Metabolomics Simultaneously Derives Benchmark Dose Estimates and Discovers Metabolic Biotransformations in a Rat Bioassay.

Benchmark dose (BMD) modeling estimates the dose of a chemical that causes a perturbation from baseline. Transcriptional BMDs have been shown to be relatively consistent with apical end point BMDs, opening the door to using molecular BMDs to derive human health-based guidance values for chemical exposure. Metabolomics measures the responses of small-molecule endogenous metabolites to chemical exposure, complementing transcriptomics by characterizing downstream molecular phenotypes that are more closely associated with apical end points. The aim of this study was to apply BMD modeling to in vivo metabolomics data, to compare metabolic BMDs to both transcriptional and apical end point BMDs. This builds upon our previous application of transcriptomics and BMD modeling to a 5-day rat study of triphenyl phosphate (TPhP), applying metabolomics to the same archived tissues. Specifically, liver from rats exposed to five doses of TPhP was investigated using liquid chromatography-mass spectrometry and 1H nuclear magnetic resonance spectroscopy-based metabolomics. Following the application of BMDExpress2 software, 2903 endogenous metabolic features yielded viable dose-response models, confirming a perturbation to the liver metabolome. Metabolic BMD estimates were similarly sensitive to transcriptional BMDs, and more sensitive than both clinical chemistry and apical end point BMDs. Pathway analysis of the multiomics data sets revealed a major effect of TPhP exposure on cholesterol (and downstream) pathways, consistent with clinical chemistry measurements. Additionally, the transcriptomics data indicated that TPhP activated xenobiotic metabolism pathways, which was confirmed by using the underexploited capability of metabolomics to detect xenobiotic-related compounds. Eleven biotransformation products of TPhP were discovered, and their levels were highly correlated with multiple xenobiotic metabolism genes. This work provides a case study showing how metabolomics and transcriptomics can estimate mechanistically anchored points-of-departure. Furthermore, the study demonstrates how metabolomics can also discover biotransformation products, which could be of value within a regulatory setting, for example, as an enhancement of OECD Test Guideline 417 (toxicokinetics).

Bilateral remote ischemic conditioning in children: A two-center, double-blind, randomized controlled trial in young children undergoing cardiac surgery.

Objective: The study objective was to determine whether adequately delivered bilateral remote ischemic preconditioning is cardioprotective in young children undergoing surgery for 2 common congenital heart defects with or without cyanosis. Methods: We performed a prospective, double-blind, randomized controlled trial at 2 centers in the United Kingdom. Children aged 3 to 36 months undergoing tetralogy of Fallot repair or ventricular septal defect closure were randomized 1:1 to receive bilateral preconditioning or sham intervention. Participants were followed up until hospital discharge or 30 days. The primary outcome was area under the curve for high-sensitivity troponin-T in the first 24 hours after surgery, analyzed by intention-to-treat. Right atrial biopsies were obtained in selected participants. Results: Between October 2016 and December 2020, 120 eligible children were randomized to receive bilateral preconditioning (n = 60) or sham intervention (n = 60). The primary outcome, area under the curve for high-sensitivity troponin-T, was higher in the preconditioning group (mean: 70.0 ± 50.9 µg/L/h, n = 56) than in controls (mean: 55.6 ± 30.1 µg/L/h, n = 58) (mean difference, 13.2 µg/L/h; 95% CI, 0.5-25.8; P = .04). Subgroup analyses did not show a differential treatment effect by oxygen saturations (pinteraction = .25), but there was evidence of a differential effect by underlying defect (pinteraction = .04). Secondary outcomes and myocardial metabolism, quantified in atrial biopsies, were not different between randomized groups. Conclusions: Bilateral remote ischemic preconditioning does not attenuate myocardial injury in children undergoing surgical repair for congenital heart defects, and there was evidence of potential harm in unstented tetralogy of Fallot. The routine use of remote ischemic preconditioning cannot be recommended for myocardial protection during pediatric cardiac surgery.

Multi-omics bioactivity profile-based chemical grouping and read-across: a case study with Daphnia magna and azo dyes.

Grouping/read-across is widely used for predicting the toxicity of data-poor target substance(s) using data-rich source substance(s). While the chemical industry and the regulators recognise its benefits, registration dossiers are often rejected due to weak analogue/category justifications based largely on the structural similarity of source and target substances. Here we demonstrate how multi-omics measurements can improve confidence in grouping via a statistical assessment of the similarity of molecular effects. Six azo dyes provided a pool of potential source substances to predict long-term toxicity to aquatic invertebrates (Daphnia magna) for the dye Disperse Yellow 3 (DY3) as the target substance. First, we assessed the structural similarities of the dyes, generating a grouping hypothesis with DY3 and two Sudan dyes within one group. Daphnia magna were exposed acutely to equi-effective doses of all seven dyes (each at 3 doses and 3 time points), transcriptomics and metabolomics data were generated from 760 samples. Multi-omics bioactivity profile-based grouping uniquely revealed that Sudan 1 (S1) is the most suitable analogue for read-across to DY3. Mapping ToxPrint structural fingerprints of the dyes onto the bioactivity profile-based grouping indicated an aromatic alcohol moiety could be responsible for this bioactivity similarity. The long-term reproductive toxicity to aquatic invertebrates of DY3 was predicted from S1 (21-day NOEC, 40 µg/L). This prediction was confirmed experimentally by measuring the toxicity of DY3 in D. magna. While limitations of this 'omics approach are identified, the study illustrates an effective statistical approach for building chemical groups.

Lipidome characterisation and sex-specific differences in type 1 and type 2 diabetes mellitus.

BACKGROUND: In this study, we evaluated the lipidome alterations caused by type 1 diabetes (T1D) and type 2 diabetes (T2D), by determining lipids significantly associated with diabetes overall and in both sexes, and lipids associated with the glycaemic state. METHODS: An untargeted lipidomic analysis was performed to measure the lipid profiles of 360 subjects (91 T1D, 91 T2D, 74 with prediabetes and 104 controls (CT)) without cardiovascular and/or chronic kidney disease. Ultra-high performance liquid chromatography-electrospray ionization mass spectrometry (UHPLC-ESI-MS) was conducted in two ion modes (positive and negative). We used multiple linear regression models to (1) assess the association between each lipid feature and each condition, (2) determine sex-specific differences related to diabetes, and (3) identify lipids associated with the glycaemic state by considering the prediabetes stage. The models were adjusted by sex, age, hypertension, dyslipidaemia, body mass index, glucose, smoking, systolic blood pressure, triglycerides, HDL cholesterol, LDL cholesterol, alternate Mediterranean diet score (aMED) and estimated glomerular filtration rate (eGFR); diabetes duration and glycated haemoglobin (HbA1c) were also included in the comparison between T1D and T2D. RESULTS: A total of 54 unique lipid subspecies from 15 unique lipid classes were annotated. Lysophosphatidylcholines (LPC) and ceramides (Cer) showed opposite effects in subjects with T1D and subjects with T2D, LPCs being mainly up-regulated in T1D and down-regulated in T2D, and Cer being up-regulated in T2D and down-regulated in T1D. Also, Phosphatidylcholines were clearly down-regulated in subjects with T1D. Regarding sex-specific differences, ceramides and phosphatidylcholines exhibited important diabetes-associated differences due to sex. Concerning the glycaemic state, we found a gradual increase of a panel of 1-deoxyceramides from normoglycemia to prediabetes to T2D. CONCLUSIONS: Our findings revealed an extensive disruption of lipid metabolism in both T1D and T2D. Additionally, we found sex-specific lipidome changes associated with diabetes, and lipids associated with the glycaemic state that can be linked to previously described molecular mechanisms in diabetes.

Simultaneously discovering the fate and biochemical effects of pharmaceuticals through untargeted metabolomics.

Untargeted metabolomics is an established approach in toxicology for characterising endogenous metabolic responses to xenobiotic exposure. Detecting the xenobiotic and its biotransformation products as part of the metabolomics analysis provides an opportunity to simultaneously gain deep insights into its fate and metabolism, and to associate the internal relative dose directly with endogenous metabolic responses. This integration of untargeted exposure and response measurements into a single assay has yet to be fully demonstrated. Here we assemble a workflow to discover and analyse pharmaceutical-related measurements from routine untargeted UHPLC-MS metabolomics datasets, derived from in vivo (rat plasma and cardiac tissue, and human plasma) and in vitro (human cardiomyocytes) studies that were principally designed to investigate endogenous metabolic responses to drug exposure. Our findings clearly demonstrate how untargeted metabolomics can discover extensive biotransformation maps, temporally-changing relative systemic exposure, and direct associations of endogenous biochemical responses to the internal dose.

Derivation of metabolic point of departure using high-throughput in vitro metabolomics: investigating the importance of sampling time points on benchmark concentration values in the HepaRG cell line.

Amongst omics technologies, metabolomics should have particular value in regulatory toxicology as the measurement of the molecular phenotype is the closest to traditional apical endpoints, whilst offering mechanistic insights into the biological perturbations. Despite this, the application of untargeted metabolomics for point-of-departure (POD) derivation via benchmark concentration (BMC) modelling is still a relatively unexplored area. In this study, a high-throughput workflow was applied to derive PODs associated with a chemical exposure by measuring the intracellular metabolome of the HepaRG cell line following treatment with one of four chemicals (aflatoxin B1, benzo[a]pyrene, cyclosporin A, or rotenone), each at seven concentrations (aflatoxin B1, benzo[a]pyrene, cyclosporin A: from 0.2048 µM to 50 µM; rotenone: from 0.04096 to 10 µM) and five sampling time points (2, 6, 12, 24 and 48 h). The study explored three approaches to derive PODs using benchmark concentration modelling applied to single features in the metabolomics datasets or annotated metabolites or lipids: (1) the 1st rank-ordered unannotated feature, (2) the 1st rank-ordered putatively annotated feature (using a recently developed HepaRG-specific library of polar metabolites and lipids), and (3) 25th rank-ordered feature, demonstrating that for three out of four chemical datasets all of these approaches led to relatively consistent BMC values, varying less than tenfold across the methods. In addition, using the 1st rank-ordered unannotated feature it was possible to investigate temporal trends in the datasets, which were shown to be chemical specific. Furthermore, a possible integration of metabolomics-driven POD derivation with the liver steatosis adverse outcome pathway (AOP) was demonstrated. The study highlights that advances in technologies enable application of in vitro metabolomics at scale; however, greater confidence in metabolite identification is required to ensure PODs are mechanistically anchored.

Invariant NKT cells metabolically adapt to the acute myeloid leukaemia environment.

Acute myeloid leukaemia (AML) creates an immunosuppressive environment to conventional T cells through Arginase 2 (ARG2)-induced arginine depletion. We identify that AML blasts release the acute phase protein serum amyloid A (SAA), which acts in an autocrine manner to upregulate ARG2 expression and activity, and promote AML blast viability. Following in vitro cross-talk invariant natural killer T (iNKT) cells become activated, upregulate mitochondrial capacity, and release IFN-γ. iNKT retain their ability to proliferate and be activated despite the low arginine AML environment, due to the upregulation of Large Neutral Amino Acid Transporter-1 (LAT-1) and Argininosuccinate Synthetase 1 (ASS)-dependent amino acid pathways, resulting in AML cell death. T cell proliferation is restored in vitro and in vivo. The capacity of iNKT cells to restore antigen-specific T cell immunity was similarly demonstrated against myeloid-derived suppressor cells (MDSCs) in wild-type and Jα18-/- syngeneic lymphoma-bearing models in vivo. Thus, stimulation of iNKT cell activity has the potential as an immunotherapy against AML or as an adjunct to boost antigen-specific T cell immunotherapies in haematological or solid cancers.

Suitability of Dried Blood Spots for Accelerating Veterinary Biobank Collections and Identifying Metabolomics Biomarkers With Minimal Resources.

Biomarker discovery using biobank samples collected from veterinary clinics would deliver insights into the diverse population of pets and accelerate diagnostic development. The acquisition, preparation, processing, and storage of biofluid samples in sufficient volumes and at a quality suitable for later analysis with most suitable discovery methods remain challenging. Metabolomics analysis is a valuable approach to detect health/disease phenotypes. Pre-processing changes during preparation of plasma/serum samples may induce variability that may be overcome using dried blood spots (DBSs). We report a proof of principle study by metabolite fingerprinting applying UHPLC-MS of plasma and DBSs acquired from healthy adult dogs and cats (age range 1-9 years), representing each of 4 dog breeds (Labrador retriever, Beagle, Petit Basset Griffon Vendeen, and Norfolk terrier) and the British domestic shorthair cat (n = 10 per group). Blood samples (20 and 40 µL) for DBSs were loaded onto filter paper, air-dried at room temperature (3 h), and sealed and stored (4°C for ~72 h) prior to storage at -80°C. Plasma from the same blood draw (250 µL) was prepared and stored at -80°C within 1 h of sampling. Metabolite fingerprinting of the DBSs and plasma produced similar numbers of metabolite features that had similar abilities to discriminate between biological classes and correctly assign blinded samples. These provide evidence that DBSs, sampled in a manner amenable to application in in-clinic/in-field processing, are a suitable sample for biomarker discovery using UHPLC-MS metabolomics. Further, given appropriate owner consent, the volumes tested (20-40 µL) make the acquisition of remnant blood from blood samples drawn for other reasons available for biobanking and other research activities. Together, this makes possible large-scale biobanking of veterinary samples, gaining sufficient material sooner and enabling quicker identification of biomarkers of interest.

Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached.

Dysregulated cellular metabolism is a cancer hallmark for which few druggable oncoprotein targets have been identified. Increased fatty acid (FA) acquisition allows cancer cells to meet their heightened membrane biogenesis, bioenergy, and signaling needs. Excess FAs are toxic to non-transformed cells but surprisingly not to cancer cells. Molecules underlying this cancer adaptation may provide alternative drug targets. Here, we demonstrate that diacylglycerol O-acyltransferase 1 (DGAT1), an enzyme integral to triacylglyceride synthesis and lipid droplet formation, is frequently up-regulated in melanoma, allowing melanoma cells to tolerate excess FA. DGAT1 over-expression alone transforms p53-mutant zebrafish melanocytes and co-operates with oncogenic BRAF or NRAS for more rapid melanoma formation. Antagonism of DGAT1 induces oxidative stress in melanoma cells, which adapt by up-regulating cellular reactive oxygen species defenses. We show that inhibiting both DGAT1 and superoxide dismutase 1 profoundly suppress tumor growth through eliciting intolerable oxidative stress.

Rapid identification of human muscle disease with fibre optic Raman spectroscopy.

The diagnosis of muscle disorders ("myopathies") can be challenging and new biomarkers of disease are required to enhance clinical practice and research. Despite advances in areas such as imaging and genomic medicine, muscle biopsy remains an important but time-consuming investigation. Raman spectroscopy is a vibrational spectroscopy application that could provide a rapid analysis of muscle tissue, as it requires no sample preparation and is simple to perform. Here, we investigated the feasibility of using a miniaturised, portable fibre optic Raman system for the rapid identification of muscle disease. Samples were assessed from 27 patients with a final clinico-pathological diagnosis of a myopathy and 17 patients in whom investigations and clinical follow-up excluded myopathy. Multivariate classification techniques achieved accuracies ranging between 71-77%. To explore the potential of Raman spectroscopy to identify different myopathies, patients were subdivided into mitochondrial and non-mitochondrial myopathy groups. Classification accuracies were between 74-89%. Observed spectral changes were related to changes in protein structure. These data indicate fibre optic Raman spectroscopy is a promising technique for the rapid identification of muscle disease that could provide real time diagnostic information. The application of fibre optic Raman technology raises the prospect of in vivo bedside testing for muscle diseases which would significantly streamline the diagnostic pathway of these disorders.

Infrared Spectroscopic Analysis in the Differentiation of Epithelial Misplacement From Adenocarcinoma in Sigmoid Colonic Adenomatous Polyps.

Purpose: The differential diagnosis of epithelial misplacement from invasive cancer in the colon is a challenging endeavour, augmented by the introduction of bowel cancer population screening. The main aim of the work is to test, as a proof-of concept study, the ability of the infrared spectroscopic imaging approach to differentiate epithelial misplacement from adenocarcinoma in sigmoid colonic adenomatous polyps. Methods: Ten samples from each of the four diagnostic groups, normal colonic mucosa, adenomatous polyps with low grade dysplasia, epithelial misplacement in adenomatous polyps and adenocarcinoma, were analysed using IR spectroscopic imaging and data processing methods. IR spectral images were subjected to data pre-processing and cluster analysis based segmentation to identify epithelial, connective tissue and stromal regions. Statistical analysis was carried out using principal component analysis and linear discriminant analysis based cross validation, to classify spectral features according to the pathology, and the diagnostic attributes were compared. Results: The combined 4-group classification model on an average showed a sensitivity of 64%, a specificity of 88% and an accuracy of 76% for prediction based on a 'single spectrum', whilst a 'majority-vote' prediction on an average showed a sensitivity of 73%, a specificity of 90% and an accuracy of 82%. The 2-group model, for the differential diagnosis of epithelial misplacement versus adenocarcinoma, showed an average sensitivity and specificity of 82.5% for prediction based on a 'single spectrum' whilst a 'majority-vote' classification showed an average sensitivity and specificity of 90%. A 92% area under the curve (AUC) value was obtained when evaluating the classifier using the Receiver Operating Characteristics (ROC) curves. Conclusions: IR spectroscopy shows promise in its ability to differentiate epithelial misplacement from adenocarcinoma in tissue sections, considered as one of the most challenging endeavours in population-wide diagnostic histopathology. Further studies with larger series, including cases with challenging diagnostic features are required to ascertain the capability of this modern digital pathology approach. In the long-term, IR spectroscopy based pathology which is relatively low-cost and rapid, could be a promising endeavour to consider for integration into the existing histopathology pathway, in particular for population based screening programmes where large number of samples are scrutinised.

Automated Sample Preparation and Data Collection Workflow for High-Throughput In Vitro Metabolomics.

Regulatory bodies have started to recognise the value of in vitro screening and metabolomics as two types of new approach methodologies (NAMs) for chemical risk assessments, yet few high-throughput in vitro toxicometabolomics studies have been reported. A significant challenge is to implement automated sample preparation of the low biomass samples typically used for in vitro screening. Building on previous work, we have developed, characterised and demonstrated an automated sample preparation and analysis workflow for in vitro metabolomics of HepaRG cells in 96-well microplates using a Biomek i7 Hybrid Workstation (Beckman Coulter) and Orbitrap Elite (Thermo Scientific) high-resolution nanoelectrospray direct infusion mass spectrometry (nESI-DIMS), across polar metabolites and lipids. The experimental conditions evaluated included the day of metabolite extraction, order of extraction of samples in 96-well microplates, position of the 96-well microplate on the instrument's deck and well location within a microplate. By using the median relative standard deviation (mRSD (%)) of spectral features, we have demonstrated good repeatability of the workflow (final mRSD < 30%) with a low percentage of features outside the threshold applied for statistical analysis. To improve the quality of the automated workflow further, small method modifications were made and then applied to a large cohort study (4860 sample infusions across three nESI-DIMS assays), which confirmed very high repeatability of the whole workflow from cell culturing to metabolite measurements, whilst providing a significant improvement in sample throughput. It is envisioned that the automated in vitro metabolomics workflow will help to advance the application of metabolomics (as a part of NAMs) in chemical safety, primarily as an approach for high throughput screening and prioritisation.

An Extensive Metabolomics Workflow to Discover Cardiotoxin-Induced Molecular Perturbations in Microtissues.

Discovering modes of action and predictive biomarkers of drug-induced structural cardiotoxicity offers the potential to improve cardiac safety assessment of lead compounds and enhance preclinical to clinical translation during drug development. Cardiac microtissues are a promising, physiologically relevant, in vitro model, each composed of ca. 500 cells. While untargeted metabolomics is capable of generating hypotheses on toxicological modes of action and discovering metabolic biomarkers, applying this technology to low-biomass microtissues in suspension is experimentally challenging. Thus, we first evaluated a filtration-based approach for harvesting microtissues and assessed the sensitivity and reproducibility of nanoelectrospray direct infusion mass spectrometry (nESI-DIMS) measurements of intracellular extracts, revealing samples consisting of 28 pooled microtissues, harvested by filtration, are suitable for profiling the intracellular metabolome and lipidome. Subsequently, an extensive workflow combining nESI-DIMS untargeted metabolomics and lipidomics of intracellular extracts with ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) analysis of spent culture medium, to profile the metabolic footprint and quantify drug exposure concentrations, was implemented. Using the synthetic drug and model cardiotoxin sunitinib, time-resolved metabolic and lipid perturbations in cardiac microtissues were investigated, providing valuable data for generating hypotheses on toxicological modes of action and identifying putative biomarkers such as disruption of purine metabolism and perturbation of polyunsaturated fatty acid levels.

In Vivo Fiber Optic Raman Spectroscopy of Muscle in Preclinical Models of Amyotrophic Lateral Sclerosis and Duchenne Muscular Dystrophy.

Neuromuscular diseases result in muscle weakness, disability, and, in many instances, death. Preclinical models form the bedrock of research into these disorders, and the development of in vivo and potentially translational biomarkers for the accurate identification of disease is crucial. Spontaneous Raman spectroscopy can provide a rapid, label-free, and highly specific molecular fingerprint of tissue, making it an attractive potential biomarker. In this study, we have developed and tested an in vivo intramuscular fiber optic Raman technique in two mouse models of devastating human neuromuscular diseases, amyotrophic lateral sclerosis, and Duchenne muscular dystrophy (SOD1G93A and mdx, respectively). The method identified diseased and healthy muscle with high classification accuracies (area under the receiver operating characteristic curves (AUROC): 0.76-0.92). In addition, changes in diseased muscle over time were also identified (AUROCs 0.89-0.97). Key spectral changes related to proteins and the loss of α-helix protein structure. Importantly, in vivo recording did not cause functional motor impairment and only a limited, resolving tissue injury was seen on high-resolution magnetic resonance imaging. Lastly, we demonstrate that ex vivo muscle from human patients with these conditions produced similar spectra to those observed in mice. We conclude that spontaneous Raman spectroscopy of muscle shows promise as a translational research tool.

struct: an R/Bioconductor-based framework for standardized metabolomics data analysis and beyond.

SUMMARY: Implementing and combining methods from a diverse range of R/Bioconductor packages into 'omics' data analysis workflows represents a significant challenge in terms of standardization, readability and reproducibility. Here, we present an R/Bioconductor package, named struct (Statistics in R using Class-based Templates), which defines a suite of class-based templates that allows users to develop and implement highly standardized and readable statistical analysis workflows. Struct integrates with the STATistics Ontology to ensure consistent reporting and maximizes semantic interoperability. We also present a toolbox, named structToolbox, which includes an extensive set of commonly used data analysis methods that have been implemented using struct. This toolbox can be used to build data-analysis workflows for metabolomics and other omics technologies. AVAILABILITY AND IMPLEMENTATION: struct and structToolbox are implemented in R, and are freely available from Bioconductor (http://bioconductor.org/packages/struct and http://bioconductor.org/packages/structToolbox), including documentation and vignettes. Source code is available and maintained at https://github.com/computational-metabolomics.

Metabolic engineering against the arginine microenvironment enhances CAR-T cell proliferation and therapeutic activity.

Hematological and solid cancers catabolize the semiessential amino acid arginine to drive cell proliferation. However, the resulting low arginine microenvironment also impairs chimeric antigen receptor T cells (CAR-T) cell proliferation, limiting their efficacy in clinical trials against hematological and solid malignancies. T cells are susceptible to the low arginine microenvironment because of the low expression of the arginine resynthesis enzymes argininosuccinate synthase (ASS) and ornithine transcarbamylase (OTC). We demonstrate that T cells can be reengineered to express functional ASS or OTC enzymes, in concert with different chimeric antigen receptors. Enzyme modifications increase CAR-T cell proliferation, with no loss of CAR cytotoxicity or increased exhaustion. In vivo, enzyme-modified CAR-T cells lead to enhanced clearance of leukemia or solid tumor burden, providing the first metabolic modification to enhance CAR-T cell therapies.

Multi-Omics Analysis of Diabetic Heart Disease in the db/db Model Reveals Potential Targets for Treatment by a Longevity-Associated Gene.

Characterisation of animal models of diabetic cardiomyopathy may help unravel new molecular targets for therapy. Long-living individuals are protected from the adverse influence of diabetes on the heart, and the transfer of a longevity-associated variant (LAV) of the human BPIFB4 gene protects cardiac function in the db/db mouse model. This study aimed to determine the effect of LAV-BPIFB4 therapy on the metabolic phenotype (ultra-high-performance liquid chromatography-mass spectrometry, UHPLC-MS) and cardiac transcriptome (next-generation RNAseq) in db/db mice. UHPLC-MS showed that 493 cardiac metabolites were differentially modulated in diabetic compared with non-diabetic mice, mainly related to lipid metabolism. Moreover, only 3 out of 63 metabolites influenced by LAV-BPIFB4 therapy in diabetic hearts showed a reversion from the diabetic towards the non-diabetic phenotype. RNAseq showed 60 genes were differentially expressed in hearts of diabetic and non-diabetic mice. The contrast between LAV-BPIFB4- and vehicle-treated diabetic hearts revealed eight genes differentially expressed, mainly associated with mitochondrial and metabolic function. Bioinformatic analysis indicated that LAV-BPIFB4 re-programmed the heart transcriptome and metabolome rather than reverting it to a non-diabetic phenotype. Beside illustrating global metabolic and expressional changes in diabetic heart, our findings pinpoint subtle changes in mitochondrial-related proteins and lipid metabolism that could contribute to LAV-BPIFB4-induced cardio-protection in a murine model of type-2 diabetes.

Characterization of colorectal mucus using infrared spectroscopy: a potential target for bowel cancer screening and diagnosis.

Biological materials presenting early signs of cancer would be beneficial for cancer screening/diagnosis. In this respect, the suitability of potentially exploiting mucus in colorectal cancer was tested using infrared spectroscopy in combination with statistical modeling. Twenty-six paraffinized colon tissue biopsy sections containing mucus regions from 20 individuals (10 normal and 16 cancerous) were measured using mid-infrared spectroscopic imaging. A digital de-paraffinization, followed by cluster analysis driven digital color-coded multi-staining segmented the infrared images into various histopathological features such as epithelium, connective tissue, stroma, and mucus regions within the tissue sections. Principal component analysis followed by supervised linear discriminant analysis was carried out on pure mucus and epithelial spectra from normal and cancerous regions of the tissue. For the mucus-based classification, a sensitivity of 96%, a specificity of 83%, and an area under the curve performance of 95% was obtained. For the epithelial tissue-based classification, a sensitivity of 72%, a specificity of 88%, and an area under the curve performance of 89% was obtained. The mucus spectral profiles further showed contributions indicative of glycans including that of sialic acid changes between these pathology groups. The study demonstrates that infrared spectroscopic analysis of mucus discriminates colorectal cancers with high sensitivity. This concept could be exploited to develop screening/diagnostic approaches complementary to histopathology.

Multi-centre Randomised Controlled Trial of a Smartphone-based Event Recorder Alongside Standard Care Versus Standard Care for Patients Presenting to the Emergency Department with Palpitations and Pre-syncope: The IPED (Investigation of Palpitations in the ED) study.

BACKGROUND: Patients with palpitations and pre-syncope commonly present to Emergency Departments (EDs) but underlying rhythm diagnosis is often not possible during the initial presentation. This trial compares the symptomatic rhythm detection rate of a smartphone-based event recorder (AliveCor) alongside standard care versus standard care alone, for participants presenting to the ED with palpitations and pre-syncope with no obvious cause evident at initial consultation. METHODS: Multi-centre open label, randomised controlled trial. Participants ≥ 16 years old presenting to 10 UK hospital EDs were included. Participants were randomised to either (a) intervention group; standard care plus the use of a smartphone-based event recorder or (b) control group; standard care alone. Primary endpoint was symptomatic rhythm detection rate at 90 days. Trial registration number NCT02783898 (ClinicalTrials.gov). FINDINGS: Two hundred forty-three participants were recruited over an 18-month period. A symptomatic rhythm was detected at 90 days in 69 (n = 124; 55.6%; 95% CI 46.9-64.4%) participants in the intervention group versus 11 (n = 116; 9.5%; 95% CI 4.2-14.8) in the control group (RR 5.9, 95% CI 3.3-10.5; p < 0.0001). Mean time to symptomatic rhythm detection in the intervention group was 9.5 days (SD 16.1, range 0-83) versus 42.9 days (SD 16.0, range 12-66; p < 0.0001) in the control group. The commonest symptomatic rhythms detected were sinus rhythm, sinus tachycardia and ectopic beats. A symptomatic cardiac arrhythmia was detected at 90 days in 11 (n = 124; 8.9%; 95% CI 3.9-13.9%) participants in the intervention group versus 1 (n = 116; 0.9%; 95% CI 0.0-2.5%) in the control group (RR 10.3, 95% CI 1.3-78.5; p = 0.006). INTERPRETATION: Use of a smartphone-based event recorder increased the number of patients in whom an ECG was captured during symptoms over five-fold to more than 55% at 90 days. This safe, non-invasive and easy to use device should be considered part of on-going care to all patients presenting acutely with unexplained palpitations or pre-syncope. FUNDING: This study was funded by research awards from Chest, Heart and Stroke Scotland (CHSS) and British Heart Foundation (BHF) which included funding for purchasing the devices. MR was supported by an NHS Research Scotland Career Researcher Clinician award.