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Fingermark patterns are one of the oldest means of biometric identification. During this last decade, the molecules that constitute the fingermark residue have gained interest among the forensic research community to gain additional intelligence regarding its donor profile including its gender, age, lifestyle or even its pathological state. In this work, the molecular composition of fingermarks have been studied to monitor the variability between donors and to explore its capacity to differentiate individuals using supervised multi-class classification models. Over one year, fingermarks from thirteen donors have been analysed by Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Imaging (n = 716) and mined by different machine learning approaches. We demonstrate the potential of the fingermark chemical composition to help differentiating individuals with an accuracy between 80% and 96% depending on the period of sample collection for each donor and size of the pool of donors. It would be premature at this stage to transpose the results of this research to real cases, however the conclusions of this study can provide a better understanding of the variations of the chemical composition of the fingermark residue in between individuals over long periods and help clarifying the notion of donorship.

Microsampling techniques became more popular in the last decades, and their use for common analyses such as trace element quantification by inductively coupled plasma mass spectrometry (ICP-MS) has been investigated. We decided to compare two of these techniques (dried blood spots and microtubes) to evaluate their potential for the analysis of 12 trace elements in human whole blood: aluminum (Al), total arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), selenium (Se) and zinc (Zn). Signal contributions from blank filter paper and instability at room temperature for several elements in the dried blood spot samples restrained our enthusiasm for the use of this technique. Conversely, microtube samples presented low background contamination and good stability under different temperature conditions. Therefore, our results demonstrate that the use of microtubes is more suitable than dried blood spots for trace element quantification in human blood, both in research and routine analysis.

Most OECD guidelines for chemical risk assessment include tests performed on animals, raising financial, ethical and scientific concerns. Thus, the development of human-based models for toxicity testing is highly encouraged. Here, we propose an in vitro multi-organ strategy to assess the toxicity of chemicals. Human induced pluripotent stem cells (hiPSCs)-derived models of the brain, blood-brain barrier, kidney, liver and vasculature were generated and exposed to paraquat (PQ), a widely employed herbicide with known toxic effects in kidneys and brain. The models showed differential cytotoxic sensitivity to PQ after acute exposure. TempO-Seq analysis with a set of 3565 probes revealed the deregulation of oxidative stress, unfolded protein response and estrogen receptor-mediated signaling pathways, in line with the existing knowledge on PQ mechanisms of action. The main advantages of this strategy are to assess chemical toxicity on multiple tissues/organs in parallel, exclusively in human cells, eliminating the interspecies bias, allowing a better evaluation of the differential sensitivity of the models representing the diverse organs, and increasing the chance to identify toxic compounds. Furthermore, although we focused on the mechanisms of action of PQ shared by the different models, this strategy would also allow for organ-specific toxicity testing, by including more cell type-specific probes for TempO-Seq analyses. In conclusion, we believe this strategy will participate in the further improvement of chemical risk assessment for human health. ispartof: TOXICOLOGY IN VITRO vol:81 ispartof: location:England status: published

BackgroundAccording to the World Health Organization, road traffic injuries lead to 1.3 million deaths each year and represent the first cause of death for young adults under 30 years old. The use of psychoactive substances, including alcohol, drugs and pharmaceuticals is a well-known risk factor for road traffic injuries. Our study aims to assess the prevalence of substances consumed by drivers in Western Switzerland. Such studies are capital to improve prevention and develop public awareness campaigns.MethodsTo assess the prevalence of psychoactive substances among drivers, roadside controls were performed in collaboration with local polices, using their classical sampling procedures to detect drivers under the influence of drugs or alcohol, over two time periods (P1: 2006-2008, P2: 2017-2020). When impaired-driving was not suspected by the police, minimal-invasive sampling strategies were performed on volunteer drivers, after a road safety survey. A posteriori analyses and statistical interpretation were then performed.ResultsAmongst the 1’605 drivers included in the study, 1’048 volunteers provided an oral fluid sample, whilst 299 provided a dried blood spot sample. The percentage of drivers testing positive to at least one substance which may impair driving ability was stable over time, with a rate of 10.5% of positivity measured over both periods. The proportion of medicinal drugs and drugs of abused showed a slight variation between both periods, with 7.6 % and 6.3 % of prescribed drugs and 3.6 % and 4.9 % of illicit drugs for P1 and P2 respectively. Regarding the consumption of illicit drugs, the highest percentage of positivity was measured in biological fluids of drivers under the age of 35, during nights and week-ends, periods which are considered particularly prone to fatal accidents for this age group. Disturbingly, the road safety survey highlighted that drivers’ perception of the risk of getting positively controlled whilst driving after drug consumption is low (3.3 on a 1-to-10 scale, N=299).ConclusionThe number of positive cases measured in voluntary drivers who passed the preliminary police check demonstrates the importance of systematic biofluid sampling strategies regarding driving under the influence of psychoactive substances. Although the number of fatal road accidents globally decreases overtime, the results of this study reveal the need for both better prevention and deterrent processes that could potentially reduce the risk of fatal road accidents associated with drug consumption.

Chronic inflammation that affects primarily metabolic organs, such as white adipose tissue (WAT), is considered as a major cause of human obesity-associated co-morbidities. However, the molecular mechanisms initiating this inflammation in WAT are poorly understood. By combining transcriptomics, ChIP-seq and modeling approaches, we studied the global early and late responses to a high-fat diet (HFD) in visceral (vWAT) and subcutaneous (scWAT) AT, the first being more prone to obesity-induced inflammation. HFD rapidly triggers proliferation of adipocyte precursors within vWAT. However, concomitant antiadipogenic signals limit vWAT hyperplastic expansion by interfering with the differentiation of proliferating adipocyte precursors. Conversely, in scWAT, residing beige adipocytes lose their oxidizing properties and allow storage of excessive fatty acids. This phase is followed by tissue hyperplastic growth and increased angiogenic signals, which further enable scWAT expansion without generating inflammation. Our data indicate that scWAT and vWAT differential ability to modulate adipocyte number and differentiation in response to obesogenic stimuli has a crucial impact on the different susceptibility to obesity-related inflammation of these adipose tissue depots. Electronic supplementary material The online version of this article (10.1007/s00018-020-03485-z) contains supplementary material, which is available to authorized users.

Cannabis is the most consumed drug of abuse, making it the primary target for identification and quantification in human whole blood regarding forensic and clinical toxicology analyses. Among biological matrices, blood is the reference for toxicological interpretation. A highly sensitive and selective liquid chromatography (LC) hyphenated with high-resolution mass spectrometry (HRMS) was developed for the quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxytetrahydrocannabinol (THC-OH), 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) and cannabidiol (CBD). Those cannabinoids were extracted from 1 mL of whole blood by a simple liquid–liquid extraction (LLE) in acidic conditions. HRMS was performed on an Orbitrap-based instrument using its trapping capabilities and increased selectivity for parallel reaction monitoring (PRM) quantification in positive polarity with a negative polarity switching for THC-OH and THC-COOH. Although selected reaction monitoring (SRM) and PRM-targeted methods have similar performance in terms of linearity, dynamic range, precision and repeatability, Orbitrap-based PRM provides a higher specificity due to the use of high-resolution mode separating background ions from the targeted molecules. The method was fully validated according to guidelines set forth by the “Société Française des Sciences et des Techniques Pharmaceutiques” (SFSTP). Trueness was measured below 107% for all tested concentrations. Repeatability and intermediate precision were found to be lower than 12% while the assay was found to be linear in the concentration range of 0.4–20 ng/mL for THC, THC-OH and CBD and of 2–100 ng/mL for THC-COOH. Recovery (RE) and matrix effect (ME) ranged from 70.6 to 102.5% and from −40 to 6.6%, respectively. The validated method provides an efficient procedure for the simultaneous and rapid quantification of cannabinoids in PRM mode providing an alternative over classical SRM.

These past years, the chemical composition of fingermarks have attracted interest of researchers to meet multiple objectives like the determination of an individual's age, gender or lifestyle or the impact of some fingermark detection processes, to cite a few. These studies have highlighted the need to investigate the consistency of the fingermark composition over time. This research explores the evolution of the secretion residue composition of thirteen donors over one year, focusing on the intravariability. The dual use of Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Imaging (MALDI-MSI) and chemometrics provided valuable data regarding the evolution of composition over time as well as the consistency of presence of hundreds of compounds.

In the last decade, there have been many scientific developments regarding the use of mass spectrometry to analyse the composition of fingermarks. In this context, the development of a dedicated quality control test strip would benefit the forensic community by providing a way to assess the reproducibility of the measures as well as to perform inter-laboratory comparisons. To accomplish this goal, the use of a chemical printer offers the possibility of combining a visual template with artificial fingerprint secretions. The design of the quality control test strip as well as the preliminary assessment of its performance with fingermark detection reagents and matrix-assisted laser desorption-ionisation combined with mass spectrometry imaging (MALDI-MSI) are presented in this paper. The chosen template combines two geometric patterns intended to help assess the chemical analysis (full square) and imaging (lined square) capabilities of the instrument. The artificial secretion is composed of two distinct solutions: artificial sweat and artificial sebum. The printing reproducibility and chemical homogeneity of the quality control test strips were assessed in two ways: (1) using MALDI-MSI, the printed pattern was analysed and the m/z values compared to the reference list based on the artificial secretion composition, and (2) using two common fingermark detection techniques, the printed pattern was processed using an amino acid reagent (ninhydrin) and a lipid stain (Oil Red O). Overall, the results highlight the potential of a printed quality control test strip for the assessment of the quality of fingermark detection techniques as well as the possibility of performing quality monitoring of mass-spectrometry-based techniques over time.

Background and purpose Individualized assessment of cytochrome P450 2D6 (CYP2D6) activity is usually performed through phenotyping following administration of a probe drug to measure the enzyme's activity. In order to avoid any iatrogenic harm (allergic drug reaction, dosing error) related to the probe drug, the development of non-burdensome tools for real-time phenotyping of CYP2D6 could significantly contribute to the expansion of precision medicine. This study focuses on the identification of markers of the CYP2D6 enzyme in human biofluids using a liquid chromatography-high-resolution mass spectrometry-based metabolomic approach. Experimental approach Plasma and urine samples from healthy volunteers were analysed before and after intake of a daily dose of paroxetine 20 mg over seven days. CYP2D6 genotyping and phenotyping, using single oral dose of dextromethorphan 5 mg, were also performed in all participants. Key results In this study, we reported four metabolites of solanidine and two unknown compounds as possible novel CYP2D6 markers. Mean relative intensities of these features were significantly reduced during the inhibition session compared to the control session (n = 37). Semi-quantitative analysis showed that the largest decrease (-85%) was observed for the ion m/z 432.3108 normalized to solanidine (m/z 398.3417). Furthermore, mean relative intensities of these ions were significantly higher in the CYP2D6 normal-ultrarapid metabolizer group (n = 37) compared to the poor metabolizer group (n = 6). Solanidine intensity was more than 15 times higher in CYP2D6-deficient individuals compared to other volunteers. Conclusion and implications The applied untargeted metabolomic strategy identified potential novel markers capable of semi-quantitatively predicting CYP2D6 activity, a promising discovery for personalised medicine.

Background HIV-infected patients display an increased risk of cardiovascular events. HIV and antiretroviral therapy (ART) have both been suggested to increase this risk but their specific role remains unclear. Autoantibodies against apolipoprotein A1 (anti-apoA1 IgG) are also associated with cardiovascular risk. The aim of this study was to analyse anti-apoA1 IgG levels and inflammatory markers as well as the metabolomic profile in a cohort of HIV-free, ART-experienced and naïve HIV-infected patients. Methods 144 participants from a South African cohort were categorised into HIV-free (n=50), HIV-infected/ART-experienced (n=50) and HIV-infected/ART-naïve (n=44) groups. The levels of anti-apoA1 IgG were assessed by homemade ELISA assays. We performed targeted metabolomic analyses using standard operating procedures and measured inflammatory biomarkers using the Meso Scale Discovery® platform. Additionally, we conducted in vitro experiments on human aortic endothelial cells treated with anti-apoA1 IgG and assessed levels of adhesion molecules. Results Inflammatory markers (IFNg, IL-6, IL-10, TNFa, CRP all p While ART treatment significantly reduced IFNg, IL-10, TNFa, VCAM-1, anti-apoA1 IgG levels, no significant differences were observed in other biomarkers between ART-experienced and naïve participants. Treatment of human aortic endothelial cells with anti-apoA1 IgG (40μg/ml) induced a significant increase in these adhesion molecules compared to cells treated with control IgG (40μg/ml). Metabolomic analyses identified 257 endogenous circulating metabolites in plasma. Among these metabolites, we found that in HIV-free and infected patients 14 and 17 metabolites were significantly correlated with anti-apoA1 IgG levels, respectively. Among these 17 metabolites, 2 were positively correlated with the tryptophan pathway; kynurenine (r=0.292, p=0.0069) and 5-hydroxyindolacetate (r=0.316, p=0.0019). Interestingly, kynurenine was not significantly correlated with anti-apoA1 IgG levels in HIV-free patients. Conclusion HIV-infected patients displayed a more proatherogenic profile compared to HIV-free subjects. Anti-apoA1 IgG induced adhesion molecules expression. Metabolomics identified metabolites that were significantly associated with anti-apoA1 IgG levels and inflammatory biomarkers known to underlie atherosclerosis burden. Funding Acknowledgement Type of funding sources: None.

For the quantification of the pineal hormone melatonin and its metabolite, 6-hydroxymelatonin, in human overnight urine, a single accurate method by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. Urine samples were deconjugated using β-glucuronidase/arylsulfatase from Helix pomatia before solid phase extraction (SPE) purification. Chromatographic separation was performed using a reverse phase C18 column with a 7-minute gradient elution. Water was used as matrix to prepare the calibration standards, and deuterated analogues of melatonin and 6-hydroxymelatonin were used as internal standards. This newly developed method was validated in terms of linearity, accuracy, repeatability, intermediate precision, recovery, matrix effect, and stability according to the guidelines of the European Medicines Agency. The method was successfully applied to the analysis of overnight urine samples from 12 healthy volunteers, showing significant correlations of urinary melatonin and 6-hydroxymelatonin excretion rates with age. The urinary 6-hydroxymelatonin to melatonin ratio was also established and will be assessed in further studies as a potential endogenous metric of CYP1A2 activity.

Addiction is a complex brain disease, whose molecular and cellular underpinnings remain largely unknown. Repeated drug use induces long-term plasticity in various brain areas that is considered to potentially trigger further compulsive use. Converging evidence has revealed that memory and addiction share both neural circuitry and molecular mechanisms. While many studies investigated the mesocorticolimbic system, the hippocampus has become a region of interest. Matrix assisted laser desorption and ionization mass spectrometry imaging (MALDI-MSI) presents a unique ability to map the toxicodynamic changes associated to drugs of abuse. In this study, the acute effect of cocaine in rat brain was investigated with or without co-administration of alcohol. Brain tissue sections for each condition were analyzed using a MALDI-MSI in negative polarity after 9-aminoacridine (9-AA) deposition on the tissue surface. Multivariate analysis including principal component analysis (PCA) and spatial shrunken centroids have been performed to differentiate the metabolic profiles in hippocampus according to the type of administration. This study offers promising results showing the potential of MSI for investigating the metabolomic changes in the brain associated with drug consumption. It may bring new insights into the study of toxicodynamic responses of cocaine consumption in reward-learning related regions.

The number of people affected by Type 2 diabetes mellitus (T2DM) is close to half a billion and is on a sharp rise, representing a major and growing public health burden. Given its mild initial symptoms, T2DM is often diagnosed several years after its onset, leaving half of diabetic individuals undiagnosed. While several classical clinical and genetic biomarkers have been identified, improving early diagnosis by exploring other kinds of omics data remains crucial. In this study, we have combined longitudinal data from two population-based cohorts CoLaus and DESIR (comprising in total 493 incident cases vs. 1360 controls) to identify new or confirm previously implicated metabolomic biomarkers predicting T2DM incidence more than 5 years ahead of clinical diagnosis. Our longitudinal data have shown robust evidence for valine, leucine, carnitine and glutamic acid being predictive of future conversion to T2DM. We confirmed the causality of such association for leucine by 2-sample Mendelian randomisation (MR) based on independent data. Our MR approach further identified new metabolites potentially playing a causal role on T2D, including betaine, lysine and mannose. Interestingly, for valine and leucine a strong reverse causal effect was detected, indicating that the genetic predisposition to T2DM may trigger early changes of these metabolites, which appear well-before any clinical symptoms. In addition, our study revealed a reverse causal effect of metabolites such as glutamic acid and alanine. Collectively, these findings indicate that molecular traits linked to the genetic basis of T2DM may be particularly promising early biomarkers.

Diagnostics of myocardial infarction in human post-mortem hearts can be achieved only if ischemia persisted for at least 6–12 h when certain morphological changes appear in myocardium. The initial 4 h of ischemia is difficult to diagnose due to lack of a standardized method. Developing a panel of molecular tissue markers is a promising approach and can be accelerated by characterization of molecular changes. This study is the first untargeted metabolomic profiling of ischemic myocardium during the initial 4 h directly from tissue section. Ischemic hearts from an ex-vivo Langendorff model were analysed using matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) at 15 min, 30 min, 1 h, 2 h, and 4 h. Region-specific molecular changes were identified even in absence of evident histological lesions and were segregated by unsupervised cluster analysis. Significantly differentially expressed features were detected by multivariate analysis starting at 15 min while their number increased with prolonged ischemia. The biggest significant increase at 15 min was observed for m/z 682.1294 (likely corresponding to S-NADHX—a damage product of nicotinamide adenine dinucleotide (NADH)). Based on the previously reported role of NAD+/NADH ratio in regulating localization of the sodium channel (Nav1.5) at the plasma membrane, Nav1.5 was evaluated by immunofluorescence. As expected, a fainter signal was observed at the plasma membrane in the predicted ischemic region starting 30 min of ischemia and the change became the most pronounced by 4 h. Metabolomic changes occur early during ischemia, can assist in identifying markers for post-mortem diagnostics and improve understanding of molecular mechanisms.

The paper describes the first three deaths reported in Europe involved in isotonitazene consumption, a potent benzimidazole derivate opioid consumed in the recreational drug scene. Isotonitazene powder and purity determination was performed on the sample collected in the first death scene by NMR, HRMS, GC-FTIR, ATR-FTIR and GC-MS. Isotonitazene purity was determined by GC-MS analysis and proton NMR, and was defined to be above 95 % and 98 %, respectively. Quantification of isotonitazene in biological samples was performed using a targeted analysis based on SPE extraction and ultra-high performance liquid chromatography tandem mass spectrometry. The isotonitazene median concentration in femoral whole blood was 1.20ng/mL. Isotonitazene concentration in hair was similar or even lower compared to that seen in fentanyl abusers. Isotonitazene distribution in tissues converges in the brain, lungs and heart, respectively. Surprisingly, isotonitazene concentration in liver is the lowest measured for all tissues and fluids analyzed. Based on circumstantial evidence, autopsy findings and the results of the toxicological analysis, the medical examiner concluded that the cause of all three deaths was an acute intoxication with isotonitazene. Since isotonitazene toxic concentration levels are very low, the consumption of this new psychoactive drug is a real hazard for human health.

Beta cell failure is one of the most important features of type 2 diabetes mellitus (T2DM). High‐density lipoprotein (HDL) has been proposed to improve β‐cell function. However, the mechanisms involved in this process are still poorly understood. The aim of this study was to investigate the contribution of sphingosine‐1‐phosphate (S1P) in the impact of HDL treatment on insulin secretion by pancreatic β‐cells and to determine its mechanisms. Primary cultures of β‐cells isolated from rat were treated with or without HDL in the presence or absence of S1P pathway inhibitors and insulin secretion response was analyzed. The S1P content of HDL (HDL‐S1P) isolated from T2DM patients was analyzed and correlated to the HDL‐induced insulin secretion. The expression of genes involved in the biosynthesis of the insulin was also evaluated. HDL as well as S1P treatment enhanced glucose‐stimulated insulin secretion (GSIS). In HDL isolated from T2DM patients, while HDL‐S1P was strongly correlated to its pro‐secretory capacity (r = 0.633, p = 0.005), HDL‐cholesterol and apolipoprotein AI levels were not. HDL‐induced GSIS was blocked by the S1P1/3 antagonist but not by the S1P2 antagonist, and was also accompanied by increased intracellular S1P in β‐cells. We also observed that HDL improved GSIS without significant changes in expression levels of insulin biosynthesis genes. Our present study highlights the importance HDL‐S1P in GSIS in T2DM patients and demonstrates that HDL induces insulin secretion by a process involving both intra‐ and extra‐cellular sources of S1P independently of an effect on insulin biosynthesis genes., HDL improves insulin secretion by beta cells S1P plays a role in HDL‐induced insulin secretion HDL increases intracellular S1P content.

In this study, we assessed the potential use of the 1β-hydroxy-deoxycholic acid (1β-OH-DCA) to deoxycholic acid (DCA) urinary metabolic ratio (UMR) as a CYP3A metric in ten male healthy volunteers. Midazolam (MDZ) 1 mg was administered orally at three sessions: alone (control session), after pre-treatment with fluvoxamine 50 mg (12 h and 2 h prior to MDZ administration), and voriconazole 400 mg (2 h before MDZ administration) (inhibition session), and after a 7-day pre-treatment with the inducer rifampicin 600 mg (induction session). The 1β-OH-DCA/DCA UMR was measured at each session, and correlations with MDZ metrics were established. At baseline, the 1β-OH-DCA/DCA UMR correlated significantly with oral MDZ clearance (r = 0.652, p = 0.041) and Cmax (r = −0.652, p = 0.041). In addition, the modulation of CYP3A was reflected in the 1β-OH-DCA/DCA UMR after the intake of rifampicin (induction ratio = 11.4, p &lt, 0.01). During the inhibition session, a non-significant 22% decrease in 1β-OH-DCA/DCA was observed (p = 0.275). This result could be explained by the short duration of CYP3A inhibitors intake fixed in our clinical trial. Additional studies, particularly involving CYP3A inhibition for a longer period and larger sample sizes, are needed to confirm the 1β-OH-DCA/DCA metric as a suitable CYP3A biomarker.

This study describes two bioanalytical methods for the quantitation of the two methadone enantiomers in dried matrix spots using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) and high performance supercritical chromatography tandem mass spectrometry (HPSFC-MS/MS). Dried matrix spots were obtained by spotting 10 µL of each sample fluid on a Whatman paper. Methadone and its main metabolite, EDDP, were extracted with 100 µL methanol and subsequently injected into the LC-MS/MS and SFC-MS/MS systems. Enantiomeric separation was achieved with AGP-column for the LC conditions and with Chiralpak IH-3 in SFC. The two methods were fully validated and 93 post-mortem samples were analysed with both analytical methods. Results from validation parameters and results obtained for all post-mortem samples were compared with a significant spearman correlation of r s = 0.9978 for R-methadone and r s = 0.9981 for S-methadone. The LC method provided better results in terms of uncertainty, retention factor and resolution, whereas SFC provides better sensitivity, with lower LOD. Median R-/S-methadone ratio in peripheral blood was found equal to 1.60 (N = 32), varying from 0.79 to 4.23. The reported values were in good agreement with previously published results. Based on the results obtained here, SFC-MS/MS can be considered a reliable alternative to the widely used LC-MS/MS for the quantitation of methadone enantiomers in bioanalysis and should be evaluated for other bioanalytical methods. Both methods can be easily and quickly used in toxicological routine analysis for the methadone quantitation in human fluids matrices, even if considering that the polysaccharide coated column IH-3 used in SFC does not allow the enantiomeric EDDP separation.

Background and Purpose: Individualized assessment of the activity of cytochrome P450 2D6 (CYP2D6), a highly variable drug-metabolizing enzyme, is performed through phenotyping during which a probe drug is administered to measure the enzyme’s activity. In order to avoid any iatrogenic harm (allergic drug reaction, dosing error) related to the probe drug, the development of non-invasive tools for real-time phenotyping of CYP2D6 could significantly contribute to the expansion of precision medicine in clinical practice. This study focuses on the identification of endogenous markers of the CYP2D6 enzyme in human biofluids using a liquid chromatography (LC)-high-resolution mass spectrometry (HRMS)-based metabolomics approach. Experimental Approach: Data from a control session were compared to data from an inhibition session. Before the latter, healthy volunteers (extensive and ultrarapid metabolizers) received a daily dose of paroxetine 20 mg over seven days. CYP2D6 genotyping and phenotyping, using single oral dose of dextromethorphan 5 mg, were also performed in all participants. Key Results: In CYP2D6 extensive and ultrarapid metabolizers (n = 37), mean relative intensities of five features were significantly reduced during the inhibition session compared to the control session (fold changes ≤ 0.67, FDR-adjusted P < 0.0001). Furthermore, mean relative intensities of these candidates were significantly higher in the CYP2D6 extensive-ultrarapid metabolizer group (n = 37) compared to the poor metabolizer group (n = 6) (fold changes ≤ 0.67, P < 0.0001). Conclusion and Implications: The applied untargeted metabolomics strategy was able to identify five CYP2D6 endogenous metabolites, a promising discovery for non-invasive phenotyping and personalised medicine.

Redundant robots can provide improved kinematic performances compared to non-redundant robots, becoming suitable to perform complex tasks in several areas of application. Improved manipulability or dexterity and a larger workspace are some of the advantages of using redundant robots. This paper aims to present the design and development of an 8-DoF redundant robotic platform for medical applications. The platform is composed of a 7-DoF anthropomorphic cobot, i.e. Franka Emika, whose base is coupled to a 1-DoF linear axis. The aim of the linear axis is to enlarge the workspace of the robot. Robot-assisted Doppler sonography is presented as an example of medical application, where the robot, employed as probe-holder, is able to traverse the whole patient’s body during the exam.

The number of people affected by Type 2 Diabetes Mellitus (T2DM) is close to half a billion and is on a sharp rise, representing a major and growing public health burden. As the case for many other complex diseases, early diagnosis is key to prevent irreversible end-organ damages. However, given its mild initial symptoms, T2DM is often diagnosed several years after its onset, leaving half of diabetic individuals undiagnosed. While several classical clinical and genetic biomarkers have been identified, improving early diagnosis by exploring other kinds of omics data remains crucial. In this study, we have combined longitudinal data from two population-based cohorts CoLaus and DESIR (comprising in total 493 incident cases vs 1’360 controls) to identify new or confirm previously implicated metabolomic biomarkers predicting T2DM incidence more than five years ahead of clinical diagnosis. Our longitudinal data have shown robust evidence for valine, leucine, carnitine and glutamic acid being predictive of future conversion to T2DM, and also confirmed to be causal by 2-sample Mendelian randomisation (based on independent data). Interestingly, for valine and leucine a strong reverse causal effect was detected, indicating that the genetic predisposition to T2DM may trigger early changes of these metabolites, which appear well-before any clinical symptoms. These findings indicate that molecular traits linked to the genetic basis of T2DM may be particularly promising early biomarkers.

Background HIV-infected patients display an increased risk of cardiovascular events. HIV and antiretroviral therapy (ART) have both been suggested to increase the risk of cardiovascular events but their specific role remains unclear. Purpose The aim of this study was to analyse the metabolomic profile in a cohort of HIV-free, ART experienced and naïve HIV-infected patients and to identify the metabolite associated with atherosclerosis development and endothelial dysfunction. The impact of ART on inflammatory and autoimmune biomarkers known to underlie atherosclerosis burden was also investigated. Methods 144 participants from a South African cohort were divided in three groups: HIV-free (n=50), HIV-infected/ART experienced (n=50) and HIV-infected/ART naïve (n=44). Targeted metabolomic analyses were performed using standard operating procedures. Atherosclerosis development was evaluated using coronary intima-media thickness (CITM) and endothelial dysfunction by flow-mediated dilation (FMD). Inflammatory biomarkers were measured with the Meso Scale Discovery® platform. The levels of autoantibodies against apolipoprotein A1 (anti-apoA1 IgG) and its specific C-terminal domain (anti-F3L1) levels were assessed by homemade ELISA assays. Results 257 endogenous metabolites were identified in the 144 samples. Regarding the association of metabolites with the vascular markers, eight metabolites were found to be associated with FMD and eleven with CIMT. Among these metabolites, cytosine was negatively correlated with FMD (r=−0.22, p=0.009), while positively with CIMT (r=0.20, p=0.026). Interestingly, from the list of these 18 metabolites, ART-experienced patients displayed an increase in kynurenine (p=0.0007) and 3–2-hydroxyphenyl-propanoate (p=0.0009) levels and a decrease in itaconate (p=0.021) levels compared to ART-naïve patients. HIV-infected patients displayed a more proatherogenic profile compared to HIV-free subjects. Inflammatory markers (IFNg, IL-6, IL-10, TNFα, CRP all p Conclusion Metabolomic analysis identified metabolites that were differentially expressed in the 3 groups. 18 metabolites were significantly correlated with FMD or CITM, among which the levels of kynurenine, 3–2-hydroxyphenyl-propanoate and itaconate were modulated by ART. HIV patients displayed increased levels of inflammatory markers, circulating adhesion molecules and anti-apoA1 IgG which was downregulated by ART. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Fondation De Reuter; Fondation Schmidheiny; Fondation Prévot; SwissLife AG; Fondation pour la lutte pour le cancer et la recherche bio-médicale

Molecular medical imaging is intended to increase the accuracy of diagnosis, particularly in cardiovascular and cancer-related diseases, where early detection could significantly increase the treatment success rate. In this study, we present mixed micelles formed from four building blocks as a magnetic resonance imaging targeted contrast agent for the detection of atheroma and cancer cells. The building blocks are a gadolinium-loaded DOTA ring responsible for contrast enhancement, a fibrin-specific CREKA pentapeptide responsible for targeting, a fluorescent dye and DSPE-PEG 2000 . The micelles were fully characterized in terms of their size, zeta potential, stability, relaxivity and toxicity. Target binding assays performed on fibrin clots were quantified by fluorescence and image signal intensities and proved the binding power. An additional internalization assay showed that the micelles were also designed to specifically enter into cancer cells. Overall, these multimodal mixed micelles represent a potential formulation for MRI molecular imaging of atheroma and cancer cells.

Background Zinc deficiency is commonly encountered in chronic kidney disease (CKD). The aims of this study were to assess whether zinc deficiency was related to increased renal excretion of zinc and to the progression of CKD. Methods Plasma and 24-h urinary zinc levels, urinary electrolytes and uromodulin were measured in 108 CKD patients and 81 individuals without CKD. Serum creatinine values were collected for 3 years to calculate the yearly change in estimated glomerular filtration rate (eGFR). Multivariable regression analysis was performed to assess the association between baseline zinc levels and yearly change in eGFR. Results CKD patients had lower circulating zinc levels and higher 24-h urinary zinc excretion than non-CKD participants (612.4 ± 425.9 versus 479.2 ± 293.0 µg/day; P = 0.02). Fractional excretion (FE) of zinc was higher and it significantly increased at more advanced CKD stages. Zinc FE was correlated negatively with 24-h urinary uromodulin excretion (r=−0.29; P Conclusions Zinc levels are lower in CKD, and not compensated by reduced renal zinc excretion. The inverse association between urinary zinc excretion and uromodulin possibly points to an impaired tubular activity, which could partly account for zinc imbalance in CKD. These data suggest that zinc status is associated with renal function decline, but further studies elucidating the underlying mechanisms and the potential role of zinc supplements in CKD are needed.

Acute physical exercise improves memory functions by increasing neural plasticity in the hippocampus. In animals, a single session of physical exercise has been shown to boost anandamide (AEA), an endocannabinoid known to promote hippocampal plasticity. Hippocampal neuronal networks encode episodic memory representations, including the temporal organization of elements, and can thus benefit motor sequence learning. While previous work established that acute physical exercise has positive effects on declarative memory linked to hippocampal plasticity mechanisms, its influence on memory for motor sequences, and especially on neural mechanisms underlying possible effects, has been less investigated.Here we studied the impact of acute physical exercise on motor sequence learning, and its underlying neurophysiological mechanisms in humans, using a cross-over randomized within-subjects design. We measured behavior, fMRI activity, and circulating AEA levels in fifteen healthy participants while they performed a serial reaction time task (SRTT) before and after a short period of exercise (moderate or high intensity) or rest.We show that exercise enhanced motor sequence memory, significantly for high intensity exercise and tending towards significance for moderate intensity exercise. This enhancement correlated with AEA increase, and dovetailed with local increases in caudate nucleus and hippocampus activity.These findings demonstrate that acute physical exercise promotes sequence learning, thus attesting the overarching benefit of exercise to hippocampus-related memory functions.

As a powerful phenotyping technology, metabolomics provides new opportunities in biomarker discovery through metabolome-wide association studies (MWAS) and identification of metabolites having regulatory effect in various biological processes. While MS-based metabolomics assays are endowed with high-throughput and sensitivity, large-scale MWAS are doomed to long-term data acquisition generating an overtime-analytical signal drift that can hinder the uncovering of true biologically relevant changes.We developed “dbnorm”, a package in R environment, which allows visualization and removal of signal heterogeneity from large metabolomics datasets. “dbnorm” integrates advanced statistical tools to inspect dataset structure, at both macroscopic (sample batch) and microscopic (metabolic features) scales. To compare model performance on data correction, “dbnorm” assigns a score, which allows the straightforward identification of the best fitting model for each dataset. Herein, we show how “dbnorm” efficiently removes signal drift among batches to capture the true biological heterogeneity of data in two large-scale metabolomics studies.

Obesity is considered as a major public health concern with strong economic and social burdens. Exposure to pollutants such as heavy metals can contribute to the development of obesity and its associated metabolic disorders, including type 2 diabetes and cardiovascular diseases. Adipose tissue is an endocrine and paracrine organ that plays a key role in the development of these diseases and is one of the main target of heavy metal accumulation. In this study, we determined by inductively coupled plasma mass spectrometry cadmium concentrations in human subcutaneous and visceral adipose tissues, ranging between 2.5 nM and 2.5 µM. We found a positive correlation between cadmium levels and age, sex and smoking status and a negative correlation between cadmium and body mass index. Based on cadmium adipose tissue concentrations found in humans, we investigated the effects of cadmium exposure, at concentrations between 1 nM and 10 µM, on adipose-derived human mesenchymal stem cells differentiated into mature adipocytes in vitro. Transcriptomic analysis highlighted that such exposure altered the expression of genes involved in trace element homeostasis and heavy metal detoxification, such as Solute Carrier Family transporters and metallothioneins. This effect correlated with zinc level alteration in cells and cellular media. Interestingly, dysregulation of zinc homeostasis and transporters has been particularly associated with the development of obesity and type 2 diabetes. Moreover, we found that cadmium exposure induces the pro-inflammatory state of the adipocytes by enhancing the expression of genes such as IL-6, IL-1B and CCL2, cytokines also induced in obesity. Finally, cadmium modulates various adipocyte functions such as the insulin response signaling pathway and lipid homeostasis. Collectively, our data identified some of the cellular mechanisms by which cadmium alters adipocyte functions, thus highlighting new facets of its potential contribution to the progression of metabolic disorders.

Driving under the influence of alcohol and drugs (DUID) is a major field of study to improve road safety. In Switzerland, during controls whether or not they follow an accident, the police can request toxicological analysis targeted either on alcohol only (ALC cases), or on drugs and alcohol (DUID cases). To evaluate both the drugs consumption on the road and whether or not these requests are well correlated with toxicological results, we built a database recording 4003 offenders (3443 males, 550 females) over a two-year period (2018–2019) in Western Switzerland. ALC case samples were then analyzed to target other substances than ethanol. We found one or more psychoactive drugs in 89% of DUID cases and alcohol alone was found in 56% of ALC cases. In ALC cases, alcohol alone was found in 72% of non-accident cases and in 52% of accident cases. This highlights an influence of accident context, inducing a too high suspicion of alcohol after accidents, and therefore an underestimation of the prevalence of other drugs. The most frequently detected drugs in DUID cases were cannabinoids (58%), ethanol (30%), cocaine (21%), benzodiazepines (11%), amphetamines (7%), opiates (6%), and antidepressants (5%). For the ALC cases, the drugs found were ethanol (84%), cannabinoids (13%), benzodiazepines (9%), antidepressants (6%), opiates (5%), cocaine (4%), methadone (3%), and amphetamines (1%). Prescription drugs, such as benzodiazepines, were common in accidents (22%) but rare in non-accidents DUID cases (5%). Thus, these drugs highly impact driving skills while being hard to suspect. This is of first concern as prescription drugs are largely found in poly-drug consumption, especially in combination with alcohol in accident cases. This emphasizes the emerging issue of prescription drugs and should motivate a strategy of prevention focused on the noxious effect of combining alcohol and prescription drugs on driving skills.

In post-mortem investigations of fatal intoxication, it is challenging to determine which drug(s) were responsible for the death, and which drugs did not. This study aims to provide post-mortem femoral blood drug levels in lethal intoxication and in post-mortem control cases, where the cause of death was other than intoxication. The reference values could assist in the interpretation of toxicological results in the routine casework. To this end, all post-mortem toxicological results in femoral blood from 2011 to 2017 in Western Switzerland were considered. A full autopsy with systematic toxicological analysis (STA) was conducted in all cases. Results take into account the cause of death classified into one of four categories (as published by Druid and colleagues): I) certified intoxication by one substance alone, IIa) certified intoxication by more than one substance, IIb) certified other causes of death with incapacitation due to drugs, and III) certified other causes of death without incapacitation due to drugs. This study includes 1 990 post-mortem cases where femoral blood was analysed. The material comprised 619 women (31%) and 1 371 men (69%) with a median age of 50 years. The concentrations of the 32 most frequently recorded substances as well as alcohol are discussed. These include 6 opioids and opiates, 3 antidepressants, 6 neuroleptics and hypnotics, 1 barbiturate, 11 benzodiazepines (and related drugs), 2 amphetamine-type stimulants, cocaine, paracetamol, and tetrahydrocannabinol (THC). The most common substances that caused intoxication alone were morphine, methadone, ethanol, tramadol, and cocaine. The post-mortem concentration ranges for all substance are categorized as I, IIa, IIb, or III. Statistical post-mortem reference concentrations for drugs are discussed and compared with previously published concentrations. This study shows that recording and classifying cases is time-consuming, but it is rewarding in a long-term perspective to achieve a more reliable information about fatal and non-fatal blood concentrations.

The association of blood pressure (BP) with manganese—an essential trace element required for human health—remains poorly studied. In 734 randomly recruited Swiss participants (mean age, 47.5 years; 51.4% women), we related ambulatory BP to 2 biomarkers, plasma manganese (pMn) and the urinary manganese (uMn) excretion. To allow for diurnal variation, we assessed BP and uMn over 24 hours and during wakefulness and sleep, using split urine samples. Twenty-four-hour, daytime, and nighttime systolic/diastolic BPs averaged 119.8/78.1, 123.8/81.2, and 107.0/68.3 mm Hg; the corresponding median uMn were 199.5, 83.0, and 51.5 μmol and median pMn, 0.52 μg/L. In analyses dichotomized by the median of the biomarkers, greater pMn was associated with higher 24-hour systolic/diastolic BP (+4.1/+2.3 mm Hg; P ≤0.0003), greater daytime uMn with lower daytime BP (−3.5/−1.9 mm Hg; P ≤0.0067), and greater nighttime uMn with higher nighttime BP (+2.9/+1.2 mm Hg; P ≤0.046). In multivariable-adjusted analyses, significance ( P ≤0.030) was retained for the positive association of 24-hour and daytime diastolic BP with pMn and for systolic BP in relation to uMn at night. The association sizes for a 2-fold increment in the biomarkers amounting to 0.77 mm Hg (95% CI, 0.08–1.47 mm Hg), 0.97 (CI, 0.20–1.76) and 1.33 (CI, 0.20–2.50 mm Hg), respectively. In conclusion, there were positive associations between diastolic BP and pMn over 24 hours and during daytime and between systolic BP and uMn at night.

Cytochrome P450 (CYP) activity can be assessed using a 'cocktail' phenotyping approach. Recently, we have developed a cocktail (Geneva cocktail) which combines the use of low-dose probes with a low-invasiveness dried blood spots (DBS) sampling technique and a single analytical method for the phenotyping of six major CYP isoforms. We have previously demonstrated that modulation of CYP activity after pre-treatment with CYP inhibitors/inducer could be reliably predicted using Geneva cocktail. To further validate this cocktail, in this study, we have verified whether probe drugs contained in the latter cause mutual drug-drug interactions. In a randomized, four-way, Latin-square crossover study, 30 healthy volunteers received low-dose caffeine, flurbiprofen, omeprazole, dextromethorphan and midazolam (a previously validated combination with no mutual drug-drug interactions); fexofenadine alone; bupropion alone; or all seven drugs simultaneously (Geneva cocktail). Pharmacokinetic profiles of the probe drugs and their metabolites were determined in DBS samples using both conventional micropipette sampling and new microfluidic device allowing for self-sampling. The 90% confidence intervals for the geometric mean ratios of AUC metabolite/AUC probe for CYP probes administered alone or within Geneva cocktail fell within the 0.8-1.25 bioequivalence range indicating the absence of pharmacokinetic interaction. The same result was observed for the chosen phenotyping indices, that is metabolic ratios at 2 hr (CYP1A2, CYP3A) or 3 hr (CYP2B6, CYP2C9, CYP2C19, CYP2D6) post-cocktail administration. DBS sampling could successfully be performed using a new microfluidic device. In conclusion, Geneva cocktail combined with an innovative DBS sampling device can be used routinely as a test for simultaneous CYP phenotyping.