Cannabis sativa L., a dioecious plant derived from China, demonstrates important medicinal properties and economic value worldwide. Cannabis properties have been usually harnessed depending on the sex of the plant. To analyse the genetic structure of Chinese Cannabis and identify sex-linked makers, genome-wide insertion-deletion (InDel) markers were designed and used.In this study, a genome-wide analysis of insertion-deletion (InDel) polymorphisms was performed based on the recent genome sequences. In total, 47,558 InDels were detected between the two varieties, and the length of InDels ranged from 4 bp to 87 bp. The most common InDels were tetranucleotides, followed by pentanucleotides. Chromosome 5 exhibited the highest number of InDels among the Cannabis chromosomes, while chromosome 10 exhibited the lowest number. Additionally, 31,802 non-redundant InDel markers were designed, and 84 primers evenly distributed in the Cannabis genome were chosen for polymorphism analysis. A total of 38 primers exhibited polymorphisms among three accessions, and of the polymorphism primers, 14 biallelic primers were further used to analyse the genetic structure. A total of 39 fragments were detected, and the PIC value ranged from 0.1209 to 0.6351. According to the InDel markers and the flowering time, the 115 Chinese germplasms were divided into two subgroups, mainly composed of cultivars obtained from the northernmost and southernmost regions, respectively. Additional two markers, “Cs-I1–10” and “Cs-I1–15”, were found to amplify two bands  in the male plants, while 389-bp or 293-bp bands were amplified in female plants. Using the two markers, the feminized and dioecious varieties could also be distinguished.Based on the findings obtained herein, we believe that this study will facilitate the genetic improvement and germplasm conservation of Cannabis in China, and the sex-linked InDel markers will provide accurate sex identification strategies for Cannabis breeding and production.The online version contains supplementary material available at 10.1186/s12864-021-07883-w. Cannabis sativa L., a member of the family Cannabinaceae, is a diploid (2n = 20) monocotyledon and one of the oldest cultivated plants. Although it originated in Central Asia, its cultivation was soon commenced worldwide for applications in folk medicine, textile fibre, oil, and recreational use [onal use . Cannabi diploid n = 20 moCannabis is a dioecious species, which includes both male and female flowers separated on different plants. The sex of the plants commonly affects economically relevant traits like fibre quality and cannabinoid (CBD) content. In general, male plants have a better fibre quality, while CBD content in female plants is higher than that in male plants. Therefore, an ideal ratio of male-to-female individuals must be maintained with different production purposes to improve economic efficiency. However, it is difficult to identify the sex of plant via the mere examination of morphological traits before flowering, and DNA molecular marker technology has been considered as an accurate and reliable method for the sex identification of dioecious plants, as it is unaffected by plant growth stages .Conventional breeding is considered the primary method for developing new varieties in Cannabis breeding programs. However, this process is extremely challenging and often spans several years . PreviouInsertion–deletions (InDels) are recognised as major sources of genetic structural variations found widely distributed across the plant genomes. InDels like SSRs are also a type of length polymorphisms originating from a single mutation event, which is generally bi-allelic and single-locus in nature. Meanwhile, InDels exhibit many desirable inherent genetic characteristics of both SNP and SSR markers, such as co-dominance, abundance, and random distribution across the genome . GeneralChina has been considered one of the putative centres of origin for Cannabis, and a region where Cannabis has been cultivated for more than 2000 years for obtaining fibre, oil, and for other purposes . Howeverftp://ftpmips.helmholtz-muenchen.de/plants/barley/public_data/. On a genome-wide basis, 47,558 InDels were identified between PK and FN in the genomic DNA sequence database . As shown in Fig. Based on the 39 alleles amplified using 14 InDels, the population structure of the 115 individuals was further estimated under the Hardy–Weinberg Equilibrium using the STRUCTURE V2.3.3 software. Delta K values were plotted against K values, and the best number of clusters was obtained via the Structure Harvester platform   , lower tThe genetic structure of different genotypes can guide breeding programs for developing varieties with a broad genetic background. The genetic diversity of the Cannabis germplasm has been analysed using two types of markers: SSR and ISSR , 30, 31.Cannabis is a short-day crop, which is sensitive to photoperiod. Flowering time is an important agronomic trait that affects cannabidiol (CBD) and fibre yield content. Consistent with the population structure analysis, PCoA analysis and UPGMA clustering results Figs. , 5 and 6Owing to the different economic values between female and male plants, a suitable ratio of females to males individuals is vital for enhancing economic efficiency. To overcome the difficulties of the accurate identification of sex through morphological methods before flowering, eight pairs of markers mainly consisted of RAPD markers were reported for sex identification in Cannabis –15. HoweIn this study, we first developed 31,802 non-redundant InDel markers with a density of 31.5/Mb in the FN genome. Of these markers, 14 InDel markers could be used to divide the 115 Chinese Cannabis cultivars into two groups by genetic diversity analysis, population structure, and PCoA analysis. Additionally, two InDel markers, Cs-I1–10 and Cs-I1–15, related to female and male plant in Cannabis have been screened out. These genome-wide InDels and data on the genetic relationships of the Chinese Cannabis germplasm would serve useful in the further molecular analysis in Cannabis, and two sex-linked markers may provide accurate sex identification strategies at the early stage of Cannabis in production and breeding program.2 population derived from a cross between a female “H4” plant and a male “Yunma 6” (Y6) plant, were used for the screening of sex-linked marker. Furthermore, 24 samples  from the “H4” variety and ten samples from the feminized Cannabis variety, “ZY1”, were used for further validation of the sex-linked marker.A total of 115 Cannabis accessions were collected from different regions in China and preserved in our institute. Detailed information on these cultivars is summarised in Table The young leaves of each sample at the flowering stage were collected for DNA extraction. A Plant Genomic DNA Kit  was used for DNA extraction. DNA quality and quantity were checked using an Eppendorf BioSpectrometer , and the DNA was further diluted to a 10 ng/L working solution.ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/003/417/725/GCA_003417725.2_ASM341772v2. The DNA sequences of PK represented the reference genome, which was compared with that of FN using MUMer  software to capture the InDel loci (≥ 4 bp). Then, based on the InDel loci data, the primers were designed using the Primer 3.0 software (http://pgrc.ipk-gatersleben.de/misa/primer3.html). One pair of primers with the highest scoring was selected in the design results for the experiments. Furthermore, all InDel markers were checked for specificity using the TBtool software by blasting with the reference genome to avoid nonspecific amplification [The genomic DNA sequences of PK and FN were obtained from fication . Only unThe 84 primer pairs evenly distributed in the FN genome were selected for polymorphism analysis. Polymerase chain reactions (PCRs) were performed using 10 μL aliquots of the reaction mixture, including 7 μL of the PCR mix solution , 1 μL of the forward primer (10 nmol/L), 1 μL of the reverse primer (10 nmol/L), and 1 μL of the DNA template. PCR was conducted as follows: an initial step at 95 °C for 5 min, followed by 32 cycles of 30 s at 94 °C, 30 s at 55 °C, 40 s at 72 °C, and a final extension of 10 min at 72 °C. Primers used for genotyping were listed in Table Similar band types of 115 Cannabis cultivars on the electropherograms amplified using the same InDel markers were considered the same allele. Each polymorphic band detected by the same given primer represented an allelic mutation. To generate molecular data matrices, clear bands for each fragment were scored in every accession for each primer pair and recorded as 1 (presence of a fragment), 0 (absence of a fragment), and 9 (complete absence of band). PowerMarker version 3.25 was used to calculate the polymorphism information content (PIC), the number of alleles (NA), major allele frequency (MAF), and gene diversity for each InDel. A clustering map was conducted based on genetic distances and the unweighted pair group method with arithmetic mean (UPGMA) using the SM functionality of the NTSYS-pc2.10e software. Principal Coordinate Analysis (PCoA) was also performed using the NTSYS-pc2.10e software to resolve clustering patterns among genotypes. STRUCTURE v2.3.4 was used to estimate the population structure of the 115 Cannabis genotypes, and the number of the sub-population (K) was set from 1 to 10 based on admixture models and correlated with band frequencies three times. IBM SPSS Statistic 19.0 was used for cluster analysis of 115 Cannabis cultivars with the longest distance method and the Euclidean distance square based on the flowering time of each cultivar.Additional file 1.0 Table S1. All InDel markers developed in this study.Additional file 2. Table S2. The primers used for screenning of sex-linked InDel markers. Table S3. The genetic admixture of 115 cultivars. Fig. S1 The physical location of 84 InDel primers on Cannabis chromosome used in this study. Fig. S2. Amplification products from 96 Cannabis cultivars using the InDel markers CS-I1–2.


An increasing number of people are in a state of stress due to social and psychological pressures, which may result in mental disorders. Previous studies indicated that mesencephalic dopaminergic neurons are associated with not only reward-related behaviors but also with stress-induced mental disorders. To explore the effect of stress on dopaminergic neuron and potential mechanism, we established stressed rat models of different time durations and observed pathological changes in dopaminergic neurons of the ventral tegmental area (VTA) through HE and thionine staining. Immunohistochemistry coupled with microscopy-based multicolor tissue cytometry (MMTC) was employed to investigate the number changes of dopaminergic neurons. Double immunofluorescence labelling was used to investigate expression changes of endoplasmic reticulum stress (ERS) protein GRP78 and CHOP in dopaminergic neurons. Our results showed that prolonged stress led to pathological alteration in dopaminergic neurons of VTA, such as missing of Nissl bodies and pyknosis in dopaminergic neurons. Immunohistochemistry with MMTC indicated that chronic stress exposure resulted in a significant decrease in dopaminergic neurons. Double immunofluorescence labelling showed that the endoplasmic reticulum stress protein took part in the injury of dopaminergic neurons. Taken together, these results indicated the involvement of ERS in mesencephalic dopaminergic neuron injury induced by stress exposure. With the rapid development of modern society, more and more people are in a state of stress as a result of increasing social competition and psychological pressures, which has resulted in a number of undesirable mental disorders and remains a major medical and public health issue , 2. Despγ-aminobutyric acid (GABA) ergic neurons. Previous studies have indicated that chronic stress leads to not only elevation of hypothalamo-pituitary-adrenal axis activity [The ventral tegmental area (VTA) is a major part of the limbic system that plays a pivotal part in reward prediction , motivatactivity , a decreactivity  and cortactivity , 9 pyramactivity , 11. Theactivity , 13.The endoplasmic reticulum (ER) is a major locale of protein synthesis, glycosylation, folding, and nascent protein transport . AlteratGiven a strong association of the mesencephalic dopamine system with stress exposure, we established different durations of stressed rat models and focused on dopaminergic neuron injury in the VTA as well as changes in ERS proteins. The purpose of the present study was to examine whether the ERS was involved in mesencephalic dopaminergic neuron injury of stressed rats.Healthy male Sprague Dawley (SD) rats weighing 220 ± 20 g were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. Rats were placed in an environment with a constant temperature of 22°C and a light/dark cycle of 12/12 hours. Experimental rats were fed for one week prior to the experiments to adapt the environment. All animal experimental procedures were approved by the Examination Committee of the Animal Experimental Institution of Hebei Medical University. The experiment was divided into 6 groups: control group, 1 day, 3 days, 7 days, 14 days, and 21 days of restraint stress plus ice-water swimming groups (RS+IS), with 10 animals in each group.ad libitum.RS+IS rat models were established according to published methods . On eachμm thick coronal sections were collected to obtain VTA region. For morphological analysis of VTA, coronal slices were cut at -4.80 mm from bregma, according to the protocol provided by Paxinos and Watson atlas [Sixty minutes after the last of RS+IS, rats were anesthetized via intraperitoneal injection of pentobarbital (150 mg/kg) and their brains were harvested and fixed with 10% formalin for 48 hours. Fixed tissues were subsequently sectioned and stained, following dehydration, clearing, and paraffin embedding. Consecutive 6 on atlas .2O2 chilled methanol for 30 min. Tissues were incubated with a blocking solution  for 1 h at room temperature. Sections were then incubated with mouse TH-specific monoclonal antibody  at 4°C overnight. The sections were then incubated with a biotinylated secondary antibody , prior to treatment for 30 min with horseradish peroxidase- (HRP-) binding biotin. Finally, 3,3′-diaminobenzidine (DAB) was used as a color developing agent to visualise antibody localisation. Nuclei were counterstained with hematoxylin.After deparaffinisation and microwave antigen extraction, sections were incubated in 3% HDouble immunofluorescence staining was performed as described previously . MonocloThe accurate identification of the largest VTA was performed based on the stereotaxic atlas . The ser+ cells exhibiting GRP78/CHOP, as described previously [n = 6/group) were selected for morphological observation and data analysis. The numbers of GRP78+-TH+ and CHOP+-TH+ cells in the largest VTA area were counted at 100x magnification. The calculation of the average number of positive cells was performed by two independent investigators who were blinded to the study's conditions.Cell counting was performed to determine the number of THeviously . Rats (nμg of protein/lane) were loaded into an SDS-PAGE gel, separated by electrophoresis, and transferred to polyvinyl difluoride (PVDF) membranes. The membranes were incubated overnight at 4°C with anti-GRP78 (1 : 200), anti-CHOP (1 : 200), and anti-GAPDH antibodies. Then, incubation with horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG was performed. For exposure of the membranes to X-ray film, the enhanced chemiluminescence system was employed.Four rats from each group were used for western blot. According to a previously described protocol , tissue P > 0.1). All data were expressed as mean ± SEM. The multiple comparison one-way analysis of variance and post hoc LSD t-test were used for statistical analysis. P < 0.05 was considered statistically significant.Data were imported into the SPSS 21.0 statistical software for statistical analysis. The Kolmogorov-Smirnov test showed normal distribution of the data in all groups  = 276.971; P < 0.001). Post hoc comparisons showed that the number of TH+-positive cells was remarkably decreased after stress exposure at 3 days (P < 0.05), 7 days (P < 0.05), 14 days (P < 0.05), and 21 days (P < 0.05), while insignificant change was found at 1 day (P > 0.05) compared with the control group.Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine synthesis  that pla+-TH+-positive cells showed significant changes after different time of stress exposure  = 216.947; P < 0.001). The number of GRP78+-TH+-positive cells was markedly increased in the stress group at 1 day (P < 0.05), peaked at 3 days (P < 0.05), and remained significantly higher at 7 days (P < 0.05) and 14 days (P < 0.05) compared with the control group; however, no significant alteration was detected at 21 days (P > 0.05). The relative level of GRP78 in the VTA analysed by western blot was consistent with the immunohistochemistry results  = 227.898; P < 0.001). Post hoc test revealed that compared with the control group, CHOP+-TH+-positive cells were significantly increased after stress treatment at 1day (P < 0.05), 3 days (P < 0.05), 7 days (P < 0.05), 14 days (P < 0.05), and 21 days (P < 0.05). Consistent with the immunohistochemistry results, the relative level of CHOP in the VTA analysed by western blot showed the same trend (Double immunofluorescence labelling showed that GRP78 and CHOP proteins were colocalized with dopaminergic neuron marker TH in the VTA Figures . As show results . As depime trend .Stress refers to natural responses of the body to different stressors including various environmental, social, and psychological factors. Moderate stress helps body to cope with external risk factors. However, a series of abnormal psychological and physiological changes will occur due to excessive stress. Of late, an increasing number of people are in a state of stress due to social and psychological pressures, leading to mental disorders such as depressed mood, suicidality, and anhedonia –27. In tMental disorder, a complex condition induced by stress, involves many neuronal circuits. Among the regulation of these neuronal circuits, mesencephalic dopamine system may play a crucial role. The dopamine system plays a critical role in reward prediction , motivatα, inhibiting mRNA transcription and promoting the translocation and activation of ATF4, thus inducing the expression of the transcription factor CHOP [The relationship between ERS and injury has attracted the attention of numerous researchers. ERS is practically involved in the mechanism of cell protection; however, excessive ERS contributes to undesirable consequences including cell damage or death. For instance, ERS could activate the caspase family, such as capase-12, and induce cell apoptosis. GRP78 is a specific marker of ERS activation that is merely expressed in the ER. GRP78 determines ERS initiation and plays a vital part in promotion of unfolded protein maturation and maintenance of ER homeostasis , 34, sugtor CHOP . CHOP setor CHOP  is poorltor CHOP , we note


Body composition measures were not associated with LVEF or LAVI. These associations were not modified by sex or mediated by inflammation. Body composition could play a role in the pathophysiology of LV hypertrophy. Future research should focus on sex differences in regional adiposity in relation with diastolic dysfunction.We investigated the prospective associations of body composition with cardiac structure and function and explored effect modification by sex and whether inflammation was a mediator in these associations. Total body (BF), trunk (TF) and leg fat (LF), and total lean mass (LM) were measured at baseline by a whole body DXA scan. Inflammatory biomarkers and echocardiographic measures were determined both at baseline and follow-up in the Hoorn Study (n = 321). We performed linear regression analyses with body composition measures as determinant and left ventricular ejection fraction (LVEF), left ventricular mass index (LVMI) or left atrial volume index (LAVI) at follow-up as outcome. Additionally, we performed mediation analysis using inflammation at follow-up as mediator. The study population was 67.7 ± 5.2 years and 50% were female. After adjustment, BF, TF and LF, and LM were associated with LVMI with regression coefficients of 2.9 g/m HFpEF is often accompanied by multiple comorbidities, such as type 2 diabetes (T2D), hypertension, chronic obstructive pulmonary disease and adiposity3. Body mass index (BMI), waist circumference and waist-to-hip ratio are the most common indicators of adiposity and higher BMI, waist circumference and waist-to-hip ratio are associated with increased risk of incident HFpEF5. A different measure of adiposity is visceral fat and higher visceral fat is also associated with increased risk of HFpEF5. In a Mendelian randomization study, participants with genetically predicted higher fat mass index (fat mass divided by height squared) had an increased risk of heart failure (HF)6. Moreover, higher adiposity is associated with risk factors of HFpEF, such as type 2 diabetes and cardiovascular diseases8. In the Helius study, a large multiethnic Dutch cohort, various body composition measures, such as BMI, waist-hip ratio and waist circumference, and fat percentage, were associated with T2D prevalence in both women and men separately7. Additionally, results from the MORGEN project, one of the two Dutch cohorts in the European Prospective Investigation into Cancer and Nutrition project, show that higher BMI (≥ 30 kg/m2) and waist circumference resulted in respectively threefold and twofold higher risk of fatal cardiovascular disease in comparison to normal BMI and waist circumference, after 10 years of follow-up8.Heart failure with preserved ejection fraction (HFpEF) is an emerging public health problem. It affects more women than men14. These echocardiographic measures are among other measures, used as diagnostic criteria to detect HFpEF15. As these indicators of adiposity differ between women and men16, difference in body composition might explain sex differences in development of HFpEF. However, only one study reported sex-stratified results for the associations between fat mass and echocardiographic measures, such as left ventricular mass, showing that a higher fat mass was associated with higher left ventricular volume in women, and with lower left ventricular volume in men9.Cross-sectional studies have shown that higher BMI, waist circumference, body and visceral fat, and lean body mass are associated with worsening of various echocardiographic measures related to cardiac structure and function such as left ventricular mass and left atrial volume17. Though sex-specific results from cross-sectional studies on various body composition measures and levels of inflammatory biomarkers are inconsistent20.A proposed mechanism by which adiposity could lead to HFpEF development is via higher levels of systemic inflammation21. Additionally, BMI cannot differentiate between the proportion of fat and lean or muscle mass and is influenced by physical activity level22. Therefore, more specific measures for adiposity than BMI should be used, such as body fat in absolute or relative numbers. Most importantly, studies determining the prospective associations between body composition measures and echocardiographic measures related to cardiac structure and function are lacking. Moreover, it is unclear whether these associations differ for women and men and whether inflammation is a mediator in these associations.However, body mass index (BMI) is an indirect measure of adiposity and introduces misclassification because it does not reflect changes in body fat that occur with ageTherefore, we aimed to determine the prospective association between body composition measures and echocardiographic measures related to cardiac structure and function. Second, we investigated whether sex was an effect modifier in these associations. Third, we determined the mediating effect of inflammation on the association between body composition measures and echocardiographic measures.23. The Hoorn Study is a prospective cohort, which started in 1989 with 2484 participants, and was initiated to study the prevalence and determinants of type 2 diabetes in the general population. In 1513 participants the baseline measurements were repeated during the first follow-up visit in 1996–1998. We included 831 participants who underwent echocardiographic measurements during the second follow-up visit in 1999–2001, considered as baseline for this study. This subgroup was oversampled for participants with impaired glucose metabolism (IGM) and T2D to enable investigation of effect-modification by glucose metabolism status23. Participants with missing information on dual-energy X-ray absorptiometry (DXA) parameters (N = 150), on low-grade inflammatory biomarkers (N = 27), on either left ventricular ejection fraction (LVEF), left ventricular mass index (LVMI) or left atrial volume index (LAVI) available (N = 16), or a combination of these three (N = 59) were excluded. After eight years of follow-up (fourth follow-up visit in 2007–2009), follow-up echocardiographic measurements were performed in 340 participants . Participants with missing information at follow-up were excluded as well: low-grade inflammatory biomarkers at follow-up (N = 3) or without either LVEF, LVMI or LAVI available (N = 16), resulting in an analytic sample of 321 participants. We did not have complete data on all three echocardiographic outcomes, which resulted in different analytic samples for each outcome measure: 244, 250 and 268 participants for LVEF, LVMI and LAVI, respectively.This study included participants with baseline and follow-up echocardiographic measurements of the Hoorn studyAll individuals gave written informed consent, and the ethical committee of the VU University Medical Centre, Amsterdam, The Netherlands approved the study. All methods were performed in accordance with the relevant guidelines and regulations.24. All DXA scans were performed and read by one investigator. In the analyses we used a percentage of total body fat, trunk and leg fat mass of respectively total trunk or leg mass , and total lean mass as the determinants.At baseline, a whole body DXA scan was performed using the fan beam technology . This equipment used an X-ray tube with a switched pulse stable dual energy radiation with two excitation voltages of 70 and 140 kV. The machine performs serial transverse scans from head to toe at 1.2 cm intervals providing a pixel size of 1.9 mm × 1.2 cm. For each pixel, this software calculates weight, bone mass and fat percentage. Lean tissue mass was indirectly calculated as weight minus bone mass and fat mass. All measurements and calculations were performed for the total body and specific regions: head, trunk, arms and legs. These regions were distinguished using anatomic landmarks as described previouslyFasting, venous blood samples were drawn at baseline and follow-up by trained nurses. Biomarkers of low-grade inflammation , were measured by a multi-array detection system based on electro-chemiluminescence technology . All serum samples were measured in duplicate. Intra- and inter-assay coefficients of variability were for CRP, 2.8 and 4.0%; for SAA, 2.7 and 11.6%; for IL-6, 5.6 and 13.0%; for IL-8, 5.6 and 12.2%; for TNF-α, 3.9 and 8.8%; for sICAM-1, 2.4 and 4.9%; respectively.26. An experienced cardiologist evaluated the echocardiograms in order to ensure the quality. Left ventricular systolic dysfunction was determined by LVEF(%). Left ventricular mass indexed to height to the power of 2.7  was determined to assess cardiac structure. For LV diastolic dysfunction left atrial volume index (LAVI) (mL/m2) was determined.Experienced ultrasound technicians unaware of the medical history of the participant performed the echocardiographic measurements at baseline and follow-up. These measurements were obtained according to a standardized protocol consisting of two-dimensional, M-mode and pulsed-wave Doppler assessment27. Estimated glomerular filtration rate (eGFR) (mL/min/1.73 m2) was calculated according to CKD-EPI 2009 [Chronic Kidney Disease Epidemiology Collaboration] formula28.Study personnel collected demographic, metabolic and other risk factors, and medication use with standardized methods during the baseline and follow-up visits. Smoking status was categorized in never smokers, former smokers and current smokers. Educational level was self-reported and was stratified into three categories: low (no or primary education), middle (secondary education) and high (tertiary education). History of cardiovascular disease (CVD) was based on self-report or medical records. Blood pressure (mmHg) was measured twice at the left upper arm in a sitting position using an oscillometric device and averaged . All participants in the Hoorn Study, except those with previously diagnosed T2D, underwent a standard oral glucose tolerance test and were classified as either NGM, IGM (either impaired fasting glucose or impaired glucose metabolism), or T2DM according to the 1999 WHO criteria)Baseline characteristics are presented as mean ± SD, median [interquartile range] in case of a skewed distribution, or number (percentages) for the total population and stratified by sex. All analyses were performed in Rstudio version 3.5.3.We performed linear regression analysis using body composition measures, in percentages, at baseline as determinant and either LVEF, LVMI or LAVI at follow-up as the outcome to determine the prospective association between body composition measures and echocardiographic measures at follow-up. These analyses were adjusted for the respective echocardiographic measures at baseline, to account for baseline differences, and follow-up duration (years). We reported unstandardized regression coefficients per ten percentage points increase of body composition measures, and their respective 95% confidence intervals for the total population and stratified by sex. An increase of ten percentage points reflects the standard deviation and is used for ease of interpretation of the regression coefficients.29.Potential confounders were selected a priori and included age, sex, smoking status,  kidney function, hypertension status, CVD and anti-inflammatory medication use, all measured at baseline. Anti-inflammatory medication can influence certain inflammatory markers and include nonsteroidal anti-inflammatory drugs (NSAIDs), platelet aggregation inhibitors (abciximab), lipid lowering agents (statins and niacins) and ACE inhibitorsIn the first model, age, sex and glucose metabolism status at baseline, were added as potential confounders. In the second model, we additionally added HbA1c, kidney function, hypertension status, history of CVD and smoking status, all measured at baseline. Additionally, sex was assessed as effect modifier by including interaction terms in all models (body composition measures × sex). ANOVA was used to determine improved model fit for the model with and without the interaction term. A P-value of < 0.05 indicated a better model fit.Individual biomarker levels were divided into a combined Z-score of low-grade inflammation, for both baseline and follow-up. When biomarker levels were not normally distributed, the levels were natural log-transformed. The Z-scores were calculated per individual as follows: /study population standard deviation. The individual Z-scores were averaged into an overall Z-score for baseline and follow-up. Higher scores indicate more inflammation. The Z-score represents the deviation from the mean per standard deviation: 0 no deviation, 1 represents 1 standard deviation (SD) larger than mean.30:We performed mediation analysis using body composition measures, in percentages, as determinant, the continuous biomarker Z-score at follow-up, as mediator, and either LVEF, LVMI or LAVI at follow-up as the outcome, as visualized by a directed acyclic graph Fig. . The fol31. We reported unstandardized regression coefficients, and their respective 95% confidence intervals. Proportion mediated was calculated by: Formula  estimateFirst, we investigated selection bias due to loss to follow-up by comparing baseline characteristics of participants and dropouts, and a sensitivity analysis was performed using inverse-probability weighting for the second model. The numerator was calculated directly from the data as the probability of being in the study. The denominator was computed using logistic regression with complete data (yes/no) as the outcome and variables associated to missing data as independent variables (Supplementary Table The study population consisted of 321 participants (Table Mean follow-up was 7.5 ± 0.5 years. Participants with follow-up measurements were younger (67.5 ± 5 years vs. 72.4 ± 7 years), less often had a history of CVD (47% vs. 59%), and T2DM (16% vs. 24%) than people without follow-up measurements g/m2.7 and 2.0 g/m2.7 per ten percentage points increase in total body, trunk or leg fat, respectively  g/m2.7 and 3.5  g/m2.7 per ten percentage points increase, respectively. In women, total body, trunk and leg fat were not associated with LVMI with regression coefficients of 2.8  g/m2.7, 2.2  g/m2.7 and 1.8  g/m2.7 per ten percentage points increase, respectively. However, these differences between men and women were not statistically significant . In the total population, total lean mass was associated with LVMI with a regression coefficient of − 2.9  g/m2.7 per ten percentage points increase in total lean mass. In men, total lean mass was associated with LVMI with a regression coefficient of − 3.7  g/m2.7 per ten percentage points increase. In women, total lean mass was, albeit not statistically significant associated with LVMI with a regression coefficient of − 2.8  g/m2.7 per ten percentage points increase. This difference between men and women was not statistically significant . All body composition measures were not associated with either LVEF or LAVI. We did not observe effect modification by sex for these associations .At baseline, mean total body fat and trunk fat were 34 ± 9%, mean leg fat was 34 ± 11% and mean total lean mass was 63 ± 9%. Mean baseline LVEF was 63 ± 8%, mean baseline LVMI was 40 ± 12 g/mTable 2.7 and − 0.6  g/m2.7, respectively. Use of trunk and leg fat relative to total body fat as determinant, resulted in similar results as the additional adjustment for total body fat with regression coefficients of 0.8  g/m2.7 and − 1.1  g/m2.7, respectively.Additional adjustment for total body fat in the associations of trunk and leg fat with LVMI altered the regression coefficients: 0.7  g/mInverse-probability weighting resulted in slightly stronger associations than the main analysis , instead of aspecific fat mass, could be crucial to differentiate between the development of HFpEF in men and women.One study reported sex-stratified results for the associations between fat and lean mass and echocardiographic measures, such as left ventricular mass and left ventricular volume17. Although high total body and trunk fat were associated with higher LVMI, confirming the potential role of adiposity in the development of LV hypertrophy and possibly HFpEF, we could not confirm the mediating role of low-grade inflammatory biomarkers in this association. This is in contrast to a cross-sectional study in the Hoorn Study that showed that higher total body and trunk fat were associated with higher low-grade inflammation levels32. However, since low-grade inflammation was not consistently associated with cardiac function, this did not explain the association of the body composition measures with LVMI. Specific fat depots, such as epicardial fat, i.e. regional fat that surrounds the myocardium, has pro-inflammatory effects and is higher in older women in comparison to men and younger women and could be one of the sex-specific mechanisms in the development of HFpEF34. Future research should focus on sex differences in regional adiposity in general populations and (a)symptomatic patients, in relation with diastolic dysfunction as a precursor of HFpEF, to further unravel the pathophysiological mechanisms and the course in the development of HFpEF. Furthermore, the role of regional adiposity in screening programs among general populations for the detection of early stages of HFpEF should be studied.Cellular mechanisms that drive the development of HFpEF, in general populations specifically, are unclear. One paradigm states that comorbidities, such as adiposity, induce pro-inflammatory cytokines that result in myocardial remodeling in the development of HFpEFOur study is the first prospective study that determined the association of body composition measures at baseline with measures of cardiac structure and function at follow-up. Additionally, to our knowledge, this is the first time that the potential mediating effect of low-grade inflammation on this association has been studied. Several strengths are the long follow-up time of approximately eight years and the standardized measurements of biomarkers at two time points. Further, we presented both stratified results by sex and mediation analyses to provide more insight in the results. Nonetheless, there are certain limitations we need to address. Due to the follow-up time, we had a high loss to follow-up that could result in survival bias. However, a sensitivity analysis using inverse-probability weighting to adjust for this selection bias gave slightly stronger results as the main analysis meaning that healthier participants were included in this study and the main analysis would be an underestimation of the actual association. Second, we combined the inflammatory biomarkers in an overall low-grade inflammation Z-score. The underlying assumption is that all separate biomarkers reflect a similar pathophysiological mechanism. However, there is no consistent evidence whether this is true in a general population. Third, the DXA measurements were performed at baseline only, so changes over time could not be determined, and we did not have data available for visceral or subcutaneous fat. Fourth, other important echocardiographic measures for diastolic dysfunction, such as strain, were not measured in this cohort.In conclusion, higher total body, trunk and leg fat at baseline was associated with higher LVMI, but not with LVEF and LAVI at follow-up. Higher total lean mass was associated with lower LVMI at follow-up. This could implicate the role of total body and trunk fat in the pathophysiology of LV hypertrophy. Low-grade inflammation is not a mediator in these associations. Effect modification by sex was not apparent in all associations. Future research should focus on sex differences in regional adiposity, in relation with diastolic dysfunction and HFpEF.Supplementary Tables.


Anemia and iron deficiency were obtained through a 15% blood volume loss, followed by major surgical intervention (femur fracture and osteosynthesis using Kirschner wire). Therapeutic intervention was obtained with an intravenous ferric carboxymaltose infusion, as follows: group II: intraoperative (n = 7), group III: 48 h after surgery (n = 7), group IV: 48 h before surgery (n = 5), and group V: seven days before surgery (n = 6). Group I (n = 5) was left anemic, while group 0 (n = 5) was nonanemic without therapeutic intervention. Results and Discussion: In group I, serum iron lower than in group 0 (27.04 ± 6.92 μg/dL versus 60.5 ± 2.34 μg/dL), as well as hemoglobin (10.4 ± 0.54 g/dL versus 14.32 ± 2.01 g/dL) and ferritin values (22.52 ± 0.53 ng/mL versus 29.86 ± 3.97 ng/mL), validated the experimental model. Regarding wound healing after surgical trauma, we observed that neovascularization was more significant in group III, followed by group V, with fewer neutrophils, a well-represented and rich in lymphomonocytes inflammatory infiltrate associated with the biggest collagen fiber dimensions. The periosteal reaction and callus area presented thicker trabeculae in groups II and III compared to the anemic group. Conclusions: This original experimental study assessed the effect of perioperative intravenous iron administration at a specific time by comparing the weight, hematological, and iron status-defining parameters, as well as histological characteristics of the included subjects. The present findings highlight that correcting the iron deficiency in emergency settings through intravenous iron administration intraoperatively or 48 h postoperatively could determine the improved bioumoral parameters, as well as a better evolution of the postoperative wound and bone healing compared to the anemic group or subjects that received therapeutic intervention 48 h before surgery.Background: Anemia and iron deficiency are two of the main public health problems worldwide, associated with negative outcomes in surgical patients. This experimental study aimed to create a model of acute iron deficiency with anemia through blood loss and extensive surgery. Afterwards, intravenous iron was administered to correct the iron deficiency and to improve the hematological parameters in distinct moments regarding the surgical time. To assess the optimum time for therapeutic intervention, experimental subjects were compared, performing clinical, paraclinical, and histological examinations, as well. Methods: Male rats ( Anemia and iron deficiency, despite being intensively explored, are two of the main public health problems worldwide, with certain negative impacts in hospitalized patients. Regarding anemia, its prevalence is about 26–75% in the preoperative period, reaching even the threshold of 90% postoperatively ,2. Iron For this reason, intravenous iron agents have been thoroughly researched and improved over the years  in orderVarious studies have shown the undisputed role of preoperative intravenous iron used for hemoglobin correction in patients undergoing elective surgeries ,9,10. CuWith reference to the healing process after a major surgical intervention, research has shown that anemia and iron deficiency impair skin healing through secondary hypoxia  and low From our knowledge, no experimental study has carried out a complex analysis of the response to iron supplementation depending on the time of therapeutic intervention in the immediate perioperative period. We performed in vivo studies to create an experimental model of acute anemia associated with an iron deficiency by blood volume losses in subjects undergoing major surgery. The primary aim of this study was to identify the best perioperative time to correct an iron deficiency in order to improve the hematological status and recovery in an emergency setting using intravenous iron pharmacological agents. Thus, the weight, bioumoral determinations, and morpho-pathological findings were compared in accordance with the outcome, wound, and bone healing. The secondary aim was to assess the influence of iron administration on kidney function, considering that iron-induced renal impairment was noticed in previous experimental studies .The animal care and experimental procedures strictly followed the regulations of the Sanitary Veterinary and Safety Food Direction of Bucharest (DSVSA), Romania, which authorized the Emergency Clinical Hospital of Bucharest Experimental Lab (No. 481/13.11.2020). The experimental study also received the approval of the Ethical Committee of the Emergency Clinical Hospital of Bucharest (No. 10404/23.11.2020). All experiments were performed according to the Animal Care and Use Committee guidelines and recommendations.rattus norvegicus , with ages between 11 and 13 months, were left to acclimatize for 7 days. All animals were housed in polycarbonate cages (maximum 4 per cage), with sawdust bedding changed every three days under controlled environmental conditions , having access to standard food and water ad libitum.Healthy male adults Wistar rats, n = 5), the anemia model group (I) (n = 5), intraoperative iron group (II) (n = 7), 48-h postoperative iron group (III) (n = 7), 48-h preoperative iron group (IV) (n = 5), and 7 days preoperative iron group (V) (n = 6). These specific perioperative times were chosen to mimic emergency settings. Group 0 is the control group for the accuracy of weight and blood tests without intervention, while group I served as the control group for comparison with the other intervention groups.After an adaptation period, thirty-five animals were randomly divided into six groups: group 0  for iron deficiency, 20 mg/kg, through internal jugular vein catheterization as follows: group II: intraoperative, group III: 48 h after surgery, group IV: 48 h before surgery, and group V: seven days before surgery  of each subject followed by major surgery. In the treatment groups (II–V), animals were treated with intravenous ferric carboxymaltose  in an induction room until reflexes abolition, followed by the intraperitoneal administration of ketamine /xylazine  40/5 mg/kg. Surgical intervention had the subsequent steps: skin incision, dissection of the anatomical planes and exposure of the femur, intraoperative femur fracture, open reduction and osteosynthesis using Kirschner wire, anatomical planes closing, and wound dressing. All the animals were operated by the same surgical team, which was blinded to the group allocation. Rigorous hemostasis was obtained. Around 1 mL of blood loss was quantified using weighted gauzes. There were about four interventions/day with a duration of 50 min.After surgery, every subject was single-housed in a cage and observed for 12 h. Weight was measured every two days after inducing anemia (day one for groups I–IV and day seven for group V) until day 7.® Vacuum Tube) for the serum separation (centrifugation for 10 min at 4 °C and stored at −20 °C until processing).At the end of the experiment, the blood samples were collected. For iron profile determination, 2.4–2.6 mL of blood were collected in special tubes (PRIMA®), as well as serum iron (A25 Biosystems®). The total iron-binding capacity (TIBC) and transferrin saturation (TSAT) were obtained through specific formulas , as w®) after the tail puncture using manual micropipettes for minimal blood loss.Hemoglobin (Hb), hematocrit (Ht), red blood cells (RBC), platelets (PLT), mean cell volume (MCV), and mean cell hemoglobin (MCH) were directly measured using a veterinary hematology analyzer (Exigo H400 System®) equipped with a digital camera. The collected data were analyzed using Olympus cellSens Dimension® software (version 1.13).Promptly after blood sampling, at the end of the experiment (day seven: groups I–IV and day fourteen: group V), animals were sacrificed, and histological samples were harvested: the postoperative healed wound and surrounding skin, operated-on femur, and kidney. The samples were introduced in special recipients containing formol  and left for fixation for 24 h. Further, the bone samples were decalcified using a nitric acid solution 15% for 12 h. Four micrometer sample slides were stained with hematoxylin and eosin (H&E) then evaluated using an electronic microscope  and collagen deposition  were graded. Collagen dimension fiber was measured in ten different zones from each sample and compared. Kidney probes were assessed to identify the presence/absence of iatrogenic lesions following intravenous iron administration. Pathologists were blinded to the group allocation.p-value less than α (α’) should be considered of statistical significance.The results of the present study were analyzed with MedCalc (version 14) and IBM SPSS Statistics 23 programs. The procedures used were descriptive statistics (for characterizing discrete and continuous variables defined in the database), graphs, parametric statistical tests . The significance level used for the one-way ANOVA test is α = 0.05. The adjusted significance level used for multiple comparisons is α’ = 0.01. According to the test used, a Three experimental subjects were excluded: one for wound dehiscence (group II), one for self-skin injuries (group III), and one for refracture of the operated femur (group V).p = 0.931)  .p-value < 0.05 and to determine which mean values were different, the Student–Newman–Keuls test was applied.At the end of the experiment (day seven or fourteen), the subject within group I presented the most modified red blood cell parameters . AccordiThe iron parameters that were investigated are presented in p = 0.004)  .The fracture site, periosteal reaction, and callus area for all the studied groups are presented in No iron deposits or any renal injuries were identified in either of the groups .This study researched the proper time to administer intravenous iron in order to correct perioperative anemia and, thus, to improve the hemoglobin level in acute settings no longer than seven days before surgery or 48 h postoperative. To achieve that, we analyzed the weight, bioumoral markers, and histological skin and bone healing after a surgical trauma, and we observed that performing this intervention seven days before surgery, intraoperatively, or 48 h after surgery led to better results compared with 48 h before surgery or not performing it at all.We obtained an iron deficit model associated with anemia by acute blood loss, a feasible experimental method according to the published data . SpecifiIron is an essential element involved in development and growth . Animal This study showed that seven days after an important blood loss, followed by major surgery, the hemoglobin, hematocrit, and red blood cells (sensitive and specific indices of anemia ) were siAnother important finding was represented by higher platelets values in group I, significantly modified compared with the other groups. An analysis published by Hung et al. showed that the risk of adverse thromboembolic events grows from 7.8% to 15.8% in patients with thrombocytosis and iron deficiency . TherefoClinical trials targeting postoperative wound healing are scarce and usually evaluate the local tissue strength ,34. In eWe observed that seven days after surgery, the neovascularization was more significant in group III, followed by groups V and II. Fewer neutrophils and a well-represented inflammatory infiltrate with lymphomonocytes were noticed in groups III, V, and II. The mean dimension values of the collagen fibers were the biggest in group V, followed by groups III and II. All of the above support the hypothesis that the wound healing process at seven days postoperative presents the best features in groups V, III, and II, while group I presents the lowest progress.In bone tissue, iron is involved in the structure of some enzymes that synthesize collagen  and in vStudies have shown that many iron-containing intravenous agents have metabolites with renal excretion, therefore having the potential of producing nephrotoxicity through oxidative stress . In ordeThe study limitations counted the small number of subjects per group that could impair the statistical power and the short duration of the experiment. Additionally, using specific markers for inflammation and infections will increase the value of the research. Further investigation of these aspects above the acute perioperative period (7 days after surgery) will help compare the long-term benefits of these interventions.This study presents an original experimental model of acute induced anemia and iron deficiency through blood loss followed by major surgery. From our knowledge, it is the first experimental study to assess the effect of perioperative intravenous iron administration in an emergency setting at a specific time by comparing the weight, hematological, and iron status-defining parameters, as well as the histological characteristics of the included subjects seven days after surgery. Rapidly correcting the iron deficiency leads to a favorable improvement of hemoglobin, iron homeostasis and clinical outcome in surgical subjects. The present findings show that, by correcting the iron deficiency through intravenous iron administration 7 days before surgery, intraoperative, or 48 h postoperatively, a better clinical outcome is achieved, including a better healing process evolution of postoperative wounds and bone fractures compared to the anemic group and subjects that received therapeutic intervention 48 h before surgery.


Burkholderia cepacia complex bacteria comprise opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. These microorganisms produce an exopolysaccharide named cepacian, which is considered a virulence determinant. To find genes implicated in the regulation of cepacian biosynthesis, we characterized an evolved nonmucoid variant (17616nmv) derived from the ancestor, Burkholderia multivorans ATCC 17616, after prolonged stationary phase. Lack of cepacian biosynthesis was correlated with downregulation of the expression of bce genes implicated in its biosynthesis. Furthermore, genome sequencing of the variant identified the transposition of the mobile element IS406 upstream of the coding sequence of an hns-like gene (Bmul_0158) encoding a histone-like nucleoid structuring (H-NS) protein, a known global transcriptional repressor. This insertion sequence (IS) element upregulated the expression of Bmul_0158 by 4-fold. Transcriptome analysis identified the global effects of this mutation on gene expression, with major changes in genes implicated in motility, pilus synthesis, type VI secretion, and chromosome-associated functions. Concomitant with these differences, the nonmucoid variant displays reduced adherence to a CF lung bronchial cell line and reduced surface hydrophobicity and forms smaller cellular aggregates but has an increase in swimming and swarming motilities. Finally, analysis of the GC content of the upstream region of differentially expressed genes led to the identification of various genomic regions, possibly acquired by horizontal gene transfer, which were transcriptionally repressed by the increased expression of the Bmul_0158 gene in the 17616nmv strain. Taken together, the results revealed a significant role for this H-NS protein in the regulation of B. multivorans persistence- and virulence-associated genes.IMPORTANCE Members of the histone-like nucleoid structuring (H-NS) family of proteins, present in many bacteria, are important global regulators of gene expression. Many of the regulated genes were acquired horizontally and include pathogenicity islands and prophages, among others. Additionally, H-NS can play a structural role by bridging and compacting DNA, fulfilling a crucial role in cell physiology. Several virulence phenotypes have been frequently identified in several bacteria as dependent on H-NS activity. Here, we describe an H-NS-like protein of the opportunistic pathogen Burkholderia multivorans, a species commonly infecting the respiratory tract of cystic fibrosis patients. Our results indicate that this protein is involved in regulating virulence traits such as exopolysaccharide biosynthesis, adhesion to biotic surfaces, cellular aggregation, and motility. Furthermore, this H-NS-like protein is one out of eight orthologs present in the B. multivorans ATCC 17616 genome, posing relevant questions to be investigated on how these proteins coordinate the expression of virulence traits. Burkholderia cepacia complex (Bcc) are a group of opportunistic pathogens affecting mostly cystic fibrosis (CF) patients but also immunocompromised persons. The predominant Bcc species isolated from infected CF patients’ lungs are Burkholderia multivorans and Burkholderia cenocepacia (Burkholderia strains have large genomes (6 to 10 Mbp) consisting of three chromosomes and several plasmids , and quorum sensing systems, among others –6. Among–taminans –11. AlthmpR gene , 13. Theentified .Enterobacteria, is mediated by the histone-like nucleoid structuring protein (H-NS). Proteins of this family function as architectural components of the nucleoid and as global regulators of gene expression  in virulence comes from a study in B. cenocepacia J2315, in which its deletion reduced bacterial accumulation and attenuated virulence in Caenorhabditis elegans  into an intergenic region, which caused increased expression of a gene encoding a protein from the H-NS family. Besides the changes observed to the mucoid phenotype, the mutant strain also displayed an altered adherence phenotype, as well as increased motility. In summary, this study describes the importance of a protein of the H-NS family in regulating the expression of B. multivorans persistence and virulence genes.This study reports the comprehensive characterization of a nonmucoid derivative of B. multivorans ATCC 17616 after prolonged stationary phase (21 days) in mannitol-salts medium (SM) . Results confirmed the decreased expression of the bce genes in the evolved 17616nmv strain  . This phium (SM) , most liium (SM) . To detev strain . AlthougB. multivorans ATCC 17616 genome sequence present at GenBank. While our analysis did not reveal any single nucleotide variants or small indels, the analysis of reads not fully matching the reference genome allowed the discovery of an insertion sequence (IS) in the chromosome 1 intergenic region between divergently transcribed genes Bmul_0157 and Bmul_0158  that, in the ancestor strain genome, is present with six copies in chromosome 1 and one copy in chromosome 3, raising the possibility that the IS element identified within the Bmul_0157/Bmul_0158 intergenic region of the evolved strain was generated by a transposition event. Confirmation of IS406 insertion at this locus was performed by PCR amplification and Sanger sequencing. As shown in Due to the mucoid phenotypic difference observed between the ancestor and the evolved colony, we sought to sequence the genome of the evolved variant, to identify mutations that could explain the observed difference. The genome was sequenced to a coverage of 65×, and reads were assembled against the mul_0158 , showing406 activation has been reported previously  and qRT-PCR was performed . Comparieviously , and a strans from plasmid pLM20-6. Growth in SM for 72 h did not restore EPS biosynthesis (data not shown), indicating that the nonmucoid phenotype of the variant is likely caused by overexpression of gene Bmul_0158.To determine whether lack of cepacian biosynthesis in the 17616nmv strain was due to the decreased expression of Bmul_0157 , this geB. multivorans strains with genome sequence available, with 100% identity at the amino acid level. The conservation level to other species of Burkholderia ranges from 78 to 98%. The overall identity at the amino acid level between the Bmul_0158 gene product and H-NS from other non-Burkholderia species is much lower , but there is conservation of the C-terminal region containing the DNA-binding domain (78SGRGKPPN85) matches the consensus sequence of this motif almost perfectly (T/SXQ/RGRXPA). Besides Bmul_0158, the genome of B. multivorans ATCC encodes additional H-NS-like proteins with lower percent identity/similarity to Bmul_0158. These are Bmul_0190  and Bmul_3279 (46/64) located in chromosome 1, Bmul_3806 (52/68) located in chromosome 2, and Bmul_5655 (57/68), Bmul_5701 (37/55), Bmul_5767 (53/68), and Bmul_6044 (34/58) in chromosome 3.The protein encoded by the Bmul_0158 gene is highly conserved between g domain . The BmuB. multivorans ATCC 17616 and confirmed by qRT-PCR for the increased expression of the Bmul_0158 gene at all time points compared with the control strain harboring the empty vector . Furthermore, visual inspection of broth cultures showed the previously reported large planktonic cellular aggregates for the wild-type strain (i and ii) confirms the larger size and denser structure of the wild-type aggregates in comparison to the evolved strain, which showed aggregates smaller than 100 μm. In contrast to the optical density measurement and CFU count, quantification of total dry biomass did not show significant differences between the two strains  of 10 bacteria per eukaryotic cell. We found significantly lower adhesion of the evolved variant compared to the ancestor , suggestBurkholderia, the acquisition of bacterial resistance against antibiotics used to treat these infections is common. To determine whether Bmul_0158 could be involved in antimicrobial resistance, we determined the susceptibility of the ancestor and variant against β-lactams ceftazidime, imipenem, aztreonam, and piperacillin plus tazobactam and the aminoglycoside kanamycin. No major differences between ancestor and evolved variant were found, although the variant is slightly more resistant to the antibiotic kanamycin . According to P < 0.001) and secondary metabolite biosynthesis, transport, and catabolism  (P < 0.001) and inorganic ion transport and metabolism .Comparative analysis of the transcriptomes of cells with differences at the genetic level may provide information about the molecular mechanisms and regulatory pathways responsible for certain physiological states. Therefore, we performed whole-genome microarray analysis to examine the effect of overexpression of the  < 0.05) . ContrasfimA, papC, and fimC) showed decreased expression. Since pili are implicated in adhesion of bacteria to surfaces, this might contribute to the experimentally observed decrease of adhesion to epithelial cells  receptor RpfR /fumarate nitrate reductase (FNR) family and is a cyclic-di-GMP effector protein, regulating the expression of genes involved in cellulose biosynthesis and in fimbria assembly , on the circular representation of the ATCC 17616 three chromosomes  of downregulated genes with a GC content below the average GC percentage of the ATCC 17616 genome-wide intergenic regions, which is 63.4% (P < 0.001). In the group of upregulated genes, we observed 1.4-fold underenrichment (P < 0.001) of the ones with a GC content of the upstream intergenic region below 63.4%, while the number of the ones above that value was the expected one  at 37°C, supplemented with antibiotics when appropriate. Antibiotics were used at the following concentrations for B. multivorans: chloramphenicol, 200 μg/ml; and gentamicin, 40 μg/ml; for E. coli, concentrations were as follows: chloramphenicol, 25 μg/ml, and kanamycin, 50 μg/ml. Growth in liquid medium was analyzed in triplicates through measurement of the optical density at 640 nm (OD640) over time. Depending on the phenotype test, Burkholderia strains were grown in salts-mannitol medium (SM)  or YEM .The bacterial strains and plasmids used are described in Burkholderia was extracted by using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s recommendations. Plasmid DNA isolation and purification, DNA restriction, agarose gel electrophoresis, DNA amplification by PCR, and E. coli transformation were performed using standard procedures . Raw paired-end reads of 2× 250 bp were filtered on the basis of Phred quality scores, with adapter contamination and ambiguous nucleotides trimmed using the fastq-mcf tool . The fil v0.7.17 , and the v0.7.17 . Predict v0.7.17 .406 element in the genome of the 17616nmv strain was done by amplification of a product with an expected size of 699 bp if IS406 is absent and 2,066 bp if it is present. Primers used were Bmul_0157/0158_IG_Fw and Bmul_0157/0158_IG-Rev , and total RNA extraction and purification were carried out using the RNeasy minikit (Qiagen), followed by treatment with DNase for 1 h at 37°C according to the manufacturer’s protocol. Total RNA concentration was assessed using a NanoDrop ND-1000 spectrophotometer.For expression profiling by using microarrays, overnight cultures of proC or sigE, which showed no variation in transcription abundance under the conditions tested, was determined. Relative quantification of gene expression was determined using the threshold cycle (ΔΔTC) method  according to the manufacturer’s instructions. The primers used to amplify the chosen genes  were des) method .Burkholderia arrays, as previously described . RNA was processed for use on Affymetrix  custom dual-species escribed .Burkholderia multivorans ATCC 17616 transcripts. Then, the 6 arrays were normalized to a baseline array with median CEL intensity by applying an invariant set normalization method  to identify regions possibly acquired by horizontal gene transfer.The CGView server  was used640 was measured for 48 h. Simultaneously, CFU were also determined by plating serial dilutions into LB solid medium. Three independent experiments were performed.Cultures were grown in SM at 37°C with 180-rpm orbital agitation, and the ODExopolysaccharide production was assessed based on the dry weight of the ethanol-precipitated polysaccharide recovered from 100-ml culture samples of the two strains grown in liquid SM for 3 days at 37°C with orbital agitation as previously described .g, 25°C, for 30 s. The supernatant obtained was transferred to a second 50-ml Falcon tube, and the loose pellet was transferred to a 2-ml Eppendorf tube, previously weighed. This was subjected to short-spin centrifugations, and the resulting pellet, corresponding to cellular aggregates, was obtained. The Falcon tube with the free cells’ supernatant was centrifuged for 10 min at 8,000 × g, 25°C. Cell sediment was transferred to a 2-ml Eppendorf tube, also previously weighed. Both Eppendorf tubes of cellular aggregates and free cells were left to dry at 60°C for 72 h and weighed again. Results are the means from data from three independent experiments.Cellular aggregates and free cells were quantified according to a previously described protocol , with a B. multivorans ancestor and the evolved variant was determined by measuring their ability to absorb n-hexadecane as described previously  by centrifugation at 10,000 × g for 5 min and resuspended in the same buffer. Bacterial suspensions were adjusted to an optical density of 0.6 (6 × 108 cells/ml) at OD640 . To one and one-half milliliters of this suspension, 400 ml of n-hexadecane  was added. This mixture was vortexed for 30 s and then incubated for 30 min at room temperature. The optical density of the cell suspensions after (ODaq) addition of n-hexadecane was measured at 640 nm, and the hydrophobicity index (HI), expressed as a percentage, was calculated using the following formula: HI (%) = [1 −  × 100]. Experiments were performed at least three times.The relative surface hydrophobicity of the eviously . Briefly2) with 0.6% (wt/vol) Bacto agar (Difco). Agar plates were spot inoculated with a 5-μl drop of a culture at an OD640 of 1.0. After inoculation, swimming plates were incubated for 24 h at 37°C while swarming plates were incubated for 48 h. Following that period, the diameter of the swarming and swimming zones was measured. Results are the means from data from at least 10 replicates of two independent experiments.Swimming and swarming motilities were carried out as described previously . Swimmin640 of 0.1. The diameters of growth inhibition were measured after 24 h of incubation at 37°C. Results are the mean values from at least three independent determinations with four replicates each.Antimicrobial susceptibility tests were based on the agar disc diffusion method against piperacillin (75 μg) plus tazobactam (10 μg), ceftazidime (30 μg), kanamycin (30 μg), imipenem (10 μg), and aztreonam (30 μg). The discs, obtained from Becton, Dickinson, were applied onto the surface of Mueller-Hinton agar plates (Difco Laboratories) that had been previously inoculated with 100-μl aliquots prepared from cultures grown overnight in LB, at 37°C, with agitation, and diluted to a standardized culture ODB. multivorans isolates were analyzed for adhesion to the bronchial epithelial cell line CFBE41o-, derived from a patient homozygous for the cystic fibrosis transmembrane conductance regulator F508del mutation. Bacterial strains were grown overnight, after which 200 μl of those cultures was grown in LB for 4 h and then used to infect epithelial cells at a multiplicity of infection (MOI) of 10 . Bacteria were applied to a 24-well plate previously seeded with CFBE41o- cells in supplemented minimal essential medium, and plates were centrifuged at 700 × g for 5 min. Plates were then incubated for 30 min at 37°C in an atmosphere of 5% CO2. Afterward, each well was washed three times with PBS to remove unbound bacteria, and cells were lysed with lysis buffer  for 20 min at 4°C. Serial dilutions were plated on LB agar, and adhesion was quantified by determining CFU counts after 48 h of incubation at 37°C. Duplicates with each strain were performed per assay, and the results presented were obtained from three independent experiments. Results are shown as the percentage of adhesion, which was calculated as the number of CFU recovered divided by the number of CFU applied to the epithelial cells multiplied by 100.P value was lower than 0.05.Error propagation was used to calculate standard errors, and one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test were performed to assess statistical significance. Enrichment analysis was performed based on the cumulative distribution function of the hypergeometric distribution. Differences were considered statistically significant if the GSE162693.Microarray data were deposited in the Gene Expression Omnibus (GEO) repository under accession number


DNA methylation, an epigenetic mechanism modulated by lifestyle and environmental factors, may be an important biomarker of complex diseases including cardiovascular diseases (CVD) and subclinical atherosclerosis.CAC and MRScarotid), and four measures of atherosclerosis , ankle–brachial index (ABI), and multi-site atherosclerosis based on gender-specific quartiles of the single-site measures). We also examined the association between four epigenetic age acceleration measures  and the four atherosclerosis measures. Finally, we characterized the temporal stability of the epigenetic measures using repeated DNA methylation measured 5 years after baseline (N = 193).DNA methylation in peripheral blood samples from 391 African-Americans from the Genetic Epidemiology Network of Arteriopathy (GENOA) was assessed at baseline, and atherosclerosis was assessed 5 and 12 years later. Using linear mixed models, we examined the association between previously identified CpGs for coronary artery calcification (CAC) and carotid plaque, both individually and aggregated into methylation risk scores (MRSAHRR), cg09935388 (GFI1), cg21161138 (AHRR), and cg18168448 (LRRC52)) were associated with multi-site atherosclerosis (FDR < 0.1). cg05575921 was also associated with AAC and cg09935388 with ABI. MRSCAC was associated with ABI , and MRScarotid was associated with both AAC  and multi-site atherosclerosis . A 5-year increase in GrimAge acceleration (~ 1 SD) was associated with a 1.6-fold (P = 0.012) increase in the Agatston score of AAC and 0.7 units (P = 0.0003) increase in multi-site atherosclerosis, all after adjusting for CVD risk factors. All epigenetic measures were relatively stable over 5 years, with the highest intraclass correlation coefficients observed for MRScarotid and GrimAge acceleration .After adjusting for CVD risk factors, four CpGs (cg05575921(We found evidence of an association between DNA methylation and atherosclerosis at multiple vascular sites in a sample of African-Americans. Further evaluation of these potential biomarkers is warranted to deepen our understanding of the relationship between epigenetics and atherosclerosis.The online version contains supplementary material available at 10.1186/s13148-022-01229-3. Cardiovascular diseases (CVD), including coronary heart disease, myocardial infarction, and peripheral artery disease, are the leading cause of death in the USA . The CVDAtherosclerosis, a chronic inflammatory age-related condition that develops over several decades, is a precursor for CVD and can occur in either the intimal or medial layers of the arterial wall –6. The iEpigenetic mechanisms, including DNA methylation (DNAm), capture both genetic influences as well as the imprints of lifestyle and environmental exposures throughout the life course, and may have potential as biomarkers of CVD risk. Epigenetic age acceleration measures are DNA methylation-based markers of biological aging that are associated with late-life onset diseases and mortality –18. EpigAHRR gene body and is a well-documented smoking marker [Only a few epidemiological studies have examined the association between DNAm and subclinical CVD or atherosclerosis , 28. A rg marker –32.N = 129).In the current study, we evaluated the association between potential epigenetic markers of atherosclerosis and single- or multi-site atherosclerosis, assessed 5 and 12 years later, in 391 African-Americans from the Genetic Epidemiology Network of Arteriopathy (GENOA). Our atherosclerosis measures included CAC, abdominal aorta calcification (AAC), and ABI. Epigenetic markers included the previously identified CpGs for atherosclerosis , methylaGENOA is a community-based study in Rochester, MN and Jackson, MS that was established to identify genes influencing blood pressure . In the N = 1482) and 75% of non-Hispanic Whites (N = 1213) from Phase I returned. At Phase III (2009–2011), 752 African-Americans returned for a third examination. Demographic information, medical history, clinical characteristics, lifestyle factors, and blood samples were collected in each phase. This study includes 391 African-American participants from 277 sibships who had their DNA methylation measured in whole blood samples collected at Phase I and were seen in Phase III. For a subset of 129 participants that were seen in Phase III, DNA methylation was measured at both Phase I and Phase II. Written informed consent was obtained from all participants and approval was granted by participating institutional review boards .At baseline (Phase I), a total of 1583 non-Hispanic Whites  and 1854 African-Americans  were enrolled. In the second phase (Phase II: 2001–2005), all participants were invited for a second examination. Eighty percent of African-Americans  was calculated as weight in kilograms divided by the square of height in meters. Smoking was categorized as current, former, or never. Resting systolic (SBP) and diastolic blood pressure (DBP) were measured by a random zero sphygmomanometer and a cuff appropriate for arm size. The second and third of three readings, after the participant sat for at least five minutes, were averaged for analysis . HyperteP value < 10–16 were considered to be successfully detected [Genomic DNA from 1106 African-American participants from Phase I and 304 from Phase II was extracted from stored peripheral blood leukocytes using AutoGen FlexStar  and DNA methylation was measured using the Infinium MethylationEPIC BeadChip. DNA methylation processing procedures have been previously described . Brieflydetected . Samplesdetected . Regressdetected . White bdetected . A totalCAC based on 15 CpGs and MRScarotid based on 62 CpGs. Two CpGs (cg07033253 and cg23661483) were associated with both CAC and carotid plaque in MESA and are included in both MRSs. Higher values of MRS correspond to greater risk of atherosclerosis.We derived two MRSs based on the regression coefficients of CpG sites associated with CAC and/or carotid plaque at FDR ≤ 0.1 in MESA . For CACOf the 75 total CpGs used to construct the two MRSs, approximately 13% are in CpG island regions, 33% in shore/shelf regions, 11% in promoter regions, 21% in enhancer regions, 50% in transcription factor binding sites, and 52% in DNase hypersensitivity sites. Detailed annotation of the individual CpGs has been previously described . To inveFor the epigenetic age acceleration estimation, methylation beta values for Phase I and Phase II DNA methylation were uploaded to the online Horvath epigenetic age calculator to calculate DNAm age . Four meGene expression levels in GENOA African-American participants (N = 1233) were measured in Epstein–Barr virus (EBV) transformed B-lymphoblastoid cell lines using the Affymetrix Human Transcriptome Array 2.0. Quality control was done using the Affymetrix Expression Console provided by Affymetrix. All array images passed visual inspection and the Affymetrix CEL files were normalized using the Robust Multichip Average algorithm in the Affymetrix Power Tool software . AdjustmComputed tomography (CT) imaging conducted at Phase III was used to quantify calcification in the coronary arteries and the abdominal aorta. CT images were read by trained technologists and the amount of calcified plaque was calculated by multiplying each lesion area weighted by attenuation based on the maximum Hounsfield units in the lesion on a TeraRecon Aquarius Workstation . The amount of calcification was quantified using the Agatston score . Both CAABI was used to quantify atherosclerosis in the peripheral arteries. Details about the ABI measurement conducted at Phase II have been previously described . BrieflyMulti-site atherosclerosis score was defined similarly to that described by Zhao et al. . Both CAOutliers beyond 5 standard deviations from the mean of the outcome and epigenetic measures were removed. We calculated the Pearson correlation coefficients between the single-site atherosclerosis measures, and among the epigenetic age acceleration measures and MRSs. We also calculated the correlations between the individual GrimAge components and the MRSs.We used linear mixed models that account for familial relatedness to assess the association between the previously identified atherosclerosis-associated CpGs in MESA and MRSs derived from these CpGs (predictors) and the single- or multi-site atherosclerosis measures (outcomes). The minimally adjusted model (Model 1) was adjusted for age at baseline, sex, first 4 genetic principal components (PCs), and time between the measures. The time covariate was calculated based on the age difference of the participants at the time of atherosclerosis assessment (Phase II or Phase III) and DNA methylation assessment (Phase I). For models of multi-site atherosclerosis, we included two time covariates—the age difference between Phase III and Phase I, and an additional covariate for the age difference between Phase III and Phase II—to account for the differences in the assessment times of ABI (at Phase II) and CAC and AAC (at Phase III). Model 2 was additionally adjusted for smoking status at baseline, and Model 3 was further adjusted for baseline CVD risk factors .For CpGs associated with at least one of the atherosclerosis measures in Model 1 at FDR < 0.1, we investigated for association with cis-gene expression (± 1 Mb for each CpG). For that, we fit a linear mixed model adjusted for age, sex, time between measures, and the first 4 genetic PCs. For genes significantly associated with DNA methylation at FDR < 0.1, we investigated their association with the atherosclerosis measures using a linear mixed model approach with the same covariates.We used a similar procedure as described above to assess the association between the four epigenetic age acceleration measures and atherosclerosis, except that genetic PCs were not included as covariates. This approach of not adjusting for genetic PCs is consistent with other studies and allows for comparability of findings. As sensitivity analysis, we also performed this analysis after adjusting for the first four genetic PCs. We additionally carried out sensitivity analyses adjusting for white blood cell counts for significant associations with PhenoAA and GrimAA to assess confounding by changes in blood cell composition , 19, 51.For the subset of individuals with repeated DNA methylation measurements, we used chi-square and t tests as appropriate to compare the demographic and clinical characteristics of the subset to individuals without the repeated measurements. We used linear mixed-effect models adjusted for age and sex to calculate the intraclass correlation coefficients (ICC) between Phases I and II for the methylation risk scores and the epigenetic age acceleration measures. Parametric bootstrapping (1000 iterations) was used to calculate the 95% confidence intervals of the ICC coefficients , 53. AddP value of < 0.025  and P < 0.0125  were considered significant. Analyses were conducted in R (Version 3.4.1) [Statistical tests were two-sided. False discovery rate (FDR)  of 0.1 wn 3.4.1) , using tn 3.4.1) , and rptn 3.4.1)  packagesCAC, one outlier beyond 5 SD from the mean was removed from the analysis.Baseline characteristics of the participants are shown in Table r = 0.57 (P < 2.2 × 10–16). ABI was negatively correlated with AAC , but only weakly correlated with CAC . Additional file r range 0.21–0.46). The two MRSs were correlated at r = 0.32 (P = 1.55 × 10–10). Most of the epigenetic age acceleration measures and MRSs were weakly correlated (r range 0.09–0.29), except for GrimAA and MRScarotid which were correlated at r = 0.68 (P = 2.2 × 10–16). Scatterplots of the MRSs against the epigenetic age acceleration measures are shown in Additional file carotid than the overall GrimAA measure , with the second highest correlation between MRScarotid and PAI-1 .CAC and AAC were correlated at AHRR), cg09935388 (GFI1), cg21161138 (AHRR), and cg18168448 (LRRC52)) remained significantly associated with multi-site atherosclerosis. CpG cg05575921 was also associated with AAC and cg09935388 with ABI (FDR < 0.1). All of these CpGs were carotid plaque-associated CpGs in MESA. Hypermethylation at all of the significant CpGs (FDR < 0.1 in Model 3) was associated with decreased multi-site atherosclerosis. Significant CpGs in Model 3 explained between 5.5% and 11.2% of the variability of multi-site atherosclerosis, cg05575921 explained 6.9% of the variability of AAC, and cg09935388 explained 11.1% of the variability in ABI.CAC, AAC, ABI, and multi-site atherosclerosis were associated with 21, 15, 14, and 42 CpGs in Model 1, respectively (FDR < 0.1). The significant regression results are shown in Additional file ALDH9A1 and increased expression of ENSG00000236364 at FDR < 0.1  and marginally higher multi-site atherosclerosis (P = 0.054). DNA methylation at cg03636183 was associated with decreased gene expression of F2RL which was associated with CAC (P = 0.017).To investigate any regulatory effects on gene expression by the significant CpGs, we examined the association between CpGs shown in Additional file CAC and MRScarotid and single- or multi-site atherosclerosis measures. The beta coefficients shown correspond to the change in the atherosclerosis measures associated with a 1-unit increase in MRS. In Model 1, MRSCAC was associated with log-transformed AAC  but the association was not significant after adjusting for CVD risk factors (Models 2 and 3). MRSCAC was associated with higher ABI, indicative of less peripheral artery disease, and the associations were significant after adjusting for CVD risk factors . The MRSCAC explained 1.2%, 1.9%, 14.8%, and 0.8% of the variability of CAC, AAC, ABI, and multi-site atherosclerosis after adjusting for CVD risk factors.Table carotid was associated with all of the atherosclerosis measures in Model 1 (Table carotid was associated 0.605 units increase in log-transformed AAC (95%CI 0.197–1.013). This is equivalent to an approximately 1.8-fold increase in the Agatston score of AAC. Similarly, the associations between MRScarotid and multi-site atherosclerosis remained significant after adjusting for CVD risk factors. A one unit increase in MRScarotid was associated with approximately 0.7 units (95%CI 0.25–1.13) increase in the multi-site atherosclerosis score after adjusting for CVD risk factors (Model 3). The MRScarotid explained 7.8%, 5.3%, 2.1%, and 9.4% of the variability of CAC, AAC, ABI, and multi-site atherosclerosis after adjusting for CVD risk factors.MRS 1 Table . AssociaP < 0.05.Associations between the epigenetic age acceleration measures and atherosclerosis are shown in Table P < 0.05). DNAm smoking pack-years was associated with each of the atherosclerosis measures and was the most significant predictor of CAC, AAC, and multi-site atherosclerosis, with consistent direction of effects across all of the measures. Compared to the corresponding model of GrimAA , had a lower multi-site atherosclerosis score  and a lower PhenoAA . All the remaining epigenetic and atherosclerosis measures were similar across both samples (P > 0.05). The mean time difference between the DNA methylation measurements was 5.5 years . All of the epigenetic measures were relatively stable between Phases I and II, with both MRScarotid and GrimAA showing the highest stability (ICC > 0.8)  and was marginally associated with multi-site atherosclerosis  compared to the full sample analysis using Phase I MRSs  and another associated with AAC (cg05575921). DNA methylation at cg18168448 was associated with After adjusting for CVD risk factors, four CpG was associated multi-site atherosclerosis at FDR < 0.1. The direction of effects between the individual CpG sites and atherosclerosis in our study were consistent with MESA, where hypomethylation was associated with increased carotid plaque . All 4 CAHRR gene body. AHRR is an aryl hydrocarbon receptor repressor, which among other roles, inhibits the metabolism of polycyclic aromatic hydrocarbons and dioxins by competing with AHR [AHRR and atherosclerosis, hypomethylation at cg05575921 (P = 3.08 × 10−10) and cg21161138 (P = 7.73 × 10−8) was significantly associated with carotid plaque score [GFI1) gene body region, has been found to be associated with smoking [GFI1 encodes a nuclear zinc finger protein that plays a role in hematopoiesis, oncogenesis, and in controlling histone modification as part of a complex with other cofactors [Three of the significant CpGs  were previously found to be associated with smoking –32. Whenwith AHR . The sigue score , by mateue score , 64, andue score . Similar smoking , 67 and  smoking , 69. GFIofactors , 71.LRRC52), which plays a role in the regulation of voltage-gated potassium channel activity. We found evidence of cis-gene expression changes at this CpG, cg18168448, in addition to cg03636183. Our findings in EBV transformed B-lymphoblastoid cell lines are complimentary to those reported previously in monocytes [ALDH9A1) and increased expression of ENSG00000236364, which is antisense to UCK2. ALDH9A1 encoded enzyme has high activity for oxidation of gamma-aminobutyraldehyde and other amino aldehydes. UCK2 encodes a pyrimidine ribonucleoside kinase, which catalyzes the phosphorylation of uridine and cytidine. DNA methylation at cg0363183 located in the body region of F2R like thrombin or trypsin receptor 3 (F2RL3) was associated with significant decreased gene expression of that gene. F2RL3 plays a role in blood coagulation and inflammation, and methylation at cg0363183 has been previously found to be associated with smoking [LRRC52 which is mainly expressed in the testis [AHRR, GF1, ALDH9A1, ENSG00000236364, and F2RL3 have at least a low level of expression in the arteries, heart, and whole blood.DNA methylation at cg18168448 was also associated with significantly higher multi-site atherosclerosis (FDR = 0.063). The direction of association was consistent for the single-site atherosclerosis measures although it did not reach statistical significance. CpG cg18168448 is located in the first exon region of the leucine-rich repeat-containing protein 52  and a cg25953130 site in the gene were associated with CAC and carotid plaque atherosclerosis measures [P = 0.029), but the effect direction was consistent across studies. In MESA, out of seven CpGs associated with CAC (FDR < 0.05) in the full sample, only 3 were significant in the African-American sub-sample, although the magnitude and direction of effect were consistent across ancestry groups. Also, despite having similar age distributions, MESA multi-ethnic participants had a higher median CAC score than African-Americans in GENOA, and MESA had also a lower percentage of females. The prevalence of CAC in GENOA was low (median: 18.7 and IQR 0–195.7) with about 40% of the sample having an Agatston score of zero. In MESA, participants had a median CAC score of 46 (IQR 0–305), and the proportion with no detectable CAC was not reported. This is consistent with evidence from epidemiological studies showing that African-Americans tend to have lower calcification in the coronary arteries compared to Whites [CAC could be related to a more extreme form of the trait versus the lower atherosclerosis burden seen in GENOA. Another difference between MESA and GENOA is that the methylation signature in MESA was measured in monocytes, which have a well-established role in atherogenesis [CAC was low, which could indicate a lower stability of methylation at these sites. This could be particularly relevant to GENOA, as we assessed the association with CAC 12 years after DNA methylation measurement, while in MESA methylation and atherosclerosis were assessed concurrently.In our study, MRSmeasures . In GENOo Whites –79. Hencogenesis , 81, whicarotid, with the smoking pack-years component of GrimAge being slightly more correlated with MRScarotid than the overall GrimAA measure. While we do not know the individual CpGs comprising GrimAge, we know of one CpG overlap, cg05575921, between GrimAge and MRScarotid, and potentially other smoking-related CpGs, which may explain the observed correlations [GrimAA was the only epigenetic age acceleration measure associated with atherosclerosis in GENOA after adjusting for CVD risk factors. Increased GrimAA, indicative of increased biological aging, was associated with higher atherosclerosis. Very few other studies have examined epigenetic age associations with subclinical measures of CVD. In a cross-sectional analysis of 2500 African-Americans from the Atherosclerosis Risk in Communities (ARIC) study, a 5-year increase in both the Horvath and the Hannum acceleration measures was associated with an approximately 0.01 mm increase in carotid intima thickness . We did elations .carotid, had moderate stability  across repeated samples taken approximately 5 years apart. The characteristic of reliability over time is an important consideration for biomarkers that may be used for risk prediction. Further studies are needed to fully characterize the longitudinal patterns of DNA methylation, especially in response to known drivers of DNA methylation changes, such as smoking. In GENOA, smoking status was unchanged for the majority of the sample between Phases I and III, with only 27 current smokers becoming former smokers. One study that looked at the longitudinal changes of fetal DNA methylation in response to maternal smoking using serial samples at birth, age 7, and age 17 found evidence of reversible methylation changes at cg09935388 (GFI1) and persistent methylation changes at cg05575921 (AHRR) [Our results show that the epigenetic measures most strongly associated with atherosclerosis, GrimAA and MRS1 (AHRR) . An epig1 (AHRR) . Most re1 (AHRR) . Little 1 (AHRR) .One strength of our study is that we examined atherosclerosis at multiple vascular sites which reflect both intimal and medial vascular changes that may manifest differentially over time. CAC has been more extensively studied because it appears in a more clinically relevant vascular site  and is mcis-gene expression associated with DNA methylation, and thus did not capture trans-regulatory effects. Finally, gene expression was assayed in EBV transformed B-lymphoblastoid cell lines, which may not accurately reflect gene expression patterns in the white blood cells in which DNA methylation was measured. Previous findings show that significant differences exist in the DNA methylation profiles of EBV transformed lymphoblastoid cell lines and peripheral blood [A limitation of our study is the attrition of participants between Phases I and III. In a previous work, we have noted that participants who were lost to follow-up had higher epigenetic age acceleration and higher CVD risk . Additioal blood –96. As sal blood , 97, andcarotid was associated with atherosclerosis in the coronary arteries and abdominal aorta suggesting common pathobiological mechanisms of atherosclerosis on a systemic level. One epigenetic aging measure, GrimAA, was also associated with multi-site atherosclerosis beyond traditional CVD risk factors. This is one of the very few studies to examine the DNA methylation signature of multiple atherosclerosis measures in a population-based cohort comprised solely of African-Americans. These results further our understanding of the relationship between epigenetics, biological aging, and atherosclerosis. Future work in this area, including in other racial groups, may lead to the identification of potential biomarkers of atherosclerosis, lead to better identification of those at increased risk for atherosclerosis, and further help delineate causes of cardiovascular disease disparities across populations.In conclusion, our study found evidence of associations between DNA methylation and atherosclerosis at multiple vascular sites after accounting for traditional CVD risk factors. DNA methylation changes were at CpGs with previously reported smoking-related changes. Despite being derived from CpGs associated with carotid plaque, MRSAdditional file 1: Supplementary material, including Supplemental Tables 1–7 and Supplemental Figures 1–4.