectively; contrast estimate (SE) = 0.0769 (0.0289), BDNF and intellectual enrichment on GM volume at baseline and change over time, but we did not identify any significant clusters.Exploratory whole-brain VBM analyses examined the interaction between MSH3 are independently associated with global cognitive function and brain structure, whereas intellectual enrichment interacts with BDNF to attenuate the deleterious effect of the Met66 polymorphism.The aim of our study was to examine the role of intellectual enrichment and genetic polymorphisms on cognitive function and brain structure in Huntington’s disease. Our results highlight the complexity of the interplay between environmental and genetic factors on behaviour and brain structure. Intellectual enrichment and genetic variation in 12 In the present work, we further show that the composite intellectual enrichment score explains additional variance in education. We also show that in participants with high DBS , those with high intellectual enrichment had a faster rate of decline over 3 years  than those with lower intellectual enrichment  stages of the disease.richment . The fasBDNF gene variation. The difference in cognitive function between individuals with low and high intellectual enrichment was greater in Met66 allele carriers than in non-carriers. Previously, comparing the blood expression levels of BDNF between 22 controls and 62 manifest gene carriers from this cohort using RNAseq, we found that Huntington’s disease gene carriers had lower levels of BDNF expression than controls (P = 0.04266).48 Post-mortem studies have also identified reduced BDNF levels in the striatum of Huntington’s disease patients. However, it is unclear whether this is due to defects in the delivery of cortical BDNF49 or to the response in the striatum.33 It is possible that carrying the Met66 allele exacerbates existing defects in the BDNF pathway. Previous research in animal models of Huntington’s disease showed that such defects can be rescued by environmental enrichment.50 Our results are in broad agreement with these findings and suggest that intellectual enrichment potentially counteracts the detrimental effect of the Met66 allele in both cognitive function and brain structure . Our findings are also in agreement with previous studies in ageing showing a significant interaction between intellectual enrichment and BDNF to predict cognitive function and decline in healthy older adults.34Further insights regarding the mechanism by which intellectual enrichment affects cognitive function are provided by the interaction between intellectual enrichment and MSH3. A recent study16 has identified MSH3 as a modifier of cognitive function in Huntington’s disease rather than motor function, while we have previously shown in this cohort that variation in MSH3, specifically carrying a 3a allele, has a protective effect on a composite score of disease progression, which included cognitive and psychomotor function.27 It is currently hypothesized that MSH3 is introducing an expansion of the HTT CAG repeat in the process of repair. Greater expansion is associated with earlier disease onset and faster progression,52 whereas carrying the 3a allele is associated with reduced expression of MSH3 and therefore reduced somatic expansion and slower progression.27 In agreement with this finding, in the present work, we further show that carrying the 3a allele in MSH3 was associated with slower GM atrophy across different regions in the cortex, including the inferior temporal and inferior frontal gyri. The absence of a significant effect in the striatum is notable given previous work, which showed that there is large somatic expansion in both the cortex  and the striatum.53 This finding could be explained by the fact that all analyses were adjusted for differences in DBS and suggests that carrying the 3a allele does not explain additional variance in striatal atrophy. Lastly, it is important to note that the protective effect of carrying the 3a allele in MSH3 is a result of reducing the expression of MSH3 and there is no evidence that it supports neuroprotective mechanisms. The effect of MSH3 on cognitive function and brain volume cannot therefore be interpreted as brain maintenance.Intellectual enrichment did not interact with any of the other genetic polymorphisms we examined, whereas the only genetic predictor with a significant effect on cognitive function was variation in MSH3, variation in FAN1 (rs3512) has also been implicated in somaticinstability52 and has been previously shown to predict delayed age of onset in Huntington’s disease.36FAN1 overexpression reduces CAG repeat expansion in human cell modes54; however, in our study, there was no significant effect of rs2140734 (or rs3512) on cognitive function. The reason for the contradictory findings is unclear. It could be due to a lack of statistical power given the relatively small sample size, but recent work suggests a differential effect of FAN1 on motor rather than cognitive function.16Similar to COMT and MAPT and cognitive function. A previous study15 showed that COMT Val158 allele carriers had slower cognitive decline compared with Met158 homozygotes in manifest Huntington’s disease patients. The number of Met158 homozygotes was low in our cohort  and our cohort included both pre-manifest and patients at early stages of the disease, which could explain the contradictory findings. Variation in the MAPT gene has also been previously shown to predict cognitive decline in Huntington’s disease,17 such that H1 homozygotes had a slower decline in cognitive function compared with H2 carriers. However, this effect was the opposite to what has been previously reported for Parkinson’s disease,55 and had a small effect size (r = −0.14). In our study, we did not find strong evidence for an association between MAPT and cognitive function; it is therefore currently unclear whether MAPT plays a role in cognitive function in Huntington’s disease.Lastly, in contrast to previous studies in Huntington’s disease, we did not find any evidence for an association between the genes MSH3, and further showed that slower cognitive decline is supported by the slowing of GM volume atrophy in the cortex, in agreement with the role of MSH3 in somatic instability. Intellectual enrichment also appears to have a protective effect on cognitive function at pre-manifest stages of the disease, but as the disease progresses, this effect is attenuated and there is faster neurodegeneration and cognitive decline. Importantly, we also observed a significant interaction between intellectual enrichment and the BDNF gene, whereby intellectual enrichment counteracted the detrimental effect of carrying the Met66 allele on cognitive function and brain structure, in agreement with the role of intellectual enrichment in enhancing brain trophic support. Future research is now needed to develop and evaluate intellectual enrichment interventions in Huntington’s disease and measure their impact on both behaviour and brain structure.In summary, we have shown that cognitive function in Huntington’s disease is affected by an interplay between genetic and environmental factors. We have replicated previous findings that cognitive decline is slower in carriers of the 3a allele in the gene fcac279_Supplementary_DataClick here for additional data file.


A weak correlation was found between PPi and CAC ; however, there was no statistically significant connection with LLAC  or a severity score . Surprisingly, we found no significant correlation between plasma alkaline phosphatase activity and PPi  or between a 10-year cardiovascular risk score and all other variables. Multivariate analysis confirmed that LLAC and CAC were strongly dependent on age, but not on PPi. Our data showed that arterial calcification is only weakly linked to circulating PPi levels and that time  appears to be the major determinant of disease severity and calcification in PXE. These data are important to better understand the natural history of this disease but also for the follow-up and management of patients, and the design of future clinical trials. Our results also show that PPi is not a good biomarker for the evaluation of disease severity and progression.Pseudoxanthoma elasticum  is an autosomal recessive metabolic disorder characterized by progressive calcification in the skin, the Bruch’s membrane, and the vasculature. Calcification in PXE results from a low level of circulating pyrophosphate (PPi) caused by ABCC6 deficiency. In this study, we used a cohort of 107 PXE patients to determine the pathophysiological relationship between plasma PPi, coronary calcification (CAC), lower limbs arterial calcification (LLAC), and disease severity. Overall, our data showed a deficit in plasma PPi in PXE patients compared to controls. Remarkably, affected females showed higher PPi levels than males, but a lower LLAC. There was a strong correlation between age and PPi in PXE patients (r = 0.423,  ABCC6 gene which encodes an ABC transporter primarily found in hepatic and renal tissues. Peripheral arterial disease is one of the major causes of morbidity in PXE [Pseudoxanthoma elasticum , is an autosomal recessive metabolic inherited disorder characterized by progressive calcification in the skin, the Bruch’s membrane, and the vasculature ,2,3. PXEy in PXE  and a hiy in PXE . In 2013Thus, ABCC6 deficiency leads to a significant deficit in circulating pyrophosphate (PPi), a major inhibitor of soft tissue calcification .Up to now, the connection between calcification susceptibility and reduced plasma PPi has essentially been studied in KO mice for ABCC6 ,12,13,14In the present study, we examined the correlation between arterial calcification, disease severity, and PPi level in a cohort of PXE patients.https://heartscore.escardio.org/2016/quickcalculator.aspx?model=EuropeLow, accessed on 1 January 2021). The renal function was determined using the MDRD-4 equation.Medical records from 232 PXE patients from the French-based PXE reference center cohort localized at the University Hospital of Angers were analyzed retrospectively. One hundred and seven PXE patients were selected from this cohort based on biological, radiological, and clinical data. All PXE patients were subjected to a comprehensive screening program including dermatological (including skin biopsy), ophthalmological, and vascular examinations every 1–3 years by trained clinicians. The diagnosis of PXE was based on the revised criteria by Plomp et al. [The study was approved by the French ethical committee (reference DC 2011-1467 and AC-2014-2329) and ClinicalTrials.gov, accessed on 1 January 2022, #NCT01446380.2 area/lesion. Lower limb arterial calcification (LLAC) scoring was performed on the volume/density data normalized to limb length . The LLAC score (expressed in HU/mm) was calculated as the sum of all segmental scores of both legs and normalized to the arterial lengths. The interobserver reliability of the analysis has been reported elsewhere [Vascular calcification from coronary and lower limb arteries was determined from a noncontrast CT scan with a 64-slice MSCT scanner . Cardiac CT angiography was performed using a collimation of 40 × 0.625 mm and a rotation time of 0.4 s. The tube current was adjusted to body weight (range 240–400 mA) and 120 kV. For the quantitative assessment of coronary artery calcification (CAC), the Agatston score was calculated (4), using a 2.5 mm CT slice thickness and a detection threshold ≥130 HU involving ≥1 mmn = 26, control cohort) showed a median value of 1.53 µmol/L (irq = 0.43 µmol/L) and a mean value of 1.63 ±0.07 µmol/L ±SEM).PPi quantification in ultrafiltrated patient plasma was performed using a modified method using ATP-sulfurylase  in the presence of APS  as previously described ,17. ValuPlasma ALP activity was assessed using paranitrophenol at alkaline pH and absorbance at 450 nm according to standard clinical protocols.p value <0.05 was considered significant, unless specified.Data are presented as mean (±SD) or median (interquartile: iqr) according to the distribution of the variables. Statistical univariate correlations between the variables were determined using the Pearson or Spearman correlation tests and multivariate analyses were performed on selected variables. Data were analyzed using Stata 12.0 software . A Data from the studied cohort are shown in Plasma PPi levels were measured twice in a subgroup of seven patients, including six females, within a three-year interval.Female patients showed higher PPi and Pi, a lower 10-year cardiovascular risk score, and significantly lower LLAC scores as compared to PXE men. However, CAC values did show differences between genders but trended down for PXE women. No significant differences in the PPi/Pi ratio, ALP, glomerular filtration, and PHENODEX score were found between men and women.p < 0.0001). Sixty-three percent of these samples  had PPi levels lower than the mean value measured in controls  plasma PPi was significantly lower in PXE patients (0.77 µmol/L (0.37 µmol/L)) than in healthy controls (1.53 µmol/L (0.43 µmol/L), rols see . Comparerols see .p = 0.018). Despite this increase, there was no significant change in Pi (1.06 mmol/L (0.27 µmol/L) vs. 1.09 mmol/L ((0.19 µmol/L), p = 0.735) in their PHENODEX score (8 (2) vs. 8 (1), p = 0.317) and ALP activity .We found that plasma PPi increased in all seven patients with repeated measurements , from an average of 0.82 µmol/L (0.21 µmol/L) to 1.14 µmol/L , LLAC , and the PHENODEX score , but not with ALP . Remarkably, the correlation between PPi level and age was only significant in females .Univariate analysis showed that plasma PPi correlated with age , though there was neither correlation with LLAC  nor with the PHENODEX score . The PHENODEX score strongly correlated with CAC  and LLAC . Surprisingly, there was no significant relationship between ALP and PPi concentration  as well as between the 10-year cardiovascular risk score and other variables. No significant association was found between PPi and age in healthy controls  were taking statins at the time of the study , and a disease severity score (PHENODEX) in a large cohort (>100) of PXE patients. These patients had low levels of plasma PPi, a major calcification inhibitor produced by the liver and kidneys which regulates ectopic calcification in soft connective tissues.Based on our methodology for PPi measurement, we found that the average plasma level was 50% lower in PXE patients than in control individuals, which is consistent with previous findings reported on PXE patients ,18. Furt−/− mice [A recent study of a pediatric cohort of 200 children who underwent blood testing for medical conditions not affecting plasma PPi levels revealed no significant difference between genders and only a decreasing trend  between age 0 to 18 . To our −/− mice ,22, but −/− mice [n = 18) reported a weak negative association between PPi and TNAP in both PXE and controls [−/− mice suggested that PPi could be freely filtered with age [The reason for plasma PPi increase with age in our PXE cohort is unknown and somewhat counter intuitive, as a previous report showed an increase in calcification and symptoms severity with age . A first−/− mice . A decrecontrols . The samcontrols . Howeverwith age . FinallyPXE is characterized by premature ectopic calcification in the wall of peripheral arteries, affecting mostly elastic fibers. This is responsible for the peripheral arterial disease associated with PXE ,26. The −/− mice model without affecting PPi level [−/− mice expressing a human ENPP1 transgene, mineralization still developed independently of PPi plasma level. Two separate studies by Pomozi et al. showed a restoration of mineralization inhibition in humanized ABCC6−/− mice without significant changes in plasma PPi [In the recent literature, there are numerous examples of studies reporting a lack of correlation between plasma PPi and calcification susceptibility in both humans and animals. Inhibition of the TNAP attenuated ectopic mineralization in the ABCC6Pi level . AnotherPi level . By contPi level  showed tasma PPi ,12. Finaasma PPi .−/− murine model [−/− mice and PXE patients [Interestingly, an increased level of calcification in the lower limb arteries was strongly associated with statin use and that was independent of PPi levels. This was not the case for coronary arteries. Furthermore, observational studies in the general population have reported that statins differentially affect the progression of arterial calcification in coronary arteries vs. the aorta ,30. Atorne model . Howeverpatients , our datAn important finding of the study was the strong correlation between age, arterial calcification, and disease severity, which was independent of plasma PPi. The age-dependency of severity has been reported in another cohort, without any contribution of gender or genotype . This fiThis retrospective analysis was a transversal study of a subgroup of PXE patients issued from a large cohort.Comparison with other studies were somewhat difficult since PPi plasma level is strongly dependent of the accuracy of measurement. However, the small longitudinal analysis performed in a subgroup of patient provided a clue to natural history of PPi within a limited three-year interval period.Although our data confirm the differential impact of the low plasmatic PPi concentration on the vasculature as previously reported , our intOur study shows, for the first time, a significant role of age on plasma PPi levels in PXE, independently of AC and disease severity, and mainly in females patients. Although plasma PPi level may provide a biological clue for the clinical diagnosis, our data shed more light on the complexity of PPi homeostasis and its role on calcification and disease severity. This highlights the importance of taking these factors into account in the design of clinical endpoints, and also the need to increase the plasma level of PPi at an early stage in the disease. Finally, we strongly suggest that the benefice impact of statins in PXE should be re-examined.


Lead is an environmental hazard that should be addressed worldwide. Over time, human lead exposure in the western world has decreased drastically to levels comparable to those among humans living in the preindustrial era, who were mainly exposed to natural sources of lead. To re-evaluate the potential health risks associated with present-day lead exposure, a two-pronged approach was applied. First, recently published population metrics describing the adverse health effects associated with lead exposure at the population level were critically assessed. Next, the key results of the Study for Promotion of Health in Recycling Lead  were summarized and put in perspective with those of the published population metrics. To our knowledge, SPHERL is the first prospective study that accounted for interindividual variability between people with respect to their vulnerability to the toxic effects of lead exposure by assessing the participants’ health status before and after occupational lead exposure. The overall conclusion of this comprehensive review is that mainstream ideas about the public and occupational health risks related to lead exposure urgently need to be updated because a large portion of the available literature became obsolete given the sharp decrease in exposure levels over the past 40 years.  Lead is an environmental toxicant. High levels of lead exposure can lead to hypertension and renal failure, as observed in the past in occupational settings or in the general population  , 2. HoweOur studies in the field of environmental medicine span four decades but did not provide convincing evidence supporting environmental lead exposure as being causally related to hypertension –12, renaLead enters the body primarily through inhalation and ingestion. Currently, adults are mainly exposed by breathing in lead-contaminated fine particulate and fumes at work or during leisure time activities that involve lead. Exposure of the general population to ambient air is generally due to respirable particles capable of deep lung penetration and deposition . Once thLead is a cumulative toxicant, 90–95% of which is stored in bone, from which it is recirculated with a half-life of 20–25 years , 23. BloRelevant publications are the NHANES III results and the articles published by the GBD consortium.The cross-sectional NHANES III survey (1988–1994) involved the collection of clinical variables, questionnaire data and biochemical measurements, including blood lead, among a representative sample of the adult population of the United States –27. Bloon = 891,896), and coronary mortality  in the United States and assuming that blood lead concentrations might be reduced to 1.0 µg/dl or less, the number of preventable deaths amounted to 412,000  for total mortality, 256,000  for cardiovascular mortality, and 185,000  for coronary mortality.In 14,289 individuals  had the highest mean blood lead level (2.20 μg/dl), compared with 3814 Hispanic men (2.18 μg/dl) and 6989 non-Hispanic White men (1.89 μg/dl). A similar ethnic gradient in blood lead was observed among women: 1.49 μg/dl in 3256 non-Hispanic Black women, 1.30 μg/dl in 4130 Hispanic women, and 1.30 μg/dl in 7078 non-Hispanic White women. In multivariable-adjusted logistic regression models [This 2018 NHANES report  is a summary metric that reflects the sum of years lived with a disability and the years of life lost. This metric therefore reflects both quality of life and premature mortality . The GBDThe GBD investigators listed among the possible limitations of their results (i) residual confounding; (ii) uncertainty as to the extent to which effect sizes were generalizable; and (iii) the impossibility of accounting for temporal changes in the exposure to risk factors. Thus, the GBD statistics fell short in accounting for the steady global decline in environmental lead exposure. This might explain why globally, despite declining environmental exposure –5, 11, ewww.osha.gov/laws-regs/regulations/standardnumber/1910/1910.1025). This standard includes regular medical monitoring, proper workplace ventilation, and the obligatory use of personal protective equipment. The coprimary endpoints for SPHERL were the changes in blood pressure and renal function [SPHERL is a longitudinal study of newly hired lead workers without known previous occupational exposure. The participants were employed at battery manufacturing and lead recycling plants in the United States. SPHERL complied with the Helsinki Declaration for investigations in humans. The Ethics Committee of the University Hospitals Leuven (Belgium) approved the study protocol (No B322201421631), which has been published in detail . The heaThe workers underwent follow-up visits 1 and 2 years after enrollment. Detailed diagrams describing the flow of participants and the number of workers excluded from the statistical analyses have been published for each endpoint –48. In mP < 0.001; Fig. In the most recently published SPHERL article focusing on renal function , the geoAt the study sites, trained nurses applied current guidelines to measure office blood pressure at the brachial artery . After tOffice blood pressure was measured in 267 participants  and the 24-ambulatory in 137 at two follow-up visits. Fully adjusted changes in systolic/diastolic blood pressure associated with a doubling of the blood lead ratio were 0.36/0.28 mmHg  for office blood pressure and 0.18/0.11 mmHg (2.09–1.74/1.05–1.27 mmHg) for 24-h blood pressure. The adjusted hazard ratios for moving up hypertension categories associated with a doubling of the blood lead concentration were 1.13 (0.93–1.38) and 0.84 (0.57–1.22) for the office and the 24-h blood pressure, respectively. Heatmaps demonstrated, as is true for all clinical measurements , that th2, respectively.The glomerular filtration rate was estimated from serum creatinine (eGFRcrt), serum cystatin C (eGFRcys) or both (eGFRcc) as defined in Table 2 lower, respectively, when determined from night shift blood samples compared with morning or evening blood samples. Both serum creatinine and serum cystatin C, from which eGFR is derived, show a diurnal rhythm with little influence of meals or meat ingestion on serum cystatin C, whereas these confounders increase serum creatinine [In addition to regression to the mean in the estimates of eGFR, the SPHERL renal function article highlighted the importance of accounting in longitudinal studies for concealed, albeit not unexpected, confounders. Figure eatinine . Along seatinine , 56. Theeatinine , the jobeatinine . StrenuoHRV was measured from 5-min ECG recordings in the supine and standing positions using Cardiax software, V.4.14.0 . The time interval between the supine and standing ECG recordings was not standardized but lasted from 1 to 3 min. Cardiax software allowed the exporting of all ECG measurements into an Excel worksheet, which was subsequently imported into SAS version 9.4, using standardized programming statements, thereby excluding any observer-induced bias. The software computes the power spectrum in the frequency domain by fast Fourier transform and autoregressive modeling and provides the low-frequency (0.04–0.15 Hz) and high-frequency (0.15–0.40 Hz) HRV components in milliseconds and the low-to-high-frequency ratio. The Fourier approach consisted of a mathematical transform that decomposes the heart rate signal changing over time into its constituent frequencies. The autoregressive approach derived HRV by regressing heart rate at a given time as a response (dependent) variable on its values during a previous period, using the Akaike information criterion as an estimator of the in-sample prediction error . NormaliThe analyses of heart rate and HRV  includedPeripheral nerve conduction velocity (NCV) is a common test of neurological function in lead-exposed workers , 68. Thep = 0.14), 0.86% , 2.14% , and 2.57% ; the p value for linear trend was 0.98. The percent changes in latency time associated with a doubling of blood lead from baseline to follow-up were −0.05%  unadjusted and ‑0.09%  fully adjusted for confounders.Peripheral NCV was assessed in 192 workers. From the lowest to the top quartile of the distribution of the follow-up-to-baseline blood lead concentration ratio, the percent changes in latency time were 2.91%  [SPHERL addressed this knowledge gap . The neu ≥ 0.12) .Fig. 3Ban = 2504) reported a geometric mean blood lead level of 4.73 µg/dl; of these participants, 42.3% had a blood lead level of 5 µg/dl or higher [Lead exposure represents an environmental risk that should be addressed worldwide. To re-evaluate the health risks possibly associated with present-day lead exposure, a two-pronged approach was applied, first assessing recently published population metrics –27 and tr higher . Finallyr higher . Cadmiumr higher .SUPPLEMENTARY MATERIALS


Uniconazole is an effective plant growth regulator that can be used in banana cultivation to promote dwarfing and enhance lodging resistance. However, the mechanisms underlying banana dwarfing induced by uniconazole are unknown. In uniconazole-treated bananas, gibberellin (GA) was downregulated compared to the control groups. An integrative analysis of transcriptomes and metabolomes was performed on dwarf bananas induced by uniconazole and control groups. The key pathways involved in uniconazole-induced dwarfism in banana were determined according to the overlap of KEGG annotation of differentially expressed genes and (DEGs) differential abundant metabolites (DAMs).Compared with the control groups, the levels of some flavonoids, tannins, and alkaloids increased, and those of most lipids, amino acids and derivatives, organic acids, nucleotides and derivatives, and terpenoids decreased in uniconazole-treated bananas. Metabolome analysis revealed the significant changes of flavonoids in uniconazole-treated bananas compared to control samples at both 15 days and 25 days post treatment. Transcriptome analysis shows that the DEGs between the treatment and control groups were related to a series of metabolic pathways, including lignin biosynthesis, phenylpropanoid metabolism, and peroxidase activity. Comprehensive analysis of the key pathways of co-enrichment of DEGs and DAMs from 15 d to 25 d after uniconazole treatment shows that flavonoid biosynthesis was upregulated.In addition to the decrease in GA, the increase in tannin procyanidin B1 may contribute to dwarfing of banana plants by inhibiting the activity of GA. The increased of flavonoid biosynthesis and the change of lignin biosynthesis may lead to dwarfing phenotype of banana plants. This study expands our understanding of the mechanisms underlying uniconazole-induced banana dwarfing.The online version contains supplementary material available at 10.1186/s12870-022-04005-w. Musa spp.), the stems of tall bananas are easily damaged by typhoons and require additional support costs. Production efficiency can be optimized by controlling grass growth in bananas. Semi-dwarf banana varieties are resistant to wind and rain damage [Dwarfing, an important agronomic trait, has been a research hotspot for breeders. Varieties with dwarfing characteristics not only have unique advantages in production management but also have great potential in terms of high yield. In the production of bananas  and compared the induced traits  were identified between the treatment and control groups at 15 d Fig. A. Among 5 d Fig. A, and th5 d Fig. B. The co5 d Fig. .Fig. 3DiAfter the removal of low-quality reads, a minimum of 40 million clean reads were obtained for each sample and mapped to the reference genome at a high mapping rate > 91%) Table . A high % Table .Differentially expressed genes (DEGs) between the treatment and control groups at 15 d were identified Fig. A were enThe common enriched pathways for both DEG and DAM between 15 d and 25 d were further examined, which included four key pathways: metabolic pathways, phenylpropanoid biosynthesis, flavonoid biosynthesis, and biosynthesis of secondary metabolites Fig. A. Of theBananas treated with uniconazole showed a significant dwarf phenotype, indicating that the dwarfing induction was successful. The physiological and biochemical indexes of the treated group were also significantly different from those of the control group, and GA was significantly reduced in the treated group.Rht1 than in high Polish wheat. These changes increase flavonoid content [Rht1 gene encodes a GA signaling repressor that reduces the response to GA and limits the elongation of wheat stems [Seashore paspalum, the dwarf phenotype of mutant T51 is considered to be closely related to the upregulation of flavonoid biosynthesis in the phenylpropanoid pathway [Tilletia controversa Kühn [In previous studies, the biosynthesis of flavonoids and alkaloids has been reported to be activated in dwarfing plants, and tannins are associated with plant dwarfing. It has previously been reported that the biosynthesis or accumulation of flavonoids changes significantly in a variety of dwarf plants compared with the higher group. The expression levels of some flavonoid biosynthesis genes are significantly different in the dwarf Polish wheat mutant  content . Rht1 geat stems , 25. In  pathway . Over-ac pathway . Isoquinrsa Kühn . In our rsa Kühn , 30. Altrsa Kühn , 31. TheRht1, leading to lignin level reduction [S. paspalum, the dwarf phenotype of mutant T51 is considered to be closely related not only to the upregulation of flavonoid biosynthesis, but also to the downregulation of lignin biosynthesis [Pt4CL1 downregulated [MYB gene has previously been reported to be involved in auxin response and endothecium lignification of anther walls [MYB transcription factors such as MYB4a-like and MYB4b-like factors was observed in transgenic bananas overexpressing MusaNAC68 [ARF and MYB genes were identified in the uniconazole-treated bananas. The mechanism of dwarfism in uniconazole-treated bananas may be related to lignin biosynthesis during stem elongation, but the specific effect of lignin biosynthesis on banana growth need to be further studied.The phenylpropanoid pathway is involved in several physiological processes. In addition to flavonoid biosynthesis, the lignin synthesis pathway is involved in the phenylpropanoid pathway –35. Preveduction . In S. pynthesis . In addiynthesis . Howeveregulated . In addiegulated , 43. Moregulated . Althougegulated . Both HCer walls . The subusaNAC68 . HoweverIn summary, in addition to a decrease in GA content, an increase in tannin procyanidin B1 content may contribute to banana dwarfing by inhibiting GA activity. Flavonoids and lignin are metabolites of the phenylpropanoid pathway, and there is a competitive relationship between them. The increase in flavonoid biosynthesis promoted the increased flow of metabolites towards flavonoid synthesis, which indirectly led to a decrease in lignin biosynthesis. Based on the above results, we propose a uniconazole-induced dwarfing mechanism hypothesis: over-accumulation of tannin inhibits the role of GA in banana growth, and abnormal lignin synthesis affects cell wall function, ultimately limiting cell expansion and causing dwarfism in uniconazole-treated bananas.Experiments were performed at the Libang Scientific Base of Guangxi Academy of Agricultural Sciences, located in Futang town in Wu Ming district, Nanning city, Guangxi province, China. The banana cultivar ‘Guijiao No.9’ was used, and the seeds were planted in January, 2021. We got the permission to collect Banana seeds. And the study protocol was complied with relevant institutional, national, and international guidelines and legislation. Uniconazole wettable powder  was applied to the plants when they grew to 16–18 leaves. The powder was diluted to obtain different concentration gradients, and then 200 mL solutions were drenched along the base of the pseudostem, resulting a gradient dosage of 0.1 g/plant, 0.3 g/plant, and 0.5 g/plant. Plants treated with water at the same stage as described above were used as controls. Twenty plants were used for each uniconazole-treated and control group and used to measure the dwarfed phenotype. Leaves were collected at 15, 20, and 25 d after treatment for both the uniconazole-induced and control groups. A total of 100 g of leaf blade (without leaf vein) was clipped from the penultimate piece of leaf for each plant and immediately frozen in liquid nitrogen or stored in a refrigerator at − 80 °C. Three independent biological replicates were conducted for the subsequent measurement of physiological indices, cDNA library construction, and RNA sequencing for each uniconazole-treated and control group.2 − •. Catalase (CAT) activity was examined by measuring H2O2 decomposition. Phenylalanine ammonia-lyase (PAL) activity was measured from the conversion of l-phenylalanine to trans-cinnamic acid. Soluble protein was quantified based on the reduction of Cu2+ to Cu1+ in an alkaline environment. PPO, SOD, CAT, PAL, and soluble protein activities were determined by measuring the absorbance at 410, 450, 240, 290, and 562 nm, respectively, and expressed as units/mg protein.GA production was estimated using a tetramethyl benzidine (TMB) detection system after incubation with an HRP-conjugated antibody. GA content was quantified by measuring the absorbance at 450 nm. Other physiological indices of the leaf samples were assessed using a spectrophotometric method. Polyphenol oxidase (PPO) activity was assayed using pyrocatechol as the substrate. Superoxide dismutase (SOD) activity was measured using the xanthine oxidase method based on the production of OThe potassium, calcium, and magnesium contents were determined by atomic absorption spectroscopy . Total phosphorus content was measured using phosphorus molybdenum blue spectrophotometry at 660 nm, and total phosphorus was expressed as the concentrations of organic and inorganic phosphorus. Total nitrogen was determined by titration with ferrous ammonium sulfate using an azotometer. Si concentration was quantified using plasma atomic emission spectroscopy (ICP-AES).Leaf samples were freeze-dried and crushed using a mixer mill . Lyophilized powder (100 mg) was dissolved in 1.2 mL of 70% methanol solution and kept at 4 °C overnight. After centrifugation at 12000 rpm for 10 min, the extracts were filtered . UPLC separation was performed using a 1.8 μm, 2.1 mm * 100 mm Agilent SB-C18 column. Linear ion trap (LIT) and triple quadrupole (QQQ) scans were acquired on an Applied Biosystems 4500 Q TRAP LC-MS/MS system, including an ESI Turbo ion–spray interface.Metabolites were extracted and identified using the Metware database . VIP values for the identified metabolites were determined by OPLS-DA analysis using the R package MetaboAnalystR. Significantly regulated metabolites between groups were determined by VIP ≥ 1 and absolute log2FC (fold-change) ≥ 1 and were then subjected to metabolite set enrichment analysis (MSEA).Total RNA was extracted from leaf samples using the Qiagen RNeasy Plant Kit , according to the manufacturer’s protocol. mRNAs were enriched by poly(A) selection from the extracted total RNA, and rRNA-depleted samples were prepared using the Illumina TruSeq RNA Sample Prep Kit to obtain a strand-specific library. Purification and size selection of cDNA were performed using AMPure XP beads, resulting in a median fragment size of 300 bp. The cDNA libraries were then checked using Qubit2.0, Agilent 2100, and sequenced using the Illumina Novaseq platform.Raw data were preprocessed using fastp (v0.19.3) with parameters “–n_base_limit 15 –qualified_quality_phred 20,” and clean reads were then aligned to the banana reference genome (NCBI accession No. GCF_000313855.2) with HISAT2 (v2.1.0). Gene expression levels were quantified using featureCounts (v1.6.1), and fragments per kilobase of transcript per million fragments mapped (FPKM) was calculated. Pearson’s correlation coefficients between samples were computed, and principal component analysis (PCA) was performed based on gene expression levels. DESeq2 (v1.22.1) was used to perform differential gene expression analysis between groups. Genes with |log2foldchang| ≥ 1 and FDR < 0.05 were identified as significantly DEG. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed for DEG using clusterProfiler (v3.10.1).Primers for qRT-PCR were designed using Primer Premier software (5.0) and were synthesized commercially . RNA was isolated using TRI Reagent Solution , according to the manufacturer’s instructions. Reverse transcription was performed using HiScript QRT SuperMix for qPCR . The primers of HCT and peroxidase genes were listed in Table Pearson correlation coefficients (PCC) between samples were calculated using the cor function in R for both transcriptomic and metabolomic data. MSEA, GO, and KEGG enrichment analyses were performed using hypergeometric tests.Additional file 1.Additional file 2.


Clostridioides difficile is one of the most important human pathogens. The identification of its possible sources is important for the understanding of C. difficile infection (CDI) epidemiology. A total of 16 water samples from wastewater and surface water in South Moravia in the Czech Republic and 82 samples of fish and gulls were collected between May and July 2019. C. difficile isolates were cultured by direct plating and after enrichment on chromogenic media. Susceptibility testing to eight antimicrobials was performed by Etest. C. difficile isolates were characterized by ribotyping, multilocus sequence typing, multilocus tandem repeats analysis, and toxin gene detection. Samples from fish and gulls were C. difficile negative; a total of 15 C. difficile isolates from 8 out of 16 water samples were cultured . Direct plating was culture positive in 6 out of 16 samples (12 isolates), and enrichment culture was positive in an additional 2 out of 16 samples (3 isolates). Twelve different ribotyping profiles and 14 sequence types of clades 1, 4, and 5 were identified. Five isolates did not carry genes for toxins, and eight isolates carried genes for toxins A and B; the remaining two isolates (RT078) carried the genes for toxins A, B, and binary. All C. difficile isolates were susceptible to amoxicillin, moxifloxacin, tetracycline, and vancomycin and resistant to ciprofloxacin. A high level of erythromycin resistance (>256 mg/L) was detected in eight isolates. Clindamycin resistance was found in 14 isolates, 6 of which showed a high level of resistance (>256 mg/L) and carried ermB. Surprisingly, one isolate  showed resistance to metronidazole (12 mg/L) with the presence of the plasmid pCD-METRO. In conclusion, a diverse spectrum of C. difficile strains was found in wastewater and surface water. A recently discovered plasmid-bound resistance to metronidazole was detected in C. difficile from the surface water sample.IMPORTANCE The combination of direct plating and culture after enrichment was used in order to gain a spectrum of C. difficile ribotypes present in the water samples. Toxigenic C. difficile ribotypes detected in surface water and in wastewater treatment plants overlapped with those derived from patients with CDI and/or animals. Importantly, a recently discovered plasmid-mediated resistance to metronidazole, a drug used for the treatment of CDI, was detected in C. difficile from river water. Clostridioides (Clostridium) difficile is a spore-forming, Gram-positive anaerobic bacterium and is the leading pathogen of health care-associated gastrointestinal infections (–C. difficile infections (CDIs) and high rates of asymptomatic carriage of C. difficile in the community have been recognized, suggesting that C. difficile reservoirs exist outside hospitals  and 82 samples from fish (n = 37) and gulls (n = 45) were collected between May and July 2019 , lake (1 isolate from 2 samples), and rivers (5 isolates from 12 samples)   and 2. Dsamples) . CultureC. difficile isolates showed 12 different ribotyping profiles and 14 sequence types (STs), respectively  carried genes for toxins A and B  did not carry any of the toxin genes; two isolates of RT078 carried genes for toxins A, B, and binary. The remaining eight  A and B .C. difficile isolates were susceptible to amoxicillin, moxifloxacin, tetracycline, and vancomycin and resistant to ciprofloxacin. A high level of erythromycin resistance (>256 mg/L) was detected in eight isolates, six of which carried the ermB gene. Clindamycin resistance was found in 14 isolates; 6 isolates showed a high level of resistance (>256 mg/L) and carried ermB (gluD) after PlasmidSafe DNase treatment .All ied ermB . Surprisied ermB  has beenC. difficile is a major pathogen of gastrointestinal infection associated with health care; however, given its occurrence outside health care settings, there is a need to understand the epidemiology of CDI, and therefore, the One Health Concept of research has to be performed. In the Czech Republic, the CDI epidemiology in health care settings has been mapped in several studies, and the predominance of RTs 001 and 176 is persistent  were surprisingly positive by direct culture of the filters than after selective enrichment with alcohol shock (5 samples versus 1 sample). Zidaric et al. also studied the occurrence of C. difficile in river water and compared three methods of detection, including culture on selective agar with and without ethanol shock and real-time PCR. In contrast to our study, culture on selective media after alcohol shock was the most sensitive method for the detection of C. difficile , but the combination of two approaches yielded two additional positive samples (3 isolates).Both two WWTP samples from two sampling days were culture positive. On the first sampling day, seven different ribotypes were detected by direct culture, but on the second sampling day, the culture was positive only after selective enrichment. In the study of Steyer et al., which is similar to our study, the direct culture of filters on selective agar yielded successful  samples . It shouibotypes . Here, iC. difficile isolates did not carry any toxin genes. Similar great diversity and the presence of nontoxigenic and toxigenic ribotypes were also found in Slovenian and Australian water samples (C. difficile was detected in at least 1 sample from 17 rivers (68.0%). A total of 154 C. difficile isolates were cultured; 110 (71.4%) of them were toxigenic, and 44 (28.6%) were nontoxigenic. A study from Australia cultured C. difficile from 47.3% (53/112) of lake/pond, 23.0% (14/61) of the river, and 20.0% (3/15) of estuary samples. In both studies, the toxigenic RT014 was the most common type, accounting for 25 isolates from 11 sampling sites in Slovenian and 10.5% (8/76) in the Australian study   was identified; however, this isolate was found to be metronidazole resistant  . In Ital78 (40%) . Whole-g and 010 . In contesistant .C. difficile ribotypes identified in our study from surface and wastewater overlap C. difficile ribotypes identified in previous studies carried out in the Czech Republic, i.e., from horses  and pigs   , 17, andand 078) . The latand 078) . HoweverC. difficile strains was found in wastewater and surface water. Recently discovered plasmid-bound resistance to metronidazole was detected in C. difficile strain cultured from the surface water sample. Genomic comparative studies are needed to confirm the relatedness of these isolates with C. difficile isolates from animals and clinical CDI strains from humans.A diverse spectrum of n = 2, near the nesting colony of gulls, from which cloacal swabs were taken) and its tributaries, the Svratka River (river 1) at six randomly selected locations, one before the WWTP and the remaining five downstream at different distances from WWTPs, and Jihlava River (river 2), with six randomly selected sampling points on its lower course that differed from the first river by the catchment area (medium-sized cities on the upper course)  and asps  from the one-off catch of consumer fish by a commercial company for retail, and cloacal swabs from nestling Caspian gulls  on the Nové Mlýny Reservoir were taken (The study area is located within the South Moravia river basin in the lowlands of the southeastern part of the Czech Republic  and was  course) ; Table 1 course) . The cat course) ; Table 1re taken ; Table 1C. difficile, samples were filtered by the membrane filtration method , passing 100-mL volumes of each water sample through a 0.22-μm-pore-size cellulose ester membrane filter (Merck Millipore). One of each membrane was placed onto ChromID C. difficile selective agar (bioMérieux) and incubated anaerobically for up to 5 days. For the detection of C. difficile present in lower numbers, enrichment cultures were performed on all water samples by placing the second membrane of 100 mL filtrate into 50 mL cycloserine-cefoxitin fructose broth  supplemented with 0.1% sodium taurocholate (Sigma-Aldrich) and incubated at 37°C for 8 days in anaerobic conditions . In the same way, the samples of intestinal contents from each caught fish (approximately 0.5 g) and cloacal swabs from nestling gulls were selectively enriched in 5 mL of CCFB. Thereafter, 1 mL of enriched sample and 1 mL of absolute ethanol were mixed and left for 1 h under occasional agitation at room temperature. Finally, tubes were centrifuged at 1,520 × g for 10 min, the supernatants were discarded, and the deposit was collected using sterile cotton-tipped swabs and plated onto the solid selective media described above. Inoculated plates from direct plating and after the enrichment were incubated under anaerobic conditions for 48 h at 37°C.Treated wastewater and surface water were sampled with the collection procedure recommended for standard water microbiological examination  into steC. difficile colonies per plate were subcultured on Columbia blood agar (Oxoid), and subcultures were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) using a MALDI Biotyper v3.0 system . The isolates from the same sample were indexed with the same number and lowercase letters  on Brucella blood agar (Oxoid) containing hemin (5 μg/mL), vitamin K1 (10 μg/mL). The MIC breakpoints for metronidazole, vancomycin, and moxifloxacin were applied as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST)  . The MIC(EUCAST) . Due to (EUCAST) .ermB) and metronidazole (pCD-METRO) were investigated with primers published before (CAADHH010000013) from the study of Boekhoud et al.  was performed with treated and nontreated DNA samples .The presence of antimicrobial-resistance determinants of clindamycin/macrolides , tcdB (toxin B), and cdtA/cdtB (binary toxin) were detected by multiplex PCR. The primers for C. difficile 16S rRNA were used as an internal control for the PCR amplification for nontoxigenic strains (The capillary electrophoresis (CE) PCR ribotyping was performed according to the consensus PCR ribotyping protocol , 35. The strains . PrimersA sampling of Caspian gulls was authorized by the local Czech nature protection authorities (permissions S-JMK78643/2018 OŽP/Ško and S-JMK 40970/2019 OŽP/Ško). Samples from the fish were obtained at a local fish processing plant by the water reservoir; thus, ethical approval for these samples was not required.