Plasma Oxylipins: A Potential Risk Assessment Tool in Atherosclerotic Coronary Artery Disease

Background: While oxylipins have been linked to coronary artery disease (CAD), little is known about their diagnostic and prognostic potential. Objective: We tested whether plasma concentration of specific oxylipins may discriminate among number of diseased coronary arteries and predict median 5-year outcomes in symptomatic adults. Methods: Using a combination of high-performance liquid chromatography (HPLC) and quantitative tandem mass spectrometry, we conducted a targeted analysis of 39 oxylipins in plasma samples of 23 asymptomatic adults with low CAD risk and 74 symptomatic adults (≥70% stenosis), aged 38–87 from the Greater Portland, Oregon area. Concentrations of 22 oxylipins were above the lower limit of quantification in >98% of adults and were compared, individually and in groups based on precursors and biosynthetic pathways, in symptomatic adults to number of diseased coronary arteries [(1) n = 31; (2) n = 23; (3) n = 20], and outcomes during a median 5-year follow-up (no surgery: n = 7; coronary stent placement: n = 24; coronary artery bypass graft surgery: n = 26; death: n = 7). Results: Plasma levels of six quantified oxylipins decreased with the number of diseased arteries; a panel of five oxylipins diagnosed three diseased arteries with 100% sensitivity and 70% specificity. Concentrations of five oxylipins were lower and one oxylipin was higher with survival; a panel of two oxylipins predicted survival during follow-up with 86% sensitivity and 91% specificity. Conclusions: Quantification of plasma oxylipins may assist in CAD diagnosis and prognosis in combination with standard risk assessment tools.


INTRODUCTION
Coronary artery disease (CAD) is the leading cause of death worldwide (1)(2)(3). Traditional CAD risk factors such as diabetes, smoking, hypertension, hyperlipidemia, and family history of premature cardiovascular disease (CVD), as well as nontraditional risk factors of rheumatic inflammatory disease, human immunodeficiency disease, and gestational diabetes can assist clinicians in decisions for CAD primary prevention but have limited efficacy in high CAD risk patient management (4). This is currently done using expensive invasive tests, such as exercise stress testing (without or with concomitant imaging for myocardial perfusion and/or function) or measuring the coronary calcium score on X-ray computed tomography (CT). Our objective is to develop a point-of-care blood test that could assist in decision making regarding CAD patient management.
Ruptured arterial plaques are a major reason for adverse cardiac events with resultant thrombus formation that partially or completely impairs blood flow to the heart (5). The progression from asymptomatic to ruptured arterial plaques involves lipid oxidation and inflammation (6,7). Conventional indicators of lipid oxidation include secondary products such as 4-hydroxynonenal (4-HNE) (8), malondialdehyde (MDA) (9), and oxidized low-density lipoproteins (Ox-LDL) (10), and its associated oxidized phospholipids (11). Technological advancements in soft ionization tandem mass spectrometry (MS/MS) have allowed multiplexed quantification of oxidized lipids in one analytical run and with it the emergence of isoprostanes and oxylipins as indicators of oxidative tissue injuries, implicating oxidative tissue injuries in the pathology of a variety of chronic diseases (3,12).
In the present study, we evaluated whether plasma oxylipins, alone or in panels, may discriminate among number of diseased coronary arteries and predict median 5-year outcomes in high CAD risk symptomatic patients and ≥70% stenosis which, to our knowledge, has not been previously published. In doing so, a point-of-care plasma oxylipin test could assist in decision making regarding CAD patient management. Here, we report results of a small study confirming this hypothesis.

Participants and Study Design
The study was approved by the Institutional Review Board of the Oregon Health and Science University (OHSU) in Portland, Oregon. We prospectively enrolled 74 individuals from the greater Portland metropolitan area from October 2012 and January 2017 (IRB00008606) who were referred to OHSU for an invasive coronary angiography because of symptoms suggestive of CAD (median age: 66 years; range: 38-87 years). Inclusion criteria were inducible myocardial ischemia during stress (either on echocardiography or single-photon computed tomography) and ≥70% coronary luminal narrowing of one or more major coronary artery or its major branch on subsequent coronary angiography. Exclusion criteria were <70% coronary stenosis on angiography, prior myocardial infarction, hemodynamically significant valvular heart disease, prior revascularization, or congestive heart failure. The CAD patients were classified as having one-vessel (n = 31), two-vessel (n = 23), or three-vessel (n = 20) CAD and were followed up until November 2019 ( Figure 2) for a median of 60 months (range: 25-84 months) for adverse events [i.e., coronary stent placement; coronary artery bypass graft (CABG) surgery; death]. Ten CAD patients were lost to follow-up (unable to contact: n = 8; declined to follow-up: n = 2).
To establish ranges of plasma oxylipin concentrations in low CAD risk populations, we established the Astoria cohort and prospectively enrolled 220 individuals from Astoria, a rural community within the same area as the CAD patients, from July 2016 to February 2017 (IRB00011193). For the current study, we selected individuals of the same age range (range: 38-71 years) that fulfilled all the exclusion criteria (n = 23). Exclusion criteria were self-reported history of hyperlipidemia, diabetes, myocardial infarction, ischemia, coronary artery revascularization surgery, coronary atherosclerosis on coronary angiography, active tobacco use, and family history of CAD. Participants from the Astoria and Portland were not age-matched.

Sample Collection and Preparation for Oxylipin Analysis
All participants fasted for at least 6 h before 4.5 mL blood was collected in tubes containing 0.01 M buffered sodium citrate and immediately placed on ice. Blood samples were collected 1-4 h FIGURE 1 | Biosynthetic pathways of plasma oxylipins from omega-6 (A) and omega-3 (B) polyunsaturated fatty acids (PUFA). Normal text designates PUFA (yellow rectangular boxes) and oxylipins. Italic text designates enzymes involved in the metabolic transformation [blue oval boxes for cyclo-oxygenases or aspirin, orange oval boxes for lipoxygenases or CYP1B1, green oval boxes for cytochrome P450, and gray rectangular boxes for soluble epoxide hydrolase (sEH)] with their quantified oxylipins in the same color and oxylipins below the limit of quantification (LOQ) in noncolored squares. See Abbreviation Index and Appendix for full names description.
Frontiers in Cardiovascular Medicine | www.frontiersin.org prior to coronary angiography of participants with CAD. Whole blood samples were then centrifuged at 3,000 rpm for 15 min in a refrigerated centrifuge at 4 • C, after which the plasma was aliquoted into 1 mL Eppendorf tubes and immediately stored at −80 • C until analysis.
Oxylipins from plasma were extracted as described in Pedersen et al. (22) with minor modifications. The internal oxylipin standards used during the extraction (Supplementary Table 1) were used to correct the recovery of the quantified oxylipins (23).

Chromatographic and Mass Spectrometric Analysis of Oxylipins
The high-performance liquid chromatography (HPLC) and mass spectrometry methods used for the analysis of plasma oxylipins was based on methods previously described for the analysis of oxylipins in liver (24). The analysis was performed using a Shimadzu Prominence HPLC system (Shimadzu, Columbia, MD) coupled to an Applied Biosystems 4000 QTRAP (AB SCIEX, Framingham, MA). Employing dynamic multireaction monitoring (dMRM), we evaluated 60 oxylipins in a targeted approach (Supplementary Figure 1). For each compound, optimal transitions were determined by flow injection of pure standards using the optimizer application, and transitions were compared with literature when available. A detailed list of MRM transitions and experimental conditions is provided in Supplementary Table 2.

Data Processing and Statistical Analysis
Raw data from targeted oxylipin analyses were imported into MultiQuant 3.0.2 software (AB SCIEX) in order to perform the alignment and integration of the peaks (obtaining peak areas). This software allows for the correction of metabolite intensity with the intensity of the internal standards. Data obtained with MultiQuant were imported into MarkerView 1.3.1 software (AB SCIEX) for initial data visualization (25).
Data were analyzed using SAS version 9.2 (SAS Ins. Inc., Cary, NC). Demographic and clinical characteristics of groups were compared using Fisher's exact test for binary data and t-test for nonbinary data. Concentrations of oxylipins were compared using Wilcoxon rank sum test. To evaluate diagnostic and predictive efficacy of oxylipins, we used logistic regression analysis and calculated receiver operating characteristic (ROC) values, including area under the curves (AUC). Our goal was to identify oxylipin panels that could achieve an ROC of 0.90 or higher. To compare diagnostic and predictive efficacy of oxylipins with current standard risk assessment tool, we compared ROC values of our best oxylipin models with those of the 10-year Framingham general CVD risk scores using the ROCCONTRAST statement in PROC LOGISTIC. We were not able to use the 10-year atherosclerotic CVD risk score of the American College of Cardiology (ACC) because 41 of 74 CAD patient scores could not be calculated. All statistical tests were two sided. Significance was declared at P ≤ 0.05.

Demographic, Clinical Characteristics, and Levels of Oxylipins of Adults With Diseased Coronary Arteries
Selected demographic and clinical characteristics of adults with diseased coronary arteries stratified by number of diseased arteries and adults of the same age range with a low CAD risk are listed in Table 1. Sixty-nine of 74 adults with CAD had multiple CVD risk factors (three CAD1 patients and one CAD2 patient had one CVD risk factor and one CAD2 patient had no CVD risk factor). Almost all adults with CAD had hypertension and hypercholesterolemia. Most adults with CAD were on aspirin, were overweight or obese, or had a history of smoking. About half adults with CAD had diabetes or a family history of CVD. Demographic and clinical characteristics of adults with CAD had a limited efficacy to diagnose number of diseased arteries. The 10-year Framingham general CVD risk score and the number of CAD risk factors increased with the number of diseased arteries; specifically, adults with multiple diseased arteries were more likely to be male, were overweight or obese, former smokers, or had lower plasma HDL cholesterol concentrations.
Ten of 22 (45%) individual oxylipin concentrations decreased with greater number of diseased arteries by at least 10%; six individual oxylipins (27%) had significantly lower concentrations in adults with three vs. one diseased artery ( Table 2). For pattern detection, oxylipins were grouped in Table 3 by (1) FA precursors (i.e., LA, ARA, EPA, and DHA), oxylipin groups (i.e., midchain HODE, EET, mid-chain HETE, EpDPA, DiHDPA), (2) enzymes involved in their synthesis [i.e., oxygenation of PUFAs by LOX followed by reduction or alternatively hydroxylation of PUFAs by CYP1B1; oxidation of PUFAs by CYP450 followed by hydroxylation of oxidized PUFAs by soluble epoxide hydrolase (sEH)], and (3) based on enzymatic product to substrate TABLE 1 | Demographic and clinical characteristics of adults with diseased coronary arteries (≥70% stenosis) and adults of the same age range with a low coronary artery disease (CAD) risk.

Characteristics
Low Number of diseased arteries Contrast CAD risk (n = 23) Age ( The superscripts denote whether averages of specific groups in a row differed at P ≤ 0.05. If none of the groups differed from each other, no superscripts are shown. If two groups differed from each other, the significantly higher group got a superscript a, and the significantly lower group got a superscript b, and the group that did not significantly differ from the higher or the lower group got a superscript ab. If three groups differed from each other, the significantly highest group got a superscript a, the significantly lower group got a superscript b, and the group that was significantly lower than the group with a superscript b, got a, c. Quantitative data were compared using Student's t-test. Total oxylipin concentrations significantly declined with number of diseased arteries, specifically omega-3 FA-derived oxylipins and within those hydroxylated DHA-epoxide The superscripts denote whether averages of specific groups in a row differed at P ≤ 0.05 using Kruskal-Wallis test. If none of the groups differed from each other, no superscripts are shown. If two groups differed from each other, the significantly higher group got a superscript a, and the significantly lower group got a superscript b, and the group that did not significantly differ from the higher or the lower group got a superscript ab. If three groups differed from each other, the significantly highest group got a superscript a, the significantly lower group got a superscript b, and the group that was significantly lower than the group with a superscript b, got a, c. DiHOME, dihydroxy-octadecenoic acid; HODE, hydroxy-octadecadienoic acid; HETE, hydroxy-eicosatetraenoic acid; EET, epoxy-eicosatrienoic acid; DiHET, dihydroxy-eicosatrienoic acid; EpDPA, epoxy-docosapentaenoic acid; DiHDPA, dihydroxy-docosapentaenoic acid. The superscripts denote whether averages of specific groups in a row differed at P ≤ 0.05 using Kruskal-Wallis test. If none of the groups differed from each other, no superscripts are shown. If two groups differed from each other, the significantly higher group got a superscript a, and the significantly lower group got a superscript b, and the group that did not significantly differ from the higher or the lower group got a superscript ab. If three groups differed from each other, the significantly highest group got a superscript a, the significantly lower group got a superscript b, and the group that was significantly lower than the group with a superscript b, got a, c. HODE, hydroxy-octadecadienoic acid; HETE, hydroxy-eicosatetraenoic acid; EET, epoxy-eicosatrienoic acid; DiHET, dihydroxy-eicosatrienoic acid; EpDPA, epoxy-docosapentaenoic acid; DiHDPA, dihydroxy-docosapentaenoic acid; LOX, lipoxygenase; CYP, cytochrome P450; sEH, soluble epoxide hydrolases. DiHDPAs, which are generated by hydroxylation of oxidized PUFAs by sEH ( Table 3). The strongest decline was observed for hydroxylation of 19,20-EpDPA to 19,20-DiHDPA.

Prediction of Outcomes in Adults With Diseased Coronary Arteries
Adults with CAD were followed up until November 2019 for a median of 5 years (range: 25-84 months) and adverse events were recorded (i.e., coronary stent placement; CABG surgery; death). Ten participants (three women and seven men; median age: 61 years; range: 51-81 years) were lost to follow-up (Figure 2). Adults with multiple diseased coronary arteries were more likely to receive a CABG. Of the remaining 12 adults with CAD, seven had no further event, two adults with one diseased artery received a coronary artery stent during follow-up, and three died (CAD1: 2; CAD2: 1). In addition, four CAD adults that had undergone open-heart surgery within 3 months (two stents and two CABG) died during follow-up (CAD1: 1; CAD2: 0; CAD3: 3). Survival was not linked to the number of diseased coronary arteries. Table 4 lists selected demographic and clinical characteristics of adults with diseased coronary arteries (≥70% stenosis) based on outcomes during follow-up. Survival was linked to lower systolic blood pressure or being a male, whereas The superscripts denote whether averages of specific groups in a row differed at P ≤ 0.05. If none of the groups differed from each other, no superscripts are shown. If two groups differed from each other, the significantly higher group got a superscript a, and the significantly lower group got a superscript b, and the group that did not significantly differ from the higher or the lower group got a superscript ab. If three groups differed from each other, the significantly highest group got a superscript a, the significantly lower group got a superscript b, and the group that was significantly lower than the group with a superscript b, got a, c. Data were compared using Student's t-test. Proportions were compared using Fisher's exact test. CABG, coronary artery bypass grafting; ACE, angiotension converting enzyme; AR, angiotension receptor; ACC, American College of Cardiology; ASCVD, Atherosclerotic Cardiovascular Disease.
survival without CABG was linked to higher plasma triacylglycerol concentrations. Unfavorable outcomes were linked to elevated oxylipin concentrations ( Table 5), specifically omega-6 FA-derived oxylipins and within those LA-derived mid-chain HODEs and ARA-derived mid-chain HETEs, which are either generated by oxygenation of lipoxygenases  The superscripts denote whether averages of specific groups in a row differed at P ≤ 0.05 using Kruskal-Wallis test. If none of the groups differed from each other, no superscripts are shown. If two groups differed from each other, the significantly higher group got a superscript a, and the significantly lower group got a superscript b, and the group that did not significantly differ from the higher or the lower group got a superscript ab. If three groups differed from each other, the significantly highest group got a superscript a, the significantly lower group got a superscript b, and the group that was significantly lower than the group with a superscript b, got a, c. CABG, coronary artery bypass grafting; DiHOME, dihydroxy-octadecenoic acid; HODE, hydroxy-octadecadienoic acid; HETE, hydroxy-eicosatetraenoic acid; EET, epoxy-eicosatrienoic acid; DiHET, dihydroxy-eicosatrienoic acid; EpDPA, epoxy-docosapentaenoic acid; DiHDPA, dihydroxy-docosapentaenoic acid. The superscripts denote whether averages of specific groups in a row differed at P ≤ 0.05 using Kruskal-Wallis test. If none of the groups differed from each other, no superscripts are shown. If two groups differed from each other, the significantly higher group got a superscript a, and the significantly lower group got a superscript b, and the group that did not significantly differ from the higher or the lower group got a superscript ab. If three groups differed from each other, the significantly highest group got a superscript a, the significantly lower group got a superscript b, and the group that was significantly lower than the group with a superscript b, got a, c. CABG, coronary artery bypass grafting; HODE, hydroxy-octadecadienoic acid; HETE, hydroxy-eicosatetraenoic acid; EET, epoxy-eicosatrienoic acid; DiHET, dihydroxy-eicosatrienoic acid; EpDPA, epoxy-docosapentaenoic acid; DiHDPA, dihydroxy-docosapentaenoic acid; LOX, lipoxygenase; CYP, cytochrome P450; sEH, soluble epoxide hydrolases.
Among individual oxylipins, survival without requiring CABG was best predicted by LA-derived 9(S)-HODE (AUC: 0.65; 95% CI: 0.52-0.79; P = 0.03; Figure 5A). The tworemaining individual oxylipins that could significantly predict survival without requiring CABG were ordered by P-value: ARA- The only single oxylipin that could significantly predict no events in CAD adults was ARA-derived 5-HETE (AUC: 0.71; 95% CI: 0.52-0.91; P = 0.03). In general, patients without follow-up events had oxylipin values similar to patients who died during follow-up or had a surgery for a full blockage. The 10-year Framingham general CVD risk score had an AUC of 0.62 (95% CI: 0.38-0.87; P = 0.33).

DISCUSSION
In the current study, we provide evidence that in adults with diseased coronary arteries (>70% stenosis), plasma oxylipin panels may discriminate among the number of diseased coronary arteries and predict median 5-year outcomes, which, to our knowledge, has not been previously reported.

Analysis of Plasma Oxylipins
Novel analytical methods for extraction, detection, and data processing allow for the separation of a large number of diverse oxylipins in a short period of time (3,12,22,23). In the present study, we detected and verified with standards 39 oxylipins of diverse origin and biosynthetic pathways in a 22-min LC-MS/MS run. Similar to inflammatory cytokines, low abundance, limited dynamic range, limited tissue specificity, very short halflife, significant daily fluctuation, and high inter-and intra-assay variation, limit the use of oxylipins as diagnostic biomarkers  (16). For diagnostic and prognostic research, a good biomarker must have a large dynamic range within the population. In the current study, 22 oxylipins had concentrations in the linear quantification range in at least 98% of sampled adults, which allowed us to evaluate the most abundant enzymatic oxylipin pathways; however, excluded pathways generated by COX or aspirin and ROS.

Diagnostic and Prognostic Efficacy of Oxylipins in CAD
Currently used risk assessment scores of CAD, such as the 10year Framingham general CVD risk score, have been developed for the general population and have shown limited efficacy in high risk CAD adult management (4). In adults with significant diseased coronary arteries, a five-oxylipin panel diagnosed three diseased arteries with 100% sensitivity and 70% specificity. During a median 5-year survival, a panel of two oxylipins predicted survival with 86% sensitivity and 91% specificity. The oxylipin panels improved three diseased artery diagnosis and survival prognosis compared with the 10-year Framingham general CVD risk score.

Clinical Relevance of Oxylipins in CAD
Coronary artery disease (CAD) limits nutrient and oxygen supply to generate sufficient energy in cardiomyocytes, which becomes an even bigger challenge as the number and severity of diseased coronary arteries increase or plaques rupture with subsequent thrombus formation (5). In the present study, adults with more diseased coronary arteries (≥70% stenosis) had lower plasma concentrations of hydroxylated omega-3 PUFA-derived epoxides, specifically we observed lower levels of 19,20-DiHDPA (Figure 6). To our knowledge, the link between oxylipin concentrations and number of diseased coronary arteries has not been previously reported. The enzyme responsible for hydroxylation of epoxides is sEH, which is induced by hypoxia and has been proposed as potential pharmacological target for CAD (26)(27)(28). However, we cannot exclude the possibility that participants with CAD were already on medication that inhibited soluble CYP450 epoxide hydrolase. However, the gradual decrease in concentrations of LA-derived 12,13-DiHOME and DHA-derived DiHDPA with increasing diseased artery number support the hypothesis that the lower concentrations are a response to the hypoxia caused by arterial occlusions (29).
Five-year survival and no CABG surgery was linked to lower concentrations of oxygenated omega-6 PUFA LA and ARA, specifically lower concentrations of LA-derived midchain HODE and ARA-derived mid-chain HETE, which are either generated by oxygenation of lipoxygenases or hydroxylation of CYP1B1 (Figure 6). In support, elevated 15-HETE concentrations and LOX-15 enzymatic activity have been reported in ischemic heart disease and hypoxic human cardiomyocytes and cardiac endothelial cells (15), supporting our hypothesis that the elevated mid-chain HETE and HODE concentrations are a response to the hypoxia caused by the arterial occlusions. High concentrations of HETE, including 5-HETE, 12-HETE, and 15-HETE, were reported in atherosclerotic plaques, especially in those that were more likely to rupture (14). Elevated circulating concentrations of 5-HETE and 12-HETE were observed in individuals after cardiac surgery (16). Elevated concentrations of 5-HETE, 12-HETE, and 15-HETE were reported in individuals with acute cardiac syndrome (17). Elevated circulating concentrations of 5-HETE, 12-HETE, and 15-HETE were reported in individuals with CAD by Xu et al. (20), whereas only numerical increases were reported by Shishebor et al. (19) and Auguet FIGURE 6 | The link between plasma oxylipins and coronary artery disease. Adults with more diseased coronary arteries (≥70% stenosis) had lower plasma concentrations of hydroxylated omega-3 fatty acid-derived epoxygenated oxylipins, which was linked to decreased soluble epoxide hydrolase (sEH) activity. Nonsurviving adults with diseased coronary arteries had higher plasma concentration of oxygenated omega-6 fatty acids, which was linked to increased lipoxygenase or CYP1B1 activity. et al. (21); the latter did not quantify 5-HETE. The role of elevated mid-chain HETE in cardiovascular dysfunction has been well documented, whereas less is known of the role of mid-chain HODE (3,30,31). Inhibition of the oxygenation step of the LOX pathway has been proposed as treatment option for CAD management (30), suggesting clinical relevance of the identified oxylipins as indicator of chronic hypoxia.

Limitations of the Study
First, the number of adults with CAD were relatively small and came from a high-risk group, which underwent coronary angiography, but in whom the presence and extent of CAD was clearly defined. Second, all but five adults with CAD were on medical therapy for treatment for hypertension, hyperlipidemia, and/or diabetes, which could have influenced the levels of oxylipins. Third, differences in collateral blood flow may have impacted oxylipin concentration, which was not assessed in this cohort. Fourth, most adults with CAD had to undergo revascularization shortly after angiography, which may impact later outcomes. Fifth, follow-up time was limited to <6 years, which impacted the number of outcomes. Sixth, diagnostic and prognostic oxylipin panels could not be validated due to the limited clearly defined population size.

SUMMARY AND CONCLUSION
In summary, we observed a link between plasma oxylipin concentrations and CAD severity. Concentrations of six oxylipins decreased with the number of diseased arteries; a panel of five oxylipins diagnosed three diseased arteries with 100% sensitivity and 70% specificity. Concentrations of five oxylipins were lower and one oxylipin was higher with survival; a panel of two oxylipins predicted survival during follow-up with 86% sensitivity and 91% specificity. Plasma oxylipins may assist in diagnosis and prognosis of CAD in high-risk adults in combination with standard risk assessment tools. Our promising results require confirmation in larger unselected populations.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
This study was approved by the Institutional Review Board of the Oregon Health and Science University (OHSU) in Portland, Oregon. The patients/participants provided their written informed consent to participate in this study. DJ, CM, and SK: supervision. DL, MG-J, GB, DJ, CM, and SK: validation. All authors contributed to the article and approved the submitted version.

FUNDING
This study was funded in part by grants (DK112360, 2R90AT008924-06, and S10RR022589) from the National Institutes of Health, Bethesda, Maryland. This work used facilities in OSU's Mass Spectrometry Center, Corvallis, Oregon. Disclaimer: The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

Conflict of Interest:
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.
Copyright © 2021 Le, García-Jaramillo, Bobe, Alcazar Magana, Vaswani, Minnier, Jump, Rinkevich, Alkayed, Maier and Kaul. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.