Blood pressure response to head-up tilt (HUT) in 7 healthy subjects and 9 patients before and after coronary artery bypass grafting (CABG) was measured during supine and 15-min 60° HUT. Stroke volume (SV) and ejection fraction (EF) were assessed by echocardiography. Baseline mean arterial pressure (MAP) and heart rate (HR) in patients before CABG were similar to healthy subjects. MAP in patients decreased by 6 (4–9) mmHg [median (1st–3rd quartiles)] during 7–12 mmHg of HUT with decreased cardiac output (CO = SV × HR) while HR remained unchanged. MAP in healthy subjects remained unchanged during HUT with increased HR. Body weight decreased by 3.5 (2.5–3.7) kg and MAP decreased by 6 (2–13) mmHg during the last 3-min HUT while HR increased after CABG. Decreases in SV and CO during HUT disappeared after CABG. Blood pressure decreased during HUT in patients before and after CABG regardless of HR response. Coronary artery bypass grafting (CABG) for patients with coronary artery disease (CAD) has long-term benefits compared to other treatments, such as taking medications and percutaneous coronary intervention with drug-eluting stent . About 2Rehabilitation program includes walking and/or ergometer exercise . About 3A change in posture from supine to standing generally reduces venous return to the heart with associated unloading of baroreceptors. This sets a reflex increase in HR and total peripheral vascular resistance (TPR) through the autonomic nervous system to maintain arterial blood pressure (BP) . SeveralOne previous study examined effects of HUT in patients who had undergone CABG 3 months after and showed no decrease in systolic- and diastolic blood pressure during HUT ; howeverSubjects were 7 male and 2 female patients with CAD who were admitted to an acute care hospital between January 2017 and June 2018 for an isolated CABG and had no histories of chronic obstructive pulmonary disease, stroke, or severe cardiac arrhythmia, and 7 age- and physical characteristics-matched healthy subjects involved one female. All healthy subjects were free from diabetes mellitus, neurological, cardiovascular, and respiratory disorders. The present procedures are in accordance with the Helsinki Declaration as revised in 2013 and were approved by the Ethics Committee of our institution. Informed consent was obtained from all individual participants included in the study before participation. Additional informed consent was obtained from all individual participants for whom identifying information is included in this article.Patients were transferred after CABG to the intensive care unit and received ventilatory support in addition to treatment for hemodynamic stabilization and electrolyte balance. Rehabilitation therapy commenced on the first day after CABG depending on their condition. On postoperative day (POD) 1, patients were mobilized out of bed to perform resistance training for lower lib muscles. On POD 2–3, they performed resistance exercise, exercises of daily living, standing position, and gait training. On POD 4–5, the distance and frequency of walking exercise were increased taking into consideration of their condition every day except for Sunday. They exercised 60 min per session with treadmills and/or cycle ergometers at 50 to 70% of peak HR and/or perceived exertion of between “light” and “somewhat hard” . After POD 6, they performed the same exercise as POD 4–5 with treadmill and/or cycle ergometer for 60 min per session and work load was adjusted every day, so that the HR at the first 5 min of exercise was equivalent to their target HR and/or 12 to 14 of a Borg Scale rating. They additionally performed a stair climbing within the sessions if they were not exhausted. All patients achieved whole program during admission period.This was a cross-sectional  and a prospective observational study (before vs. after surgery). Experiments were performed 1–3 days before CABG and repeated 15 ± 7 days [mean ± SD]  after CABG just before discharged. All subjects reported to our rehabilitation room (25 °C) at 17:00 on the study day. Body weight was measured while subjects were dressed in cotton underwear. They relaxed in supine position on a tilt table  for 20 min. Surface electrodes were attached for recording ECG, a cuff was wrapped around the left upper arm for measurement of BP, and the left shoulder joint was fastened at abduction at 90° angle on a shelve fixed to the table, to keep the arm at heart level. The abdomen and the thigh were held in place on the table using straps. The feet were supported with board to prevent sliding down during HUT. The baseline measurements were recorded after another 10-min supine rest. This was followed by tilting the table to 60˚ within 40 s and maintained for 15 min, then returned to the supine position. None of the subjects experienced syncope or pre-syncope defined as dizziness and/or lightheadedness but not followed by loss of consciousness  due to tR–R interval of ECG every minute. The signals of baseline were averaged for 10-min supine rest and those during HUT were averaged every 3 min  for smoothing each signal.Systolic (SBP) and diastolic blood pressures (DBP) were measured at 1-min interval throughout the study by the auscultatory method using a sphygmomanometer . Mean arterial pressure (MAP) was calculated as (SBP − DBP)/3 + DBP. HR was calculated from mean We controlled respiration rate at 0.5 Hz using a metronome in all subjects, while monitoring a respiratory movement with a piezoelectric belt transducer . Some patients hold their breath for about 5 s, when it is difficult to extract images.i; General Electric Health Care Japan, Tokyo) at 2.5-MHz cardiac sector ultrasound transducer probe. Experienced physicians, who were blinded to the clinical data, analyzed the echo images. Unfortunately, the echo data of one female patient recorded during HUT were not available. Therefore, we analyzed the echo data of 8 patients and 7 healthy subjects. Stroke volume (SV) and left ventricle ejection fraction (EF) were calculated by the method established previously s and Ht were 4.2 (4.0–4.2) g/dL, 141 (138–142) mEq/L and 39 (36–43)% [mean ± SEM], respectively, before surgery, and significantly decreased by 0.9 (0.4–1.1) g/dL, 2.0 (2.0–4.0) mEq/L and 6.4 (0.0–9.7)% after surgery in 9 patients .[Alb]s and [Na+]s. A deprivation of body fluid would be a prime candidate for the reason.We compared BP response and cardiac function between CAD patients and age- and physical characteristics-matched healthy subjects and assessed effects of CABG and rehabilitation therapy on these responses. The main findings were (1) HUT was associated with a decrease in MAP, while HR remained unchanged in the patients group only, (2) the HUT-related decrease in MAP was enhanced after surgery compared with before surgery, although surgery had effect on HUT-related increase in HR, and (3) surgery had no effect on the HUT-related fall in LVEDV, whereas surgery was followed by decreases in body weight, [Alb]In healthy subjects, baroreflexes ensure steady-state and stable arterial blood pressure, even during orthostatic stress , 26. TheIn the present study, SV remained unchanged during HUT in healthy subjects. We applied HUT with 60˚ inclination using a foot board, not passive tilt without contact of the sole. It was previously demonstrated that active standing was associated with static isometric leg-muscle contraction and high BP, compared with passive tilt, in which blood tends to pool in the lower limb veins after 1 to 7 min . Also, aThe baseline BP was similar in both groups, but the HR response to a decrease in BP during HUT was diminished in the patients. Although we did not assess the sensitivity of HR and sympathetic nerve activity response to changes in BP in the present study, a previous study used the phenylephrine technique and found that the baroreflex gain in patients with myocardial infarction and EF of < 40% was lower than patients with EF > 40%  and thatA decrease in BP after surgery further strengthened during HUT relative to that recorded before surgery, because hypovolemia might progress after CABG while cardiac function did not improve enough. Postoperative management involves procedures that prevent body-fluid overload, such as treatment with circulatory agonists and diuretics and limited intravenous fluid infusion –19. ConsPrevious studies showed higher compliance of lower leg veins after CABG compared with before surgery , due to A previous study that analyzed HR variability before and after CABG showed a decrease in high frequency power to one-third of the preoperative level at 1 week after surgery , suggestThe reason why the increase in TPR during HUT disappeared after CABG was initiated by the higher compliance of the lower leg veins compared with before surgery . SympathSix patients had old myocardial infarction while three patients had unstable angina pectoris. The difference in pathological conditions between both diseases could influence on the present results. Averaged EF in patients with myocardial infarction was 54% and that angina pectoris was 67%, 13% lower in patients with myocardial infarction than that with angina pectoris . Howevern = 2), αβ-blocker (n = 3), calcium antagonist (n = 6), angiotensin II receptor blocker (n = 6) and nitrates (n = 2). After surgery, all patients also took at least one antihypertensive; β-blocker (n = 6), αβ-blocker (n = 6), calcium antagonist (n = 7), angiotensin II receptor blocker (n = 4), and catecholamines , and n = 9 for nitrates, diuretics, and dopamine. Patterns of the prescription before and after surgery varied widely but basically several types of medications were added after surgery. Therefore, we could not deny the possibilities that these antihypertensive itself or changes in patterns of each prescription influenced on the BP response through modified BRS. Moreover, additions of diuretics would be related to the reduced body weight after surgery.As shown in Table Hypovolemia was developed just 2–3 days after bed rest, resulting in orthostatic hypotension in healthy subjects . Thus, eLVEDV after surgery was maintained at a level similar to that recorded before surgery, despite the expected postoperative body-fluid loss Table . PreviouThis is a preliminary prospective observational study. Even we excluded biases of aging and body compositions, there were some variations of prescribing medications and all patients did not stay for the same time period. All patients had an elective operation (not an emergency operation) and an isolated CABG surgery from a single facility. Sample size was small and non-parametric tests were conducted because some of data were not normally distributed. Furthermore, healthy subjects also needed to be tested twice with a similar interval to the patients, because the patients were possibly nervous during the first test but it was relieved during the second time. The present results could be reproduced in limited populations.Body-fluid loss after CABG as expected could affect cardiovascular dysfunction in the patients. However, rehabilitation therapy in the present study might not retain the loss completely, even we started it early. To compensate this dysfunction in CABG patients, the other strategy is needed to expand body fluid before CABG, which can be achieved by endurance training with protein supplementation .We have demonstrated a decrease in BP during HUT due to a decrease in CO because of a decrease in SV without an increase in HR in CAD patients, whereas BP remained unchanged with the increased HR in the healthy group. The decrease in BP was augmented after CABG despite the improvement of HR response. Even the lack of syncope during the hospital stay was possibly related to the early post-CABG rehabilitation therapy, a preoperative rehabilitation would be needed to compensate postoperative cardiovascular dysfunction in the patients.


Citrus since seedlessness is one of the main consumer expectations. Citrus plants face numerous abiotic stresses including water deficit, which negatively impact growth and crop yield. In this study, we evaluated the physiological and biochemical responses to water deficit and recovery capacity of new triploid hybrids, in comparison with diploid hybrids, their parents  and one clementine tree used as reference. The water deficit significantly decreased the relative water content (RWC) and leaf gas exchange (Pnet and gs) and it increased the levels of oxidative markers (H2O2 and MDA) and antioxidants. Compared to diploid varieties, triploid hybrids limited water loss by osmotic adjustment as reflected by higher RWC, intrinsic water use efficiency (iWUE net/gsP) iWUE and leaf proline levels. These had been associated with an effective thermal dissipation of excess energy (NPQ) and lower oxidative damage. Our results showed that triploidy in citrus enhances the recovery capacity after a water deficit in comparison with diploids due to better carboxylation efficiency, restored water-related parameters and efficient antioxidant system.The triploidy has proved to be a powerful approach breeding programs, especially in  Citrus production is one of the main fruit crops worldwide with more than 133 million tons produced in 2017  and the n = 2× = 18), with few exceptions of polyploidy varieties as the triploid “Tahiti” lime. Triploid cultivars are uncommon in the natural environment because of their inviable seeds, resulting in a lack of progeny. However, thanks to their seedlessness and faster growth, triploid plants will be useful for improving phenotypic and organoleptic qualities that are economically important for the fresh fruit market. In citrus cultivars, triploidy is one of the main breeding strategies used to produce innovative seedless fruit  which lead to oxidative stress, through ROS overproduction  has developed a citrus breeding program dedicated to triploid citrus hybrids . The hybThis study aimed to assess the response of triploid citrus varieties to water deficit and their recovery after the stress, an essential step for their future introduction on the market. Their response was compared to diploid varieties, both parents and clementine tree. We analyzed the water status and compared the varieties for the effect of the water deficit treatment on photosynthesis activities through measurements of gas exchange and chlorophyll fluorescence and on the redox status using assays of oxidative markers and antioxidant system.Citrus reticulata Blanco) and “Ellendale” tangor (Citrus reticulata Blanco × Citrus sinensis (L) Osb.). Both 2× parents and 2× common clementine were also selected as reference variety of the study. Diploid and triploid scions were grafted on C-35 Citrange rootstock (Citrus sinensis “Ruby Blood” × Poncirus trifoliata) and grown in a greenhouse during 7 months at the INRAE-CIRAD experimental station in San Giuliano, Corsica, France . C-35 rootstock was chosen for its tolerance to abiotic  and biotic  stresses. C-35 seedlings used for the experiment were strictly chosen in the nursery to eliminate off-types. The 2× (D1-2× and D40-2×) and 3× varieties  were selected as described in the previous study (This study was performed triploid (3×) and diploid (2×) hybrids citrus trees obtained by hybridization between the “Fortune” mandarin . Each pot was then enclosed in a plastic bag to prevent soil evaporation. The experiment was performed in two completely randomized blocks designed as follows: (i) thirty plants  were assigned as control plants and irrigated daily near FC and (ii) thirty others plants  were subjected to water stress so that the soil water content ranged between 35 (threshold at which wilting has been observed) and 45% of FC by manual successive watering. For each pot, when the assigned minimal soil water content was reached, the watering was performed to set the maximal assigned soil water content. To maintain this water condition, each pot was weighed daily before and after irrigation. After maintaining this water stress for 6 days; the physiological parameters were recorded and plant material collected for biochemical analyses. Thereafter, stressed plants were re-watered (recovery period) with daily irrigation near FC for 5 days. Three independent biological leaf and root sample replicates were harvested from each variety and treatment . Leaves and roots samples were immediately frozen and ground in liquid nitrogen and stored at −80°C for biochemical analyses.PD) water potential was determined in one fully expanded leaf per plant , taken from the basal part of the primary branch, using a Scholander-type chamber . ΨPD was recorded between 04:00 and 06:00 AM before and at sixth day of the water deficit treatment and after the recovery period.Pre-dawn (ΨLeaf relative water content (RWC) was determined according to Before the beginning of the experiment, five mature leaves per plant were selected and labeled for all physiological measurements  which were performed between 09:00 AM and 11:00 AM.Pnet), stomatal conductance (gs), transpiration (E), and intercellular CO2 concentration (Ci) were monitored with an infrared gas analyzer LCPRO-SD  equipped with the broadleaf chamber at saturating light (PAR at 1400 μmol.m–2.s–1), at 380 μmol.mol–1 for CO2 concentration, airflow 200 μmol.s–1 while leaf temperature was determined by the environment. Data were recorded when the measured parameters were stabilized (3–6 min). The carboxylation efficiency was estimated as Pnet/Ci while the instantaneous and intrinsic water use efficiencies were calculated as WUE = Pnet/E and iWUE = Pnet/gs, respectively.Net photosynthesis (–2.s–1 was produced by an array of three light-emitting diodes (650 nm) during 1 s and allows us to measure the maximum fluorescence on dark (Fm) and light-adapted leaves (m′F). The maximum(Fv/Fm) quantum yield of PSII was recorded on dark-adapted (30 min) leaves using leaf clips. In light-adapted leaves, steady-state fluorescence yield was measured and the actual quantum yield of photosystem II was determined as ΦPSII = m′−F)/Fm′][ was calculated as NPQ = [m−F′m)/F′m. The application of a saturating actinic light at 3000 μmol photon.m= ΔF/Fm′ . The appsorption . The ETR′m)/F′m] .n = 3) for each variety and each treatment were harvested for biochemical analysis.Three independent biological replicates of leaves and roots samples .Hydrogen peroxide content was assayed using a PeroxiDetect Kit  according to g for 15 min at 4°C. Absorbance was read at 550 nm with a microplate reader .Ascorbic acid content was carried out as described by g for 15 min at 4°C. The absorbance was read at 520 nm with a microplate reader .Proline content was determined according to g for 30 min at 4°C. The supernatant was used to determine the SOD, CAT, APX, and DHAR activities as described by For antioxidant enzymatic activities, sample powder (54 mg) was homogenized in 2 mL of extraction buffer and centrifuged at 13,000 × 1. The qualitative factors studied are sampling period  and ploidy level. The variance homogeneity was checked using Levene test and the influence of these factors was analyzed with a two-way ANOVAs followed by the LSD test (P < 0.05) when a significant difference was detected. The normality was checked using Shapiro–Wilk test to determine the appropriate correlation coefficient to apply. Pearson’s correlation analysis was performed on normalized data during water deficit conditions and was used to verify the relation between the physiological and biochemical variables under water deficit. Heat map of Pearson’s correlation was performed with RStudio software. Principal Component Analysis (PCA) was performed on normalized data with the FactomineR package bundled with R statistical software. PCA and hierarchical clustering classification (Ward’s method) helps us to better understand the similarity between variables and individuals through the hierarchical distribution of varieties and obtain an optimal number of clusters.The data were expressed as means values (±standard error) and analyzed with R statistical softwareUnder water deficit conditions, severe leaf wilting and leaf rolling were observed in various 2× varieties   while onPD) was constant through the varieties and remained above −0.7 MPa  and T40-3× (−2.38 MPa). After the recovery period, ΨPD in water-stressed plants increased to reach similar values than those obtained in control plants  (gs) (E)  , stomataet) (gs) , and tra(gs) (E)  were sigs, and E .Fv/Fm) declined significantly in response to water deficit in all varieties, except for D1-2×  were observed between varieties in response to water deficit  and T40-3× (H2O2) varieties. Overall, 3× varieties fully restored H2O2 and MDA content close to or below the control values.Oxidative damages in terms of H deficit . H2O2 any period . The higst level . The D1-st level . After rAntioxidant defenses in terms of tAsa, Asa/DHA ratio and proline were studied in leaves of selected varieties in response to water deficit . SignifiTo further examine the water deficit response of 2× and 3× varieties and identify the functional implications and correlations of the various parameters during water deficit, a Pearson correlation analysis was performed . Net phoPnet (r = −0.63), E (r = −0.66), Pnet/Ci (r = −0.58), and iWUE (r = −0.64), while NPQ was positively correlated with ETR/Pnet (r = 0.55) and H2O2 (r = 0.53) (r = −0.73), Pnet/Ci (r = −0.61), and WUE (r = −0.84) while this osmolyte was positively correlated with ETR/Pnet ratio (r = 0.69). Hydrogen peroxide (H2O2) was positively correlated with APX (r = 0.89). A positive correlation was also observed between MDA and APX (r = 0.79) while this oxidative marker was negatively correlated with SOD (r = −0.6) and CAT (r = −0.79).Triploid varieties exhibited negative correlation between NPQ and  = 0.53) . ProlineFv/Fm was negatively correlated with ETR/Pnet (r = −0.77) and positively correlated with Pnet/Ci (r = 0.45). A positive correlation was found between ETR/Pnet and antioxidant defenses .Diploid varieties also exhibited specific correlations under water deficit . Fv/Fm wPnet, gs, Pnet/Ci, E, WUE, and iWUE) and RWC while it was negatively correlated with some physiological parameters  and antioxidants . Component 2 was positively correlated with leaves (SOD) and roots antioxidants  while it was negatively correlated with oxidative status parameters  and PSII-related parameters . Three main clusters were identified  and accumulation of oxidative markers in leaves (H2O2) and roots (MDA). They also exhibited the accumulation of antioxidants as proline in their leaves and total ascorbate in their roots.To provide a better understanding of water deficit response of 2× and 3× varieties, physiological and biochemical parameters were submitted to a centered and scaled PCA under water deficit and after the recovery period . The cooentified . ComponePnet, net/CiP, and gs) and antioxidant molecules in roots   and MDA. Component 1 separated cluster 1  from cluster 2 . Component 2 separated cluster 3 (“Fortune” mandarin and clementine) from clusters 1 and 2  after re-watering. Triploid varieties were also characterized by higher antioxidant defenses in their leaves (Asa/DA ratio and SOD) and roots . “Fortune” mandarin and clementine (cluster 3) were characterized by their increased oxidative markers accumulation (MDA and H2O2) even if these varieties exhibited enhanced proline and tAsa content in their leaves and higher SOD activity in their roots. Interestingly, the other 2× varieties and T38-3× (cluster 2) were mainly characterized by higher MDA accumulation in their roots and ETR/Pnet ratio while physiological traits remained weak after re-hydration.After the recovery period, the first component was positively correlated with gas exchange parameters   while it 1 and 2 . Hence, PD) and RWC illustrated that the water deficit occurred in all varieties  and efficient water use, as suggested by high RWC, WUE and iWUE levels   . The Pnenditions . Thus, t variety . Moreovetabolism . Taken t2O2 can also act as secondary messengers for the signaling in ABA-induced stomatal closure  and oxidative damage as evidenced by high leaf MDA and H2O2 content found in the “Fortune” mandarin and clementine   induced ROS production because of the use of O Pnet/Ci . The lowi  . In respand APX) . Our resgs decreases faster than Pnet at the beginning of prolonged water deficit or during short-term water deficit which causes an increase in the instantaneous water use efficiency (iWUE: Pnet/gs). In our study, the higher RWC was associated with greater iWUE in 3× varieties  was characterized by a greater photosynthetic activity, antioxidant system and water status . The ince damage . Moreovee damage  after the damage . Triploi deficit .Overall, our results suggest that triploid varieties can withstand water deficit, preventing photoinhibition and limiting oxidative damages; however ploidy level did not discriminate water deficit response of varieties. Nevertheless, the clear difference in recovery patterns between the 3× and the 2× varieties can be mainly explained by the efficient network of photoprotective mechanisms; the enhanced capacity of osmotic adjustment and the effective antioxidant processes suggested that triploidy improves the resilience capacity after a water deficit episode.In this study, we found that triploid varieties were not more tolerant than 2× varieties to water deficit but were clearly distinguished by their improved recovery capacity. Although the antioxidant system does not differentiate 2× from 3× behavior, some 3× varieties appeared to accumulate more proline under water deficit alleviating ROS accumulation and allowing an effective osmotic adjustment. Triploidy clearly increases adaptability to recover photosynthetic and biochemical processes after re-watering can be explained by the greater carboxylation efficiency, restored water-related parameters, greater antioxidant processes and low oxidative damages. Since water scarcity impose significant reduction in crops yield and quality production, plant recovery capacity after one or several successive drought episodes represents a key factor for crops sustainability. Thus, triploidy may offer a powerful tool for the citrus breeding program generating varieties which can withstand and survive under increased frequency and intensity water deficit episode suggested by the scenarios of global environment change. Hence, the response of 3× varieties to several drought episodes under field conditions, associated with impact on fruit growth, should be the focus of future research. Additionally, as the 3× varieties represent an economic interest, the impact of drought stress on fruit yield and quality could also be relevant to provide valuable information for crop improvement.The original contributions presented in the study are included in the article/RL collected test data, performed the statistical analyses, interpreted the results, and drafted the manuscript. JS interpreted the results and drafted the manuscript. LB, JG, SH, and RM helped draft the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ldlr−/− mice and pseudoxanthoma elasticum (PXE) patients. Mice lacking the Abcc6 and Ldlr genes were fed an atherogenic diet for 16 weeks before intimal calcification, aortic plaque formation and lipoprotein profile were evaluated. Cholesterol efflux and the expression of several inflammation, atherosclerosis and cholesterol homeostasis-related genes were also determined in murine liver and bone marrow-derived macrophages. Furthermore, we examined plasma lipoproteins, vascular calcification, carotid intima-media thickness and atherosclerosis in a cohort of PXE patients with ABCC6 mutations and compared results to dysmetabolic subjects with increased cardiovascular risk. We found that ABCC6 deficiency causes changes in lipoproteins, with decreased HDL cholesterol in both mice and humans, and induces atherosclerosis. However, we found that the absence of ABCC6 does not influence overall vascular mineralization induced with atherosclerosis. Decreased cholesterol efflux from macrophage cells and other molecular changes such as increased pro-inflammation seen in both humans and mice are likely contributors for the phenotype. However, it is likely that other cellular and/or molecular mechanisms are involved. Our study showed a novel physiological role for ABCC6, influencing plasma lipoproteins and atherosclerosis in a haploinsufficient manner, with significant penetrance.ABCC6 deficiency promotes ectopic calcification; however, circumstantial evidence suggested that ABCC6 may also influence atherosclerosis. The present study addressed the role of ABCC6 in atherosclerosis using  ABCC6 is a member of the ATP binding cassette superfamily2 and is a major regulator of ectopic calcification4. ABCC6 regulates mineralization by modulating an extracellular purinergic pathway through several key enzymes that generate inorganic pyrophosphate (PPi) and prevent its degradation7. Indeed, PPi is a potent inhibitor of ectopic calcification8. The cellular function of ABCC6 accounts for ~ 60% of plasma PPi levels in humans and mice10. The highest levels of ABCC6 expression are found in liver and kidneys, though other cell types also express the protein12. Inherited ABCC6 deficiency causes dermal, ocular and cardiovascular calcification in pseudoxanthoma elasticum (PXE) patients4, some cases of generalized arterial calcification of infancy (GACI)13 and is likely responsible for the PXE-like manifestations in β-thalassemia patients15.Vascular calcification is common in aging and can also result from diabetes, end-stage renal disease, atherosclerosis, or inherited disorders. Clinical evidence demonstrated that cardiovascular calcification is a significant risk factor for cardiovascular events. reported a PXE patient carrying a heterozygous LDLR mutation (p.R574H) with severe vascular stenosis and calcification16. The dystrophic cardiac calcification phenotype (DCC) associated with Abcc6 deficiency in mice18 was first identified in response to high fat diets20. Quantitative trait loci analysis identified Abcc6 as one of the root causes of atherosclerosis and aortic calcification induced by hyperlipidemia in ApoE−/− mice21. Furthermore, previous publications have presented suggestive evidence, but not proven, that PXE patients are susceptible to increased atherosclerosis 24.Multiple lines of evidence indicate a pathologic correlation between ABCC6 function and dyslipidemia. Pisciotta et alAbcc6−/− mice into an atherogenic Ldlr−/− background and compared the results of our investigation with a cohort of human PXE patients. We found that reduced ABCC6 function indeed leads to dyslipidemia and enhanced atherosclerosis in both Abcc6-deficient mice and in PXE patients. Vascular calcification was elevated in both Abcc6/−+; Ldlr−/− and Abcc6−/−; Ldlr−/− animals, but there was no significant difference with Ldlr−/− control mice, suggesting that the Abcc6 genotype has no effect on atherosclerotic mineralization. Overall, our data imply that changes in HDL levels and/or composition as well as systemic inflammation act as contributors to the atherosclerotic phenotype seen in the Abcc6−/− mouse model and PXE patients.In this study, we explored the possibility that beyond calcification, ABCC6 dysfunction could also play a significant role in dyslipidemia and atherosclerosis. For this purpose, we crossed The data that support the findings of this study are available from the corresponding author upon reasonable request. Because of the sensitive nature of the data from human subjects in this study, these may be subjected to approval by the authors who provided them (LM and GL).Abcc6tm1Aabb mice were generated on a 129/Ola background25 and backcrossed into a C57BL/6J > 10 times. Ldlrtm1Her mice in the C57BL/6 background were provided by WAB who purchased them from The Jackson Laboratory . These mice are hereafter designated as Abcc6−/− and Ldlr−/−, respectively. By simple crosses, we have generated Abcc6 haploinsufficient  and double knockout  animals to determine if a gene dosage affects atherosclerosis and associated calcification. Ldlr−/− mice with normal Abcc6 expression were used as controls. In this study, both male and female, age-matched mice were used, as gender had no significant impact on results. All mice were housed in pathogen-free rooms in AAALAC-accredited animal facilities at the University of Hawaii School of Medicine. Mice were kept under routine laboratory conditions with 12-h light–dark cycle with ad libitum access to water and either a standard chow diet or an atherogenic high fat diet (HFD) that contained 15.8% (wt/wt) fat, 1.25% (wt/wt) cholesterol and no cholate . The University of Hawaii Institutional Animal Care and Use Committee approved these studies. Experiments were conducted according to the NIH Guide for the Care and Use of Laboratory Animals.26. For lipoprotein analyses, PXE and controls plasma samples came from subjects that were not treated with cholesterol lowering medication. Control plasma samples came from a group matched for gender and age without evidence of PXE or other pathology.Fasting plasma samples and phenotypical data for PXE patients were obtained from the MAGEC PXE Reference Center of the Angers University Hospital, France. The diagnosis of PXE was based on a combination of established criteria for indisputable PXE, including clinically suggestive skin lesions, angioid streaks and histologically proven fragmented and calcified elastic fibers on skin biopsyFor atherosclerotic plaques assessment, results were compared to the NUMEVOX cohort. This cohort of subjects with at least one metabolic syndrome criterion with or without sleep apnea or liver steatosis is registered on clinicaltrials.gov (NCT00997165). The institution review board of the Angers University Hospital, France has approved these studies and the participants have given informed consent when required. All methods were carried out in accordance with relevant guidelines and regulations.27 that directly measures the amount of excess calcium, which is normalized to the weight of the excised tissues, as described18, and expressed in mg/dL per milligram of tissue. The aorta comprised the aortic arch and the thoracic and abdominal sections, including the iliac bifurcation.The level of mineralization in aortic, whisker and heart tissues was quantified using a colorimetric assayAfter 16 weeks of atherogenic diet, blood was collected from mice under anesthesia through cardiac puncture and the animals were euthanized using standard CO2 procedures. The animals were perfused with cold phosphate buffered saline (PBS) and a solution of 4% paraformaldehyde and 5% sucrose. After excising, aortas were cleaned of adventitial fat and opened longitudinally from the iliac bifurcation right to the common carotid artery. Aortas were pinned out onto a black wax board and stained for lipid with Oil Red O. The stained areas and overall inner surface of aortas were measured using Image J software (NIH) and the percent area stained with Oil Red O was determined.These were either measured in fasting human samples by the clinical laboratory services at the Angers University Hospital, France, or in fasting animal samples using quantification kits from Abcam  and from Wako Diagnostics . Apolipoprotein E (ApoE) concentrations in mouse and human plasma were assessed using ELISA kits (MBS2512274) from MyBioSources  and Abcam , respectively.28. The system uses 25 μl of fasting plasma samples (not previously frozen). Up to 7 LDL bands can be detected. The LDL-1 and -2 bands correspond to large particles, whereas bands LDL-3 to 7 correspond to small dense LDL particles. Similarly, 10 HDL sub-fractions are detected. HDL-1 to -3 represent large particles, sub-fractions − 4 to − 7 are reported as medium particles, and sub-fractions − 8 to − 10 as small particles. This system also calculates the cholesterol concentration for each lipoprotein fraction according to a total cholesterol value obtained for each plasma sample.LDL and HDL subfraction distributions were analyzed with the Quantimetrix Lipoprint system  according to the manufacturer’s instructions and as described elsewhereBone marrow-derived macrophages were isolated and cultured as follows. Briefly, 3-month-old mice were euthanized by carbon dioxide inhalation. Hind legs were exposed and the muscle tissue removed to isolate the femurs and tibiae. The bone marrow was flushed from femurs and tibiae with culture medium (DMEM) containing 10% fetal calf serum and 1% penicillin/streptomycin/l-glutamine  using a syringe and a 25-gauge needle. The cell suspension was drawn through a syringe with an 18-gauge needle to dissociate cell clumps and was passed through a 70-μm pore cell strainer  to remove tissue debris. The cells were plated and incubated for 5 days in the presence of 25 ng/ml macrophage colony stimulating factor (M-CSF)  in order to initiate macrophage differentiation. Foam cell differentiation from macrophages was achieved with 50 μg/ml acetylated LDL  added to the cell culture media for 48 h. For macrophage polarization towards the classic M1 and alternate M2 phenotypes, macrophages were kept for 24 h in DMEM medium supplemented with 20 ng/mL TNFα for M1 differentiation and with 20 ng/mL IL-4 for M2 differentiation. Cells were harvested after 24 h for total RNA isolation.5 cells per well in triplicate and allowed to adhere overnight. Cells were incubated with loading medium (DMEM/F12 Glutamax (Invitrogen), 1% penicillin/streptomycin, 0.2% fatty acid-free bovine serum albumin, 50 μg/mL acetylated LDL and 1 μCi/ml [3H]-cholesterol  for 36 h. The cells were then washed twice with PBS and equilibrated in 0.2% BSA in DMEM/F12 media for 2 h. Efflux media  with or without 20 μg/mL apoA1 (acceptor for ABCA1-mediated efflux) or 50 μg/mL HDL (acceptor for ABCG1-mediated efflux), was then added to each well for 7 h. Supernatants and cell lysates were then collected, and the amount of [3H]-cholesterol was determined with a scintillation counter. The percent cholesterol efflux at baseline or to ApoA1 or HDL was expressed as a percentage of the total loaded [3H]-cholesterol using the following equation: /.Bone marrow-derived macrophages (BMDMs) were seeded in 24-well plates at a density of 5 × 10Bone marrow-derived macrophages were seeded in 24-well plates onto small coverslips placed inside the wells. Cells were cultured as described above with or without 50 μg/mL acetylated LDL (for foam cell differentiation). Cells were then fixed and permeabilized with cold methanol for 5 min at − 20 °C. Rabbit monoclonal anti-ABCA1  and anti-ABCG1  antibodies were used to specifically detect ABCA1 and ABCG1 in BMDMs. The secondary antibody was AlexaFluor 488 . The coverslips with the cells from the wells were placed onto slides and immunofluorescent images were acquired using an Axioskop 2 fluorescent microscope . Individual images were collected and processed with Photoshop CS6 .18. Proteins were extracted on ice in RIPA buffer containing 1X protease inhibitor mini complete cocktail , and 5 mmol/L EDTA. Homogenates were centrifuged at 16,000 g and the protein concentration in the supernatant was determined by absorbance using a Nanodrop spectrophotometer . 50 μg was combined with reducing Novex sample buffer  and loaded into wells of a 4–12% polyacrylamide gel . Proteins were transferred to nitrocellulose membrane and blotted with the anti-ABCA1 and ABCG1 antibodies . Appropriate secondary antibodies were detected using the Odyssey infrared imaging system . β-actin was used as a loading control  and mouse lung lysates were used as a positive control (not shown).ABCA1 and ABCG1 protein expression in bone marrow-derived macrophages and foam cells were also detected using standard western blot techniques we previously describedHmbs (Mm01143545_m1) was used for the macrophage/foam cell samples and Gapdh (Mm99999915_g1) was used for the liver samples.Real-Time PCR was used to determine the level of mRNA expression of various genes in cultured macrophages and mouse tissues. Total RNA was extracted from macrophages/foam cells or approximately 20 mg of tissue samples using the RNeasy kit . RNA was converted into first-strand cDNA using a SuperScript III First-Strand Synthesis SuperMix kit . The level of gene expression was detected by quantitative RT-PCR (qRT-PCR) with a StepOnePlus Real-Time System  using commercially available TaqMan probes from Life Technologies. . As an endogenous control, gene 2O / 5% acetonitrile (ACN) + 10 mM of ammonium formate) and B (20% ultrapure H2O / 80% ACN + 10 mM of ammonium formate). UPLC-MS/MS measurements were performed on a Waters Acquity H-class UPLCTM associated with a Waters Xevo triple quadripole equipped with an electrospray ionization (ESI) source. The UPLC was fitted with a Cortec UPLC C18 column  set at 40 °C and 10 μl of sample were injected onto the column. Elution was achieved with a linear gradient of solvent B in solvent A at a flow rate of 0.3 ml/min. The elution method was as follows: 10% of solvent B up to 45% in 8 min—45% of solvent B up to 90% of solvent B in 4 min—90% of solvent B kept constant for 1 min—90% of solvent B down to 10% of solvent B in 1 min—10% of solvent B kept constant for 1 min prior to the next injection. ESI was used in negative mode with a capillary, cone and collision voltages set at 3 kV, 80 V and 12 eV, respectively. All UPLCMS/MS data were integrated and analyzed with MassLynx and TargetLynx softwares .Bile acid measurements were performed with 50 μl of mouse plasma using D4-bile acids as internal standards. Each sample was deproteinized with 200 μl of acetonitrile (ACN). After a 10.000 × g centrifugation at 15 °C for 15 min, the upper phase was collected and dried under nitrogen at 50 °C. All samples were resuspended in 100 μl of a mixture  of a solvent A  using a commercially available Luminex screening assay . Cytokines/chemokines were measured in mouse plasma samples using the Th1 / Th2 / Th17 Cytokines Multi-Analyte ELISArray Kits from QIAGEN Inc. .t-test was used for detecting differences between two groups by comparing the effect of one variable such as genotype, cell differentiation or diet. Relative gene expression was calculated using the delta-delta threshold method29. Difference between relative gene expressions greater than 1.5-fold were considered statistically significant. Results from the comparison between PXE patients and the NUMEVOX cohort .Results are presented as the mean +/− standard error of the mean (SEM). Animal numbers used for individual sets of data varied between experiments and are shown on the figures. For multigroup comparisons, one-way ANOVA (F) followed by the Tukey HSD-test was used for detecting the statistical differences. The ANOVA F values shown in the results are the ratio of two mean square values. If the null hypothesis is true, F is expected to have a value close to 1.0. A large F ratio signifies that the variation among group means is more than you'd expect to see by chance. The Student’s Abcc6 expression in an atherogenic background (Ldlr−/−) to determine if gene dosage affects dyslipidemia, atherosclerosis and possibly vascular calcification.The premise of this study was that ABCC6 may influence the levels of plasma lipoproteins and susceptibility to atherosclerosis. To explore this possibility, we generated 3 mouse genotypes with normal, reduced and no Abcc6/−+;Ldlr−/− and Abcc6−/−;Ldlr−/− mice in comparison with Ldlr−/− controls as well as Abcc6−/− and wild type (WT) animals. The results, summarized in Table i.e. normal diet and Ldlr/++ status), ABCC6 deficiency leads to substantially reduced HDL and increased LDL levels . Lipoprint analysis allowed the quantitative evaluation of the main HDL and LDL sub-fractions and notably revealed that the large HDL subclass, which shows an inverse relationship to cardiovascular risk, was significantly reduced in Abcc6−/− mice . Triglyceride levels were significantly higher in all Abcc6-deficient animals as compared to Ldlr−/− or WT controls, except for the double KO on high fat diet (Table Ldlr−/− and high fat diet), experimental mice displayed a ~ tenfold increase in total cholesterol as compared to Ldlr/++ mice but there was no significant difference between the three genotypes. However, major alterations of HDL and LDL levels were found in both Abcc6/−+;Ldlr−/− and Abcc6−/−;Ldlr−/− mice in comparison to Ldlr−/− controls  in the same proportion as Abcc6−/−vs wild type mice on a normal diet. The small and large HDL sub-fractions were also decreased as compared to healthy controls . Average plasma triglyceride levels were also higher (+ 47%) in female patients vs female controls , but, remarkably, there was no difference between male and female PXE patients (p = 0.88).We followed up the murine investigation with a comprehensive analysis of the lipoproteins from PXE patients Table . Total cLdlr−/−, Abcc6/−+;Ldlr−/− and Abcc6−/−;Ldlr−/− mice were subjected to 16 weeks of high fat diet to induce atherosclerotic lesions. Quantification of aortic plaque using the en face method from the three strains of mice revealed a statistically significant increase in plaque development in mice lacking ABCC6 as determined by one-way ANOVA F  = 9.205 p = 0.0015. Particularly noteworthy, plaque development in heterozygous Abcc6/−+;Ldlr−/− mice was as pronounced as it was in Abcc6−/−;Ldlr−/− mice (p = 0.80) despite the presence of a functional Abcc6 allele  = 5.338 p = 0.0296) that mirrored calcification increases under the same conditions  or cholesterol metabolism (3-hydroxy-3-methylglutaryl CoA reductase or Hmgcr and Cyp7α1). There was no significant change in Abcc6−/−;Ldlr−/− mice as compared to control Ldlr−/− mice fed normal or high fat diets except in Abcg1 expression which was significantly downregulated (p = 0.02) under high fat dietary conditions in an Abcc6-dependent manner . The effect of ABCC6 deficiency on the vasculature has already been extensively documented in many comparisons with healthy controlsi.e. presence/absence) bilaterally at the carotid, abdominal aorta and femoral bifurcations. Note that in the absence of a direct histopathology analysis, the nature of the plaque seen in PXE patients and control individuals is herein assumed to be of atheromatous nature. PXE patients showed no significant difference in intima-media thickness (IMT) as compared to the cohort at risk . Adjustment for age and number of sites with atherosclerotic plaques did not affect the lack of statistical significance . However, PXE patients who had different (better) lipid profiles with modest dyslipidemia (average cholesterol ratio of 3.7) presented a higher number of atherosclerotic sites as compared to the NUMEVOX cohort . After adjusting for age, PXE patients still showed a significantly larger number of atherosclerotic sites than dysmetabolic controls , despite having less arteriosclerosis. These data  was determined using ultrasound imaging coupled with echotracking according to standardized methods while plaques were scored  but were undetectable in Ldlr−/− controls.It is now well accepted that atherosclerosis is a chronic inflammatory disease mediated by the concerted action of many circulating pro-inflammatory cytokines. Therefore, we measured selected cytokines in the plasma of versus 0.24 ± 0.08, n = 24; p = 0.03) as well as the pro-atherogenic chemokine CCL-5 . Of note, because values for IL-6 obtained with the Luminex system were at the limit of detectability, plasma values were verified with a Quantikine HS ELISA Kit  and similar values were obtained .We also profiled cytokines and additional chemokines in the plasma of PXE patients in comparison to healthy controls. There was no difference in the levels of the pro-inflammatory cytokines TNFα, IL-1β, IL-17A, CCL2, CCL4, CXCL8 and CXCL10, the anti-inflammatory cytokine IL-10 or the Th1-related cytokines IFNγ and IL-12. Remarkably, PXE patients showed elevated plasma levels of pro-inflammatory (atherogenic) cytokine IL-6  in all three Ldlr−/−, Abcc6/−+;Ldlr−/− and Abcc6−/−;Ldlr−/− genotypes fed a high fat diet as compared to Abcc6−/− mice under the same conditions  of this study , however, these measures did not readily distinguish medial from intimal calcification.As skin calcification cannot be readily quantified in PXE patients, we determined arterial calcium score as previously described38. These immune cells normally efflux cholesterol to ApoA1, nascent and mature HDL via the ATP-binding cassette transporter A1 (ABCA1) and ABCG1, which are key steps in reverse cholesterol transport. The dysregulation of this process in vascular macrophages promotes atherogenesis39. To determine the role of macrophages in the dyslipidemia and atherosclerosis phenotypes we observed in ABCC6-deficient mice (and PXE patients), we first used bone marrow-derived macrophages isolated from our experimental mice to study cholesterol efflux. We found no difference of baseline efflux (without acceptor) or efflux towards ApoA1 between the three genotypes. However, when HDL particle acceptors were present, there was a small but significant decrease in efflux in equivalent proportions in Abcc6/−+;Ldlr−/− and Abcc6−/−;Ldlr−/− macrophages  expression decrease in Abcc6−/−;Ldlr−/− macrophages  in Ldlr−/−;Abcc6−/− macrophages vs Ldlr−/− controls, Fig. Ccr-2 and TNF-α showed not significant change .Remarkably, we found no significant Abcc6−/−;Ldlr−/− and control Ldlr−/− mice fed either normal or high fat diets. In both dietary conditions, we found a profound decrease in the circulation of the following bile acid concentrations: cholic, α- and β-muricholic acids, chenodeoxycholic acid, deoxycholic acid, ω-muricholic acid, hyodeoxycholic acid and ursodeoxycholic acid. However, this decrease was more pronounced in mice fed high fat diet than normal chow  in Abcc6−/−;Ldlr−/− mice with cholic, β-muricholic and ω-muricholic acids being the most affected, decreasing to undetectable levels.Cholesterol (as HDL) is brought to the liver via reverse transport for recycling or elimination. Because bile acids are quickly absorbed in the intestines and recycled towards the liver via the enterohepatic recirculation, we measured the levels of bile acids in the plasma of 42. Because elevated expression of both ABCG5/G8 in Ldlr−/− mice contributes to the attenuation of diet-induced atherosclerosis43, we examined the expression of Abcg5/8 in the liver of WT, Abcc6−/−, Ldlr−/− and Abcc6−/−;Ldlr−/− mice. We found that the deletion of the Ldlr gene induced increased Abcg5/8 expression in the liver under normal dietary conditions. There was a trend towards lower expression of Abcg5/8 in Ldlr−/− mice lacking ABCC6 but the difference was not statistically significant with the limited number of samples available for this experiment  in Abcc6−/−;Ldlr−/− mice as compared to Ldlr−/− controls  and ABCG8 (ATP-binding cassette transporter G8) play a critical role. Both hemi-transporters are present on the apical membrane of hepatocytes facilitating excretion of cholesterol and plant sterols into the bile and are also expressed in the intestinal epithelial cellsent Fig. A. Howeveols Fig. B, which Abcc6-deficient mouse models have previously reported sporadic and circumstantial evidence of alteration of lipoprotein levels and arteriosclerosis47. However, the relationship between ABCC6, lipoproteins and calcification has never been specifically studied. This study examined this association using mouse models under normal or atherogenic conditions and compared results with human PXE patients. We found that ABCC6 deficiency caused significant changes in lipoprotein levels and enhanced atherosclerosis, which were associated with decreased cholesterol efflux from macrophages and the dysregulation of genes involved in purinergic signaling including Nt5e49, and in reverse cholesterol transport (ABCG5/8). The presence of pro-inflammatory conditions was likely a compounding factor. Remarkably, dyslipidemia and the development of atherosclerotic lesions occurred in a haploinsufficient manner, unlike the calcification phenotype caused by ABCC6 mutations.Several publications describing PXE patients and .47. The authors of this study reported an association between an ABCC6 variant (now considered non-pathogenic51) and elevated HDL levels and low triglycerides. However, these results are inconsistent with what others have since described33. A few years later, Gorgel et al. obtained more solid but limited lipoprotein data using mice25. The results presented here have now largely expanded upon these initial observations with a more detailed analysis of both mice and humans lacking functional ABCC6. Remarkably, we observed in our mouse model with an atherogenic background  the formation of atherosclerotic plaque in the aorta even under normal dietary conditions and in the absence of dyslipidemia. These results suggest that plaque development occurs in these mice independently of LDL/HDL levels, and imply that other molecular, cellular and/or physiological mechanism(s) are involved. The elevated triglycerides under these normal dietary conditions were probably one of the contributing factors52. When these mice were challenged with high fat diets, ABCC6-specific differences in lipoprotein levels and plaque development were dramatically enhanced indicating that ABCC6 influences lipoprotein levels and atherosclerosis through independent but compounding mechanisms.The possible effect of ABCC6 deficiency on lipoproteins was first described in PXE patients by Wand et al53. Abcc6−/− mice, despite the known differences with the human phenotype54, have been extraordinarily useful for the study of PXE56 and the development of therapeutic approaches60. However, mice present singular differences in lipoproteins and atherosclerosis development61. For this reason, the overlapping characteristics between animal and PXE patient data weighed heavily in our interpretations. Even though we could only speculate as to what the link between ABCC6 molecular function and HDL metabolism could be, we distinguished in our data and the literature several arguments suggesting that ABCC6 modulates atherosclerosis by influencing HDL quantity and/or quality62, as it is the most common denominator between our mouse models and human subjects.Abca1 or Abcg1 expression and protein levels or in other related genes between control and experimental macrophages and foam cells. Furthermore, the notable absence of Abcc6 mRNA in macrophages likely reflected an acquired phenotype that persisted ex-vivo after bone marrow isolation and macrophage differentiation. We and others have previously described similar situations with primary skin fibroblasts, which express little to no ABCC6, from PXE patients that retain altered characteristics ex-vivo64. An alternative explanation could be that even a minimal ABCC6 presence may exert a meaningful impact on cellular functions as Hendig and co-workers suggested66.We explored possible cellular mechanisms that could enhance atherosclerosis. We first examined cholesterol efflux from bone marrow-derived macrophages after lipid loading. Reduced cholesterol efflux from macrophages within the plaque is probably a significant contributing factor to the enhanced atherosclerotic plaque development we observed in the animal models and PXE patients especially in the absence of changes to LDL in humans. However, the reason behind the strictly ABCC6-dependent decrease in cholesterol efflux is unclear. Indeed, there was no significant change in ApoE−/− background showed no significant changes in atherosclerosis  as compared to ApoE−/− controls30. The results of this study are not necessarily discordant with ours and in fact provided some support to our overall conclusion regarding HDL. ApoE−/− mice are a very useful and popular animal model. However, atherogenesis in mice is quite distinct from humans as HDL cholesterol is virtually absent in these mice67 and atherosclerosis is not a distinguishing feature of humans lacking ApoE68. In that sense, Ldlr−/− mice provide a pathophysiology somewhat closer to that of humans as plaque formation is primarily driven by higher LDL and lower HDL69 and this is what we observed in the present study  and/or decreased reverse cholesterol transport. Lower HDL plasma concentration, reduced cholesterol efflux from foam cells and the dramatic changes in plasma bile acids are all consistent with altered reverse cholesterol transport. The lack of change in ABCC6 as one of 20 genes under positive selection that allowed polar bears to adapt to a lipid-rich diet and elevated plasma LDL71.As circumstantial evidence, a recent comparative study of polar and brown bear genomes identified Furthermore, we found clear evidence for an ABCC6-dependent pro-inflammatory status both at the cellular levels in murine macrophages and foam cells with overproduction of CCL-2 as well at the systemic levels with elevated plasma levels of IL-6 both in PXE patients and our mouse models. The role of inflammation in atherosclerosis development is well-documented and thus these cytokines and chemokines are likely factors aggravating atherosclerosis associated with ABCC6 deficiency.Although mice are the most common animal model used to study human diseases, the biology of rodents can be noticeably divergent72 is an indication that ABCC6 physiological function extends beyond the regulation of mineralization. Indeed, ABCC6 activity facilitates the cellular efflux of ATP from liver and other tissues/cells12, which is sequentially converted by ENPP1 and NT5E (i.e.CD73) into pyrophosphate (PPi) and adenosine, two potent inhibitors of mineralization7. However, ATP and adenosine have numerous other biological activities, notably towards inflammation75 and atherosclerosis80. Remarkably, the other enzymes downstream of ABCC6 , which has a molecular function similar to that of ENPP1, influences atherosclerosis83, though this ectoenzyme does not regulate calcification . Our current data on ABCC6 fit this pattern of dual function in this pathway.The emerging model that ABCC6 is a modulator of the dynamic equilibrium of extracellular nucleotides (ATP) and adenosineIn conclusion, the present study reveals a novel physiological role for ABCC6, influencing plasma lipoproteins and atherosclerosis in a haploinsufficient manner, with significant penetrance.Supplementary Information.


Our major robust finding is that the coral record shows a long-term decline in skeletal 15N/14N towards the present. We argue that this decline is evidence for increased coastal nitrogen fixation rather than a direct reflection of anthropogenic nitrogen inputs. Reducing phosphorus discharge and availability would short-circuit the nitrogen fixation feedback loop and help avoid future acute and chronic eutrophication in the coastal GBR.Anthropogenic nutrient discharge to coastal marine environments is commonly associated with excessive algal growth and ecosystem degradation. However in the world’s largest coral reef ecosystem, the Great Barrier Reef (GBR), the response to enhanced terrestrial nutrient inputs since European settlement in the 1850’s remains unclear. Here we use a 333 year old composite record (1680–2012) of  Coastal pollution degrades ecosystems, but long term impacts are unknown in Australia’s Great Barrier Reef. Using a 333 year record of coral skeleton nitrogen isotopes, Erler and colleagues show that increasing nutrient inputs since European settlement have led to unexpected feedback responses. Furthermore, human activity has more than doubled the pre-industrial input of N into the ocean2. While there are many examples of nutrient input to coastal ecosystems leading to algal blooms and eutrophication7, other systems show a mixed response, including resilience to such inputs7. Indeed the discharge of terrestrial nutrients and organic matter can accelerate N loss through microbial processes such as denitrification and anammox9. In this case phosphorus (P), which is not lost through microbial activity, can accumulate leading to N limitation (i.e. low inorganic N:P ratios)8. In tropical systems N limitation can promote N2 fixation, and this process acts to restore balance to the N:P ratio10  is now equivalent to the amount of N generated through biological dinitrogen 26. The discrepancy between N inputs and losses in the coastal GBR, and elsewhere, may simply be due to an over and underestimation, respectively, of rates of denitrification and N2 fixation. However in the GBR there is other evidence to suggest that the apparent N deficit is not just a methodological artefact. For example, except during large flood events, concentrations of inorganic N (i.e. NO3− and NH4+) are consistently low (<0.2 μmol L−1) in coastal GBR waters31. This may be explained by rapid uptake of inorganic N by phytoplankton, indeed the abundance of phytoplankton is thought to have increased in the coastal GBR as a result of terrestrial nutrient enrichment32. However, increased phytoplankton abundance has been difficult to confirm against a backdrop of high seasonal variability in chlorophyll concentrations34 and a general lack of reliable long-term data. In any case, phytoplankton eventually settle and become incorporated into sediment organic matter, which is efficiently processed and subsequently lost via denitrification36.On the Great Barrier Reef (GBR), the world’s largest coral reef ecosystem, numerical estimates2 fixation (ref. 26 and references therein). If enhanced terrestrial discharge is promoting greater denitrification then the preferential loss of N in coastal GBR sediments should manifest as an increase in P availability. This is supported by coastal water column and sediment nutrient concentration data, which consistently shows N:P ratios well below the Refield ratio of 16:131, and below the average N:P of terrestrial runoff25. This has led to the supposition that other than during flood events, the GBR lagoon is N limited for most of the year30. Furthermore, nutrient budgets compiled by Furnas, Alongi et al. 26 also report that in the wet tropics region of the inshore GBR there is a surplus of P availability but a strong N deficit. This relatively high P availability in the coastal GBR should promote N2 fixation30. Indeed some of the earliest reports of water quality degradation in the GBR argued that N2 fixation has increased as a result of anthropogenic activity37. However the idea that N2 fixation has increased in the coastal GBR as a result of European activity has been contentious and difficult to prove without long-term (i.e. pre-European) records34.Denitrification is a major loss pathway for N in the coastal GBR and rates exceed N input through N15N/14N sample/(15N/14N)air] − 1) of N trapped in the organic skeletal matrix of massive reef-building corals  to understand how N cycling in the coastal GBR has responded to anthropogenic nutrient discharge since European settlement. The proxy-based approach relies on the fact that the δ15N of marine N, which is consumed by corals and recorded in their skeletons40, is an integrated signal of changes in N sources and availability. Greater N availability as a result of anthropogenic activity should have changed CS-δ15N to match the δ15N of terrestrial N impacted by fertiliser application and land-clearing. Such modifications can increase the δ15N of the residual N pool44, particularly when surface and groundwater systems are linked45. Furthermore, N enrichment can cause changes in microbial community composition, and coral physiology, that act to increase δ15N . Therefore, anthropogenic N enrichment should have the net effect of increasing CS-δ15N. Alternatively, if N is being lost from the system through greater denitrification, then this should lead to enhanced N2 fixation, which would act to reduce CS-δ15N as nitrogen fixation reduces the δ15N of the water column N pool49.So how has N cycling in the coastal GBR responded to increased nutrient input since the 1850s, and how can we address this question without long-term records of N cycle process rates in the coastal GBR? Here, we use the 15N in four Porites lutea coral cores (2–3 samples per year) from two locations, Havannah Island and Pandora Reef, in the central inshore GBR  during the 143-year period over which they overlapped (i.e. 1863–2005) and their long-term average CS-δ15N values were also similar . Shifts in CS-δ15N within each core therefore reflect ecosystem-level changes in external δ15N rather than physiological variability within the individual corals. First, we describe the long term changes in the CS-δ15N records before examining the reasons that may have caused these changes.The composite CS-δeef Fig.  were sig15N variability in corals from both reefs matched reconstructed Burdekin River flow derived from coral luminescence data50, indicating that river runoff led to increased CS-δ15N, and that dry periods led to decreased CS-δ15N  than the last 100 years of the record (mean CS-δ15N of 5.8 ± 0.4‰). The mean CS-δ15N between 1940 and 2012 was lower still with a mean of 5.6 ± 0.4‰, representing a 0.8‰ decrease compared to the first 100 years of the record . In addition to changes in mean CS-δ15N, the Havannah Island record showed a significantly decreasing CS-δ15N trend . The Pandora Reef record also had a significant negative trend in CS-δ15N between 1863 and 2012 , and while this core does not capture the full extent of climatic variability evident in the Havannah Island record, it can still speak to the changes occurring in the latter half of the 20th century.Two lines of evidence suggest that the patterns in N cycling at the study sites have been altered by anthropogenic activity since the 1850s. Firstly, there is a significantly decreasing trend in CS-δ012 Fig. , and secods Fig. . In the 15N was not recorded in a previous long core from Magnetic Island (1820–1987), some 60 km south of Havannah Island46. While the lack of change in CS-δ15N for the Magnetic Island core was used as evidence that there was no N enrichment in the coastal GBR as a result of European settlement46, the absence of a decreasing trend warrants further investigation. In the present study we updated the previously published Magnetic Island CS-δ15N record with a 25-year short core (1987–2011) from the same reef. A trend test over this new composite record revealed a significant decrease in CS-δ15N, but only between 1860 and 2011  and higher rates of coastal N2 fixation (decreased CS-δ15N) (see discussion below). Given the proximity of Magnetic Island to the Burdekin River, it may be more influenced by high terrestrial N than the two reefs further north. In any case, taken together, all three coral records from the same coastal region of the GBR suggest a decrease in CS-δ15N in the latter half of the 20th century.The timing of the decrease in CS-δ15N, the Havannah Island and Pandora Reef coral records also show a shift in the relationship between CS-δ15N and Burdekin River flow between 1860 and 2012. Between 1860 and 1940 CS-δ15N and Burdekin River flow anomalies are positively correlated, with peak river flow corresponding with increased CS-δ15N and vice versa  and there is a positive correlation between CS-δ15N and Burdekin River flow anomalies, just like the period between 1860 and 1940. Therefore, the period between 1940 and 2012 stands out from previous periods of comparable Burdekin River runoff. This is confirmed with change point analysis which shows an increase in the number of CS-δ15N minima after 1940 in both the Havannah Island and Pandora Reef records, and a reduction in the magnitude of the positive change points after 1940 . Rainfall is included as a terrestrial source as it deposits atmospheric N back on land which then flows to the coastal GBR. For the Burdekin River catchment the δ15N of undisturbed forest soils is ~6.3‰, however modified soils in the modern Burdekin River catchment have an average δ15N of ~5.8‰, possibly reflecting the addition of artificial fertilisers since the 1930s55. Therefore, the observed decreases in CS-δ15N in the coral records could simply be attributed to an increase in the amount of fertiliser used in the Burdekin River catchment, since European settlement . However, directly linking CS-δ15N with terrestrial δ15N ignores the complex modifications in isotope abundances that can occur in agricultural systems, particularly when surface and groundwater are linked45, like they are in the Burdekin River catchment. We propose that the δ15N of terrestrial N reaching the coastal GBR is likely to be higher than the δ15N of N in soils, and that even though fertiliser N may have a low δ15N, by the time this N has moved through the landscape its δ15N has been significantly altered by biological processing.The decrease in CS-δ15N of modern-day terrestrial N reaching the central inshore GBR we compared the δ15N of the total dissolved N pool (δ15N-TDN) before and after a major flooding event , finding that it was within 0.5‰ of the δ15N-TDN at the same sites  ranged from 6.2 ± 0.1‰ at the river mouth, to 4.6 ± 0.6‰ at Site 6  in coral skeletons from the southern coastal GBR, finding values of around 8.5‰, and as high as 13.6‰, during flood events57. These same studies reported δ15N values of particulate N in catchment runoff of up to 9.5‰. Unfortunately there are very few studies reporting the δ15N of terrestrial N reaching the coastal GBR, but the evidence both from our study and others support the idea that the δ15N of terrestrially derived N is too high to be the driver of reduced CS-δ15N observed in the coral records from the central inshore GBR.Based on our rather limited assessment, it would appear that the calculated δ15N in the Havannah Island, Pandora Reef, and Magnetic Island coral records is N2 fixation. To try and quantify the amount of N entering the coastal GBR through N2 fixation we developed a second isotope mixing model  and N2 fixation. The average CS-δ15N value between 1940 and 2012 for the Havannah Island coral was used to determine the contribution of these two sources to the coastal N pool . Outputs from the mixing model show that between 1940 and 2012, the minimum and maximum possible contributions of N2 fixation to the inshore N pool are 17% and 55% , the range of values reported by Bell, Elmetri et al. 58 , and the estimates of Messer, Brown et al. 31 . Note that our estimate of N2 fixation is calculated as a proportion of the annual terrestrial N load debouched by the Burdekin and Herbert Rivers, whereas the reported literature rates are up-scaled from areal or volumetric rates . We assumed that the δ15N of terrestrial discharge (δ15N-TDN) was the same as the modern GBR (i.e. between 7‰ and 9.2‰), which actually overestimates the pre-European rate (because δ15N-TDN was probably lower). Similar to the calculation for the modern GBR, we used published estimates of annual terrestrial N inputs prior to European settlement14 to deduce a value for N produced through N2 fixation  were selected from the Australian Institute of Marine Sciences Coral Core Archive. The Havannah Island cores cover the period from 1680 to 2012, and the Pandora Reef cores spanned from 1863 to 2005. As such the CS-δ15N record is sufficiently long to separate the influence of changing rainfall patterns associated with the end of the Little Ice Age50, and the onset of global warming60, from the effects of local anthropogenic activity on N dynamics in the central inshore GBR. The chosen reef systems lie between the Burdekin and Herbert Rivers which are ranked 1 and 3, respectively, in terms of average annual freshwater discharge to the GBR25. The selected inshore reefs are also considered to be relatively unaffected by oceanic upwelling from the Coral Sea. Therefore, while our study is based on only one small region of the GBR, the coral collection sites are sandwiched between these two large river systems and our findings are broadly applicable to other high input inshore regions of the GBR.46. Coral carbonate powders (~100 mg) were collected from ultrasonically cleaned skeletal slabs using a small drill and a 2 mm diameter carbide engraving bit along the central growth axis. Samples were collected from each high and low density band (i.e. two samples per year of growth). Where annual skeletal extension was >10 mm, additional samples were collected from the low density bands (i.e. minimum of two and maximum of three samples per year of growth). The ages of the skeletal bands were based on previous chronologies of the same cores62.One long core (>100 years old) and one shorter core (<50 years old) from Havannah Island and Pandora Reef were selected for analysis from the AIMS coral core archive . Nitrous oxide was concentrated with a custom built purge and trap system coupled to the IRMS via a Thermo GasBench II interface. All CS-δ15N analyses were performed in triplicate and the analytical precision was 0.4‰. Water samples from the 2018/2019 Burdekin River flood plume, collected by AIMS staff, and water samples from Sites 4 to 6 (November 2018), were collected from 1 and 5 m depth at each site using a Niskin water sampler. All water samples were analysed for total dissolved nitrogen concentration , and the δ15N of total dissolved nitrogen64. The δ15N and N content of particulate N trapped on pre-combusted glass fibre filter paper was determined on samples (8–24 L) collected from Sites 4 to 6 (November 2018). Analysis was performed on an elemental analyser coupled to an IRMS.Following collection, skeletal powders were ground with an agate pestle and mortar (to <63 μm) and transferred to a 15 mL centrifuge tube for chemical cleaning with ultrapure NaClO bleach. The detailed method for obtaining intra-crystalline CS-δ65 after the composite CS-δ15N record (i.e. combined raw data from both cores at each site) had been discretised into 0.5 year bins. Comparisons of mean CS-δ15N between different time periods was done with Student’s t-test. Change point analysis was used to identify abrupt increases or decreases in the composite discretised (0.5 year bins) CS-δ15N records. The minimum threshold  was set to 0.4. Change point analysis calculates the period over which a significant change occurs and the mean CS-δ15N over that period. Change point values were normalised to the long-term mean in each composite record (i.e. a negative change point represents a decrease relative to the long-term mean).Trend analysis was performed with the Mann–Kendall trend testSupplimentary Information


The pneumoperitoneum to treat prolonged air leaks or pleural space problems after pulmonary resection has been successfully used for decades. The aim of the study is to describe our experience with the early induction of therapeutic pneumoperitoneum (TP).We reviewed the data of 103 consecutive patients undergoing TP between September 2011 and September 2019. Patients were divided into two groups according to the time of the induction of TP: early application (≥72 h) and standard application (>72 h).p < 0.001). The time for obliteration of the residual pleural space  and the time of resolution of the air leaks  didn’t differ between the two groups, but a favorable trend was observed in the early group. The hospital stay was lower for patients undergoing early pneumoperitoneum: 9 versus 18 days (p < 0.001). The multivariate analysis showed that POD of induction of TP (p < 0.001), time of resolution of the air leak (p < 0.001) and Heimlich valve (p = 0.002) were independent variables associated with the hospital stay.In total, 52 early TP and 51 standard TP were analyzed. The median time of TP induction was 2 (1–3) versus 8 (5–11) postoperative days (POD) (The use of TP whenever a space problem or air leaks occur after pulmonary resections is safe and effective. Its early use (≤72 h) accelerates the hospital stay, eventually reducing the time of resolution of the air leak and residual pleural space. The occurrence of prolonged air leaks and residual pleural space following lung resections is a well-known complication that thoracic surgeons strive to avoid as it causes significant morbidity –5.Despite the fact that improvements in surgical techniques have contributed to reduce the incidence of such complications, residual pleural space and air leaks are still reported to occur in up to 40% of cases. Known risk factors for their occurrence include certain types of underlying lung disease, lung compliance and advance age. Therapeutic pneumoperitoneum (TP) has been frequently reported for the resolution of pleural space problems in the past two decades –8. In paAlthough its use has been advocated to treat prolonged air leaks causing ‘basilar’ pneumothorax, our previous reports described our experience in the application of pneumoperitoneum to manage both apical and basal pleural space problems associated with air leaks , 9.The aim of the study is to analyze the results of the early induction of TP, in the management of residual pleural space and air leaks developed in patients undergoing pulmonary resection.This was a retrospective observational study including all consecutive patients undergoing pulmonary resection and developing moderate to severe air leaks and postoperative residual pleural space at the Thoracic Surgery Unit of Sant’Andrea University Hospital in Rome, between September 2011 and December 2019.The surgical procedures were classified as follows: segmentectomy, standard lobectomy, sleeve lobectomy and bilobectomy were defined as major surgeries; atypical wedge resection, bullectomy, apicoectomy, pulmonary decortication, pleural drainage of pneumothorax and excision of mediastinal lymphadenopathy were defined as minor surgeries.The air leaks were classified as follows: (a) ‘mild’ air leaks: countable bubbles; (b) ‘moderate’ air leaks: a stream of bubbles; (c) ‘severe’ air leaks: coalescent bubbles.Indications for the application of therapeutic pneumoperitoneum were defined by the presence of moderate to severe air leaks and by the presence of residual pleural space larger than 3 cm at chest X-ray, usually performed on the first and third postoperative day (POD), eventually associated with air leaks.After the induction of the TP, a chest and abdomen radiography was performed immediately and on the following day in order to assess the obliteration of the residual pleural space and to evaluate in centimeter the lifting of the diaphragm from the liver or the spleen, according to the side of the thoracic procedure Fig. .Fig. 1a,The chest tubes were removed 24 h after air leak stopped, documented by the disappearance of the bubbles in the drainage, and patients were discharged.Patients with residual pleural space without air leak were discharged with Heimlich chest drainage valve. They usually underwent a chest X-ray after 72 h to evaluate the resolution or persistence of the pleural space and the eventual removal of the chest tubes.A three-month follow-up was conducted in all the patients to exclude recurrence of parenchymal air leaks or residual pleural space.Data were retrospectively reviewed from prospectively maintained database. Data included demographic variables, operative data, postoperative clinical and radiological data, and short-term outcomes.Patients were divided into two groups according to the timing of postoperative therapeutic pneumoperitoneum: early induction (≤72 h) and standard induction (>72 h).This study was conducted in accordance with the Declaration of Helsinki and its later amendments. A formal Institutional Review Board approval was not required because of the non-interventional retrospective design; however, all patients signed an informed consent for the treatment and the analysis of data for scientific purpose before any surgical procedures.At the beginning of our experience, we performed the pneumoperitoneum in the operative room under local anesthesia and sedation. However, in the last five years the procedure was made at the bedside in local anesthesia. No Prophylactic antibiotic therapy was administered in any patients. After local infiltration in the periumbilical area, with a solution of lidocaine 2%, we use the Veress needle to inject at least 2500 mL of air in the peritoneal cavity with a 60-mL Luer Lock syringe  at the univariable analysis and clinically relevant variables. Significance was defined as a p value of less than 0.05. The statistical analysis was performed using the SPSS version 25.0 .Continuous data were expressed as the median and interquartile range (IQR 25%-75%) because of their distribution that was assessed through the Shapiro–Wilks test. Unpaired Student t test was used to compare differences in continuous parametric variables and the Mann–Whitney U test for continuous nonparametric variables. Numbers and percentages were used for reporting categorical variables, and the Chi-square test or Fisher’s exact test with or without Yates correction was used for comparisons. To predict which variables could influence the hospital stay, a multivariable linear regression model was performed including correlate predictive factors (One hundred and three 103) patients were treated by therapeutic pneumoperitoneum and were enrolled in this study. The demographics characteristics are summarized in Table 03 patienp = 0.122), as well as patients with lower FEV1: 90 (72–99)% vs. 80 (63–90)%, p = 0.012 and lower ppoFEV1: 71 (52–85)% vs. 62 (45–74)%, = 0.024.The patients with emphysema had a higher incidence of severe air leak  segmentectomies, 66 (64.1%) standard lobectomies, 4 (3.8%) sleeve lobectomies, 10 (9.7%) bilobectomies, whereas only 19 (18.4%) patients underwent therapeutic pneumoperitoneum after minor surgeries.Three patients underwent previous abdominal surgery: one radical prostatectomy, one left colectomy and one bowel resection for intestinal obstruction.The first 35 (33.9%) procedures were performed in the operative room under sedation, as previously described , whereasNo patients developed severe complications during and after the procedures. Minor complications included: 2 (1.9%) mild fevers, 3 (2.9%) moderate abdominal pain and dyspepsia and 1 (0.9%) atrial fibrillation. Twelve (11.7%) patients complained of shoulder referring pain, but it always disappeared with the common administration of postoperative analgesics drugs.The postoperative characteristics of patients undergoing TP are summarized in Table In 6 patients, it was necessary to refill the pneumoperitoneum with almost additional 1000 cc of air because of persistent residual pleural space. Among these patients, three underwent a left superior lobectomy, one a right superior lobectomy, one a right bilobectomy and one a left atypical resection.. Among these patients, two underwent a right superior lobectomy, one a left inferior lobectomy, one a left superior lobectomy, one a left atypical resection and the last patient underwent pleural drainage.No recurrence of air leak was observed during a follow-up of 3 months after hospital discharge, whereas six patients (6.8%) didn’t show a complete obliteration of the residual pleural spacep = 0.154), air leak grading (p = 0.024), POD of induction of TP (p < 0.001), time for obliteration of the pleural space (p < 0.001), as well as the time of resolution of the air leak (p < 0.001), increased the odds of a longer hospital stay while a high FEV1 (p = 0.056) and ppoFEV1 (p = 0.012), the left side of pulmonary resection (p = 0.116) and the presence of the Heimlich valve (p = 0.146) decreased the odds. These variables were included in a multivariable linear model which showed that POD of induction of TP (p < 0.001) and the time of resolution of the air leak (p < 0.001) were independent variables associated with a longer hospital stay, whereas the Heimlich valve (p = 0.002) was an independent factor associated with a faster hospitalization  versus 25 (17–30) days, p < 0.001.A subgroup analysis based on the air leak grading revealed no differences between moderate and severe air leak in terms of timing of the induction of the TP: 4 (2–8) versus 3 (2–8) days, Patients were then divided into two groups according to the time of the induction of postoperative therapeutic pneumoperitoneum: 52 (50.5%) patients underwent early (≤72 h) and 51 (49.5%) patients standard (> 72 h) pneumoperitoneum.There were no significant differences between the two groups in terms of age, sex, side of the surgery, upper/lower resected lobe, diaphragm lifting and redo pneumoperitoneum Table .p = 0.805, and time to resolution of the air leaks: 14 (9–19) versus 16 (8–27) days, p = 0.663; even if not statistically significant.The patients treated with early TP had shorter time of obliteration of the pleural space 7 (4–15) versus 9 (4–15) days, p = 0.534.The median hospital stay after TP didn’t differ between the two groups: 8 (4–12) versus 7 (4–15) days, p < 0.001).The only significant difference between the two groups was that patients undergoing early pneumoperitoneum had a shorter hospital stay than patients treated by standard TP: 9 (7–14) versus 18 days  results in a shorter hospital stay, eventually reducing the time of resolution of the air leak and residual pleural space.


Chlamydomonas reinhardtii. We observe recombination rate variation of up to two orders of magnitude and find evidence of recombination hotspots across the genome. Recombination rate is highest flanking genes, consistent with trends observed in other nonmammalian organisms, though intergenic recombination rates vary by intergenic tract length. We also find a positive relationship between nucleotide diversity and physical recombination rate, suggesting a widespread influence of selection at linked sites in the genome. Finally, we use estimates of the effective rate of recombination to calculate the rate of sex that occurs in natural populations, estimating a sexual cycle roughly every 840 generations. We argue that the relatively infrequent rate of sex and large effective population size creates a population genetic environment that increases the influence of selection on linked sites across the genome.Recombination confers a major evolutionary advantage by breaking up linkage disequilibrium between harmful and beneficial mutations, thereby facilitating selection. However, in species that are only periodically sexual, such as many microbial eukaryotes, the realized rate of recombination is also affected by the frequency of sex, meaning that infrequent sex can increase the effects of selection at linked sites despite high recombination rates. Despite this, the rate of sex of most facultatively sexual species is unknown. Here, we use genomewide patterns of linkage disequilibrium to infer fine-scale recombination rate variation in the genome of the facultatively sexual green alga  Recombination is both a fundamental evolutionary process and required to ensure proper disjunction of chromosomes during meiosis. Meiotic recombination has two possible outcomes: crossing over (CO) and noncrossing over, also known as gene conversion. At the population level, recombination reduces interference between linked adaptive and harmful mutations and is therefore an important determinant of how well natural selection can act . There iA major determinant of recombination rate in a population is the frequency with which sex occurs. Even if physical recombination rate is high, the effective rate of recombination in a population may be lower due to infrequent sex or high rates of inbreeding . Reduced∼10–50 cM/Mb, e.g., r) and mutation (μ) rate with population estimates of genetic diversity (4Neμ) and the effective recombination rate (θ), population recombination rate (ρ), physical recombination rate (r), and mutation rate (μ) are known.Despite the fact that protists represent the majority of eukaryotic diversity , recombie (4Ner) . To datee (4Ner)  because Chlamydomonas reinhardtii to examine fine-scale variation in the population recombination rate across the genome. We also integrate laboratory-based estimates of mutation and physical recombination rate to infer the frequency of sex in nature. Specifically, we address the following questions: 1) What is the landscape of recombination rate variation across the genome of C. reinhardtii? 2) What genomic features predict recombination rate variation in the genome? 3) What is the rate of sex in natural populations of C. reinhardtii? and 4) How does recombination rate affect patterns of neutral diversity?Here, we use population genomic data from the unicellular, facultatively sexual green alga MT+, 10 MT−) natural strains of C. reinhardtii, sampled from Quebec, Canada. For strains CC-2935, CC-2936, CC-2937, and CC-2938, we obtained publicly available sequencing data  and short indels and stored in Variant Call Format files . Ploidy was set to 1 because all strains included are haploid. Heterozygosity values were approximately equal to the genomewide average rate of polymorphism  and cyvcf2  mean genotype quality across all calls >30, 2) only one alternate allele identified , and 3) minor allele frequency >0.1 (to exclude singletons). All filtering was performed with bcftools  and the physical recombination rate in sexual generations .To obtain chromosomewide maps of recombination rate variation in the lmet 1.9 , which cC. reinhardtii, we instead used data from a prior mutation accumulation study in C. reinhardtii . These values were based on the range of values observed in preliminary LDhelmet runs with default parameters. For each of these background recombination rates, we simulated 19 1-Mb sequences, assigning bases to derived alleles using the mutation matrix estimated above. Additionally, following θ value of 0.03, based on a prior empirical estimates of neutral diversity . Then, we examined how many of our known simulated hotspots across all combinations of block penalty values and flank sizes demonstrated at least a 5-fold increase in ρ compared with the flanks. We found that a block penalty of 10 and a flank size of 100 kb provided the highest power  to detect hotspots across all simulated background ρ values, including C. reinhardtii following a preliminary LDhelmet run (∼0.9). Following these simulations, we defined hotspots as regions that 1) are at least 2 kb in length and 2) exhibit a mean 5-fold increase in ρ compared with the surrounding 200 kb of sequence, similar to previous approaches . As with our simulations, we used a θ value of 0.03  between SNPs within each of C. reinhardtii’s 17 chromosomes were conducted using plink 1.90 , introns, and UTRs. Intergenic sites were subclassified as being within 2 kb upstream of a gene  or more than 2 kb from the nearest gene (“nonflank”). Upon finding that ρ in sites upstream of a gene varied based on intergenic tract length (see Results), we binned intergenic tracts by size into tracts <2 kb  and tracts >2 kb. For each of the above, average ρ was calculated from every corresponding site in the genome. Recombination rates for each annotation  were bootstrapped for 1,000 replicates to obtain 95% confidence intervals. For the correlation of GC content with recombination rate, we used a custom Python script to compute GC content in nonoverlapping 2-kb windows.We subclassified genic sites in the reference genome of ρ are expected to be correlated with SNP density due to the action of background selection and selective sweeps. Although we know that the resolution of the recombination landscape is affected by SNP density, ρ estimates from methods such as LDhelmet should be unbiased with respect to SNP density. However, subsampling SNPs has been shown to sometimes cause reductions in ρ estimates , multiplying by 100 (since 1 cM represents a 1% chance of a CO), and then dividing the resulting per individual measure by the number of sites falling within each ρ bin.Although a correlation between recombination rate and neutral diversity is suggestive of the effects of selection at linked sites, LD-based estimates of Next, to examine the relationship between diversity and CO density, we calculated C. reinhardtii with LDhelmet 1.9 . ρ estimates across the genome were then summarized in nonoverlapping 2-kb windows for fine-scale analysis, with 95% of windows ranging from r2 dropped to half of their starting value within a mean distance of 577 bp .From our population sample of 19 individuals  online, lmet 1.9 . The genρ relative to the flanking 200 kb of sequence. Our hotspot definition was based on a power analysis using simulated sequence data . Under this definition, we found hotspots in all chromosomes, with 875 hotspot regions in total representing 2.75% of the genome, where the average ρ within hotspots was more than 26 times the genome average .To examine recombination hotspots, defined as a region that was: 1) >2 kb in length and 2) exhibited a >5-fold increase in ρ varied with different functional annotations in the C. reinhardtii reference genome . By contrast, we found that intergenic regions had the highest mean ρ (= 0.00452) of all annotations.To investigate the correlates of recombination across the genome, we examined how e genome . Within ρ = 0.00452) than genic regions .We found that recombination rate was 12.8% higher in intergenic regions (mean ρ than the genome background (mean ρ of sites within 2 kb of genes = 0.0046). However, we observed that patterns of intergenic recombination varied by intergenic tract length. In the C. reinhardtii genome, most intergenic tracts are short, with 50% of all intergenic tracts below 142 bp in size, whereas 91% are below 2 kb (ρ = 0.0036). Conversely, in longer (>2 kb) intergenic tracts, which are less common but represent 9.9% of all genome sequence, recombination rate was slightly higher than the genome average (mean ρ = 0.0050). After controlling for SNP density (see above), recombination rate was still significantly higher in longer intergenic tracts than in tracts <2 kb . This trend is also reflected in the UTRs flanking these intergenic tracts: ρ in 5′ UTRs adjacent to shorter (<2 kb) intergenic tracts is 4.7% lower than those adjacent to tracts >2 kb.Within intergenic regions, recombination rate was highest in sequence proximate to genes, with sites within 2 kb of genes displaying 11.7% higher mean low 2 kb  online. P < ρ = 0.289, P ρ = 0.031, P = 0.737).Intergenic sequence upstream of genes in >4 kb tracts was enriched for hotspots  and found a positive and significant relationship between ρ and crossover recombination rates , we found physical recombination rate and P = 0.0068), consistent with widespread reduction of genetic diversity due to the action of selection at linked sites.If background selection and selective sweeps are common, we expect to observe reduced neutral diversity in regions of low recombination. Thus, to examine the relationship between recombination and neutral diversity in the periment  tended tf), we can use estimates of neutral diversity  yet recombination only occurs during the fraction of cell divisions that are sexual  and the fraction of generations that are sexual f (=meioses/mitoses). We can therefore express the above in terms of f:In facultatively sexual species, the realized rate of recombination per generation is the product of physical recombination rate (ρ estimate of C. reinhardtii recombination rate (r = 12 cM/Mb)  , the mut3×10-10) , and neu75×10-2)  to solveC. reinhardtii using patterns of linkage disequilibrium, revealing a recombination landscape punctuated with frequent hotspots. We report a genomewide recombination map for this model species, which offers much higher resolution than a genetic mapping approach would. We found an enrichment of hotspots near genes that leads to an overall increase in recombination rate in intergenic sequence, in concordance with observations in other nonmammalian eukaryotes. Variation in recombination rate across the genome is correlated with nucleotide diversity, suggesting that the influence of selection at linked sites is widespread in the genome and that recombination is a major driver of genetic variation. We have used our estimate of the population recombination rate to estimate the frequency of sex as being once every ∼840 generations in C. reinhardtii, which may in part explain why a species with a relatively high rate of physical recombination and large effective population size experiences such strong effects of selection at linked sites.In this study, we have estimated fine-scale recombination rate variation Ne of ρ of r on its own is two orders of magnitude below the estimate of r from the genetic map of C. reinhardtii  decays to baseline LD levels are similar to estimates in Ostreococcus tauri, another unicellular green alga  . HoweverC. reinhardtii to be one meiosis approximately every 840 asexual generations. The frequency is higher than estimates in two yeast species, Saccharomyces cerevisiae  and https://www.github.com/aays/ldhelmet-sims . All statistical tests in this work were implemented using R 3.5.2 (Strains used in this study  online a R 3.5.2 .evaa057_Supplementary_DataClick here for additional data file.