•Non-targeted LC/MS/MS in forensic toxicological analysis•Principles of non-targeted data acquisition strategies•Principles of compound identification•Performance of tandem mass spectral libraries
A ...core task of any forensic toxicology lab is providing comprehensive information on the chemical composition of evidence. This mission can only be accomplished by combining efficient detection techniques with reliable identification procedures. A competent approach for the sensitive detection of a large variety of potentially toxic compounds is liquid chromatography/tandem mass spectrometry (LC/MS/MS). The concept of non-targeted analysis is realized by applying either data-dependent or data-independent acquisition strategies. Non-targeted LC/MS/MS produces informative features for subsequent compound identification. Of particular importance is the availability of fragmentation information. Additionally, retention times and isotopic distributions may be used. During the identification process, extracted features are matched against data sets obtained from reference standards. Reference data is often stored in databases. State-of-the-art tandem mass spectral databases enable automated compound identification with high sensitivity and specificity. Nevertheless, the final decision on identity is still taken by an expert who carefully reviews the provided arguments.
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•Chemicals of Emerging Concern (CECs) are a growing concern in HBM.•Suspect and non-targeted screening (NTS) approaches offer new capabilities for capturing CECs in human ...matrices.•These new approaches applied to HBM come with a number of technical and scientific issues to be addressed.•Harmonisation is required at international level to reinforce NTS methods comparability and performance assessment.
Large-scale suspect and non-targeted screening approaches based on high-resolution mass spectrometry (HRMS) are today available for chemical profiling and holistic characterisation of biological samples. These advanced techniques allow the simultaneous detection of a large number of chemical features, including markers of human chemical exposure. Such markers are of interest for biomonitoring, environmental health studies and support to risk assessment. Furthermore, these screening approaches have the promising capability to detect chemicals of emerging concern (CECs), document the extent of human chemical exposure, generate new research hypotheses and provide early warning support to policy. Whilst of growing importance in the environment and food safety areas, respectively, CECs remain poorly addressed in the field of human biomonitoring. This shortfall is due to several scientific and methodological reasons, including a global lack of harmonisation. In this context, the main aim of this paper is to present an overview of the basic principles, promises and challenges of suspect and non-targeted screening approaches applied to human samples as this specific field introduce major specificities compared to other fields. Focused on liquid chromatography coupled to HRMS-based data acquisition methods, this overview addresses all steps of these new analytical workflows. Beyond this general picture, the main activities carried out on this topic within the particular framework of the European Human Biomonitoring initiative (project HBM4EU, 2017–2021) are described, with an emphasis on harmonisation measures.
Nicotinamide phosphoribosyltransferase (NAMPT, also referred to as pre-B cell colony-enhancing factor or visfatin) is critically required for the maintenance of cellular nicotinamide adenine ...dinucleotide (NAD) supply catalysing the rate-limiting step of the NAD
. NAMPT is strongly upregulated in inflammation including IBD and counteracts an increased cellular NAD turnover mediated by NAD-depleting enzymes. These constitute an important mechanistic link between inflammatory, metabolic and transcriptional pathways and NAD metabolism.
We investigated the impact of NAMPT inhibition by the small-molecule inhibitor FK866 in the dextran sulfate sodium (DSS) model of colitis and the azoxymethane/DSS model of colitis-associated cancer. The impact of NAD depletion on differentiation of mouse and human primary monocytes/macrophages was studied in vitro. Finally, we tested the efficacy of FK866 compared with dexamethasone and infliximab in lamina propria mononuclear cells (LPMNC) isolated from patients with IBD.
FK866 ameliorated DSS-induced colitis and suppressed inflammation-associated tumorigenesis in mice. FK866 potently inhibited NAMPT activity as demonstrated by reduced mucosal NAD, resulting in reduced abundances and activities of NAD-dependent enzymes including PARP1, Sirt6 and CD38, reduced nuclear factor kappa B activation, and decreased cellular infiltration by inflammatory monocytes, macrophages and activated T cells. Remarkably, FK866 effectively supressed cytokine release from LPMNCs of patients with IBD. As FK866 was also effective in Rag1
mice, we mechanistically linked FK866 treatment with altered monocyte/macrophage biology and skewed macrophage polarisation by reducing CD86, CD38, MHC-II and interleukin (IL)-6 and promoting CD206, Egr2 and IL-10.
Our data emphasise the importance of NAD immunometabolism for mucosal immunity and highlight FK866-mediated NAMPT blockade as a promising therapeutic approach in acute intestinal inflammation.
After completion of the human genome sequence the search for differences among individual genomes has become the centre of focus for geneticists. Two different types of polymorphism--single ...nucleotide polymorphisms (SNPs) and short tandem repeats (STRs)--are major sources of genetic diversity and are of widespread use in genetic analysis. A plethora of genotyping techniques have been developed, and mass spectrometry (MS) is among the most widely used analytical platforms. The most striking advantage of mass spectrometric genotyping assays over others is the use of the measured molecular mass information for allele calling. The molecular mass is less error-prone than other sequence-specific parameters, including migration times, retention times, or hybridization yields, as it represents an intrinsic property of a nucleic acid molecule that is directly related to its nucleotide composition. Mass spectrometric assays can roughly be divided into two major groups--matrix-assisted laser desorption/ionization (MALDI)-based and electrospray ionization (ESI)-based assays. An important subdivision of ESI-based genotyping methods are approaches that originate from the hyphenation of liquid chromatography (LC) to MS. The principles of these three classes of mass spectrometric genotyping techniques are summarized in this review. Possibilities and limitations are critically discussed to assist scientists, especially non-experts in MS, in choosing the appropriate mass spectrometric assay for genotyping a genetic marker of interest. graphic removed
Mass spectral library annotation of liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) data is a reliable approach for fast identification of organic contaminants and ...toxicants in complex environmental and biological matrices. While determining the exposure of humans or mammals, it is indispensable to include phase I and phase II metabolites (conjugates) along with the parent compounds, but often, tandem mass spectra for these are unavailable. In this study, we present and evaluate a strategy for annotating glucuronide conjugates in LC-HRMS/MS scans by applying a neutral loss search for detection, then truncating the spectra which we refer to as in silico deconjugation, and finally searching these against mass spectral libraries of the aglycones. The workflow was tested on a dataset of in vitro
–
generated glucuronides of reference standard mixtures and a dataset of 51 authentic urine samples collected from patients with known medication status, acquired on different instrumentations. A total number of 75 different glucuronidated molecular structures were identified by in silico deconjugation and spectral library annotation. We also identified specific molecular structures (sulfonamides, ether bonds, di-glucuronides), which resulted in slightly different fragmentation patterns between the glucuronide and the unconjugated compound. This led to a decreased spectral matching score and in some cases to a false-negative identification. Still, by applying this method, we revealed a reliable annotation of most common glucuronides, leading to a new strategy reducing the need for deconjugation steps or for recording many reference glucuronide spectra for screening approaches.
Graphical Abstract
Eminent in pandemic management is accurate information on infection dynamics to plan for timely installation of control measures and vaccination campaigns. Despite huge efforts in diagnostic testing ...of individuals, the underestimation of the actual number of SARS-CoV-2 infections remains significant due to the large number of undocumented cases. In this paper we demonstrate and compare three methods to estimate the dynamics of true infections based on secondary data i.e., (a) test positivity, (b) infection fatality and (c) wastewater monitoring. The concept is tested with Austrian data on a national basis for the period of April 2020 to December 2022. Further, we use the results of prevalence studies from the same period to generate (upper and lower bounds of) credible intervals for true infections for four data points. Model parameters are subsequently estimated by applying Approximate Bayesian Computation-rejection sampling and Genetic Algorithms. The method is then validated for the case study Vienna. We find that all three methods yield fairly similar results for estimating the true number of infections, which supports the idea that all three datasets contain similar baseline information. None of them is considered superior, as their advantages and shortcomings depend on the specific case study at hand.
Abstract
Desflurane, isoflurane and sevoflurane, three halogenated ethers, are commonly used inhaled anesthetics, both in the operating room and in the intensive care unit (ICU). The potency and ...dosage of these drugs is expressed by the MAC value (minimum alveolar concentration). Their interaction with hemoglobin and its affinity for oxygen, best described by the oxygen dissociation curve (ODC), has already been investigated, with conflicting results. Altered by many factors, the ODC can be shifted to the left or to the right, therefore increasing or decreasing hemoglobin oxygen (Hb-O
2
) affinity. In venous blood samples of 22 healthy participants (11 female, 11 male) ODC were measured with a high-throughput method in vitro. Blood samples were either exposed to control or to three different concentrations of desflurane, isoflurane or sevoflurane prior to and during measurements (low, medium and high corresponding to MAC 0.5, MAC 1.0 and MAC 2.0). With increasing concentrations from control to medium, desflurane and isoflurane significantly decreased Hb-O
2
affinity by shifting the ODC to the right (p = 0.016 and p < 0.001) but sevoflurane showed no effects. When further increasing concentrations from medium to high, all three inhaled anesthetics shifted the ODC back to the left (p < 0.001). Comparing only controls to high concentrations, a significant increase in Hb-O
2
affinity for desflurane (p = 0.005) and sevoflurane (p < 0.001) was detected. Our study shows a varying effect at different doses of inhaled anesthetics on Hb-O
2
affinity. While the underlying mechanisms remain unclear, these results show an effect which needs to be further investigated to determine if patients undergoing anesthesia may potentially benefit or get disadvantage from this slightly increased (e.g. impaired pulmonary oxygen uptake), or decreased Hb-O
2
affinity (e.g. arterial vascular disease).
Trial registration:
This study is registered with clinicaltrials.gov (NCT04612270).
Acute graft-versus-host disease (aGVHD) and tumor relapse remain major complications after allogeneic hematopoietic stem cell transplantation. Alloreactive T cells and cancer cells share a similar ...metabolic phenotype to meet the bioenergetic demands necessary for cellular proliferation and effector functions. Nicotinamide adenine dinucleotide (NAD) is an essential co-factor in energy metabolism and is constantly replenished by nicotinamide phosphoribosyl-transferase (Nampt), the rate-limiting enzyme in the NAD salvage pathway. Here we show, that Nampt blockage strongly ameliorates aGVHD and limits leukemic expansion. Nampt was highly elevated in serum of patients with gastrointestinal GVHD and was particularly abundant in human and mouse intestinal T cells. Therapeutic application of the Nampt small-molecule inhibitor, Fk866, strongly attenuated experimental GVHD and caused NAD depletion in T-cell subsets, which displayed differential susceptibility to NAD shortage. Fk866 robustly inhibited expansion of alloreactive but not memory T cells and promoted FoxP3-mediated lineage stability in regulatory T cells. Furthermore, Fk866 strongly reduced the tumor burden in mouse leukemia and graft-versus-leukemia models. Ex vivo studies using lymphocytes from GVHD patients demonstrated potent antiproliferative properties of Fk866, suggesting potential clinical utility. Thus, targeting NAD immunometabolism represents a novel approach to selectively inhibit alloreactive T cells during aGVHD with additional antileukemic efficacy.
It is becoming increasingly evident that genetic variants contribute to the development of opioid addiction. An elucidation of these genetic factors is crucial for a better understanding of this ...chronic disease and may help to develop novel therapeutic strategies. In recent years, several candidate genes were implicated in opioid dependence. However, most study findings have not been replicated and additional studies are required before reported associations can be considered robust. Thus, the major objective of this study was to replicate earlier findings and to identify new genetic polymorphisms contributing to the individual susceptibility to opioid addiction, respectively. Therefore, a candidate gene association study was conducted including 142 well-phenotyped long-term opioid addicts undergoing opioid maintenance therapy and 142 well-matched healthy controls. In both study groups, 24 single nucleotide polymorphisms predominantly located in pharmacogenetic candidate genes have been genotyped using an accurate mass spectrometry based method. The most significant associations with opioid addiction (remaining significant after adjustment for multiple testing) were observed for the rs948854 SNP in the galanin gene (GAL, p = 0.001) and the rs2236861 SNP in the delta opioid receptor gene (OPRD1, p = 0.001). Moreover, an association of the ATP binding cassette transporter 1 (ABCB1) variant rs1045642 and the Mu Opioid receptor (OPRM1) variant rs9479757 with opioid addiction was observed. The present study provides further support for a contribution of GAL and OPRD1 variants to the development of opioid addiction. Furthermore, our results indicate a potential contribution of OPRM1 and ABCB1 SNPs to the development of this chronic relapsing disease. Therefore it seems important that these genes are addressed in further addiction related studies.
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