Sequential Orbitrap Secondary Ion Mass Spectrometry and Liquid Extraction Surface Analysis-Tandem Mass Spectrometry-Based Metabolomics for Prediction of Brain Tumor Relapse from Sample-Limited Primary Tissue Archives.

We present here a novel surface mass spectrometry strategy to perform untargeted metabolite profiling of formalin-fixed paraffin-embedded pediatric ependymoma archives. Sequential Orbitrap secondary ion mass spectrometry (3D OrbiSIMS) and liquid extraction surface analysis-tandem mass spectrometry (LESA-MS/MS) permitted the detection of 887 metabolites (163 chemical classes) from pediatric ependymoma tumor tissue microarrays (diameter: <1 mm; thickness: 4 µm). From these 163 classes, 60 classes were detected with both techniques, whilst LESA-MS/MS and 3D OrbiSIMS individually allowed the detection of another 83 and 20 unique metabolite classes, respectively. Through data fusion and multivariate analysis, we were able to identify key metabolites and corresponding pathways predictive of tumor relapse, which were retrospectively confirmed by gene expression analysis with publicly available data. Altogether, this sequential mass spectrometry strategy has shown to be a versatile tool to perform high-throughput metabolite profiling on sample-limited tissue archives.

Combined hydrophilic interaction liquid chromatography-scanning field asymmetric waveform ion mobility spectrometry-time-of-flight mass spectrometry for untargeted metabolomics.

Untargeted metabolite profiling of biological samples is a challenge for analytical science due to the high degree of complexity of biofluids. Isobaric species may also not be resolved using mass spectrometry alone. As a result of these factors, many potential biomarkers may not be detected or are masked by co-eluting interferences in conventional LC-MS metabolomic analyses. In this study, a comprehensive liquid chromatography-mass spectrometry workflow incorporating a fast-scanning miniaturised high-field asymmetric waveform ion mobility spectrometry separation (LC-FAIMS-MS) is applied to the untargeted metabolomic analysis of human urine. The time-of-flight mass spectrometer used in the study was scanned at a rate of 20 scans s-1 enabling a FAIMS CF spectrum to be acquired within a 1-s scan time, maintaining an adequate number of data points across each LC peak. The developed method is demonstrated to be able to resolve co-eluting isomeric species and shows good reproducibility (%RSD < 4.9%). The nested datasets obtained for fresh, aged, and QC urine samples were submitted for multivariate statistical analysis. Seventy unique biomarker ions showing a statistically significant difference between fresh and aged urine were identified with optimal transmission CF values obtained across the full CF spectrum. The potential of using FAIMS to select ions for in-source collision-induced dissociation is demonstrated for FAIMS-selected methylxanthine ions yielding characteristic fragment ion species indicative of the precursor. Graphical abstract.

Evaluation of postmortem biochemical markers: Completeness of data and assessment of implication in the field.

Throughout the years an increase has been observed in research output on biochemical markers for determining the postmortem interval (PMI). However, to date, a complete overview is missing on the results of postmortem biochemical markers (PBM's) for PMI estimation. In this paper, literature was reviewed in order to identify the knowledge lacunae of PBM research from a practical point of view. A three-step approach was undertaken in order to achieve the set goal. Literature was collected, the PBM's were evaluated for completeness by means of a scorings index based on set criteria, and PBM's were subsequently evaluated in light of the Daubert &Frye criteria for scientific evidence in court. Seven PBM's were found to be well investigated, from which potassium had the highest completion score. However, none of these PBM's could be qualified as suitable for court evidence. Further, this study revealed that the majority of PBM's (94%) is not well investigated. Consequently, these PBM's did not meet Daubert &Frye criteria. In order to improve the assessment for use of PBM's as evidence in court regarding PMI estimation, PBM's should be investigated more thoroughly and data should be made readily available.

Improved Extraction Repeatability and Spectral Reproducibility for Liquid Extraction Surface Analysis-Mass Spectrometry Using Superhydrophobic-Superhydrophilic Patterning.

A major problem limiting reproducible use of liquid extraction surface analysis (LESA) array sampling of dried surface-deposited liquid samples is the unwanted spread of extraction solvent beyond the dried sample limits, resulting in unreliable data. Here, we explore the use of the Droplet Microarray (DMA), which consists of an array of superhydrophilic spots bordered by a superhydrophobic material giving the potential to confine both the sample spot and the LESA extraction solvent in a defined area. We investigated the DMA method in comparison with a standard glass substrate using LESA analysis of a mixture of biologically relevant compounds with a wide mass range and different physicochemical properties. The optimized DMA method was subsequently applied to urine samples from a human intervention study. Relative standard deviations for the signal intensities were all reduced at least 3-fold when performing LESA-MS on the DMA surface compared with a standard glass surface. Principal component analysis revealed more tight clusters indicating improved spectral reproducibility for a human urine sample extracted from the DMA compared to glass. Lastly, in urine samples from an intervention study, more significant ions (145) were identified when using LESA-MS spectra of control and test urine extracted from the DMA. We demonstrate that DMA provides a surface-assisted LESA-MS method delivering significant improvement of the surface extraction repeatability leading to the acquisition of more robust and higher quality data. The DMA shows potential to be used for LESA-MS for controlled and reproducible surface extraction and for acquisition of high quality, qualitative data in a high-throughput manner.

Technical note: unsafe rectal temperature measurements due to delayed warming of the thermocouple by using a condom. An issue concerning the estimation of the postmortem interval by using Henßge's nomogram.

In some cases, in the Netherlands, an additional layer is being added to the thermocouple, used to measure the rectal temperature in medicolegal death investigations. Because of this deviation from the standard method, questions arose regarding the accuracy and precision of the measured temperature. Therefore, a cooling experiment was carried out on a round body made of agar with an average thermal conductivity of 0.454 W/(m °C) while measuring the temperature with and without an additional layer around the thermocouple for three different starting temperatures: 36, 30, and 27 °C. The results show a significant difference between the measured values for the first 5 min when comparing with and without the additional layer. Further, a decrease in precision is present within the first minutes when using an additional layer. Therefore, it is concluded that it is best to measure the rectal temperature without an additional layer around the thermocouple and caution should be taken when measuring with an additional layer.

The experimental design of postmortem studies: the effect size and statistical power.

PURPOSE: The aim is of this study was to show the poor statistical power of postmortem studies. Further, this study aimed to find an estimate of the effect size for postmortem studies in order to show the importance of this parameter. This can be an aid in performing power analysis to determine a minimal sample size. METHODS: GPower was used to perform calculations on sample size, effect size, and statistical power. The minimal significance (α) and statistical power (1 - ß) were set at 0.05 and 0.80 respectively. Calculations were performed for two groups (Student's t-distribution) and multiple groups (one-way ANOVA; F-distribution). RESULTS: In this study, an average effect size of 0.46 was found (n = 22; SD = 0.30). Using this value to calculate the statistical power of another group of postmortem studies (n = 5) revealed that the average statistical power of these studies was poor (1 - ß < 0.80). CONCLUSION: The probability of a type-II error in postmortem studies is considerable. In order to enhance statistical power of postmortem studies, power analysis should be performed in which the effect size found in this study can be used as a guideline.

Real-Time Non-Invasive Monitoring of Short-Chain Fatty Acids in Exhaled Breath.

Short-chain fatty acids (SCFAs) are important metabolites produced by the gut microbiome as a result of the fermentation of non-digestible polysaccharides. The most abundant SCFAs are acetic acid, propionic acid, and butyric acid which make up 95% of this group of metabolites in the gut. Whilst conventional analysis SCFAs is done using either blood or fecal samples, SCFAs can also be detected in exhaled breath using proton transfer reaction-time-of-flight- mass spectrometry (PTR-ToF-MS) using H3O+ for ionization. However, no investigation has been performed to characterize the reactions of SCFAs with H3O+ and with other reagent ions, such as O2 + and NO+. Gas-phase samples of acetic acid, propionic acid, and butyric acid were analyzed with SRI/PTR-ToF-MS under dry and humid conditions. The ions generated and their distribution was determined for each reagent ion. It was found the humidity did not influence the product ion distribution for each SCFA. Using H3O+ as a reagent ion, SRI/PTR-ToF-MS analysis of an exhaled breath sample was performed in real-time to demonstrate the methodology. The presence of SCFAs in exhaled breath was confirmed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). Breath sampling repeatability was within acceptable limits (<15%) for an analytical methodology for each investigated SCFA. Nutritional intervention studies could potentially benefit from real-time monitoring of exhaled SCFAs as an alternative to measuring SCFAs invasively in blood or fecal samples since it is non-invasive, and requires minimal time investment from participants.

Non-Invasive Monitoring of Inflammation in Inflammatory Bowel Disease Patients during Prolonged Exercise via Exhaled Breath Volatile Organic Compounds.

The aim of this study was to investigate volatile organic compounds (VOCs) in exhaled breath as possible non-invasive markers to monitor the inflammatory response in inflammatory bowel disease (IBD) patients as a result of repeated and prolonged moderate-intensity exercise. We included 18 IBD patients and 19 non-IBD individuals who each completed a 30, 40, or 50 km walking exercise over three consecutive days. Breath and blood samples were taken before the start of the exercise event and every day post-exercise to assess changes in the VOC profiles and cytokine concentrations. Proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) was used to measure exhaled breath VOCs. Multivariate analysis, particularly ANOVA-simultaneous component analysis (ASCA), was employed to extract relevant ions related to exercise and IBD. Prolonged exercise induces a similar response in breath butanoic acid and plasma cytokines for participants with or without IBD. Butanoic acid showed a significant correlation with the cytokine IL-6, indicating that butanoic acid could be a potential non-invasive marker for exercise-induced inflammation. The findings are relevant in monitoring personalized IBD management.

Discovery of a Novel Polymer for Xeno-Free, Long-Term Culture of Human Pluripotent Stem Cell Expansion.

Human pluripotent stem cells (hPSCs) can be expanded and differentiated in vitro into almost any adult tissue cell type, and thus have great potential as a source for cell therapies with biomedical application. In this study, a fully-defined polymer synthetic substrate is identified for hPSC culture in completely defined, xenogenic (xeno)-free conditions. This system can overcome the cost, scalability, and reproducibility limitations of current hPSC culture strategies, and facilitate large-scale production. A high-throughput, multi-generational polymer microarray platform approach is used to test over 600 unique polymers and rapidly assess hPSC-polymer interactions in combination with the fully defined xeno-free medium, Essential 8 (E8). This study identifies a novel nanoscale phase separated blend of poly(tricyclodecane-dimethanol diacrylate) and poly(butyl acrylate) (2:1 v/v), which supports long-term expansion of hPSCs and can be readily coated onto standard cultureware. Analysis of cell-polymer interface interactions through mass spectrometry and integrin blocking studies provides novel mechanistic insight into the role of the E8 proteins in promoting integrin-mediated hPSC attachment and maintaining hPSC signaling, including ability to undergo multi-lineage differentiation. This study therefore identifies a novel substrate for long-term serial passaging of hPSCs in serum-free, commercial chemically-defined E8, which provides a promising and economic hPSC expansion platform for clinical-scale application.

Exhaled Breath Reflects Prolonged Exercise and Statin Use during a Field Campaign.

Volatile organic compounds (VOCs) in exhaled breath provide insights into various metabolic processes and can be used to monitor physiological response to exercise and medication. We integrated and validated in situ a sampling and analysis protocol using proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) for exhaled breath research. The approach was demonstrated on a participant cohort comprising users of the cholesterol-lowering drug statins and non-statin users during a field campaign of three days of prolonged and repeated exercise, with no restrictions on food or drink consumption. The effect of prolonged exercise was reflected in the exhaled breath of participants, and relevant VOCs were identified. Most of the VOCs, such as acetone, showed an increase in concentration after the first day of walking and subsequent decrease towards baseline levels prior to walking on the second day. A cluster of short-chain fatty acids including acetic acid, butanoic acid, and propionic acid were identified in exhaled breath as potential indicators of gut microbiota activity relating to exercise and drug use. We have provided novel information regarding the use of breathomics for non-invasive monitoring of changes in human metabolism and especially for the gut microbiome activity in relation to exercise and the use of medication, such as statins.

Comment on "Promising blood-derived biomarkers for estimation of the postmortem interval" by I. Costa, F. Carvalho, T. Magalhães, P. G. de Pinho, R. Silvestre & R. J. Dinis-Oliveira. (Toxicol. Res., 2015, 4, 1443-1452).

Recently, Costa et al. published an article about promising biomarkers for estimating the postmortem interval. Instead of postmortem blood, antemortem blood was putrefied in vitro by exposing the blood to a temperature gradient. However, in this way several other influencing factors were excluded, hence, the accuracy of the proposed model is doubtful. Therefore, the aim of this comment is to discuss the methodology, results and shortcomings of the study of Costa et al.