Postmortem metabolomics: influence of time since death on the level of endogenous compounds in human femoral blood. Necessary to be considered in metabolome study planning?

INTRODUCTION: The (un)targeted analysis of endogenous compounds has gained interest in the field of forensic postmortem investigations. The blood metabolome is influenced by many factors, and postmortem specimens are considered particularly challenging due to unpredictable decomposition processes. OBJECTIVES: This study aimed to systematically investigate the influence of the time since death on endogenous compounds and its relevance in designing postmortem metabolome studies. METHODS: Femoral blood samples of 427 authentic postmortem cases, were collected at two time points after death (854 samples in total; t1: admission to the institute, 1.3-290 h; t2: autopsy, 11-478 h; median ∆t = 71 h). All samples were analyzed using an untargeted metabolome approach, and peak areas were determined for 38 compounds (acylcarnitines, amino acids, phospholipids, and others). Differences between t2 and t1 were assessed by Wilcoxon signed-ranked test (p < 0.05). Moreover, all samples (n = 854) were binned into time groups (6 h, 12 h, or 24 h intervals) and compared by Kruskal-Wallis/Dunn's multiple comparison tests (p < 0.05 each) to investigate the effect of the estimated time since death. RESULTS: Except for serine, threonine, and PC 34:1, all tested analytes revealed statistically significant changes between t1 and t2 (highest median increase 166%). Unpaired analysis of all 854 blood samples in-between groups indicated similar results. Significant differences were typically observed between blood samples collected within the first and later than 48 h after death, respectively. CONCLUSIONS: To improve the consistency of comprehensive data evaluation in postmortem metabolome studies, it seems advisable to only include specimens collected within the first 2 days after death.

Factors influencing the successful implementation of a novel digital health application to streamline multidisciplinary communication across multiple organisations for emergency care.

RATIONALE: Delivering optimal patient health care requires interdisciplinary clinician communication. A single communication tool across multiple pre-hospital and hospital settings, and between hospital departments is a novel solution to current systems. Fit-for-purpose, secure smartphone applications allow clinical information to be shared quickly between health providers. Little is known as to what underpins their successful implementation in an emergency care context. AIMS: To identify (a) whether implementing a single, digital health communication application across multiple health care organisations and hospital departments is feasible; (b) the barriers and facilitators to implementation; and (c) which factors are associated with clinicians' intentions to use the technology. METHODS: We used a multimethod design, evaluating the implementation of a secure, digital communication application (Pulsara™). The technology was trialled in two Australian regional hospitals and 25 Ambulance Victoria branches (AV). Post-training, clinicians involved in treating patients with suspected stroke or cardiac events were administered surveys measuring perceived organisational readiness (Organisational Readiness for Implementing Change), clinicians' intentions (Unified Theory of Acceptance and Use of Technology) and internal motivations (Self-Determination Theory) to use Pulsara™, and the perceived benefits and barriers of use. Quantitative data were descriptively summarised with multivariable associations between factors and intentions to use Pulsara™ examined with linear regression. Qualitative data responses were subjected to directed content analysis (two coders). RESULTS: Participants were paramedics (n = 82, median 44 years) or hospital-based clinicians (n = 90, median 37 years), with organisations perceived to be similarly ready. Regression results (F(11, 136) = 21.28, p = <0.001, Adj R2 = 0.60) indicated Habit, Effort Expectancy, Perceived Organisational Readiness, Performance Expectancy and Organisation membership (AV) as predictors of intending to use Pulsara™. Themes relating to benefits (95% coder agreement) included improved communication, procedural efficiencies and faster patient care. Barriers (92% coder agreement) included network accessibility and remembering passwords. PulsaraTM was initiated 562 times. CONCLUSION: Implementing multiorganisational, digital health communication applications is feasible, and facilitated when organisations are change-ready for an easy-to-use, effective solution. Developing habitual use is key, supported through implementation strategies (e.g., hands-on training). Benefits should be emphasised (e.g., during education sessions), including streamlining communication and patient flow, and barriers addressed (e.g., identify champions and local technical support) at project commencement.

Overcoming the clinical challenges of traditional ayahuasca: a first-in-human trial exploring novel routes of administration of N,N-Dimethyltryptamine and harmine.

Recently, the Amazonian plant medicine "ayahuasca"-containing the psychedelic compound N,N-dimethyltryptamine (DMT) and numerous ß-carboline alkaloids, such as harmine-has been suggested to exhibit beneficial effects in patients with affective and other mental health disorders. Although ayahuasca ingestion is considered safe, its pharmacokinetics/pharmacodynamics and tolerability profile pose some challenges and may limit the clinical applicability in vulnerable patient populations. While overdosing and the admixture of intolerable plant constituents may explain some of the common adverse reactions, the peroral route of administration may represent another relevant source of gastro-intestinal intolerabilities and unpredictable pharmacokinetics across users. To overcome these challenges, the present work aimed at creating ayahuasca-analogue formulations with improved pharmacokinetics and tolerability profiles. To this end, we developed peroral formulas and compared them with parenteral formulas specifically designed to circumvent the gastro-intestinal tract. In more detail, peroral administration of a capsule (containing purified DMT and harmine) was tested against a combined administration of an oromucosal harmine tablet and an intranasal DMT spray at two dose levels in an open-label within-subject study in 10 healthy male subjects. Pharmacokinetic and pharmacodynamic profiles were assessed by means of continuous blood sampling, vital sign monitoring, and psychometric assessments. Common side effects induced by traditional herbal ayahuasca such as nausea, vomiting, and diarrhea were significantly attenuated by our DMT/harmine formulations. While all preparations were well tolerated, the combined buccal/intranasal administration of harmine and DMT yielded substantially improved pharmacokinetic profiles, indicated by significantly reduced variations in systemic exposure. In conclusion, the combined buccal/intranasal administration of harmine and DMT is an innovative approach that may pave the way towards a safe, rapid-acting, and patient-oriented administration of DMT/harmine for the treatment of affective disorders. Clinical Trial Registration: clinicaltrials.gov, identifier NCT04716335.

Untargeted Metabolomics Profiling for Determination of the Time since Deposition of Biofluids in a Forensic Context: A Proof-of-Concept for Urine, Saliva, and Semen in Addition to Blood.

In a criminal trial, the reconstruction of a crime is one of the fundamental steps of the prosecution process. Common questions, such as what happened, where and how it happened, and who made it happen, need to be solved. Biological evidence at crime scenes can be crucial in the determination of these fundamental questions. One of the more challenging riddles to solve is the when? A trace left at a crime scene can prove a person's presence at the crime scene. Knowledge about when it was deposited there, the time since deposition (TsD), would allow linking the person in space and time to the site. This could fortify allegations against a suspect or discharge accusations if proven to be outside of the temporal boundaries where a suspected crime had occurred. Determining the TsD has yet to become routine forensic casework, despite recent research efforts, especially for blood traces. However, next to blood, other biological traces are also commonly encountered in crime scenes. We here present a study to profile the metabolomes of artificially aged dried body fluid spots of blood, semen, saliva, and urine over 4 weeks by liquid chromatography high-resolution mass spectrometry and data-dependent acquisition. All four body fluids (BFs) exhibited diverse time-dependent changes, and a large number of molecular features (MF) were associated with TsD. Still, significant differences between the BFs were observed, limiting universal interpretability independent of the BF and facilitating a need to further study time-dependent changes of different BFs individually toward the goal of TsD estimation.

Urinary concentrations of GHB and its novel amino acid and carnitine conjugates following controlled GHB administration to humans.

Gamma-hydroxybutyrate (GHB) remains a challenging clinical/forensic toxicology drug. Its rapid elimination to endogenous levels mainly causes this. Especially in drug-facilitated sexual assaults, sample collection often occurs later than the detection window for GHB. We aimed to investigate new GHB conjugates with amino acids (AA), fatty acids, and its organic acid metabolites for their suitability as ingestion/application markers in urine following controlled GHB administration to humans. We used LC-MS/MS for validated quantification of human urine samples collected within two randomized, double-blinded, placebo-controlled crossover studies (GHB 50 mg/kg, 79 participants) at approximately 4.5, 8, 11, and 28 h after intake. We found significant differences (placebo vs. GHB) for all but two analytes at 4.5 h. Eleven hours post GHB administration, GHB, GHB-AAs, 3,4-dihydroxybutyric acid, and glycolic acid still showed significantly higher concentrations; at 28 h only GHB-glycine. Three different discrimination strategies were evaluated: (a) GHB-glycine cut-off concentration (1 µg/mL), (b) metabolite ratios of GHB-glycine/GHB (2.5), and (c) elevation threshold between two urine samples (> 5). Sensitivities were 0.1, 0.3, or 0.5, respectively. Only GHB-glycine showed prolonged detection over GHB, mainly when compared to a second time- and subject-matched urine sample (strategy c).

Determination of the Time since Deposition of blood-traces in a forensic context: Application of untargeted LC-HR-MS/MS metabolomics profiling.

Being able to attest when a bloodstain was deposited at a crime scene can be invaluable to a prosecution process, and methods to provide that information have long been desired. Determining the Time since Deposition (TsD) of a trace would allow placing a subject both in space and time to the crime scene-or prove that a trace left by that person was unrelated to it because it was deposited before or after the time the crime had occurred. To this day, no method for TsD determination has made its way into routine forensic casework, mainly because of the numerous challenges that await when trying to understand and account for all the influencing and confounding factors that affect the aging process (such as, e.g., temperature, UV-light exposure, or humidity). Here, we present an untargeted metabolomics-based study using liquid chromatography high-resolution mass spectrometry (LC-HR-MS) and data-dependent acquisition to analyze blood samples aged under two distinctly different storage conditions over 48 weeks. Global differences in age- and storage-dependent changes in blood metabolomes were described, and TsD-classification strategies based on qualitative and quantitative assessment of molecular features (MFs) have been proposed. Based on the selected criteria to best predict the TsD, the dipeptide Phenylalanylalanine (PheAla) can be considered as a promising candidate for TsD prediction. In essence, changes in the blood metabolome dynamics showed a strong association with increasing TsD, but significant differences depending on storage conditioning were observed, facilitating the need to study further the influence of individual influencing factors on TsD determination.

Nocturnal sodium oxybate increases the anterior cingulate cortex magnetic resonance glutamate signal upon awakening.

Clinical guidelines recommend sodium oxybate (SXB; the sodium salt of γ-hydroxybutyrate) for the treatment of disturbed sleep and excessive daytime sleepiness in narcolepsy, yet the underlying mode of action is elusive. In a randomised controlled trial in 20 healthy volunteers, we aimed at establishing neurochemical changes in the anterior cingulate cortex (ACC) following SXB-enhanced sleep. The ACC is a core neural hub regulating vigilance in humans. At 2:30 a.m., we administered in a double-blind cross-over manner an oral dose of 50 mg/kg SXB or placebo, to enhance electroencephalography-defined sleep intensity in the second half of nocturnal sleep (11:00 p.m. to 7:00 a.m.). Upon scheduled awakening, we assessed subjective sleepiness, tiredness and mood and measured two-dimensional, J-resolved, point-resolved magnetic resonance spectroscopy (PRESS) localisation at 3-Tesla field strength. Following brain scanning, we used validated tools to quantify psychomotor vigilance test (PVT) performance and executive functioning. We analysed the data with independent t tests, false discovery rate (FDR) corrected for multiple comparisons. The morning glutamate signal (at 8:30 a.m.) in the ACC was specifically increased after SXB-enhanced sleep in all participants in whom good-quality spectroscopy data were available (n = 16; pFDR < 0.002). Further, global vigilance (10th-90th inter-percentile range on the PVT) was improved (pFDR < 0.04) and median PVT response time was shorter (pFDR < 0.04) compared to placebo. The data indicate that elevated glutamate in the ACC could provide a neurochemical mechanism underlying SXB's pro-vigilant efficacy in disorders of hypersomnolence.

Metabolomics-based Sleepiness Markers for Risk Prevention and Traffic Safety (ME-SMART): a monocentric, controlled, randomized, crossover trial.

BACKGROUND: Too little sleep and the consequences thereof are a heavy burden in modern societies. In contrast to alcohol or illicit drug use, there are no quick roadside or workplace tests for objective biomarkers for sleepiness. We hypothesize that changes in physiological functions (such as sleep-wake regulation) are reflected in changes of endogenous metabolism and should therefore be detectable as a change in metabolic profiles. This study will allow for creating a reliable and objective panel of candidate biomarkers being indicative for sleepiness and its behavioral outcomes. METHODS: This is a monocentric, controlled, randomized, crossover, clinical study to detect potential biomarkers. Each of the anticipated 24 participants will be allocated in randomized order to each of the three study arms (control, sleep restriction, and sleep deprivation). These only differ in the amount of hours slept per night. In the control condition, participants will adhere to a 16/8 h wake/sleep regime. In both sleep restriction and sleep deprivation conditions, participants will accumulate a total sleep deficit of 8 h, achieved by different wake/sleep regimes that simulate real-life scenarios. The primary outcome is changes in the metabolic profile (i.e., metabolome) in oral fluid. Secondary outcome measures will include driving performance, psychomotor vigilance test, d2 Test of Attention, visual attention test, subjective (situational) sleepiness, electroencephalographic changes, behavioral markers of sleepiness, changes in metabolite concentrations in exhaled breath and finger sweat, and correlation of metabolic changes among biological matrices. DISCUSSION: This is the first trial of its kind that investigates complete metabolic profiles combined with performance monitoring in humans over a multi-day period involving different sleep-wake schedules. Hereby, we aim to establish a candidate biomarker panel being indicative for sleepiness and its behavioral outcomes. To date, there are no robust and easily accessible biomarkers for the detection of sleepiness, even though the vast damage on society is well known. Thus, our findings will be of high value for many related disciplines. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT05585515, released on 18.10.2022; Swiss National Clinical Trial Portal SNCTP000005089, registered on 12 August 2022.

Do dried blood spots have the potential to support result management processes in routine sports drug testing?-Part 3: LC-MS/MS-based peptide analysis for dried blood spot sampling time point estimation.

Along with the recent acknowledgement of the World Anti-Doping Agency to use dried blood spot (DBS) samples for routine doping control purposes, there have been propositions to use DBS as a matrix that allows regular proactive remotely supervised self-sampling, providing potential longitudinal monitoring of an athlete's exposure to doping agents. However, several organizational aspects have to be considered before implementation, such as the verification of the sample collections time point. Based on a previous untargeted proteomics workflow utilizing liquid chromatography-high-resolution mass spectrometry (LC-HRMS) to identify protein/peptide markers to define the time since deposition of a bloodstain, the aim of the current study was to develop a targeted LC-HRMS/MS analytical method for promising peptidic target analytes. A long-term DBS storage experiment was carried out over a 3-month period (sample collection time points: 0, 2, 4, 7, 14, 21, 28, 42, 56, 70, 84 and 91 days) with DBS samples of 10 volunteers for longitudinal investigation of signal abundance changes of targeted peptide sequences at different storage temperatures (room temperature [RT], 4°C and -20°C). Prior to experimental analysis, LC-HRMS/MS method characteristics were successfully assessed, including intraday precision, carryover and sample extract stability. For estimation of DBS sample collection time points, ratios of two peptides that originate from the same protein prior to tryptic digestion were created. Two targeted peptide area ratios were found to significantly increase after being stored at RT for 28 days, representing potential markers for future use in routine doping controls that contribute to advancing complementary avenues in anti-doping.

New gamma-hydroxybutyric acid (GHB) biomarkers: Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of GHB amino acid, carnitine, and fatty acid conjugates in urine.

Gamma-hydroxybutyric acid (GHB) represents an important drug in clinical and forensic toxicology, particularly in the context of drug-facilitated crimes. Analytically, GHB remains a major challenge given its endogenous occurrence and short detection window. Previous studies identified a number of potential interesting novel conjugates of GHB with carnitine, amino acids (AA, glutamate, glycine, and taurine), or fatty acids. As a basis for comprehensive studies on the suitability of these novel biomarkers, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in human urine. Additionally, already known markers 2,4-dihydroxy butyric acid (2,4-DHB), 3,4-DHB, glycolic acid, succinic acid, succinylcarnitine, and GHB glucuronide were included. The method was fully validated according to (inter)national guidelines. Synthetic urine proved suitable as a surrogate matrix for calibration. Matrix effects were observed for all analytes with suppression effects of about 50% at QC LOW, and approximately 20% to 40% at QC HIGH, but with consistent standard deviation of <25% at QC LOW and <15% at QC HIGH, respectively. All analytes showed acceptable intra- and inter-day imprecision of below 20%, except for inter-day variation of GHB taurine and FA conjugates at the lowest QC. Preliminary applicability studies proved the usefulness of the method and pointed towards GHB glycine, followed by other AA conjugates as the most promising candidates to improve GHB detection. FA conjugates were not detected in urine samples yet. The method can be used now for comprehensive sample analysis on (controlled) GHB administration to prove the usefulness of the novel GHB biomarkers.

TiO2 nanoparticles abrogate the protective effect of the Crohn's disease-associated variation within the PTPN22 gene locus.

OBJECTIVE: Inflammatory bowel disease (IBD) is a multifactorial condition driven by genetic and environmental risk factors. A genetic variation in the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene has been associated with autoimmune disorders while protecting from the IBD subtype Crohn's disease. Mice expressing the murine orthologous PTPN22-R619W variant are protected from intestinal inflammation in the model of acute dextran sodium sulfate (DSS)-induced colitis. We previously identified food-grade titanium dioxide (TiO2, E171) as a neglected IBD risk factor. Here, we investigate the interplay of the PTPN22 variant and TiO2-mediated effects during IBD pathogenesis. DESIGN: Acute DSS colitis was induced in wild-type and PTPN22 variant mice (PTPN22-R619W) and animals were treated with TiO2 nanoparticles during colitis induction. Disease-triggering mechanisms were investigated using bulk and single-cell RNA sequencing. RESULTS: In mice, administration of TiO2 nanoparticles abrogated the protective effect of the variant, rendering PTPN22-R619W mice susceptible to DSS colitis. In early disease, cytotoxic CD8+ T-cells were found to be reduced in the lamina propria of PTPN22-R619W mice, an effect reversed by TiO2 administration. Normalisation of T-cell populations correlated with increased Ifng expression and, at a later stage of disease, the promoted prevalence of proinflammatory macrophages that triggered severe intestinal inflammation. CONCLUSION: Our findings indicate that the consumption of TiO2 nanoparticles might have adverse effects on the gastrointestinal health of individuals carrying the PTPN22 variant. This demonstrates that environmental factors interact with genetic risk variants and can reverse a protective mechanism into a disease-promoting effect.

Single sample preparation for the simultaneous extraction of drugs, pharmaceuticals, cannabinoids and endogenous steroids in hair.

Recently, we published a multi-analyte method for the simultaneous analysis of 116 drugs and pharmaceuticals including different substance groups like opioids, stimulants, benzodiazepines, z-drugs, antidepressants and neuroleptics based on a single sample workup followed by a single analytical measurement with LC-MS/MS. However, in some cases, additional analysis of further substance groups, such as cannabinoids and endogenous steroids, is required, which are analyzed in our laboratory using separate sample preparation and separate analytical methods. The goal of this study was to use the knowledge from the different sample preparations and combine them into a single sample preparation and extraction workflow for the simultaneous extraction of drugs, pharmaceuticals, cannabinoids, and endogenous steroids to be analyzed with the appropriate analytical methods. A partial validation of selected parameters such as selectivity, linearity, limit of quantification (LOQ), accuracy, precision and robustness for the different analytical methods was carried out and revalidated. In addition, comparative measurements of quality controls and authentic pools were performed and statistically evaluated using the unpaired t-test or the non-parametric Mann-Whitney test. The results using the newly established sample preparation and extraction were in good agreement with the original data. In conclusion, the newly established sample preparation is suitable for the combined extraction of drugs, pharmaceuticals, cannabinoids and endogenous steroids, and gives reliable results for quantification of various substances.

The renin-angiotensin-aldosterone system, neurohumoral axis and cardiovascular mortality in LURIC.

Although neurohormones and Renin-Angiotensin-Aldosterone-System (RAAS) components are important predictors of cardiovascular mortality (CVM), their importance for predicting outcomes in patients with/without RAAS-blockers and different degrees of arterial stiffness is less understood. We therefore analyzed long-term data from the Ludwigshafen Risk and Cardiovascular Health (LURIC) study in 3316 patients subdivided according to pulse pressure (PP) and RAAS-blocker use. Patients on RAAS-inhibition had higher renin and noradrenaline, lower aldosterone and aldosterone/renin quotient (ARQ). Renin and noradrenaline significantly predicted CVM in patients without RAAS-blocker (HR = 1.17, 1.15) and in patients receiving angiotensin-converting-enzyme (ACE) inhibitors (HR = 1.17, 1.29), whereas aldosterone predicted CVM only in patients receiving ACE-inhibitors (HR = 1.13). CVM was predicted independently from PP by renin, noradrenaline and angiotensin II. Independently from RAAS inhibition renin decreased and ARQs increased with rising PP. Furthermore, noradrenaline increased with PP, but only without ACE-inhibition. The HR for CVM in the ACE-inhibitor group were 1.29, 1.28, 1.29 for renin in the first, second and third PP quartiles and 1.22, and 1.19 for aldosterone in the second and fourth quartile. Furthermore, we showed that noradrenaline predicts CVM in all PP quartiles in patients with ACE-inhibition. In the RAAS-blocker-free group, the HR for renin for CVM were 1.36 and 1.18 in the third and fourth PP quartiles, but neither aldosterone nor noradrenaline were predictive for CVM within the PP quartiles. Renin and noradrenaline are strong predictors of CVM regardless of RAAS blockade, whereas aldosterone is predictive only in the ACE-inhibitor group. Catecholamines but not renin are associated with rising PP.

Inhaled mosliciguat (BAY 1237592): targeting pulmonary vasculature via activating apo-sGC.

BACKGROUND: Oxidative stress associated with severe cardiopulmonary diseases leads to impairment in the nitric oxide/soluble guanylate cyclase signaling pathway, shifting native soluble guanylate cyclase toward heme-free apo-soluble guanylate cyclase. Here we describe a new inhaled soluble guanylate cyclase activator to target apo-soluble guanylate cyclase and outline its therapeutic potential. METHODS: We aimed to generate a novel soluble guanylate cyclase activator, specifically designed for local inhaled application in the lung. We report the discovery and in vitro and in vivo characterization of the soluble guanylate cyclase activator mosliciguat (BAY 1237592). RESULTS: Mosliciguat specifically activates apo-soluble guanylate cyclase leading to improved cardiopulmonary circulation. Lung-selective effects, e.g., reduced pulmonary artery pressure without reduced systemic artery pressure, were seen after inhaled but not after intravenous administration in a thromboxane-induced pulmonary hypertension minipig model. These effects were observed over a broad dose range with a long duration of action and were further enhanced under experimental oxidative stress conditions. In a unilateral broncho-occlusion minipig model, inhaled mosliciguat decreased pulmonary arterial pressure without ventilation/perfusion mismatch. With respect to airway resistance, mosliciguat showed additional beneficial bronchodilatory effects in an acetylcholine-induced rat model. CONCLUSION: Inhaled mosliciguat may overcome treatment limitations in patients with pulmonary hypertension by improving pulmonary circulation and airway resistance without systemic exposure or ventilation/perfusion mismatch. Mosliciguat has the potential to become a new therapeutic paradigm, exhibiting a unique mode of action and route of application, and is currently under clinical development in phase Ib for pulmonary hypertension.

Endocannabinoid and steroid analysis in infant and adult nails by LC-MS/MS.

A common method to quantify chronic stress is the analysis of stress markers in keratinized matrices such as hair or nail. In this study, we aimed to validate a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the combined quantification of steroid hormones and endocannabinoids (eCBs) in the keratinized matrix nail. Furthermore, we aimed to investigate the suitability of the nail matrix for the detection of these stress markers in a pilot study. An LC-MS/MS method was used for the simultaneous identification and quantification of four eCBs (2-arachidonoylglycerol (2-AG), anandamide (AEA), oleoylethanolamide (OEA), palmitoylethanolamide (PEA)) and five steroid hormones (cortisol, cortisone, androstenedione, progesterone, testosterone) in human nails using a surrogate analyte method for each analyte. The method was validated in terms of selectivity, response factor, linearity, limit of quantification (LOQ), precision, accuracy, matrix effect, recovery, robustness, and autosampler stability. Nail samples were extracted for 1 h with methanol following a clean-up with a fully automated supported liquid extraction (SLE). The influence of nail weight on the quantification was investigated by using 0.5-20 mg of nail sample. As a proof of concept, nail samples (N = 57) were analyzed from a cohort representing newborns (1 month old), children (between 1 and 10 years), and adults (up to 43 years). It could be shown that the established workflow using a 1 hour extraction and clean-up by SLE was very robust and resulted in a short sample preparation time. The LC-MS/MS method was successfully validated. Matrix effects with ion enhancement occurred mainly for 2-AG. Sample weights below 5 mg showed variations in quantification for some analytes. Certain analytes such as PEA and progesterone could be accurately quantified at a sample weight lower than 5 mg. This is the first study where steroids and eCBs could be simultaneously detected and quantified in infant and adult nails. These results show that nails may serve as an alternative keratinized matrix (compared to hair) for the retrospective monitoring of cumulative eCB and steroid hormone levels. The combined assessment of eCBs and steroids from nails could provide a new approach to gain new insights into stress exposure in newborns and adults.

Determination of the Time since Deposition of Blood Traces Utilizing a Liquid Chromatography-Mass Spectrometry-Based Proteomics Approach.

Knowledge about when a bloodstain was deposited at a crime scene can be of critical value in forensic investigation. A donor of a genetically identified bloodstain could be linked to a suspected time frame and the crime scene itself. Determination of the time since deposition (TsD) has been extensively studied before but has yet to reach maturity. We therefore conducted a proof-of-principle study to study time- and storage-dependent changes of the proteomes of dried blood stains. A bottom-up proteomics approach was employed, and high-resolution liquid-chromatography-mass-spectrometry (HR-LC-MS) and data-independent acquisition (DIA) were used to analyze samples aged over a 2 month period and two different storage conditions. In multivariate analysis, samples showed distinct clustering according to their TsD in both principal component analysis (PCA) and in partial least square discriminant analysis (PLS DA). The storage condition alters sample aging and yields different separation-driving peptides in hierarchical clustering and in TsD marker peptide selection. Certain peptides and amino acid modifications were identified and further assessed for their applicability in assessing passed TsD. A prediction model based on data resampling (Jackknife) was applied, and prediction values for selected peptide ratios were created. Depending on storage conditions and actual sample age, mean prediction performances ranges in between 70 and 130% for the majority of peptides and time points. This places this study as a first in investigating LC-MS based bottom-up proteomics approaches for TsD determination.

Real-world, feasibility study to investigate the use of a multidisciplinary app (Pulsara) to improve prehospital communication and timelines for acute stroke/STEMI care.

OBJECTIVES: To determine if a digital communication app improves care timelines for patients with suspected acute stroke/ST-elevation myocardial infarction (STEMI). DESIGN: Real-world feasibility study, quasi-experimental design. SETTING: Prehospital (25 Ambulance Victoria branches) and within-hospital (2 hospitals) in regional Victoria, Australia. PARTICIPANTS: Paramedics or emergency department (ED) clinicians identified patients with suspected acute stroke (onset <4.5 hours; n=604) or STEMI (n=247). INTERVENTION: The Pulsara communication app provides secure, two-way, real-time communication. Assessment and treatment times were recorded for 12 months (May 2017-April 2018), with timelines compared between 'Pulsara initiated' (Pulsara) and 'not initiated' (no Pulsara). PRIMARY OUTCOME MEASURE: Door-to-treatment (needle for stroke, balloon for STEMI) Secondary outcome measures: ambulance and hospital processes. RESULTS: Stroke (no Pulsara n=215, Pulsara n=389) and STEMI (no Pulsara n=76, Pulsara n=171) groups were of similar age and sex (stroke: 76 vs 75 years; both groups 50% male; STEMI: 66 vs 63 years; 68% and 72% male). When Pulsara was used, patients were off ambulance stretcher faster for stroke (11(7, 17) vs 19(11, 29); p=0.0001) and STEMI (14(7, 23) vs 19(10, 32); p=0.0014). ED door-to-first medical review was faster (6(2, 14) vs 23(8, 67); p=0.0001) for stroke but only by 1 min for STEMI (3 (0, 7) vs 4 (0, 14); p=0.25). Door-to-CT times were 44 min faster (27(18, 44) vs 71(43, 147); p=0.0001) for stroke, and percutaneous intervention door-to-balloon times improved by 17 min, but non-significant (56 (34, 88) vs 73 (49, 110); p=0.41) for STEMI. There were improvements in the proportions of patients treated within 60 min for stroke (12%-26%, p=0.15) and 90 min for STEMI (50%-78%, p=0.20). CONCLUSIONS: In this Australian-first study, uptake of the digital communication app was strong, patient-centred care timelines improved, although door-to-treatment times remained similar.

Towards a New Qualitative Screening Assay for Synthetic Cannabinoids Using Metabolomics and Machine Learning.

BACKGROUND: Synthetic cannabinoids (SCs) are steadily emerging on the drug market. To remain competitive in clinical or forensic toxicology, new screening strategies including high-resolution mass spectrometry (HRMS) are required. Machine learning algorithms can detect and learn chemical signatures in complex datasets and use them as a proxy to predict new samples. We propose a new screening tool based on a SC-specific change of the metabolome and a machine learning algorithm. METHODS: Authentic human urine samples (n = 474), positive or negative for SCs, were used. These samples were measured with an untargeted metabolomics liquid chromatography (LC)-quadrupole time-of-flight-HRMS method. Progenesis QI software was used to preprocess the raw data. Following feature engineering, a random forest (RF) model was optimized in R using a 10-fold cross-validation method and a training set (n = 369). The performance of the model was assessed with a test (n = 50) and a verification (n = 55) set. RESULTS: During RF optimization, 49 features, 200 trees, and 7 variables at each branching node were determined as most predictive. The optimized model accuracy, clinical sensitivity, clinical specificity, positive predictive value, and negative predictive value were 88.1%, 83.0%, 92.7%, 91.3%, and 85.6%, respectively. The test set was predicted with an accuracy of 88.0%, and the verification set provided evidence that the model was able to detect cannabinoid-specific changes in the metabolome. CONCLUSIONS: An RF approach combined with metabolomics enables a novel screening strategy for responding effectively to the challenge of new SCs. Biomarkers identified by this approach may also be integrated in routine screening methods.

Diabetes in ischaemic stroke in a regional Australian hospital: uncharted territory.

BACKGROUND: Stroke and diabetes mellitus (DM) are significant interrelated healthcare issues but there is a dearth of data on the prevalence of DM among Australia's regional stroke population. AIMS: We aimed to determine the prevalence of DM in stroke patients at a large regional centre, including subanalyses on stroke subtypes, glycaemic control and renal function in ischaemic stroke (IS). METHODS: We conducted a retrospective analysis of all patients (n = 323) with IS or transient ischaemic attack (TIA) admitted to Ballarat Base Hospital from January 2015 to December 2016. Demographic data, cardiovascular risk factors, aetiology/territory of IS, pre-morbid DM status, indicators of glycaemic control and renal impairment were recorded. RESULTS: DM was present in 28.5% of IS and TIA patients, including 4% being newly diagnosed. Among diabetic IS patients, 45.3% had poor glycaemic control (HbA1c ≥7.0%) while 16% had moderate to severe renal impairment (estimated glomerular filtration rate of <30). The majority of IS were partial anterior circulation stroke (53.4%) and cardioembolism was the commonest mechanism (43.5%). We found no significant association between DM and a specific stroke location or mechanism. CONCLUSIONS: Almost one-third of IS/TIA patients had DM, with a significant proportion showing poor glycaemic control. The DM prevalence in our cohort was comparable with reported rates from other developed countries. Although we found no association between DM and a particular stroke type or mechanism, it is likely a reflection of our cohort size. Our study demonstrated that DM, as a significant risk factor in IS, warrants early detection and better management strategies.

Alterations of Stress-Related Glucocorticoids and Endocannabinoids in Hair of Chronic Cocaine Users.

BACKGROUND: Previous research in animals and humans has demonstrated a potential role of stress regulatory systems, such as the hypothalamic-pituitary-adrenal (HPA) axis and the endocannabinoid (eCB) system, in the development of substance use disorders. We thus investigated alterations of HPA and eCB markers in individuals with chronic cocaine use disorder by using an advanced hair analysis technique. METHODS: We compared hair concentrations of glucocorticoids (cortisone, cortisol) and the eCBs 2-arachidonylglycerol, anandamide (AEA), oleoylethanolamide (OEA), and palmitoylethanolamide (PEA) between 48 recreational cocaine users (RCU), 25 dependent cocaine users (DCU), and 67 stimulant-naïve controls. Self-reported substance use and hair concentrations of substances were also assessed. RESULTS: Significantly higher concentrations of hair cortisone were found in RCU and DCU compared with controls. Hair concentrations of OEA and PEA were significantly lower in DCU compared with RCU and controls. Additionally, within cocaine users, elevated cocaine hair concentration was a significant predictor for increased glucocorticoid and decreased OEA hair levels. Moreover, higher 3,4-methyl​enedioxymethamphetamine hair concentration was correlated with elevated cortisone and AEA, OEA, and PEA levels in hair within cocaine users, whereas more self-reported cannabis use was associated with lower eCBs levels in hair across the total sample. CONCLUSION: Our findings support the hypothesis that the HPA axis and eCB system might be important regulators for substance use disorders. The mechanistic understanding of changes in glucocorticoid and eCB levels in future research might be a promising pharmacological target to reduce stress-induced craving and relapse specifically in cocaine use disorder.