Noninvasive hemodynamic indices of vascular aging: an in silico assessment.

Vascular aging (VA) involves structural and functional changes in blood vessels that contribute to cardiovascular disease. Several noninvasive pulse wave (PW) indices have been proposed to assess the arterial stiffness component of VA in the clinic and daily life. This study investigated 19 of these indices, identified in recent review articles on VA, by using a database comprising 3,837 virtual healthy subjects aged 25-75 yr, each with unique PW signals simulated under various levels of artificial noise to mimic real measurement errors. For each subject, VA indices were calculated from filtered PW signals and compared with the precise theoretical value of aortic Young's modulus (EAo). In silico PW indices showed age-related changes that align with in vivo population studies. The cardio-ankle vascular index (CAVI) and all pulse wave velocity (PWV) indices showed strong linear correlations with EAo (Pearson's rp > 0.95). Carotid distensibility showed a strong negative nonlinear correlation (Spearman's rs < -0.99). CAVI and distensibility exhibited greater resilience to noise compared with PWV indices. Blood pressure-related indices and photoplethysmography (PPG)-based indices showed weaker correlations with EAo (rp and rs < 0.89, |rp| and |rs| < 0.84, respectively). Overall, blood pressure-related indices were confounded by more cardiovascular properties (heart rate, stroke volume, duration of systole, large artery diameter, and/or peripheral vascular resistance) compared with other studied indices, and PPG-based indices were most affected by noise. In conclusion, carotid-femoral PWV, CAVI and carotid distensibility emerged as the superior clinical VA indicators, with a strong EAo correlation and noise resilience. PPG-based indices showed potential for daily VA monitoring under minimized noise disturbances.NEW & NOTEWORTHY For the first time, 19 noninvasive pulse wave indices for assessing vascular aging were examined together in a single database of nearly 4,000 subjects aged 25-75 yr. The dataset contained precise values of the aortic Young's modulus and other hemodynamic measures for each subject, which enabled us to test each index's ability to measure changes in aortic stiffness while accounting for confounding factors and measurement errors. The study provides freely available tools for analyzing these and additional indices.

The glucose tolerance test in mice: Sex, drugs and protocol.

AIM: To establish the impact of sex, dosing route, fasting duration and acute habituation stress on glucose tolerance test (GTT) measurements used in the preclinical evaluation of potential glucose-modulating therapeutics. METHODS: Adult male and female C57Bl/6J mice, implanted with HD-XG glucose telemetry devices, were fasted for 16 hours or 6 hours following acute habituation stress due to whole cage change, cage change with retention of used bedding or no cage change prior to intraperitoneal (IP) GTTs. To evaluate protocol refinement and sex on the ability of the GTT to detect drug effects, we administered 250 mg/kg oral metformin or 10 nmol/kg IP exendin-4 using optimized protocols. RESULTS: Female mice were less sensitive to human intervention when initiating fasting. Following a 6-hour fast, retention of bedding whilst changing the cage base promotes quicker stabilization of basal blood glucose in both sexes. Prolonged fasting for 16 hours resulted in an exaggerated GTT response but induced pronounced basal hypoglycaemia. Following GTT protocol optimization the effect of exendin-4 and metformin was equivalent in both sexes, with females showing a more modest but more reproducible GTT response. CONCLUSIONS: Variations in GTT protocol have profound effects on glucose homeostasis. Protocol refinement and/or the use of females still allows for detection of drug effects, providing evidence that more severe phenotypes are not an essential prerequisite when characterizing/validating new drugs.

The use of mice in diabetes research: The impact of experimental protocols.

Mice are used extensively in preclinical diabetes research to model various aspects of blood glucose homeostasis. Careful experimental design is vital for maximising welfare and improving reproducibility of data. Alongside decisions regarding physiological characteristics of the animal cohort (e.g., sex, strain and age), experimental protocols must also be carefully considered. This includes choosing relevant end points of interest and understanding what information they can provide and what their limitations are. Details of experimental protocols must, therefore, be carefully planned during the experimental design stage, especially considering the impact of researcher interventions on preclinical end points. Indeed, in line with the 3Rs of animal research, experiments should be refined where possible to maximise welfare. The role of welfare may be particularly pertinent in preclinical diabetes research as blood glucose concentrations are directly altered by physiological stress responses. Despite the potential impact of variations in experimental protocols, there is distinct lack of standardisation and consistency throughout the literature with regards to several experimental procedures including fasting, cage changing and glucose tolerance test protocol. This review firstly highlights practical considerations with regard to the choice of end points in preclinical diabetes research and the potential for novel technologies such as continuous glucose monitoring and glucose clamping techniques to improve data resolution. The potential influence of differing experimental protocols and in vivo procedures on both welfare and experimental outcomes is then discussed with focus on standardisation, consistency and full disclosure of methods.

Rethinking animal models of sepsis - working towards improved clinical translation whilst integrating the 3Rs.

Sepsis is a major worldwide healthcare issue with unmet clinical need. Despite extensive animal research in this area, successful clinical translation has been largely unsuccessful. We propose one reason for this is that, sometimes, the experimental question is misdirected or unrealistic expectations are being made of the animal model. As sepsis models can lead to a rapid and substantial suffering - it is essential that we continually review experimental approaches and undertake a full harm:benefit impact assessment for each study. In some instances, this may require refinement of existing sepsis models. In other cases, it may be replacement to a different experimental system altogether, answering a mechanistic question whilst aligning with the principles of reduction, refinement and replacement (3Rs). We discuss making better use of patient data to identify potentially useful therapeutic targets which can subsequently be validated in preclinical systems. This may be achieved through greater use of construct validity models, from which mechanistic conclusions are drawn. We argue that such models could provide equally useful scientific data as face validity models, but with an improved 3Rs impact. Indeed, construct validity models may not require sepsis to be modelled, per se. We propose that approaches that could support and refine clinical translation of research findings, whilst reducing the overall welfare burden on research animals.

Extracting new information from old waveforms: Symmetric projection attractor reconstruction: Where maths meets medicine.

NEW FINDINGS: What is the topic of this review? Symmetric Projection Attractor Reconstruction (SPAR) is a relatively new mathematical method that can extract additional information pertaining to the morphology and variability of physiological waveforms, such as arterial pulse pressure. Herein, we describe the potential utility of the method for more sensitive quantification of cardiovascular changes. What advances does it highlight? We use a simple example of a human tilt table to illustrate these concepts. SPAR can be used on any approximately periodic waveform and may add value to experimental and clinical settings, where such signals are collected routinely. ABSTRACT: Periodic physiological waveform data, such as blood pressure, pulse oximetry and ECG, are routinely sampled between 100 and 1000 Hz in preclinical research and in the clinical setting from a wide variety of implantable, bedside and wearable monitoring devices. Despite the underlying numerical waveform data being captured at such high fidelity, conventional analysis tends to reside in reporting only averages of minimum, maximum, amplitude and rate, as single point averages. Although these averages are undoubtedly of value, simplification of the data in this way means that most of the available numerical data are discarded. In turn, this may lead to subtle physiological changes being missed when investigating the cardiovascular system over time. We have developed a mathematical method (symmetric projection attractor reconstruction) that uses all the numerical data, replotting and revisualizing them in a manner that allows unique quantification of multiple changes in waveform morphology and variability. We propose that the additional quantification of these features will allow the complex behaviour of the cardiovascular system to be mapped more sensitively in different physiological and pathophysiological settings.

Platelet Depletion Impairs Host Defense to Pulmonary Infection with Pseudomonas aeruginosa in Mice.

Platelets have been implicated in pulmonary inflammatory cell recruitment after exposure to allergic and nonallergic stimuli, but little is known about the role of platelets in response to pulmonary infection with Pseudomonas aeruginosa. In this study, we have investigated the impact of the experimental depletion of circulating platelets on a range of inflammatory and bacterial parameters, and their subsequent impact on mortality in a murine model of pulmonary infection with P. aeruginosa. P. aeruginosa infection in mice induced a mild, but significant, state of peripheral thrombocytopenia in addition to pulmonary platelet accumulation. Increased platelet activation was detected in infected mice through increased levels of the platelet-derived mediators, platelet factor-4 and ß-thromboglobulin, in BAL fluid and blood plasma. In mice depleted of circulating platelets, pulmonary neutrophil recruitment was significantly reduced 24 hours after infection, whereas the incidence of systemic dissemination of bacteria was significantly increased compared with non-platelet-depleted control mice. Furthermore, mortality rates were increased in bacterial-infected mice depleted of circulating platelets. This work demonstrates a role for platelets in the host response toward a gram-negative bacterial respiratory infection.

A Novel α-Calcitonin Gene-Related Peptide Analogue Protects Against End-Organ Damage in Experimental Hypertension, Cardiac Hypertrophy, and Heart Failure.

BACKGROUND: Research into the therapeutic potential of α-calcitonin gene-related peptide (α-CGRP) has been limited because of its peptide nature and short half-life. Here, we evaluate whether a novel potent and long-lasting (t½ ≥7 hours) acylated α-CGRP analogue (αAnalogue) could alleviate and reverse cardiovascular disease in 2 distinct murine models of hypertension and heart failure in vivo. METHODS: The ability of the αAnalogue to act selectively via the CGRP pathway was shown in skin by using a CGRP receptor antagonist. The effect of the αAnalogue on angiotensin II-induced hypertension was investigated over 14 days. Blood pressure was measured by radiotelemetry. The ability of the αAnalogue to modulate heart failure was studied in an abdominal aortic constriction model of murine cardiac hypertrophy and heart failure over 5 weeks. Extensive ex vivo analysis was performed via RNA analysis, Western blot, and histology. RESULTS: The angiotensin II-induced hypertension was attenuated by cotreatment with the αAnalogue (50 nmol·kg-1·d-1, SC, at a dose selected for lack of long-term hypotensive effects at baseline). The αAnalogue protected against vascular, renal, and cardiac dysfunction, characterized by reduced hypertrophy and biomarkers of fibrosis, remodeling, inflammation, and oxidative stress. In a separate study, the αAnalogue reversed angiotensin II-induced hypertension and associated vascular and cardiac damage. The αAnalogue was effective over 5 weeks in a murine model of cardiac hypertrophy and heart failure. It preserved heart function, assessed by echocardiography, while protecting against adverse cardiac remodeling and apoptosis. Moreover, treatment with the αAnalogue was well tolerated with neither signs of desensitization nor behavioral changes. CONCLUSIONS: These findings, in 2 distinct models, provide the first evidence for the therapeutic potential of a stabilized αAnalogue, by mediating (1) antihypertensive effects, (2) attenuating cardiac remodeling, and (3) increasing angiogenesis and cell survival to protect against and limit damage associated with the progression of cardiovascular diseases. This indicates the therapeutic potential of the CGRP pathway and the possibility that this injectable CGRP analogue may be effective in cardiac disease.

Dimethylarginine dimethylaminohydrolase-2 deficiency promotes vascular regeneration and attenuates pathological angiogenesis.

Ischemia-induced angiogenesis is critical for tissue repair, but aberrant neovascularization in the retina causes severe sight impairment. Nitric oxide (NO) has been implicated in neovascular eye disease because of its pro-angiogenic properties in the retina. Nitric oxide production is inhibited endogenously by asymmetric dimethylarginines (ADMA and L-NMMA) which are metabolized by dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2. The aim of this study was to determine the roles of DDAH1, DDAH2, ADMA and L-NMMA in retinal ischemia-induced angiogenesis. First, DDAH1, DDAH2, ADMA and L-NMMA levels were determined in adult C57BL/6J mice. The results obtained revealed that DDAH1 was twofold increased in the retina compared to the brain and the choroid. DDAH2 expression was approximately 150 fold greater in retinal and 70 fold greater in choroidal tissue compared to brain tissue suggesting an important tissue-specific role for DDAH2 in the retina and choroid. ADMA and L-NMMA levels were similar in the retina and choroid under physiological conditions. Next, characterization of DDAH1(+/-) and DDAH2(-/-) deficient mice by in vivo fluorescein angiography, immunohistochemistry and electroretinography revealed normal neurovascular function compared with wildtype control mice. Finally, DDAH1(+/-) and DDAH2(-/-) deficient mice were studied in the oxygen-induced retinopathy (OIR) model, a model used to emulate retinal ischemia and neovascularization, and VEGF and ADMA levels were quantified by ELISA and liquid chromatography tandem mass spectrometry. In the OIR model, DDAH1(+/-) exhibited a similar phenotype compared to wildtype controls. DDAH2 deficiency, in contrast, resulted in elevated retinal ADMA which was associated with attenuated aberrant angiogenesis and improved vascular regeneration in a VEGF independent manner. Taken together this study suggests, that in retinal ischemia, DDAH2 deficiency elevates ADMA, promotes vascular regeneration and protects against aberrant angiogenesis. Therapeutic inhibition of DDAH2 may therefore offer a potential therapeutic strategy to protect sight by promoting retinal vascular regeneration and preventing pathological angiogenesis.

In vivo pharmacological activity and biodistribution of S-nitrosophytochelatins after intravenous and intranasal administration in mice.

S-nitrosophytochelatins (SNOPCs) are novel analogues of S-nitrosoglutathione (GSNO) with the advantage of carrying varying ratios of S-nitrosothiol (SNO) moieties per molecule. Our aim was to investigate the in vivo pharmacological potency and biodistribution of these new GSNO analogues after intravenous (i.v.) and intranasal (i.n.) administration in mice. SNOPCs with either two or six SNO groups and GSNO were synthesized and characterized for purity. Compounds were administered i.v. or i.n. at 1 µmol NO/kg body weight to CD-1 mice. Blood pressure was measured and biodistribution studies of total nitrate and nitrite species (NOx) and phytochelatins were performed after i.v. administration. At equivalent doses of NO, it was observed that SNOPC-6 generated a rapid and significantly greater reduction in blood pressure (∼60% reduction compared to saline) whereas GSNO and SNOPC-2 only achieved a 30-35% decrease. The reduction in blood pressure was transient and recovered to baseline levels within ∼2 min for all compounds. NOx species were transiently elevated (over 5 min) in the plasma, lung, heart and liver. Interestingly, a size-dependent phytochelatin accumulation was observed in several tissues including the heart, lungs, kidney, brain and liver. Biodistribution profiles of NOx were also obtained after i.n. administration, showing significant lung retention of NOx over 15 min with minor systemic increases observed from 5 to 15 min. In summary, this study has revealed interesting in vivo pharmacological properties of SNOPCs, with regard to their dramatic hypotensive effects and differing biodistribution patterns following two different routes of administration.

Dimethylarginine dimethylaminohydrolase 2 regulates nitric oxide synthesis and hemodynamics and determines outcome in polymicrobial sepsis.

OBJECTIVE: Nitric oxide is a key to numerous physiological and pathophysiological processes. Nitric oxide production is regulated endogenously by 2 methylarginines, asymmetric dimethylarginine (ADMA) and monomethyl-L-arginine. The enzyme that specifically metabolizes asymmetric dimethylarginine and monomethyl-L-arginine is dimethylarginine dimethylaminohydrolase (DDAH). The first isoform dimethylarginine dimethylaminohydrolase 1 has previously been shown to be an important regulator of methylarginines in both health and disease. This study explores for the first time the role of endogenous dimethylarginine dimethylaminohydrolase 2 in regulating cardiovascular physiology and also determines the functional impact of dimethylarginine dimethylaminohydrolase 2 deletion on outcome and immune function in sepsis. APPROACH AND RESULTS: Mice, globally deficient in Ddah2, were compared with their wild-type littermates to determine the physiological role of Ddah2 using in vivo and ex vivo assessments of vascular function. We show that global knockout of Ddah2 results in elevated blood pressure during periods of activity (mean [SEM], 118.5 [1.3] versus 112.7 [1.1] mm Hg; P=0.025) and changes in vascular responsiveness mediated by changes in methylarginine concentration, mean myocardial tissue asymmetric dimethylarginine (SEM) was 0.89 (0.06) versus 0.67 (0.05) µmol/L (P=0.02) and systemic nitric oxide concentrations. In a model of severe polymicrobial sepsis, Ddah2 knockout affects outcome (120-hour survival was 12% in Ddah2 knockouts versus 53% in wild-type animals; P<0.001). Monocyte-specific deletion of Ddah2 results in a similar pattern of increased severity to that seen in globally deficient animals. CONCLUSIONS: Ddah2 has a regulatory role both in normal physiology and in determining outcome of severe polymicrobial sepsis. Elucidation of this role identifies a mechanism for the observed relationship between Ddah2 polymorphisms, cardiovascular disease, and outcome in sepsis.

Pharmacological inhibition of DDAH1 improves survival, haemodynamics and organ function in experimental septic shock.

The aim of the present study was to investigate the therapeutic effects of pharmacological inhibition of DDAH1 (dimethylarginine dimethylaminohydrolase 1), an enzyme that metabolizes endogenously produced nitric oxide synthase inhibitors, principally ADMA (asymmetric dimethylarginine). The present study employs a series of rodent models to evaluate the effectiveness a DDAH1-selective inhibitor (L-257). Short-term models involved the development of endotoxaemia using lipopolysaccharide and long-term models involved the intraperitoneal administration of faecal slurry. In order to generate the most relevant model possible, following induction of severe sepsis, animals received appropriate fluid resuscitation and in some models vasopressor therapy. The effects of L-257 on survival, haemodynamics and organ function were subsequently assessed. Survival was significantly longer in all L-257 treatment groups (P<0.01) and no adverse effects on haemodynamics and organ function were observed following L-257 administration to either animals with sepsis or naïve animals. Haemodynamic performance was preserved and the noradrenaline dose required to maintain target blood pressure was reduced in the treated animals (P<0.01). Animals receiving L-257 had significantly increased plasma ADMA concentrations. Plasma nitrite/nitrate was reduced as was severity of sepsis-associated renal dysfunction. The degree of tachycardia was improved as were indices of tissue and microvascular perfusion. The results of the present study show that the selective DDAH-1 inhibitor L-257 improved haemodynamics, provided catecholamine sparing and prolonged survival in experimental sepsis. Further studies will determine its potential utility in human septic shock.

Disruption of methylarginine metabolism impairs vascular homeostasis.

Asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA) are endogenously produced amino acids that inhibit all three isoforms of nitric oxide synthase (NOS). ADMA accumulates in various disease states, including renal failure, diabetes and pulmonary hypertension, and its concentration in plasma is strongly predictive of premature cardiovascular disease and death. Both L-NMMA and ADMA are eliminated largely through active metabolism by dimethylarginine dimethylaminohydrolase (DDAH) and thus DDAH dysfunction may be a crucial unifying feature of increased cardiovascular risk. However, despite considerable interest in this pathway and in the role of ADMA as a cardiovascular risk factor, there is little evidence to support a causal role of ADMA in pathophysiology. Here we reveal the structure of human DDAH-1 and probe the function of DDAH-1 both by deleting the DDAH1 gene in mice and by using DDAH-specific inhibitors which, as we demonstrate by crystallography, bind to the active site of human DDAH-1. We show that loss of DDAH-1 activity leads to accumulation of ADMA and reduction in NO signaling. This in turn causes vascular pathophysiology, including endothelial dysfunction, increased systemic vascular resistance and elevated systemic and pulmonary blood pressure. Our results also suggest that DDAH inhibition could be harnessed therapeutically to reduce the vascular collapse associated with sepsis.

Advanced waveform analysis of the photoplethysmogram signal using complementary signal processing techniques for the extraction of biomarkers of cardiovascular function.

Introduction: Photoplethysmogram signals from wearable devices typically measure heart rate and blood oxygen saturation, but contain a wealth of additional information about the cardiovascular system. In this study, we compared two signal-processing techniques: fiducial point analysis and Symmetric Projection Attractor Reconstruction, on their ability to extract new cardiovascular information from a photoplethysmogram signal. The aim was to identify fiducial point analysis and Symmetric Projection Attractor Reconstruction indices that could classify photoplethysmogram signals, according to age, sex and physical activity. Methods: Three datasets were used: an in-silico dataset of simulated photoplethysmogram waves for healthy male participants (25-75 years old); an in-vivo dataset containing 10-min photoplethysmogram recordings from 57 healthy subjects at rest (18-39 or > 70 years old; 53% female); and an in-vivo dataset containing photoplethysmogram recordings collected for 4 weeks from a single subject, in daily life. The best-performing indices from the in-silico study (5/48 fiducial point analysis and 6/49 Symmetric Projection Attractor Reconstruction) were applied to the in-vivo datasets. Results: Key fiducial point analysis and Symmetric Projection Attractor Reconstruction indices, which showed the greatest differences between groups, were found to be consistent across datasets. These indices were related to systolic augmentation, diastolic peak positioning and prominence, and waveform variability. Both fiducial point analysis and Symmetric Projection Attractor Reconstruction techniques provided indices that supported the classification of age and physical activity, but not sex. Conclusions: Both fiducial point analysis and Symmetric Projection Attractor Reconstruction techniques demonstrated utility in identifying cardiovascular differences between individuals and within an individual over time. Future research should investigate the potential utility of these techniques for extracting information on fitness and disease, to support healthcare-decision making.

Genetic and pharmacological inhibition of dimethylarginine dimethylaminohydrolase 1 is protective in endotoxic shock.

OBJECTIVE: The overproduction of vascular NO contributes toward the circulatory collapse observed in patients with septic shock. Dimethylarginine dimethylaminohydrolase (DDAH), which has 2 isoforms, metabolizes asymmetrically methylated arginines (asymmetric mono- or di-methylarginine), endogenously produced NO synthase inhibitors. We wished to investigate whether reducing DDAH1 activity, using genetic and pharmacological approaches, is protective during lipopolysaccharide-induced endotoxic shock. METHODS AND RESULTS: Experiments were conducted in DDAH1 heterozygous knockout mice (DDAH1(+/-)) or naive rats treated with a synthetic pharmacological DDAH inhibitor (L-257). We demonstrate for the first time that L-257 is DDAH1 selective using recombinant human DDAH proteins. DDAH1 mRNA was expressed in aortic but not macrophage cDNA, and consistent with this expression profile, L-257 selectively inhibited NO production from lipopolysaccharide-treated aorta but not macrophages, in culture. Conscious and anesthetized cardiovascular hemodynamics were monitored using implanted radiotelemetry devices or invasive catheters, respectively. Lipopolysaccharide was administered intravenously to model endotoxemia, and all animals presented with circulatory shock. DDAH1(+/-) mice or L-257-treated rats displayed attenuation in the rate of developed hypotension compared with wild-type littermates or vehicle control animals, respectively. CONCLUSIONS: Pharmacological and genetic reduction of DDAH1 activity is protective against the vascular changes observed during endotoxic shock.

Novel breathing pattern analysis: Symmetric Projection Attractor Reconstruction improves identification of impending COPD re-exacerbations - a retrospective cohort analysis.

Respiratory waveforms can be reduced to simple metrics, such as rate, but this may miss information about waveform shape and whole breathing pattern. A novel analysis method quantifying the whole waveform shape identifies AECOPD earlier. https://bit.ly/3M6uIEB.

Enhancing Techniques for Determining Inflammatory Edema Formation and Neutrophil Accumulation in Murine Skin.

Inflammatory edema formation and polymorphonuclear leukocyte (neutrophil) accumulation are common components of cutaneous vascular inflammation, and their assessment is a powerful investigative and drug development tool but typically requires independent cohorts of animals to assess each. We have established the use of a mathematical formula to estimate the ellipsoidal-shaped volume of the edematous wheal or bleb after intradermal injections of substances in mice pretreated intravenously with Evans blue dye (which binds to plasma albumin) to act as an edema marker. Whereas previous extraction of Evans blue dye with formamide is suitable for all strains of mice, we report this quicker and more reliable assessment of edema volume in situ. This therefore allows neutrophil accumulation to be assessed from the same mouse using the myeloperoxidase assay. Importantly, we examined the influence of Evans blue dye on the spectrometry readout at the wavelength at which myeloperoxidase activity is measured. The results indicate that it is feasible to quantify edema formation and neutrophil accumulation in the same mouse skin site. Thus, we show techniques that can assess edema formation and neutrophil accumulation at the same site in the same mouse, allowing paired measurements and reducing the total use of mice by 50%.

Cardiovascular waveforms - can we extract more from routine signals?

Cardiovascular waveforms such as blood pressure, ECG and photoplethysmography (PPG), are routinely acquired by specialised monitoring devices. Such devices include bedside monitors, wearables and radiotelemetry which sample at very high fidelity, yet most of this numerical data is disregarded and focus tends to reside on single point averages such as the maxima, minima, amplitude, rate and intervals. Whilst, these measures are undoubtedly of value, we may be missing important information by simplifying the complex waveform signal in this way. This Special Collection showcases recent advances in the appraisal of routine signals. Ultimately, such approaches and technologies may assist in improving the accuracy and sensitivity of detecting physiological change. This, in turn, may assist with identifying efficacy or safety signals for investigational new drugs or aidpatient diagnosis and management, supporting scientific and clinical decision making.

Detecting paroxysmal atrial fibrillation from normal sinus rhythm in equine athletes using Symmetric Projection Attractor Reconstruction and machine learning.

Background: Atrial fibrillation (AF) is a common cardiac arrhythmia in both human and equine populations. It is associated with adverse outcomes in humans and decreased athletic performance in both populations. Paroxysmal atrial fibrillation (PAF) presents with intermittent, self-terminating AF episodes, and is difficult to diagnose once sinus rhythm resumes. Objective: We aimed to detect PAF subjects from normal sinus rhythm equine electrocardiograms (ECGs) using the Symmetric Projection Attractor Reconstruction (SPAR) method to encapsulate the waveform morphology and variability as the basis of a machine learning classification. Methods: We obtained ECG signals from 139 active equine athletes (120 control, 19 with a PAF diagnosis). The SPAR method was applied to 9 short (20-second) ECG strips for each subject. An optimal SPAR feature set was determined by forward feature selection for input to a machine learning model ensemble of 3 different classifiers (k-nearest neighbors, linear support vector machine, and radial basis function kernel support vector machine). Imbalanced data were handled by upsampling the minority (PAF) class. A final subject classification was made by taking a majority vote over results from the 9 ECG strips. Results: Our final cross-validated classification for a subject gave an accuracy of 89.0%, sensitivity of 94.8%, specificity of 87.1%, and receiver operating characteristic area under the curve of 0.98, taking PAF as the positive class. Conclusion: The SPAR method and machine learning generated a final model with high sensitivity, suggesting that PAF can be discriminated from short equine ECG strips. This preliminary study indicated that SPAR analysis of human ECG could support patient monitoring, risk stratification, and clinical decision-making.

S-nitrosophytochelatins: investigation of the bioactivity of an oligopeptide nitric oxide delivery system.

This study investigates the in vitro bioactivity of S-nitrosophytochelatins (SNOPCs), oligopeptide analogues of S-nitrosoglutathione (GSNO), and their mechanisms of nitric oxide (NO) delivery. SNOPCs were more potent than GSNO in inhibiting platelet aggregation and stimulating vasorelaxation. Their potency was related to the number of S-nitrosated moieties per mole compound. Transnitrosation reactions with cell membrane surface components were shown to be the primary mode of NO delivery to intracellular targets for SNOPCs, while delivery via γ-glutamyl transpeptidase was unique to GSNO. Due to rapid NO release, larger SNOPCs elicited a more transitory effect compared to smaller compounds. The duration of effect was influenced by compound molecular weight, NO release kinetics, ability to undergo transnitrosation, and incubation time with tissues. In summary, a new oligopeptide NO delivery system based on SNOPCs was shown to be biologically active and can be used to investigate the mechanisms of NO delivery to intracellular targets.

Symmetric Projection Attractor Reconstruction: Sex Differences in the ECG.

Background: The electrocardiogram (ECG) is a key tool in patient management. Automated ECG analysis supports clinical decision-making, but traditional fiducial point identification discards much of the time-series data that captures the morphology of the whole waveform. Our Symmetric Projection Attractor Reconstruction (SPAR) method uses all the available data to provide a new visualization and quantification of the morphology and variability of any approximately periodic signal. We therefore applied SPAR to ECG signals to ascertain whether this more detailed investigation of ECG morphology adds clinical value. Methods: Our aim was to demonstrate the accuracy of the SPAR method in discriminating between two biologically distinct groups. As sex has been shown to influence the waveform appearance, we investigated sex differences in normal sinus rhythm ECGs. We applied the SPAR method to 9,007 10 second 12-lead ECG recordings from Physionet, which comprised; Dataset 1: 104 subjects (40% female), Dataset 2: 8,903 subjects (54% female). Results: SPAR showed clear visual differences between female and male ECGs (Dataset 1). A stacked machine learning model achieved a cross-validation sex classification accuracy of 86.3% (Dataset 2) and an unseen test accuracy of 91.3% (Dataset 1). The mid-precordial leads performed best in classification individually, but the highest overall accuracy was achieved with all 12 leads. Classification accuracy was highest for young adults and declined with older age. Conclusions: SPAR allows quantification of the morphology of the ECG without the need to identify conventional fiducial points, whilst utilizing of all the data reduces inadvertent bias. By intuitively re-visualizing signal morphology as two-dimensional images, SPAR accurately discriminated ECG sex differences in a small dataset. We extended the approach to a machine learning classification of sex for a larger dataset, and showed that the SPAR method provided a means of visualizing the similarities of subjects given the same classification. This proof-of-concept study therefore provided an implementation of SPAR using existing data and showed that subtle differences in the ECG can be amplified by the attractor. SPAR's supplementary analysis of ECG morphology may enhance conventional automated analysis in clinically important datasets, and improve patient stratification and risk management.