The study aimed to analyze anemia management in non-pregnant, and non-menopausal women aged from 18 to 50 years old, in a French primary care setting.An observational descriptive prospective study was conducted between November 2018 and February 2019. Inclusion criteria were as followed: anemia diagnosed in women aged from 18 to 50, not pregnant and not menopausal. Quantitative and qualitative data were anonymized and collected through an electronic survey. Investigating general practitioners completed the questionnaire for each newly diagnosed woman. Mean values and medians were calculated for the quantitative data. Answers to the open questions were encoded manually and proportions of the different modalities have been calculated.Altogether, 43 women with anemia were ascertained. Moderate microcytic anemia, due to an iron deficiency in a context of menorrhagia, was the most observed anemia profile. The mean value of hemoglobin was 10.5 ± 1 g/dl. Among these women: 32 (74%) presented an iron deficiency, 17 (53%) had inappropriate intakes, and 9 (28%) reported menorrhagia. For 17 (40%) women, unnecessary or inappropriate exams were prescribed. The investigations did not allow to establish a differential diagnosis for 12 women (28%). Even for similar clinical situations, anemia management was variable. Among the women who presented iron deficiency, 15 (47%) were informed about an iron-rich diet and received a daily iron supplementation of ferrous sulfate between 80 mg and 160 mg.Our study highlights that, in the absence of specific national guidelines for anemia management in non-pregnant, non-menopausal women in primary care settings, French GPs undergo various clinical management strategies leading to a heterogeneous, sometimes inappropriate follow-up.Women with iron deficiency were prescribed higher daily iron supplementation than recommended, according to new evidence, suggesting a maximal daily dose of 50 mg of elementary iron in a context of Hepcidin up-regulation in the case of an iron overload.Additional longitudinal studies with a bigger sample size and randomized controlled trials are needed to confirm our results and to elaborate national guidelines. The World Health Organization (WHO) defines anemia in women as a hemoglobin concentration below to 12 g/dl  which isAnemia is considered as an independent risk factor of morbid mortality regardless of age or gender . Its conThe majority of anemia cases are diagnosed and managed in primary care . DespiteWe conducted an exploratory study in a primary care setting to analyze GP’s anemia management in non-menopausal women between 18 and 50 years old, non-pregnant, from diagnosis to treatment in the case of iron deficiency anemia (IDA). The study was observational, descriptive, prospective, and conducted between November 12th, 2018 and February 10th, 2019.Hauts-de-France and Pays de la Loire. GPs were randomly selected from the telephone directory in each region in order to include a number between 30 and 40 investigators. The GPs were then individually informed about the study protocol, first by e-mail and followed by a phone call if necessary, some of them were met physically. Their socio-demographic characteristics were assessed: age, gender, region, and terms of exercise, such as urban or rural area, and working as a GP or a surrogate.The inclusion criteria for the investigating GPs were as followed: Being a GP surrogate, or an installed GP, working in the following French regions: EPI info software. We used non parametrical tests in the context of a small sample:  Kruskall-Walis test, and fixed IC at 95% (p < 0.05).The data were collected and anonymized through an auto-administered questionnaire created by the Lime Survey® software. Each investigating GP completed a questionnaire for each new included woman presenting anemia. The GPs had access to their questionnaire via a code to view complementary exam results, and treatments related to the anemia management. Each GP had the possibility to complete 5 patient files. Before the study started, the questionnaire was tested by 6 GPs. The questionnaire included 5 questions: “initial biological data”, “initial demographical and clinical data”, “terms of prescription of biological exams”, “your decision and action at the moment of results’ reception “, and “in case of complementary exams, or specialized expert opinion”. Inclusion criteria for patients were as followed: age between 18 and 50 years old, non-menopausal, newly diagnosed anemia (according to the WHO criteria). Pregnancy was a non-inclusion criterion. To encourage the GPs’ participation in the study, they were called by phone every 3 weeks. The questionnaire data were collected and transcribed in an Excel sheet. Quantitative data were calculated as means and averages. Qualitative data, (free answers to the open questions) were encoded by a global content analysis. New categories were created manually during the data collection along with the needs, according to the answers which were progressively returned by the GPs. We calculated the percentage proportion of the present or absent different terms using the , pallor 19% (n = 8), menstruation disorders 12% (n = 5), dander disorders  9% (n = 4), and anxious-depressive syndromes 5% (n = 2). Anemia was diagnosed during a routine check-up, a pre-therapeutic evaluation, or follow-up for 12% (n = 5) of the women.The average age of the GPs was 39 years old, and 19 (50%) were women. Thirty GPs (81%) worked in an urban area, and 17 (46%) had their own office. The women’s blood test prescriber was for 35 their GP (82%) or the GP surrogate, and for 7 (16%) another substitute. One woman declared her gynecologist as her prescribing physician. Initial symptoms leading to the blood test prescription were asthenia 72% (n = 6) of the patients were between 18 and 25 years old, 35% (n = 15) between 26 and 33 years, 25.5% (n = 11) between 34 and 41 years old, and 25.5% (N = 11) between 42 and 50 years old. Thirty-three per cent (n = 13) had no medical history. Twenty-three per cent (N = 10) had prior cardiovascular history, 14% (n = 6) had psychiatric disorders, and 12% (n = 5) had a prior gynecological history. Thirty-seven per cent (n = 16) took no medication. Twenty-one percent (n = 9) were taking a birth control pill (40% (n = 6) of the 26–33 years old women).Fourteen per cent (3 (± 8.6). Anemia was estimated mild in 40% (n = 17) of the cases, moderate in 58% (n = 25) of the cases, and severe in 2% (n = 1) of the cases. We did not observe any significant differences neither regarding the hemoglobin level nor the severity of anemia according to the different age groups .The average hemoglobin blood level was 10.5 g/dl (± 1), mean: 10.6 g/dl . The average mean corpuscular volume (MCV) was 81μmn = 29) of the cases of anemia were microcytic, 26% (n = 11) normocytic, and 7% (n = 3) macrocytic. There were no significant differences between the MCV according to the different age groups (p = 0.07). However, the distribution of the three forms of anemia:  varied in a significant way according to the different age groups (p = 0.003)  of the patients came for a follow-up appointment, one patient had a phone call, and two patients did not come to their appointment. When the patients were not available for a consultation when the GP received the blood test results, the GP did not call the patient to inform them immediately in 44% (n = 11) of the cases. The reasons were: to encourage the patient’s autonomy in 36% (n = 4) of the cases, and the absence of urgent nature of the result in 90% (n = 10) of the cases. The patients were contacted in 56% (n = 14) of the cases: in order to inform, and reassure them in 64% (n = 9) of the cases, to evaluate their clinical tolerance in 21% (n = 3) of the cases, to prescribe complementary exams in 28% (n = 4) of the cases, or to inform the patient that a treatment was necessary in 43% (n = 6) of the cases.Ninety-three per cent (n = 32) of the cases: 84% (n = 27) of these clinical exams were a quantification of menstruation, 31% (n = 10) were a gynecological exam, and 81% (n = 26) were an evaluation of the cardiological tolerance and/or an exam of the digestive system. The GPs initially based their diagnosis on other elements of the complete blood count (CBC) in 49% (n = 21) of the cases and identified an eventual regenerative character of the present anemia in 30% (n = 13) of the cases.After receiving the blood test results, a clinical exam was performed in 74% (n = 21) of the cases (cf. Table n = 17) of the patients, the prescribed exams were not indicated. The vitamin B12 and B9 blood levels were prescribed respectively in 35% (n = 15), and 37% (n = 16) of the cases. These dosages were not justified in 67% (n = 10), and 69% (n = 11) of the cases. In fact, B12 and B9 blood levels were physiological standard in 100% of the cases, or their dosages were not recommended. In case of justified prescriptions, their serum levels were low for B12 in 20% (n = 1) of the cases and for B9 in 40% (n = 2) of the cases. The serum level of Thyroid Stimulating Hormone (TSH) was prescribed in association to the anemia blood test in 17% (n = 7) of the cases, which was never recommended. Finally, the TSH was physiological standard in 100% of the cases.  of the cases. The gastroenterologists and gynecologists were solicited in 44% (n = 7) for an expert opinion each, and a hematologist, and a digestive surgeon were solicited, once each. All requested expert opinions were motivated by an organ specific symptomatology and were completely justified.An expert opinion was requested in 37% (n = 33), folate deficiency 5% (n = 2), vitamin B12 5% (n = 2), dysthydroidism 2% (n = 1), cirrhosis 2% (n = 1), heterocygote β-thalassemia 2% (n = 1), hemolytic anemia Minkowski Chauffard 2% (n = 1). The frequency of etiologies according to profiles of anemia are summarized in Table The etiologies of anemia were: iron deficiency 77%  were microcytic, 25% (n = 8) normocytic, and 3% macrocytic (n = 1). Among the 9 cases of non-microcytic anemia: 2 of them were associated to a vitamin B12 deficiency, 1 was associated to kidney failure and vitamin B9 deficiency, and in 6 other cases the biological explorations did not allow the physician to exclude an intrinsic origin. The biological explorations were insufficient to judge if a single etiology was responsible for anemia in 28% (n = 12) of the cases. Ferritin was dosed in 84% (n = 36) of the cases, and that even before the diagnosis of anemia in 51% (n = 22) of the cases. The average Ferritin level in the blood was 9.5 μg/l (± 9.4), and the mean was 10.6 . Seventy-four per cent of the patients presented an ID. Regarding the serum iron reserve: 2.3% were low, 7% were normal, and 16% among them had no Ferritin dosage. Five IDA were diagnosed in absence of a Ferritin dosage, and 4 diagnosis were based on a low iron blood level. One anemia was diagnosed despite of a normal Ferritin blood level. In these cases, the diagnosis of ID was not retained.Among the IDA: 72% (The observed serum iron reserves according to severity of anemia and age groups are summarized in Table n = 19) of the cases of iron deficiency. Seven gastric fibroscopies (GF) were performed, and 86% (n = 6) justified: 83% (n = 5) because of digestive symptoms or digestive medical histories, and 17% (n = 1) by the severity of anemia. Eight GF were indicated , but not realized. Two indicated coloscopies were realized, for which the results were normal. Five, pelvic ultrasounds, were performed and justified by present menstruation disorders. Four indicated pelvic ultrasounds were not performed.The biological etiological assessment was complete in 59% (n = 11) of the cases: menorrhagia in 82% (n = 9) of the cases, , digestive bleeding in 18% (n = 2) of the cases: (1 esophagitis and 1 benign gastric ulcer due to Helicobacter pylori). Two cases of ID were associated to malabsorption . Coeliac disease was never investigated in this primary care setting, except once after a gastroenterological expert opinion, but coeliac disease was not diagnosed.Iron deficiency was associated to bleeding in 26% (n = 17) of the cases of ID, and especially for 2 women who followed a restrictive diet. Eighty-eight percent of the patients (n = 38) took an iron supplementation. When the first blood test results were received, 70% (n = 30) of the patients were prescribed an iron medical supplementation. The supplementation was not indicated in 33% (n = 10) of the cases because ID was not proven. After the reception of additional exams, eight new patients were supplemented, which was justified because the ID was confirmed. The ferrous sulfate was the most prescribed molecule and concerned 89% of the patients (n = 34).After evaluating the patient’s nutritional status, GPs noticed a low iron intake in 53% (n = 10) of the cases, between 80 and 160 mg in 42% (n = 16) of the cases, > 160 mg/d in 18% (n = 7) of the cases, and the dose was not specified in 13% (n = 5) of the cases. An associated supplementation was prescribed in 16% of the cases of ID. Vitamin C was associated in 11% (n = 4) and vitamin B9 was in 5% (n = 4) of the cases. The initial treatment duration was 3 months in 71% (N = 27) of the cases, 1 month in 11% (n = 4) of the cases, 6 months for a single patient. Forty-one per cent (n = 15) of the patients received information by their GP. An iron-rich diet was advised in 93% (n = 14) and explanations about inhibitors of iron intake, such as tea, were given in 20% (n = 3) of the cases.The daily dose of iron was < 80 mg/d in 26%  published a report in 2011 to remind healthcare professionals of the rational choice of exploring of patient’s biological iron status . In 2008Regarding anemia management, our participating GPs were self-directed. The etiological assessment was correctly conducted and succeeded to a correct etiological diagnosis in 72% of the cases. When expert opinions were solicited, they were justified and led to changings in clinical management. At the opposite, some prescribed complementary exams did not seem appropriate. Moreover, the prescription of useless exams for frequent conditions such as anemia present a significant additional cost. Nowadays, the supervision of public costs related to health systems is increasing. A Spanish study estimates the annual extra cost due to inappropriate complementary exams during anemia clinical management in a primary care setting at 6 million euros . The absStrengths: Our study is original because the design was prospective, and GPs participated in their real-life primary care setting which provided insight into their authentic clinical practice.Limits: To encourage the investigating GPs to participate in that study, during which their clinical practice was evaluated, an anonymous online questionnaire was used to include the women. Therefore, we can’t deduce the number of enrolled women by each GP. Few statistical tests were conducted because of the small sample size due to the prospective recruitment and the small number of returned completed questionnaires. The obtained results, thanks to the statistical tests used, are acceptable only for our sample.Moderate microcytic anemia, due to an iron deficiency in a context of menorrhagia, was the most observed anemia profile. Less than a half of the women were informed about iron-rich-diet and received a daily iron supplementation of ferrous sulfate between 80 mg and 160 mg which is not recommended according to new evidence, suggesting a maximal daily dose of 50 mg of elementary iron in a context of Hepcidin up-regulation in the case of an iron overload.Our study highlights that, in the absence of precise national guidelines for anemia management in non-pregnant, non-menopausal women in primary care settings, French GPs undergo various clinical management strategies leading to a heterogeneous, sometimes inappropriate follow-up.Additional longitudinal studies with a bigger sample size and randomized controlled trials are needed to confirm our results and to elaborate national guidelines.


Linoleic acid is the major fatty acid moiety of cardiolipin, which is central to the assembly of components involved in mitochondrial oxidative phosphorylation (OXPHOS). Although linoleic acid is an essential nutrient, its excess intake is harmful to health. On the other hand, linoleic acid has been shown to prevent the reduction in cardiolipin content and to improve mitochondrial function in aged rats with spontaneous hypertensive heart failure (HF). In this study, we found that lower dietary intake of linoleic acid in HF patients statistically correlates with greater severity of HF, and we investigated the mechanisms therein involved.n = 45), II (n = 93), and III (n = 15), were analyzed regarding their dietary intakes of different fatty acids during the one month prior to the study. Then, using a mouse model of HF, we confirmed reduced cardiolipin levels in their cardiac myocytes, and then analyzed the mechanisms by which dietary supplementation of linoleic acid improves cardiac malfunction of mitochondria.HF patients, who were classified as New York Heart Association (NYHA) functional class I , as well as assembly of CII subunits and the CIII2/CIV complex statistically correlated with cardiolipin levels in cultured cardiomyocytes, respirasome assembly was not notably restored by dietary linoleic acid in HF mice. Therefore, although linoleic acid may significantly improve both CI-based and CII-based respiration of cardiomyocytes, respirasomes impaired by HF were not easily repaired by the dietary intake of linoleic acid.The dietary intake of linoleic acid was significantly lower in NYHA III patients, as compared to NYHA II patients. In HF model mice, both CI-based and CII-based OXPHOS activities were affected together with reduced cardiolipin levels. Silencing of Dietary supplement of linoleic acid is beneficial for improving cardiac malfunction in HF, but is unable to completely cure HF. The prevalence of heart failure (HF) has increased globally, particularly in developed countries in recent decades . Advancefacp−/−) prevented the reduction of cardiolipin content and the exacerbation of mitochondrial function  in cardiac mitochondria, is significantly lower in HF mice 2:61]3(18n = 4] vs HF [n = 5]: %FS (%), 45.5 ± 0.9 vs 12.9 ± 3.4, p < 0.05; LV end-diastolic diameter, (mm), 3.25 ± 0.14 vs 5.04 ± 0.26, p < 0.05; LV end-systolic diameter (mm), 1.78 ± 0.10 vs 4.42 ± 0.39, p < 0.05).Similar to our earlier studies, we herein confirmed using these HF mice that cardiac function (%fraction shortening), as analyzed by echocardiography, was significantly decreased and that the LV was larger than control mice at 4 weeks after surgery 3(18:1)1], in cardiac mitochondria were significantly improved by linoleic acid supplementation, to levels almost equal to those of healthy mice , and measured cardiolipin content and OXPHOS activities in their heart mitochondria after 4 weeks on a continued linoleic acid diet. Total cardiolipin content, including the molecular species composition of cardiolipin [18:2)0 μg/day,0 μg/day,ved Fig. a,b.Fig.2/CIV supercomplex.Linoleic acid is an essential omega-6 fatty acid, and a prospective study demonstrated that a high conjugated linoleic acid concentration is associated with a lower risk of HF incidence in older men , whereas2/IV respirasome, whereas in cultured cells, cardiolipin content was statistically associated with the assembly of the CI/III2/IV respirasome, CII-subunits, and the CIII2/CIV supercomplex. Therefore, it is likely that dysregulation of respirasome assembly that occurs in HF cannot be easily repaired by the enhanced intake of linoleic acid or by enhanced cardiolipin content. We have no clear explanation for this, but it is possible that this phenomenon is simply a reflection of the possible ordered assembly of OXPHOS components. Nevertheless, whether other factors or events that are involved in respirasome assembly, or other aspects of mitochondrial function, are specifically affected by HF should be further investigated. The mechanisms by which cardiolipin content are reduced by HF, and whether cardiolipin content can be restored to a certain extent in HF mice by linoleic acid supplementation, also needs to be clarified.In our animal experiments, dietary intake of linoleic acid did not improve the assembly of the CI/IIIWe hence concluded that dietary supplementation of linoleic acid is beneficial for improving cardiac malfunction in HF, but is unable to completely cure HF.


Resource partitioning varied temporally, with C. albimarginatus feeding on more pelagic prey during October to January, potentially attributable to an influx of pelagic prey from outside the MPA at that time. Reef sharks may therefore be affected by processes outside an MPA, even if the sharks do not leave the MPA.Stable‐isotope analyses (δ To complement carbon and nitrogen SIA, sulphur (δ34S) was also examined. Few δ34S data exist in truly marine environments but, as seawater (δ34S c. 22.9‰) and sediments (δ34S c. 1‰) have distinct δ34S signatures within the marine environment, it can help discriminate between organisms feeding on pelagic  v. benthic and lagoon‐dependent prey  and muscle tissue extracted from below the dorsal fin. Sharks were caught using baited hand‐lines or short long‐lines (<20 hooks) with barbless circle hooks that were checked hourly. Once caught, species, sex and morphological measurements  were recorded and samples of first dorsal fin, muscle , and blood were collected before returning the sharks to the water. Blood samples were taken from the caudal vein using a heparinized needle and syringe. Blood samples were spun immediately in a portable centrifuge until the blood fractionated. The resultant plasma and RBC layers were separated by pipette. Samples were stored on ice during transport, immediately frozen (−20°C) on return to the laboratory, and then processed and analysed for δ13C, δ15N and δ34S following standard procedures .Sampling was conducted between the 17th and 23rd January 2015 around Diego Garcia within the BIOT. Teleost fishes were caught using baited hand‐lines, measured . Maximum likelihood standard ellipses were created for the δ13C and δ15N values for all tissues and for both shark species following Jackson et al.  and standard ellipse areas corrected for small sample size (SEAc), traditional convex hulls were also estimated . Bayesian carbon isotope mixing models were used to quantify the probable contribution of reef v. pelagic derived food items to the diet of both shark species. End members were set as the most 13C depleted (pelagic) and most 13C enriched (reef) of the teleosts sampled  and muscle tissue  and δ15N of fin tissue , indicating an ontogenetic shift in foraging location and trophic position, respectively ; whereas the highest mean δ13C values were found in reef associated trevally  to sharks. To do this we used a δ13C discrimination factor of 0.00‰ and a standard deviation of ± 0.33 . Bayesian mixing models indicate that C. amblyrhynchos derive c. 78% of their biomass from reef sources compared with just c. 60% for C. albimarginatus  which derive the majority (c. 86%) of their biomass from pelagic sources  suggest they are heavily dependent on pelagic productivity  and the largest range  than C. albimarginatus . This timeframe matches the historical peak pelagic fishing season (October–January) within BIOT when a purse‐seine fishery targeted migrating yellowfin tuna Thunnus albacares (Bonnaterre 1788)  and are therefore directly spatially protected from the effects of legal fishing vessels, although the illegal poaching of sharks occurs within the MPA (Ferretti et al.,e.g., spawning grounds; Green et al.,C. amblyrhynchos, C. albimarginatus are more dependent on pelagic prey, some of which may migrate into BIOT MPA from outside. Thus, sharks, particularly C. albimarginatus, within the MPA may therefore be indirectly affected by processes such as fisheries beyond its borders.For all tissue types, SEAc for δe et al.  that C. File S1.Click here for additional data file.


All‐optical helicity‐dependent switching in ferromagnetic layers has revealed an unprecedented route to manipulate magnetic configurations by circularly polarized femtosecond laser pulses. In this work, rare‐earth free synthetic ferrimagnetic heterostructures made from two antiferromagnetically exchange coupled ferromagnetic layers are studied. Experimental results, supported by numerical simulations, show that the designed structures enable all‐optical switching which is controlled, not only by light helicity, but also by the relative Curie temperature of each ferromagnetic layer. Indeed, through the antiferromagnetic exchange coupling, the layer with the larger Curie temperature determines the final orientation of the other layer and so the synthetic ferrimagnet. For similar Curie temperatures, helicity‐independent back switching is observed and the final magnetic configuration is solely determined by the initial magnetic state. This demonstration of electrically‐detected, optical control of engineered rare‐earth free heterostructures opens a novel route toward practical opto‐spintronics. All‐optical helicity‐dependent switching in synthetic ferrimagnetic samples made of two exchanged coupled ferromagnetic layers is shown to depend on the respective Curie temperature of each layer. By varying the CoFeB layer thickness, its Curie temperature can be tuned. Consequently, the switching is set not only by the light helicity but also by the CoFeB thickness. One method for magnetic reversal that holds great promise is the use of circularly polarized optical laser pulses:Despite the numerous systems where AO‐HDS has been observed, in all cases any helicity dependence is intrinsic to the material composition under investigation and cannot be tuned, limiting both our ability to understand the mechanisms at play during reversal, and their ultimate use in technological applications. Synthetic ferrimagnets (SFi), where a composite ferrimagnet is fabricated from two (or more) antiferromagnetically (AF) exchange coupled thin film FM layers, are a potential solution to this issue.TC. AO‐HDS due to irradiation by circularly polarized femtosecond laser pulses is investigated using anomalous Hall effect (AHE) measurements of SFi microbars  acting as a magneto‐optical field on the FM layer,TC, we demonstrate a simple means to achieve all‐optical control of magnetic heterostructures composed of commonly used materials, which may provide a promising route to practical opto‐spintronics.In this paper, we demonstrate direct control of AO‐HDS in RE‐free SFi's through manipulation of the Curie temperature, 2tCo and tCoFeB. Varying thickness allows us to control the TC of each layerz (−z)), with magnetization, M, of each FM uniformly aligned out‐of‐plane and antiparallel to one another . In the SFi Hall bars, AHE measurements, VHall  provide an electrical measurement sensitive to Mz of each layer, and confirm remnant states I and II are maintained in the patterned films.We perform measurements on micrometer‐scale SFi Hall bars consisting of two perpendicularly magnetized, AF‐coupled FM layers, termed FM1 (Co/Pt) and FM2 (CoFeB/Pt/CoFeB) and with variable thickness + and σ−), focused to an ≈46 µm spot, is swept along the x‐axis arm. VHall is monitored as the laser passes over the Hall cross. Figure VHall versus relative beam position, x, for each incident polarization, and normalized to the remnant value, VHallAP, for tCo = 0.73 nm and tCoFeB = 0.54 nm. Here, one can clearly see that VHall decreases toward zero as the laser beam becomes centered on the cross. This is attributed to an initial helicity‐independent demagnetization, as observed in some SFi's and single FM layers,TC, causing loss of long‐range order. With no locally favored state (I or II), upon cooling the irradiated film reorders multidomain. This demagnetized state is stable, persisting after removal of the laser beam, and so is presumed to occur only when T > TC for both individual layers, TC1 and TC2. As the laser spot passes the cross a helicity‐dependent remagnetization occurs, that is, net magnetization is restored, with the σ+ beam switching to final State I, while σ− polarized beam results in State II. It is apparent from this behavior that AO‐HDS occurs in these SFi's not within the beam center (which remains demagnetized), but at the trailing edge of the beam profile. Changing the initial state from State II to State I, in Figure Figure T > CT and thermal demagnetization occurs, with multiple domains nucleated. A ring around the central region exists where T < TC (so the film remains magnetized), but exceeds the threshold for AO‐HDS.TC1 ≠ TC2, as is the case here. For these materials, while the central beam may still demagnetize the bar, a region exists where, for example, TC1 < T < TC2, that is, FM2 remains magnetized while FM1 has lost long‐range order. Should this also be sufficiently high that AO‐HDS can occur, within this ring the laser fluence and so any HD effects (such as magnetic circular dichroism (MCD), or the inverse Faraday effect (IFE)) now act not on the composite SFi as a whole, but on an individual FM layer (in this case FM2). Consequently, AO‐HDS is intimately linked with the precise ordering of the SFi on cooling, and tuning each TC offers the opportunity to tailor any helicity dependence.On first inspection, the composite SFi appears to behave as a single FM layer (such as Pt/Co/Pt):TC in each layer. To minimize the effect of changing other parameters, we maintain the same materials for FM1 and FM2 and instead tune TC by varying FM thickness. Figure TC2 as a function of tCoFeB, and (f) MstCoFeB, the layer moment per unit area. Reducing tCoFeB reduces TC2 from bulk, due to a transition toward 2D magnetic behavior,MstCoFeB . For comparison, in Figure TC1 and MstCo are shown for two different tested thicknesses of FM1, tCo = 0.49 nm and tCo = 0.73 nm. Examining the two plots, one can easily see for a given tCo the critical FM2 thicknesses where TC2 exceeds TC1, which in the case of tCo = 0.73 nm occurs above tCoFeB ≈ 0.6 nm.To test this hypothesis, we independently vary FiguretCo = 0.73 nm, a pattern emerges: At low tCoFeB  a complete loss of AO‐HDS is surprisingly found, with helicity‐independent reversion to the initialized state. Finally, further increasing tCoFeB until TC1 < TC2, for example, for tCoFeB = 0.77 nm  which, in theory, should be reflected in any crossover of HD. Figure VHall/VHallAP(x) for tCoFeB = 0.42, 0.66, and 0.78 nm, respectively. Again, when TC1 = TC2  helicity‐independent reversion occurs, while for TC2 > TC1 identical AO‐HDS is found for all samples, with σ+ leading to final State II , and the layer begins to remagnetize. As the laser pulse is removed and the sample cools, the reordering of FM2 and restoration of AF coupling forces FM2 antiparallel to FM1: The SFi ordering is dictated by FM1. Should TC2 > TC1, the situation becomes reversed and the helicity dependence of FM2 dictates ordering of the SFi. When TC1 ≈ TC2 the ring width collapses, no domain orientation direction favored , and no, or at least weak, AO‐HDS occurs. Simple checks of the reversal symmetry of the SFi stack with TC1 > TC2, as compared with single Co FM layers , confirm a consistent helicity dependence between the two. Furthermore, measurements replacing the circular polarization with linearly polarized light and an externally applied field (HZ ≈ 3 Oe) shows an equivalent symmetry to AO‐HDS . We interpret the interaction of the circularly polarized light with M to be equivalent to an effective field in this case, potentially as a result of the IFE.These results cannot be understood by current models of AO‐HDS in single FMs, where switching is intrinsically set by the light helicity and is VHall/VHallAP(x), which are in good qualitative agreement with the experimental results . In FigureTC1 > TC2. The trailing edge of the σ− beam restores the initial magnetic state and results in no‐switching .M1 = M2. This point plays an important role for single‐shot switching in GdFeCotCoFeB for SFi moment compensation is readily seen. Throughout our measurements the compensation point is systematically at higher tCoFeB than the HD inversion thickness, suggesting a limited role of magnetization compensation in this system. Further numerical simulations for parameters at and around the compensation point (varying both TC and MStCoFeB for the two FMs) also show a dominant role of Tc in determining AO‐HDS in these SFi's, over any compensation point .An interesting property of a SFi is that by appropriate engineering of layer structure one can control the so‐called magnetization compensation point, that is, create a synthetic AF, where tCo = 0.73 nm and tCoFeB = 0.54 nm  reduces as the beam fluence is decreased, effectively indicating that the switching is focused to the center of the beam where the laser provides sufficient energy to create a demagnetized state. With the beam fluence reduced to 1.5 mJ cm−2, a minimum in the demagnetization area and subsequent helicity‐dependent switching is observed. Using this laser fluence, the response of the SFi to light pulses using a fixed beam method is also investigated . The results show that as the laser pulse number increases up to 1 × 103 pulses, VHall decreases toward zero. Partial helicity‐dependent remagnetization occurs when the pulse number exceeds 5 × 103 but, in general, a sweeping beam is required to ensure complete HD‐AOS.We further studied the response of the SFi to light pulses with different laser fluence using the sweeping beam method . In this case, TC1 ≈ TC2. When TC1 = TC2, the AO‐HDS ring width collapses, resulting in a similar thermal response for each layer. Strong AF coupling remains and consequently no net expansion of one domain structure over the other should take place. Still, a heat gradient is present along the cross arms, due to the localized laser pulse, which is predicted to drive domain walls toward the hotter region .TC1 = TC2. While in the horizontal wire we obtained a multidomain state, the vertical arm breaks this randomizing effect and restores the initial magnetic state of the cross position. Additional simulations without the vertical arm  show multiple domain states as the laser beam is swept over the wire, confirming that the cross geometry is essential for the backswitching effect.As a final point, we address the origin of the helicity‐independent back switching that occurs when TC, at which point light helicity can induce a symmetry‐breaking reversal mechanism, such as the IFE, and lead to switching.In addition to explaining AO‐HDS effects in SFi's, the presented results provide valuable information on the AO‐HDS mechanism, in general. Indeed, a picture prevails where the laser pulse must provide sufficient heat for the FM to reach a temperature approaching 3TC of each layer. When the Curie temperatures of each layer are matched, we observe a helicity‐independent backswitching, with the final magnetic configuration determined only by the initial state. In most materials to‐date helicity dependence is intrinsic, with backswitching demonstrations limited to select rare‐earth alloys,To summarize, we studied all‐optical helicity‐dependent switching in planar Hall crosses made from rare‐earth free synthetic ferrimagnetic heterostructures . We demonstrate that the AOS not only depends on the light helicity but also on the relative Curie temperature of each ferromagnetic layer. Indeed, by varying the thickness of the individual FM layer constituting the SFi, the helicity dependence of magnetic switching can be modulated. This behavior can be explained by the interaction between the laser polarization and the net moment at switching temperatures, which is governed by the relative 4Sample Fabrication: Samples were grown on glass substrates at room temperature by DC magnetron sputtering, base pressure 6 × 10−8 mbar. Two sets of samples were prepared: (a) single FM layers, Ta (3 nm)/Pt (4 nm)/FM1(2)/Pt capping (4 nm), where FM1 = Co (tCo = 0.49 or 0.73 nm), and FM2 = CoFeB (tCoFeB)/Pt (0.4 nm)/CoFeB (tCoFeB) (0.30 ≤ tCoFeB ≤ 0.78 nm). In FM2, both CoFeB layers are FM‐coupled and act as a single FM; (b) SFi samples with structure: Ta (3 nm)/Pt (4 nm)/FM1/Pt (0.4 nm)/Ru (0.9 nm)/Pt (0.4 nm)/FM2/Pt capping (4 nm). The Pt/Ru/Pt stack provides the antiferromagnetic Ruderman–Kittel–Kasuya–Yosida (RKKY) interlayer exchange coupling between the adjacent FMs.−3 mbar Ar partial pressure.Measurements: Magnetic properties of the films were studied by vibrating sample magnetometry. The films were patterned into electrical Hall devices by optical lithography and ion milling. The Hall voltage was measured along the y‐direction, while a DC current was injected along the x‐direction. The width of the current carrying wire is ≈5 µm, giving a DC current density of ≈6 × 109 A m−2. A Ti:sapphire fs‐laser was used with wavelength 800 nm, pulse duration 43 fs, and laser repetition rate of 5 kHz. The Gaussian laser beam spot was focused with a full‐width at half‐maximum, FWHM ≈ 46 µm. A quarter‐wave plate was used to create circularly [right‐(σ+) and left‐handed (σ−)] or linearly polarized (π) beam. The laser fluence is 3.9 mJ cm−2, unless otherwise stated, with a half‐wave plate used to adjust beam power. For all experiments, the initial magnetic configuration (state I or II) was set by applying an external saturating magnetic field (±z‐direction), before returning to remanence. Hall voltage was constantly monitored (0.2 s per point), and unless stated otherwise, no magnetic field was applied during the measurement. For each initial state, the measurement was repeated using each circular polarization of the incoming light, σ+ and σ−. Switching was investigated via two methods: (1) Sweeping beam method—the laser beam was swept in discrete steps across the Hall device in the x‐direction, at a step rate of 1.4 µm s‐1; (2) Fixed beam method—beam position is fixed at the center of the cross, with the number of incident pulses controlled by a pulse picker.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


To evaluate the effect of the optical zone diameter (OZ) in orthokeratology contact lenses regarding the topographical profile in patients with high myopia (−4.00 D to −7.00 D) and to study its effect over the visual quality.  Twelve patients (18 eyes) were fitted with overnight orthokeratology (OrthoK) with a randomized 6 mm or 5 mm OZ lens worn for 2 weeks, followed by a 2-week washout period, between both designs. Keratometry (K) readings, optical zone treatment diameter (OZT), peripheral ring width (PRW), higher-order aberrations (HOA), high (HC) and low contrast (LC) visual acuity, and subjective vision and comfort were measured at baseline and after 2 weeks of OrthoK lens wear of each contact lens. p value > 0.05). There was no difference between OZ lens designs found in refraction, subjective vision or comfort, and HC and LC visual acuity. Contrast sensitivity was decreased in the 5 mm OZ lens design compared with 6 mm OZ design . 5 mm OZ design provoked a greater flattening, more powerful midperipheral ring and 4th-order corneal and total spherical aberration than the 6 mm OZ design, being statistically significant after 7 days, for corneal aberration, and 15 days, for corneal and total, of wearing the lens . The OZT obtained were 2.8 ± 0.2 mm and 3.1 ± 0.1 mm for 5 mm and 6 mm OZ design, respectively . Regarding PRW, the 5 mm OZ design had a wider ring width in both the nasal and temporal zones .  No significant differences were found between any measurements for the same subject at both baselines . A smaller diameter optical zone (5 mm) in orthokeratology lenses produces a smaller treatment area and a larger and more powerful midperipheral ring, increasing the 4 Currently, orthokeratology (OrthoK) has become a clinically reliable and effective method to correct refractive errors using specialty gas permeable contact lenses –3. OrthoThe tear film under the OrthoK lens reshapes the cornea in closed eye conditions by applying a positive push pressure over the central cornea and a negative pull pressure in the midperiphery . This prOrthokeratology was originally prescribed for adults to correct myopia during the day without glasses or contact lens wear . Currentth-order spherical aberration and coma [Together with peripheral defocus, higher-order aberrations have been associated with myopia control, mainly 4and coma , 23. Farand coma . Many stand coma .Thus, newer OrthoK lens designs are trying to increase the peripheral myopic defocus and take into account pupil size dependence in higher-order aberrations. These lenses are being developed with a smaller optical zone (OZ) in attempts to achieve a smaller treatment zone and a steeper, more power midperipheral ring closer to the pupil. There are few studies published that have studied the effect of lens design over the cornea –28. The A prospective, longitudinal, and randomized pilot study has been conducted. Twelve healthy subjects  were recruited in the Faculty of Optics and Optometry . The mean age of patients was 25.01 ± 6.91 years (range 18–27 years old) and mean spherical refractive error −4.72 ± 0.36 diopters (D) (range −4.00 D to −7.00 D). Each subject signed an informed consent after the study protocol, and risk and benefits of the treatment were explained. Participants were free to leave the study at any time without any reason. This study obtained ethical approval from the Ethical Committee of the Complutense University of Madrid and followed the tenets of the Declaration of Helsinki .Inclusion criteria were myopia between −4.00 diopters (D) and −7.00 D and with astigmatism less or equal to −1.50 D. Exclusion criteria were history of ocular disease or systemic disease that could affect visual system or pregnancy. Contact lens wearers were asked to stop wearing their habitual contact lens one week before the examination day. All subjects were fit with Paragon CRT™ contact lenses  in HDS 100 material  according to manufacturer guidelines.The study was divided into two phases, in which the patient used two different types of OrthoK lenses: a lens with 6 mm OZ diameter and another lens with 5 mm OZ . PatientRefraction without cycloplegia, high (HC) and low (LC) contrast uncorrected visual acuity (UCVA), HC and LC best-corrected visual acuity (BCVA), contrast sensitivity, corneal topography, anterior corneal and total wavefront aberration, and Visual Analogue Scale (VAS) questionnaire were performed. All measurements were performed at baseline for pretreatment records (PRE), at 1 day (after the first night of lens wear), at 7 days, and at 15 days for both lens designs, except for total wavefront aberration, contrast sensitivity, and LC BCVA and UCVA. All measurements were taken early in the morning, so that the patient had slept with the lenses for at least 6 hours. A slit lamp examination was performed in all visits to verify the ocular surface integrity.X-axis from 4 mm of distance to the apex in both nasal and temporal directions. In addition, optical zone treatment diameter (OZT) and peripheral ring width (PRW) were defined. The OZT was defined as the central zone of corneal flattening from baseline after OrthoK wear. Margins of this zone were marked from comparison map topography data, considering the OZT diameter where the difference between before and after orthokeratology wear was zero. The corneal zone where the corneal radius was steepened during OrthoK lens wear from baseline reading was defined as PRW. PRW margins were considered as the width between OZT margins and where the corneal radius returns (from steepening) the same before and after OrthoK lens wear. PRW was measured in the nasal and temporal zones of X-axis . Parameters obtained with Pentacam included flat keratometry (flat k), steep keratometry (steep k), and corneal radii in f X-axis .2) using the ETDRS test form Chart Display VX24  with HC (contrast level 100%) and LC (contrast level 10%) at 4 meters. LC BCVA and UCVA were measured at baseline and 15 days after each lens design; HC BCVA and UCVA were measured at all visits. Contrast sensitivity was measured using the Pelli-Robson test at 1 meter, in which spatial frequency corresponds to 1 cycle per degree. VX110  was used to determine the changes in 4th-order spherical aberration for corneal and total spherical aberrations.Subjective refraction was performed based on the patients' current spectacle prescription, and a fogging method was created for obtaining the final subjective refraction. The astigmatism was adjusted by crossed cylinder technique. The main objective was to find the BCVA with the maximum positive sphere. UCVA and BCVA were measured monocularly in photopic luminance conditions .Statistical analysis was performed using the SPSS Statistics 23 software . Sample size calculations were performed with statistical software Granmo 6.0 . A statistical power of 80% was considered. Considering the horizontal corneal radius as the main variable, with an accepted two-sided statistical significant threshold of 0.05 and a risk of 0.20, for a standard deviation of 0.06 units to the mean and in order to detect a difference of 0.05 units, at least 12 subjects were needed to find statistically significant differences. Normality of samples was analyzed using the Kolgomorov–Smirnov test. To analyze the differences between the baseline and additional visits and also between both lens designs in the same visit, a Student's p value > 0.05; Student's t-test for paired samples). Refraction improved from the first day of OrthoK lens wear , being −2.51 ± 0.35 D and −2.87 ± 0.97 D for 1 day of wear and −0.31 ± 0.80 D and −0.35 ± 0.51 D at 15 days of wearing for 5 mm OZ and 6 mm OZ designs, respectively. No differences in refraction were found between designs .All patients completed the study without drop outs. No significant differences were found between any baseline measurements for the same subject in the two different lenses . However, after seven days of wearing, both designs showed HC UCVA improvement, reaching the BCVA at the baseline . In addition, no differences were found between designs for any visit studied . Regarding LC visual acuity, 6 mm OZ lenses showed a decrease in LC UCVA after 15 days of wear compared with LC BCVA at the baseline . Meanwhile, with the 5 mm OZ design, LC UCVA was slightly worse than LC BCVA but not statistically significant . As HC visual acuity, no differences were found for LC visual acuity between designs for any visit studied .p value > 0.05; Student's t-test for paired samples). However, during 5 mm OZ wearing, CS statistically decreased . While the 6 mm OZ wearing provoked a slight increase in CS, this was not statistically significant .The contrast sensitivity (CS) was also evaluated. No differences were found between orthokeratology lens designs . The 5 mm OZ design had a statistically significant greater flattening than 6 mm OZ design after 7 and 15 days of wearing the lens . The horizontal and vertical corneal radius flattening differences between lens designs after 15 days of wearing were 0.13 ± 0.02 mm and 0.14 ± 0.06 mm, respectively.Regarding corneal topography, p value < 0.05; Student's t-test for paired samples). X-axis. The treatment size of the 5 mm OZ lens design was 2.8 ± 0.2 mm and 3.1 ± 0.1 mm with 6 mm OZ lens design, with a statistically difference  . These differences provoke a very different keratometric profile in the X-axis, with the changes between central cornea and peripheral ring more abrupt. This demonstrates that for 1.00 D of anterior corneal power difference (between the center and the periphery), the 5 mm OZ design measured 1.3 mm to the center. The 6 mm OZ design is a wider measurement of 2.1 mm. Likewise, to reach a power difference of 1.50 D from the center and the periphery, the 5 mm OZ lens design was measured at 2.1 mm and 2.4 mm for the 6 mm OZ designs.The keratometric or topography profile of the cornea for each OZ lens design before 15 days of lens wear is displayed in samples) . Regardith-order corneal and total spherical aberrations (Z12) had a statistical significant difference for all follow-up visits compared with baseline for both OZ lens designs , trending towards greater positive corneal spherical aberration. Comparing both designs, no statistical differences were found at baseline and wearing OrthoK after 1 day; however, the 5 mm OZ design showed greater positive spherical aberration than the 6 mm OZ design after 7 and 15 days of wearing the lens, representing a statistical significant difference for both corneal and total spherical aberrations . See Corneal and total spherical aberration with a 5 mm pupil diameter was also evaluated. Both 4p > 0.05; Student's t-test for paired samples). However, less corneal staining was observed in 37.5% of eyes with 6 mm OZ diameter compared to 62.5% with 5 mm OZ diameter (Regarding VAs, no significant differences were found between either OrthoK OZ designs for subjective comfort and vision (diameter .The current study had the aim to analyze the effect of different OZ diameters of OrthoK lenses over the topography profile in high myopia patients. The 5 mm OZ lens design is able to generate a greater midperipheral curvature and central flattening than 6 mm OZ design. This profile difference means that smaller OZ produces a narrower treatment area and a wider and steeper peripheral ring, closing the steepening ring to the pupil center.Currently, the scientific literature regarding the effect of lens design, and in particular the optical zone diameter, in OrthoK lenses is very weak. Few studies have been published in this interesting research field –28. KangChanges in the topographic profile, including a wider, steeper midperipheral ring, and closer to the pupil center, has been hypothesized as an important factor in improving the efficacy of myopia control with OrthoK lenses , 27, 32.th-order spherical aberration found between both designs studied could be the most probable reason to observe lower contrast sensitivity with smaller OZ diameter in OrthoK.Visual quality is another factor to consider when patients are fit in OrthoK lenses. There are a lot of studies published describing a decrease in visual quality during OrthoK treatment , 34–37. Given the nature of OrthoK treatment which produces a molding on the corneal surface, it is expected that changes occur with corneal aberrations, and consequently total aberrations , 42. Higth-order spherical aberration that affects only the contrast sensitivity, but without differences in terms of visual acuity and subjective vision, compared to a lens design with a larger OZ diameter. More studies are needed to understand if these outcomes only represent corneal modifications or if a smaller treatment zone and more powerful midperipheral ring, closer to the pupil center, play an important role in increasing the efficacy of myopia control in an OrthoK lens.In conclusion, a smaller diameter OZ in OrthoK lenses produces a smaller treatment area and a larger and more powerful midperipheral ring, increasing the 4


Plant neighbor detection and response strategies are important mediators of interactions among species. Despite increasing knowledge of neighbor detection and response involving plant volatiles, less is known about how soil-borne signaling chemicals may act belowground in plant–plant interactions. Here, we experimentally demonstrate neighbor detection and allelopathic responses between wheat and 100 other plant species via belowground signaling. Wheat can detect both conspecific and heterospecific neighbors and responds by increasing allelochemical production. Furthermore, we show that (-)-loliolide and jasmonic acid are present in root exudates from a diverse range of species and are able to trigger allelochemical production in wheat. These findings suggest that root-secreted (-)-loliolide and jasmonic acid are involved in plant neighbor detection and allelochemical response and may be widespread mediators of belowground plant-plant interactions. Plant growth and physiology respond to the presence of neighboring plants. Here the authors show that the chemicals (-)-loliolide and jasmonic acid, which were present in the root exudates of a range of plant species, can be detected by wheat and induce allelochemical production. Plant neighbor detection involves both physical and chemical signals, including far-red light reflection, alteration of nutrient availability and plant-released secondary metabolites. These signals trigger complex plant response strategies such as shade avoidance, root foraging, and chemical defense6. Much of the research into chemical-mediated neighbor detection and signaling interactions has dealt with volatiles induced by herbivory or other environmental stressors in initiating defensive responses9. This focus is primarily driven by the accessibility of aerial tissues and availability of reliable techniques to detect and identify volatile chemicals. Nevertheless, plant neighbor detection and signaling interactions take place both aboveground and belowground. Aboveground signaling interactions are well established and mediated by air-borne chemicals including methyl jasmonate, salicylate, benzoate, and indole, as well as ethylene and several volatile terpenes13. However, the identity of soil-borne chemicals involved in belowground signaling interactions is largely unknown.Plants are capable of detecting and responding to neighboring plants, generating consequences for plant performance and playing important roles in plant coexistence and community assembly16. In particular, root detection and placement patterns may be mediated through root-secreted chemicals20. These belowground signals are potentially conducted by physical contact of root hairs and tips or conveyed through associated mycorrhizal hyphae22. However, it is extremely difficult to unambiguously isolate chemical-mediated belowground signaling interactions due to the complexity of plant-soil interactions. Specifically, it has been difficult to identify root-secreted signaling chemicals and determine whether their delivery is dependent on root contact or common mycorrhizal networks.In contrast to aboveground signaling chemicals which move freely in air, the transduction of belowground chemicals requires root-soil interactions. A great deal of recent attention has been paid to belowground signaling interactions among plant species at the root level24. In particular, neighbors influence plant defensive biochemistry in a species-specific fashion, altering the production of defensive metabolites such as allelochemicals25. Allelochemicals can have profound effects on the performance of neighboring plants, i.e., allelopathy, an interference mechanism in which neighbor plants are chemically suppressed through the release of allelochemicals from focal plants26. However, allelopathy research has primarily focused only on focal plants and their allelochemicals rather than on the signaling interactions between plants that mediate the interaction29. Therefore, allelopathic interference and neighbor detection usually are studied separately, despite their necessary linkage in nature.When neighboring plants are detected, focal plants may respond to neighbors in morphological and biochemical ways30. This pattern may arise through the production and release of signaling chemicals that induce the production of defensive allelochemicals. Accordingly, the signaling chemicals may ideally be common to most potential competitors. Alternatively, allelochemical production may be constitutive, produced whether or not neighbors are present, potentially wasting valuable plant resources26.For two species that coexist, plants first may detect and potentially recognize their neighbors, and then initiate allelopathic interference to regulate inter-specific or intra-specific interactions. Neighbor detection and allelochemical response are two inseparable processes when one or more plants occur together and interact34, by allelopathic wheat (Triticum aestivum) as a model system. We reported that production of DIMBOA was induced by a diverse range of neighboring plants in a density-dependent manner30. Root exudates from a range of species were able to induce DIMBOA production and the plant hormone jasmonate, and to a lesser extent salicylate, were able to induce DIMBOA production30. Despite these findings, the identity of the soil-borne chemical signals that are responsible for neighbor detection and allelochemical response in belowground signaling interactions has not been exhaustively characterized and whether such soil-borne chemical signals are common to most plant species remains unknown.In previous studies we used production of the putative allelochemical DIMBOA , which has a charcterized metabolic profil in soilHere, we show that, using new data and data from previous studies we show that wheat can respond to at least 100 other plant species via belowground interactions. We show that (-)-loliolide and jasmonic acid, present in root exudates are sufficient to induce this response and suggesting that they can mediate this response.30. We investigated density-dependence of the DIMBOA response further by co-cultivating wheat with eight weed species (four of which were examined previously30). When wheat was paired with itself and eight common weeds  that often come into contact with wheat, allelochemical DIMBOA concentration varied with the density of the neighbors. Significantly increased concentrations of the allelochemical were observed at the 5:4–5:9 (wheat: neighbor) proportions  increased DIMBOA concentration in all treatments other than those completely separated with plastic film . Although these four neighbors did not significantly induce DIMBOA production at a 5:5 mixture, significant induction occurred at a 5:8 mixture, even under belowground segregation with the 0.45 μm mesh , and salicylic acid (SA) when supplied at higher concentration, were sufficient to induce the production of DIMBOA30. Here, we extended this analysis by re-analyzing those samples and adding four additional species. The root exudates from neighbor species were applied to the soil at varying concentrations. When wheat was exposed to the root exudates after 6 h, the concentration of allelochemical DIMBOA varied with neighbor species identity in a dose-dependent manner , but such a significant correlation was not found for JA, SA or luteolin . Four potential chemical signals jasmonic acid (JA), salicylic acid (SA), (-)-loliolide and luteolin were identified  and salicylic acid (SA)45. JA and SA are two ubiquitous signaling chemicals which elicit the production of defensive plant metabolites against microbes, herbivores, or competitors47. However, information on other signaling chemicals involved in belowground plant–plant signaling interactions has remained scarce. Root-secreted strigolactones were described as signaling chemicals between parasitic plants and their hosts several decades ago48. Strigolactones also act as chemical signals for fungal symbionts and parasitic weeds in plant roots49. However, strigolactones do not appear to have a universal role in belowground plant-plant signaling interactions and there may be other, more specific, root-secreted signaling chemicals awaiting identification.Plants may detect neighbors through plant volatiles as air-borne signals45. A few studies have documented root-secreted JA and SA and their roles in rhizosphere signaling interactions44. In this study, besides the commonly studied JA and SA, (-)-loliolide and luteolin contributed to neighbor-induced allelochemical responses. In particular, (-)-loliolide and JA strongly induced allelochemical production at very low concentrations, whereas luteolin and SA required greater concentrations. Although these concentrations were greater when compared with the quantities of (-)-loliolide and JA as determined in the root exudates and rhizosphere soils from interacting weeds tested, we propose that such bioactive phytochemicals provided over a long time period at low concentrations might be able to induce substantial effects. Additionally, soil extractions would have diluted the chemical signals over large soil volumes while the concentrations of (-)-loliolide and JA detected would be locally much higher in intact soils. Furthermore, there was a joint action in the mixture of (-)-loliolide and JA at low concentrations. Thus, even if the actual concentration of (-)-loliolide and JA in soil was still substantially lower than the necessary concentration to elicit wheat allelochemical production, an effect would still be expected.JA and SA may play some roles in belowground chemical communications. However, most studies have tested this with exogenous addition of JA and SA at arbitrary concentrations, rather than through quantification in the root exudates and rhizosphere soils51. However, (-)-loliolide has never been reported in participating in any plant–plant or plant–microbe signaling interactions. More importantly, (-)-loliolide was detected in all of the 101 plant species tested while luteolin only occurred in 79 plant species. Actually, (-)-loliolide is the most ubiquitous lactone that occurs in many plant families and marine alga52. In the current study, (-)-loliolide was found in every monocot and dicot plant species tested and could be secreted from roots into rhizosphere soils. (-)-loliolide had a higher soil mobility and could easily be moved from the rhizosphere to bulk soil. Therefore, (-)-loliolide may be a ubiquitous, soil-borne signaling chemical that can trigger plant defensive responses in belowground plant–plant interactions.Similar to SA, phenolic luteolin is a signal in the resistance response of plant to microbes35. It is now clear that plant-derived signaling chemicals contribute to plant neighbor detection and defensive responses. In general, chemicals produced by the competitor species that are xenobiotic to the focal plants might represent a better, species-specific signal, just like strigolactones as signaling chemicals of parasitic plants to the host49. However, (-)-loliolide and JA found in this study are ubiquitous signaling chemicals among plants, and are produced by the focal allelopathic wheat as well. In fact, signaling chemicals such as ethylene, JA, SA, and their formylates are ubiquitous plant–plant signals rather than xenobiotic chemicals that may indicate identity11. Plant neighbor detection and allelochemical response in generating coexistence heavily depended on neighbor density rather than neighbor identity, reminiscent of plant neighbor detection mediated in a dose-dependent fashion.Growth-inhibiting allelochemicals and signaling chemicals involved in plant–plant interactions profoundly affect the performance of plants, altering the consequences of intra-specific and inter-specific interactions in ecosystemsThe (-)-loliolide or JA secreted from either conspecific or heterospecifc roots could elicit the production of allelochemicals and appears to be a signal for belowground competition, potentially common to all species. We speculate that variation in level of soil signaling chemicals produced across species has the potential to lead to variable responses in natural systems, though these chemical signals could still be general indicators of competition. We assume that there is an additional, species-specific signaling chemical that inhibits the induction of allelochemicals in conspecific interactions. This could be a much less common soil chemical, one more representative of the species identity and also much less likely to be detected by our methods which focused on chemicals that generated an allelochemical response. Fractionation-guided bioassays that focus on the inhibition of (-)-loliolide responses would be necessary to identify this soil signal.42. From a model system of allelopathic wheat and 100 interacting plant species, this study suggests that plant neighbor detection and allelochemical response mediated by root-secreted signaling chemicals are a general phenomenon. In particular, this study identified (-)-loliolide as a soil–borne chemical signal that could act to signify the presence of competitors and enhance allelochemical production.The importance of belowground signaling interactions to plant neighbor detection and response strategies, as well as the mechanisms underlying such communications, has been a major focus of the science of plant interaction in recent yearsIn conclusion, the discovery of (-)-loliolide as a general soil-borne signaling chemical common to all the plants tested here, improves our understanding of plant neighbor detection and response strategies. Studies on the behavior of (-)-loliolide in the soil and its molecular biology, could lead to new insights into plant sensing and communication. Although a generic response to (-)-loliolide was detected here, there are likely to be additional signaling mechanisms that could allow species-specific responses. In addition, the ability of the signaling chemical to induce allelopathic responses in plants needs to be explored in other plant systems to test if they respond similarly to allelopathic wheat.53. Accordingly, a DIMBOA-rich winter wheat cultivar (Jing411) was used in this study and its seeds obtained from the Chinese wheat germplasm collection. Combining data from our previous study29 with new data, wheat plants were challenged with 100 plant species  is a putative and dominant allelochemicalThree experiments for wheat–neighbor interactions were carried out in plastic pots (11 cm diameter × 12 cm height) that contained a central cylinder  where a barrier could be inserted  at different densities. Pre-germinated seeds of wheat and neighbor pairs were each sown into the plastic pot containing 800 g of soil with no barrier. Five wheat seeds were spaced uniformly in the central cylinder of each pot, while neighbor seeds were sown surrounding the wheat. Proportions of wheat to eight common neighbor species ranged from 5:1 to 5:10 per pot (wheat:neighbor), while trials between wheat and the remaining 92 species . The CH2Cl2 extract was concentrated, and subjected to silica gel column chromatography (5 cm × 80 cm) by eluting stepwise with a series of mixtures of PE, CH2Cl2 and MeOH . The fractions were screened using a bioassay-driven fractionation approach56 as described in the soil incubation section below, resulting in four fractions that induced the production of allelochemical DIMBOA in wheat. The first fraction was further purified by silica gel column chromatography (2.5 cm × 40 cm) with a mixture of CH2Cl2 and MeOH , and yielded luteolin. The second fraction was further purified by Sephadex LH-20  with MeOH, resulting in liquid jasmonic acid (JA). The third fraction was further purified by ODS  with H2O containing increasing amounts of MeOH to obtain a green solid that was recrystallized with CH2Cl2 to yield (-)-loliolide. The fourth fraction was further purified by silica gel column chromatography (2.5 cm × 40 cm) with a mixture of CH2Cl2 and MeOH , resulting a crude solid that was recrystallized with n-hexane-MeOH  mixture and gave salicylic acid (SA). All four chemicals, JA, SA, (-)-loliolide and luteolin, were identified by spectroscopic analysis (Supplementary Table The dried root exudates (100 g) were successively partitioned three times with petroleum ether (PE), followed by methylene chloride . The container was placed in a sterile environmental chamber at 28 ± 1 °C with a 12 h photoperiod. The hydroponic solution in the container was kept at a constant level by adding distilled water daily. After 7 days the hydroponic solution was filtered with sterile filter papers, and the filtrate was collected to yield the root exudates.29. Wheat seedlings were collected for the quantification of DIMBOA as described in the quantitative analysis section below.Ten wheat seeds were sown into a series of 5 cm × 5 cm plastic pots with 100 g of soil. At the 2-leaf stage these were thinned to fivee plants per pot. The root exudates described above, one of the fractions or mixtures and potential signaling components -loliolide or luteolin) that were isolated and identified as describe in the section above, were each added to the treated pots at different concentrations. Control pots received distilled water only. All treatments and controls were replicated three times and were placed in an environmental chamber with a temperature of 25 °C and 65–90% relative humidity. The pots were randomly sampled at 6 h after soil-incubation. This sampling time was based on a significant increase in wheat DIMBOA observed in an incubation experiment involving the addition of weed root exudates58. Soils as described above were ground and sieved to 125 μm. The soil was suspended in a dioxane/water  solvent to make a slurry which was then spread as a 0.7 mm thick layer on a 10 cm × 20 cm glass plate. The plates were air-dried at 20–25 °C and stored in a desiccating chamber until used for chromatographic tests. Four potential signaling chemicals, JA, SA, (-)-loliolide or luteolin were each sampled with a microsyringe at 2.5 cm from the bottom edge of the plates. Distilled water in sampling served as the control. After the spots had been deposited, the plates were allowed to develop in a closed glass chamber using distilled water as solvent. A sheet of filter paper dipping into the developing water fed water continuously to the substrate at the base of the plate, thus leading to a relatively uniform flow. During development with water, the whole device was held in a horizontal position. Water migration occurred at a distance 17.5 cm from the baseline. The migration lasted between 1 h and 5 h depending on the samples. The plates were dried at 20–25 °C, and the soil layer of drying the developed TLC plates with three replicates for each chemical was cut into segments of 1.5 cm each. To avoid microbial degradation and transformation, chemical residue in each segment was quantified immediately by ultra performance liquid chromatography (UPLC) described below. Mobility factor (Rf) value of each chemical was calculated according to the formulae Rf = ∑Ri × Mi /Rw × ∑Mi, where, Rw was remove distance of water from start point, i was number of segments, Ri was distance of segment i from start point, Mi was chemical content in segment i59.Soil TLC was performed by a combination of two methods2O mixture (60:40 v/v) and then loaded onto reverse-phase C18 Sep-Pak cartridges  equilibrated with water, which eluted with acidified MeOH (1% HOAC) and then MeOH. The MeOH fraction was concentrated with nitrogen gas to a final volume of 100 μl. The concentrated samples were subsequently subjected to an HPLC-1260 instrument  equipped with a C18 reverse-phase column  and a diode array UV detector at 280 nm. Elution was performed with a mixture of 0.5% acetic acid and MeOH  at a constant flow rate of 1.0 ml min−1 at 40 °C. The peak of DIMBOA was identified by its retention time (9.8 min) and coelution with an authentic standard . DIMBOA was quantified by regression analysis of the peak areas against standard concentrations .Quantification of the allelochemical DIMBOA was performed by liquid extraction/solid-phase extraction, followed by high-performance liquid chromatography (HPLC). The wheat tissues (roots or shoots) were freeze-dried and ground with liquid nitrogen. Then 500 mg of the resulting powder was homogenized with 50 ml of 25% aqueous MeOH and extracted by ultrasonic oscillator for 30 min at a temperature of 25 °C. The extract was filtered, and the filtrate was evaporated to dryness individually under nitrogen gas. Dry residues were dissolved in 2.5 ml of 0.05% acetic acid (HOAC) in a MeOH-H2O-HOAC mixture , vortexed for 5 min at 25 °C and then NaCl was added and immediately vortexed vigorously for 1 min. After the solution was centrifuged at 2800×g for 10 min, the supernatant was filtered with a 0.22 μm nylon syringe filter . Analyses of four chemicals were carried out on a triple–quadrupole mass spectrometer  equipped with an electrospray ionization (ESI) source operating in positive mode for (-)-loliolide and luteolin and negative mode for JA and SA. Instrument control and data acquisition were performed using MassLynx software (version 4.1). Chromatographic separation was performed using an Acquity UPLC-BEH C18 column  at 40 °C. The injection volume was 5 μl. The elution gradient was carried out with a binary solvent system consisting of 0.2% HOAC in H2O (solvent A) and MeCN (solvent B) at a constant flow rate of 0.3 ml min−1. Simultaneous separations were completed using a gradient elution of 0.0 min/90% A, 2 min/10% A, 3.0 min/10% A, 4 min/90% A, and 5.0 min/90% A. Separation and stabilization were achieved in 5.0 min. The typical conditions were capillary voltage, 3.0 kV; source temperature, 120 °C; and desolvation temperature, 350 °C. The cone and desolvation gas were set at flow rates of 50 and 600 l h−1, respectively. Multi-reaction monitoring (MRM) mode was operated for each chemical. All parameters for the MRM transitions, cone voltage and collision energy were optimized to obtain the highest sensitivity and resolution. For JA: retention time, 1.60 min; parent ion, m/z 209; product ion, m/z 58.9; cone voltage, 35 V. For SA: retention time, 1.40 min; parent ion, m/z 136.8; product ions, m/z 92.9; cone voltage, 26 V. For (-)-loliolide: retention time, 1.25 min; parent ion, m/z 197; product ion, m/z 178.7; cone voltage, 23 V. For luteolin: retention time, 1.35 min; parent ion, m/z 287; product ion, m/z 153; cone voltage, 20 V. Quantification of four chemicals was each conducted by the addition method with authentic standards. Limit of detection (LOD) for the chemicals is the concentration that produces S/N  = 3, estimated from the chromatogram corresponding to the lowest concentration used in the calibration, whereas limit of quantification (LOQ) is defined based on S/N = 10. The LOD were estimated at 0.006 nmol g−1 for loliolide, 0.004 nmol g−1 for luteolin, 0.004 nmol g−1 for JA and 0.006 nmol g−1 for SA. The LOQ were estimated at 0.02 nmol g−1 for loliolide, 0.01 nmol g−1 for luteolin, 0.01 nmol g−1 for JA and 0.02 nmol g−1 for SA. The recoveries of the four chemicals ranged in 86.2–102.6%  at the spike level of 0.01–150 nmol g−1.Four potential signaling chemicals JA, SA, (-)-loliolide and luteolin were quantified by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The plant tissues, root exudates or rhizosphere soils were each freeze-dried and ground with liquid nitrogen. An amount of 250 mg of the resulting powder was extracted with 10 ml of MeCN (acetonitrile)-Ht-test or analysis of variance (ANOVA). Tukey post-hoc tests were used for multiple comparisons when ANOVA terms were significant using SPSS 16.0 for Windows . Regression lines were fitted as linear or quadratic model results using Sigmaplot 10.0 . A simple Pearson’s correlation was used to test whether the allelochemical DIMBOA induction across samples correlated with signaling components of the root exudates also using SPSS. While we did not control for multiple comparisons in the response survey of 100 species, we calculated binomial probabilities for obtaining the number of significant individual tests. To provide a single overview of this survey, we calculated Ln-transformed response ratios relative to the wheat:wheat treatment in each species for both the DIMBOA concentration and biomass of roots and shoots. From these 100 ratios, we present means ±95% confidence intervals as a measure of effect size.For the survey of 100 species, data were presented as means ± standard error (SE) from each of independent experiments with three replicates. The data were analyzed using Student’s Supplementary Information


The variant is lethal in utero when in the homozygous state and is the only ADPKD variant known in cats. Affected cats have a wide range of progression and disease severity. However, cats are an overlooked biomedical model and have not been used to test therapeutics and diets that may support human clinical trials. To reinvigorate the cat as a large animal model for ADPKD, the efficacy of imaging modalities was evaluated and estimates of kidney and fractional cystic volumes (FCV) determined.Approximately 30% of Persian cats have a c.10063C > A variant in Three imaging modalities, ultrasonography, computed tomography (CT), and magnetic resonance imaging examined variation in disease presentation and disease progression in 11 felines with ADPKD. Imaging data was compared to well-known biomarkers for chronic kidney disease and glomerular filtration rate. Total kidney volume, total cystic volume, and FCV were determined for the first time in ADPKD cats. Two cats had follow-up examinations to evaluate progression.FCV measurements were feasible in cats. CT was a rapid and an efficient modality for evaluating therapeutic effects that cause alterations in kidney volume and/or FCV. Biomarkers, including glomerular filtration rate and creatinine, were not predictive for disease progression in feline ADPKD. The wide variation in cystic presentation suggested genetic modifiers likely influence disease progression in cats. All imaging modalities had comparable resolutions to those acquired for humans, and software used for kidney and cystic volume estimates in humans proved useful for cats.Routine imaging protocols used in veterinary medicine are as robust and efficient for evaluating ADPKD in cats as those used in human medicine. Cats can be identified as fast and slow progressors, thus, could assist with genetic modifier discovery. Software to measure kidney and cystic volume in human ADPKD kidney studies is applicable and efficient in cats. The longer life and larger kidney size span than rodents, similar genetics, disease presentation and progression as humans suggest cats are an efficient biomedical model for evaluation of ADPKD therapeutics.The online version of this article (10.1186/s12882-019-1448-1) contains supplementary material, which is available to authorized users. PKD1, which encodes the protein known as polycystin-1 [PKD1 are catalogued in the Autosomal Dominant Polycystic Kidney Disease Mutation Database: PKDB [PKD1 variant or cohorts with the same genetic backgrounds are limited in humans, which inhibits the power of studies focused on genetic modifiers that would influence interfamilial, intrafamilial and sex differences in disease progression and responses to therapeutics. Genetic screening is also complicated in humans since PKD1 includes 46 exons, has a large, ~ 14 kb mRNA [Autosomal dominant polycystic kidney disease (ADPKD) is one of the most commonly inherited disorders in humans, with an estimated prevalence of 1:400 to 1:1,000 –4 and iscystin-1 –7. Most se: PKDB . Because kb mRNA  spanning kb mRNA , 11.PKD1 variant (c.10063C > A) causes a stop codon at position 3284 in exon 29 (C3284X) [PKD1 variants [ADPKD is widely recognized as the most commonly inherited renal disease in the domestic cat, specifically cats of the Persian breed –16. The (C3284X)  and is t(C3284X) –19. Hepa(C3284X) , 16, 20,(C3284X) , and oth(C3284X) . These yvariants . Cats shUS, CT, and MRI are commonly used imaging modalities to evaluate disease progression and the therapeutic efficacy in humans with ADPKD , howeverThree imaging modalities, US, CT, and MRI, are used to examine variation in disease presentation and disease progression in feline ADPKD. Imaging was compared to well-known biomarkers for CKD, such as urine specific gravity (USG), blood urea nitrogen (BUN), serum creatinine (sCr), glomerular filtration rate (GFR) and symmetric dimethylarginine (SDMA), which is a novel biomarker for feline CKD at the earlier stage . Total kPKD1 c.10063C > A that causes feline ADPKD (data not shown) [The cats represented an ADPKD cat colony housed at the University of Missouri (MU), which has been maintained for over 20 years. Five Persian cats with ADPKD were originally donated by private owners to establish the colony. All animal procedures were conducted in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and were approved by the MU Animal Care and Use Committee (protocol #8787). Cat housing and husbandry was overseen by MU Office of Animal Research. No cats were euthanized as part of this study. However, cats were euthanized by barbiturate overdose after sedation as dictated by poor health . All cats were genotyped for the t shown) , 27. DiaUS examinations were performed using an 8 MHz micro-convex transducer on a dedicated ultrasound unit . Each kidney was scanned in sagittal and transverse planes by either a first-year veterinary radiology resident (K.L.S.) or a board-certified veterinary radiologist (J.S.M.). The length was measured, using internal digital calipers, as the longest point between the cranial and caudal poles in the sagittal plane. Width and height were measured in the transverse plane. TKV was calculated using the prolate ellipsoid formula .99mTc-diethylenetriaminepentaacetic acid was administered intravenously. Images were obtained using a gamma camera  with a low energy all-purpose collimator and using a 256 × 256 matrix. The GFR was calculated for each kidney by a single observer (K.L.S.), using Mirage software and a previously described method for scintigraphic uptake [GFRs were determined on the same day as the US imaging. Approximately 3 mCi of c uptake .MRI was performed using a 3 T unit  and a transmit/receive coil. Scans obtained included dorsal T2  weighted imaging (T2WI) with 2 mm slices, a 0.2 mm interspace gap, matrix = 320 × 320–352, and number of acquisitions = 1–3. The scans were performed using respiratory gating to eliminate respiratory motion.CT scans were performed using a third generation 64 slice instrument  under the same anesthetic event as the MRI scan. Follow-up imaging was performed for two cats at 12 months and 15 months, which were thought to have fast and slow cyst progression , respectively.TKVs were calculated from US as described above. TKVs as determined by CT and MRI was measured using a planimetry method with Fiji software common to veterinary practice . To deteP values < 0.05 were considered statistically significant. Data are expressed as mean ± standard deviation (SD).Means and standard deviations were calculated for 11 ADPKD cats for variables imaging indexes. All statistical analyses were performed using R software . Comparisons of kidney volumes between male and female cats were analyzed using Mann–Whitney U test. The Spearman’s rank correlation test was used for the correlation analyses of imaging parameters and clinical data such as age, body weight and serum biomarkers. Kendall’s coefficient of concordance was calculated to evaluate the reliability between or among each modality. High agreement is indicated when Kendall’s coefficient of concordance (W) is higher than 0.75. PKD1 variant (c.10063C > A) , a positive correlation for sCr with BUN , and a positive correlation for SDMA with BUN .Thirteen cats were examined, 11 cats were positive for the feline http://www.iris-kidney.com/guidelines/staging.html, accessed May 13, 2018). This cat was re-evaluated after 15 months and had mildly elevated levels of sCr (2.4 mg/dL) and BUN (36 mg/dL) and lower USG (1.015) indicating advancing renal failure. At 19 months after the initial imaging, this cat rapidly declined within 2 weeks, reducing from normal weight of 3.9 kg to 2.7 kg and was euthanized due to end stage renal disease. All other APDKD cats had cysts and were classified as feline CKD Stage 1.The oldest ADPKD cat  had an SDMA level of 15 μg/dL, sCr level of 1.6 mg/dL, and the lowest USG of 1.028. This cat was categorized as feline CKD Stage 2 based on the guidelines for feline CKD  . US estimates were more strongly correlated with CT (MIROS)  than MRI , and were fairly consistent between the planimetry and MIROS methods for CT    and TCV (MRI-based) . Additionally, concentration of BUN was mildly associated with MRI-based TCV . GFR was not significantly correlated with TKV, TCV or FCV  and MRI (1 and MRI9) Table . The aver > 0.8, p < 0.05) for both MRI-based and CT-based TCV and FCV estimates  was an average of 0.19% per month (CT-based), if progression is considered linear, and 0.29% per month (MRI-based), when excluding the fastest outlier. The fast progression “outlier” cat Case 3) had an expected FCV per month of 1.08% by CT and 1.84% by MRI. Case 6 was a slowly progressing cat with an expected FCV per month of 0.10% by CT and 0.16% by MRI. These two cats were re-evaluated after 12.6 and 15.0 months, respectively  . The rank of FCV showed W = 0.82 (p = 0.11) with Kendall’s coefficient of concordance   was greater in cats older than 3 years old, and especially in those older than 7 years. In contrast, a few cats aged ≥9 years had low plasma Cre concentrations (≤1.6 mg/dl) [Eleven ADPKD cats were examined by different modalities to quantify the variation in the cats and to compare imaging modalities. Ten of eleven cats had no suggestion of renal compromise other than the presence of cysts, thus renal biomarkers, including SDMA, sCr and BUN, are not predictive risk indicators of feline ADPKD and disease severity. GFR was also not indicative of kidney disease in the ADPKD cats. Significant parenchymal loss is likely to be required before abnormal GFRs are observed in APDKD cats . The GFR6 mg/dl) . TherefoTKV, rather than renal function, is suggested as the more appropriate biomarker for monitoring and predicting disease progression in human medicine . In huma3, and US and CT imaging from 14.8 ± 2.9 ml to 19.01 ± 7.55 ml, respectively [US, CT and MRI were conducted in ADPKD cats to estimate TKV. Normal cat kidney volumes have been estimated using water displacement at 18.99 ± 7.68 cmectively , 41. Forectively , ~ 30% lectively  were an PKD1 c.10063C > A), suggesting other factors modify disease expression and progression.In one study, cyst growth in humans is reported as relatively symmetrically and at a steady rate . HoweverPKD1 c.10063C > A, considering progression variability, disease progression of feline ADPKD is likely influenced by other genetic and/or environmental factors, such as those identified in humans [PKD1 mutation [Cats as a large animal model are clearly an asset to evaluate the efficacy and safety of the development of drugs and gene therapies , 46. Altn humans –51. In amutation . IdentifIn conclusion, TKV, TCV, and FCV estimations are valuable for evaluating disease status of feline ADPKD and could lead to renal failure risk classifications analogous to humans , 53 . ThAdditional file 1:Table S1. Correlation of CKD biomarkers and GFRs with total kidney volume. Table S2. Estimations of individual kidney volumes and TKVs for each ADPKD and control cat. Table S3. Correlation of kidney imaging parameters by imaging modalities. Table S4. Rank of kidney volumes estimated by different modalities. Figure S1.GFRs at different ages in 11 cats with ADPKD. Figure S2. Post-mortem kidneys of cats with ADPKD. (DOCX 1170 kb)Supplementary Methods.