The ADEM2 project: early pathogenic mechanisms of preschool wheeze and a randomised controlled trial assessing the gain in health and cost-effectiveness by application of the breath test for the diagnosis of asthma in wheezing preschool children.

BACKGROUND: The prevalence of asthma-like symptoms in preschool children is high. Despite numerous efforts, there still is no clinically available diagnostic tool to discriminate asthmatic children from children with transient wheeze at preschool age. This leads to potential overtreatment of children outgrowing their symptoms, and to potential undertreatment of children who turn out to have asthma. Our research group developed a breath test (using GC-tof-MS for VOC-analysis in exhaled breath) that is able to predict a diagnosis of asthma at preschool age. The ADEM2 study assesses the improvement in health gain and costs of care with the application of this breath test in wheezing preschool children. METHODS: This study is a combination of a multi-centre, parallel group, two arm, randomised controlled trial and a multi-centre longitudinal observational cohort study. The preschool children randomised into the treatment arm of the RCT receive a probability diagnosis (and corresponding treatment recommendations) of either asthma or transient wheeze based on the exhaled breath test. Children in the usual care arm do not receive a probability diagnosis. Participants are longitudinally followed up until the age of 6 years. The primary outcome is disease control after 1 and 2 years of follow-up. Participants of the RCT, together with a group of healthy preschool children, also contribute to the parallel observational cohort study developed to assess the validity of alternative VOC-sensing techniques and to explore numerous other potential discriminating biological parameters (such as allergic sensitisation, immunological markers, epigenetics, transcriptomics, microbiomics) and the subsequent identification of underlying disease pathways and relation to the discriminative VOCs in exhaled breath. DISCUSSION: The potential societal and clinical impact of the diagnostic tool for wheezing preschool children is substantial. By means of the breath test, it will become possible to deliver customized and high qualitative care to the large group of vulnerable preschool children with asthma-like symptoms. By applying a multi-omics approach to an extensive set of biological parameters we aim to explore (new) pathogenic mechanisms in the early development of asthma, creating potentially interesting targets for the development of new therapies. TRIAL REGISTRATION: Netherlands Trial Register, NL7336, Date registered 11-10-2018.

Practical and Robust NMR-Based Metabolic Phenotyping of Gut Health in Early Life.

While substantial efforts have been made to optimize and standardize fecal metabolomics for studies in adults, the development of a standard protocol to analyze infant feces is, however, still lagging behind. Here, we present the development of a hands-on and robust protocol for proton 1H NMR spectroscopy of infant feces. The influence of extraction solvent, dilution ratio, homogenization method, filtration, and duration of centrifugation on the biochemical composition of infant feces was carefully evaluated using visual inspection of 1H NMR spectra in combination with multivariate statistical modeling. The optimal metabolomics protocol was subsequently applied on feces from seven infants collected at 8 weeks, 4, and 9 months of age. Interindividual variation was exceeding the variation induced by different fecal sample preparation methods, except for filtration. We recommend extracting fecal samples using water with a dilution ratio of 1:5 feces-to-water to homogenize using bead beating and to remove particulates using centrifugation. Samples collected from infants aged 8 weeks and 4 months showed elevated concentrations of milk oligosaccharide derivatives and lactic acid, whereas short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) were higher in the 9 month samples. The established protocol enables hands-on and robust analyses of the infant gut metabolome. The wide-ranging application of this protocol will facilitate interlaboratory comparison of infants' metabolic profiles and finally aid in a better understanding of infant gut health.

SCD1 regulates the AMPK/SIRT1 pathway and histone acetylation through changes in adenine nucleotide metabolism in skeletal muscle.

Stearoyl-CoA desaturase (SCD) is a rate-limiting enzyme that catalyzes the synthesis of monounsaturated fatty acids. It plays an important role in regulating skeletal muscle metabolism. Lack of the SCD1 gene increases the rate of fatty acid ß-oxidation through activation of the AMP-activated protein kinase (AMPK) pathway and the upregulation of genes that are related to fatty acid oxidation. The mechanism of AMPK activation under conditions of SCD1 deficiency has been unclear. In the present study, we found that the ablation/inhibition of SCD1 led to AMPK activation in skeletal muscle through an increase in AMP levels whereas muscle-specific SCD1 overexpression decreased both AMPK phosphorylation and the adenosine monophosphate/adenosine triphosphate (AMP/ATP) ratio. Changes in AMPK phosphorylation that were caused by SCD1 down- and upregulation affected NAD+ levels following changes in NAD+ -dependent deacetylase sirtuin-1 (SIRT1) activity and histone 3 (H3K9) acetylation and methylation status. Moreover, mice with muscle-targeted overexpression of SCD1 were more susceptible to high-fat diet-induced lipid accumulation and the development of insulin resistance compared with wild-type mice. These data show that SCD1 is involved in nucleotide (ATP and NAD+ ) metabolism and suggest that the SCD1-dependent regulation of muscle steatosis and insulin sensitivity are mediated by cooperation between AMPK- and SIRT1-regulated pathways. Altogether, the present study reveals a novel mechanism that links SCD1 with the maintenance of metabolic homeostasis and insulin sensitivity in skeletal muscle.

Exhaled biomarkers and gene expression at preschool age improve asthma prediction at 6 years of age.

RATIONALE: A reliable asthma diagnosis is difficult in wheezing preschool children. OBJECTIVES: To assess whether exhaled biomarkers, expression of inflammation genes, and early lung function measurements can improve a reliable asthma prediction in preschool wheezing children. METHODS: Two hundred two preschool recurrent wheezers (aged 2-4 yr) were prospectively followed up until 6 years of age. At 6 years of age, a diagnosis (asthma or transient wheeze) was based on symptoms, lung function, and asthma medication use. The added predictive value (area under the receiver operating characteristic curve [AUC]) of biomarkers to clinical information (assessed with the Asthma Predictive Index [API]) assessed at preschool age in diagnosing asthma at 6 years of age was determined with a validation set. Biomarkers in exhaled breath condensate, exhaled volatile organic compounds (VOCs), gene expression, and airway resistance were measured. MEASUREMENTS AND MAIN RESULTS: At 6 years of age, 198 children were diagnosed (76 with asthma, 122 with transient wheeze). Information on exhaled VOCs significantly improved asthma prediction (AUC, 89% [increase of 28%]; positive predictive value [PPV]/negative predictive value [NPV], 82/83%), which persisted in the validation set. Information on gene expression of toll-like receptor 4, catalase, and tumor necrosis factor-α significantly improved asthma prediction (AUC, 75% [increase of 17%]; PPV/NPV, 76/73%). This could not be confirmed after validation. Biomarkers in exhaled breath condensate and airway resistance (pre- and post- bronchodilator) did not improve an asthma prediction. The combined model with VOCs, gene expression, and API had an AUC of 95% (PPV/NPV, 90/89%). CONCLUSIONS: Adding information on exhaled VOCs and possibly expression of inflammation genes to the API significantly improves an accurate asthma diagnosis in preschool children. Clinical trial registered with www.clinicaltrial.gov (NCT 00422747).

The optimization and comparison of two high-throughput faecal headspace sampling platforms: the microchamber/thermal extractor and hi-capacity sorptive extraction probes (HiSorb).

Disease detection and monitoring using volatile organic compounds (VOCs) is becoming increasingly popular. For a variety of (gastrointestinal) diseases the microbiome should be considered. As its output is to large extent volatile, faecal volatilomics carries great potential. One technical limitation is that current faecal headspace analysis requires specialized instrumentation which is costly and typically does not work in harmony with thermal desorption units often utilized in e.g. exhaled breath studies. This lack of harmonization hinders uptake of such analyses by the Volatilomics community. Therefore, this study optimized and compared two recently harmonized faecal headspace sampling platforms:High-capacity Sorptive extraction (HiSorb) probesand theMicrochamber thermal extractor (Microchamber). Statistical design of experiment was applied to find optimal sampling conditions by maximizing reproducibility, the number of VOCs detected, and between subject variation. To foster general applicability those factors were defined using semi-targeted as well as untargeted metabolic profiles. HiSorb probes were found to result in a faster sampling procedure, higher number of detected VOCs, and higher stability. The headspace collection using the Microchamber resulted in a lower number of detected VOCs, longer sampling times and decreased stability despite a smaller number of interfering VOCs and no background signals. Based on the observed profiles, recommendations are provided on pre-processing and study design when using either one of both platforms. Both can be used to perform faecal headspace collection, but altogether HiSorb is recommended.

Stemness properties of SSEA-4+ subpopulation isolated from heterogenous Wharton's jelly mesenchymal stem/stromal cells.

Background: High heterogeneity of mesenchymal stem/stromal cells (MSCs) due to different degrees of differentiation of cell subpopulations poses a considerable challenge in preclinical studies. The cells at a pluripotent-like stage represent a stem cell population of interest for many researchers worldwide, which is worthy of identification, isolation, and functional characterization. In the current study, we asked whether Wharton's jelly-derived MSCs (WJ-MSCs) which express stage-specific embryonic antigen-4 (SSEA-4) can be considered as a pluripotent-like stem cell population. Methods: SSEA-4 expression in different culture conditions was compared and the efficiency of two cell separation methods were assessed: Magnetic Activated Cell Sorting (MACS) and Fluorescence Activated Cell Sorting (FACS). After isolation, SSEA-4+ cells were analyzed for the following parameters: the maintenance of the SSEA-4 antigen expression after cell sorting, stem cell-related gene expression, proliferation potential, clonogenicity, secretome profiling, and the ability to form spheres under 3D culture conditions. Results: FACS allowed for the enrichment of SSEA-4+ cell content in the population that lasted for six passages after sorting. Despite the elevated expression of stemness-related genes, SSEA-4+ cells neither differed in their proliferation and clonogenicity potential from initial and negative populations nor exhibited pluripotent differentiation repertoire. SSEA-4+ cells were observed to form smaller spheroids and exhibited increased survival under 3D conditions. Conclusion: Despite the transient expression of stemness-related genes, our findings could not fully confirm the undifferentiated pluripotent-like nature of the SSEA-4+ WJ-MSC population cultured in vitro.

Volatile organic compounds in headspace characterize isolated bacterial strains independent of growth medium or antibiotic sensitivity.

INTRODUCTION: Early and reliable determination of bacterial strain specificity and antibiotic resistance is critical to improve sepsis treatment. Previous research demonstrated the potential of headspace analysis of volatile organic compounds (VOCs) to differentiate between various microorganisms associated with pulmonary infections in vitro. This study evaluates whether VOC analysis can also discriminate antibiotic sensitive from resistant bacterial strains when cultured on varying growth media. METHODS: Both antibiotic-sensitive and -resistant strains of Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumonia were cultured on 4 different growth media, i.e. Brain Heart Infusion, Marine Broth, Müller-Hinton and Trypticase Soy Agar. After overnight incubation at 37°C, the headspace air of the cultures was collected on stainless steel desorption tubes and analyzed by gas chromatography time-of-flight mass spectrometry (GC-tof-MS). Statistical analysis was performed using regularized multivariate analysis of variance and cross validation. RESULTS: The three bacterial species could be correctly recognized based on the differential presence of 14 VOCs (p<0.001). This discrimination was not influenced by the different growth media. Interestingly, a clear discrimination could be made between the antibiotic-resistant and -sensitive variant of Pseudomonas aeruginosa (p<0.001) based on their species-specific VOC signature. CONCLUSION: This study demonstrates that isolated microorganisms, including antibiotic-sensitive and -resistant strains of Pseudomonas aeruginosa, could be identified based on their excreted VOCs independent of the applied growth media. These findings suggest that the discriminating volatiles are associated with the microorganisms themselves rather than with their growth medium. This study exemplifies the potential of VOC analysis as diagnostic tool in medical microbiology. However, validation of our results in appropriate in vivo models is critical to improve translation of breath analysis to clinical applications.

The effect of expectancy versus actual gluten intake on gastrointestinal and extra-intestinal symptoms in non-coeliac gluten sensitivity: a randomised, double-blind, placebo-controlled, international, multicentre study.

BACKGROUND: Many individuals without coeliac disease or wheat allergy reduce their gluten intake because they believe that gluten causes their gastrointestinal symptoms. Symptoms could be affected by negative expectancy. Therefore, we aimed to investigate the effects of expectancy versus actual gluten intake on symptoms in people with non-coeliac gluten sensitivity (NCGS). METHODS: This randomised, double-blind, placebo-controlled, international, multicentre study was done at the University of Leeds (Leeds, UK), Maastricht University (Maastricht, the Netherlands), and Wageningen University and Research (Wageningen, the Netherlands). People aged 18-70 years with self-reported NCGS (ie, gastrointestinal symptoms within 8 h of gluten consumption) without coeliac disease and wheat allergy were recruited. Participants had to follow a gluten-free or gluten-restricted diet for at least 1 week before (and throughout) study participation and had to be asymptomatic or mildly symptomatic (overall gastrointestinal symptom score ≤30 mm on the Visual Analogue Scale [VAS]) while on the diet. Participants were randomly assigned (1:1:1:1; blocks of eight; stratified by site and gender) to one of four groups based on the expectation to consume gluten-containing (E+) or gluten-free (E-) oat bread for breakfast and lunch (two slices each) and actual intake of gluten-containing (G+) or gluten-free (G-) oat bread. Participants, investigators, and those assessing outcomes were masked to the actual gluten assignment, and participants were also masked to the expectancy part of the study. The primary outcome was overall gastrointestinal symptom score on the VAS, which was measured at and corrected for baseline (before breakfast) and hourly for 8 h, with lunch served after 4 h, and analysed per-protocol. Safety analysis included all participants incorporated in the per-protocol analysis. The study is registered at ClinicalTrials.gov, NCT05779358, and has ended. FINDINGS: Between Oct 19, 2018, and Feb 14, 2022, 165 people were screened and 84 were randomly assigned to E+G+ (n=21), E+G- (n=21), E-G+ (n=20), or E-G- (n=22). One person in the E+G+ group was excluded due to not following test day instructions, leaving 83 participants in the per-protocol analysis. Median age was 27·0 years (IQR 21·0-45·0), 71 (86%) of 83 people were women, and 12 (14%) were men. Mean overall gastrointestinal symptom score was significantly higher for E+G+ (16·6 mm [95% CI 13·1 to 20·0]) than for E-G+ (6·9 mm [3·5 to 10·4]; difference 9·6 mm [95% CI 3·0 to 16·2], p=0·0010) and E-G- (7·4 mm [4·2 to 10·7]; difference 9·1 mm [2·7 to 15·6], p=0·0016), but not for E+G- (11·7 mm [8·3 to 15·1]; difference 4·9 mm [-1·7 to 11·5], p=0·28). There was no difference between E+G- and E-G+ (difference 4·7 mm [-1·8 to 11·3], p=0·33), E+G- and E-G- (difference 4·2 mm [-2·2 to 10·7], p=0·47), and E-G+ and E-G- (difference -0·5 mm [-7·0 to 5·9], p=1·0). Adverse events were reported by two participants in the E+G- group (itching jaw [n=1]; feeling lightheaded and stomach rumbling [n=1]) and one participant in the E-G+ group (vomiting). INTERPRETATION: The combination of expectancy and actual gluten intake had the largest effect on gastrointestinal symptoms, reflecting a nocebo effect, although an additional effect of gluten cannot be ruled out. Our results necessitate further research into the possible involvement of the gut-brain interaction in NCGS. FUNDING: Government of the Netherlands Topsector Agri & Food Top Consortium for Knowledge and Innovation, AB Mauri Global Bakery Ingredients, Baking Industry Research Trust, Borgesius-Albert Heijn, CSM Innovation Centre, the International Maize and Wheat Improvement Center (CIMMYT), DSM Food Specialties, Fazer, Healthgrain Forum, the International Association for Cereal Science and Technology, the International Wheat Gluten Association, Lantmännen, Mondelez International, Nederlands Bakkerij Centrum, Nutrition & Santé, Puratos, Rademaker, Sonneveld Group, and Zeelandia HJ Doeleman.

Promising Markers in the Context of Mesenchymal Stem/Stromal Cells Subpopulations with Unique Properties.

The heterogeneity of the mesenchymal stem/stromal cells (MSCs) population poses a challenge to researchers and clinicians, especially those observed at the population level. What is more, the lack of precise evidences regarding MSCs developmental origin even further complicate this issue. As the available evidences indicate several possible pathways of MSCs formation, this diverse origin may be reflected in the unique subsets of cells found within the MSCs population. Such populations differ in specialization degree, proliferation, and immunomodulatory properties or exhibit other additional properties such as increased angiogenesis capacity. In this review article, we attempted to identify such outstanding populations according to the specific surface antigens or intracellular markers. Described groups were characterized depending on their specialization and potential therapeutic application. The reports presented here cover a wide variety of properties found in the recent literature, which is quite scarce for many candidates mentioned in this article. Even though the collected information would allow for better targeting of specific subpopulations in regenerative medicine to increase the effectiveness of MSC-based therapies.

Corrigendum: Identification of volatile organic compounds in extremophilic bacteria and their effective use in biocontrol of postharvest fungal phytopathogens.

[This corrects the article DOI: 10.3389/fmicb.2021.773092.].

Maternal fatty acid status during pregnancy versus offspring inflammatory markers: a canonical correlation analysis of the MEFAB cohort.

The development of inflammatory lung disorders in children may be related to maternal fatty acid intake during pregnancy. We therefore examined maternal fatty acid (FA) status during pregnancy and its associations with inflammatory markers and lung conditions in the child by analyzing data from the MEFAB cohort using multivariate canonical correlation analysis (CCA). In the MEFAB cohort, 39 different phospholipid FAs were measured in maternal plasma at 16, 22 and 32 weeks of pregnancy, and at day of birth. Child inflammatory markers and self-reported doctor diagnosis of inflammatory lung disorders were assessed at 7 years of age. Using CCA, we found that maternal FA levels during pregnancy were significantly associated with child inflammatory markers at 7 years of age and that Mead acid (20:3n-9) was the most important FA for this correlation. To further verify the importance of Mead acid, we examined the relation between maternal Mead acid levels at the day of birth with the development of inflammatory lung disorders in children at age 7. After stratification for the child's sex, maternal Mead acid levels at day of birth were significantly related with self-reported doctor diagnosis of asthma and lung infections in boys, and bronchitis and total number of lung disorders in girls. Future studies should investigate whether the importance of Mead acid in the relation between maternal FA status and inflammation and lung disorders in the child is due to its role as biomarker for essential fatty acid deficiency or due to its own biological function as pro-inflammatory mediator.

The Detection of Primary Sclerosing Cholangitis Using Volatile Metabolites in Fecal Headspace and Exhaled Breath.

Up to 5% of inflammatory bowel disease patients may at some point develop primary sclerosing cholangitis (PSC). PSC is a rare liver disease that ultimately results in liver damage, cirrhosis and liver failure. It typically remains subclinical until irreversible damage has been inflicted. Hence, it is crucial to screen IBD patients for PSC, but its early detection is challenging, and the disease's etiology is not well understood. This current study aimed at the early detection of PSC in an IBD population using Volatile Organic Compounds in fecal headspace and exhaled breath. To this aim, fecal material and exhaled breath were collected from 73 patients (n = 16 PSC/IBD; n = 8 PSC; n = 49 IBD), and their volatile profile were analyzed using Gas Chromatography-Mass Spectrometry. Using the most discriminatory features, PSC detection resulted in areas under the ROC curve (AUCs) of 0.83 and 0.84 based on fecal headspace and exhaled breath, respectively. Upon data fusion, the predictive performance increased to AUC 0.92. The observed features in the fecal headspace relate to detrimental microbial dysbiosis and exogenous exposure. Future research should aim for the early detection of PSC in a prospective study design.

Exogenous Volatile Organic Compound (EVOC®) Breath Testing Maximizes Classification Performance for Subjects with Cirrhosis and Reveals Signs of Portal Hypertension.

Background: Cirrhosis detection in primary care relies on low-performing biomarkers. Consequently, up to 75% of subjects with cirrhosis receive their first diagnosis with decompensation when causal treatments are less effective at preserving liver function. We investigated an unprecedented approach to cirrhosis detection based on dynamic breath testing. Methods: We enrolled 29 subjects with cirrhosis (Child-Pugh A and B), and 29 controls. All subjects fasted overnight. Breath samples were taken using Breath Biopsy® before and at different time points after the administration of 100 mg limonene. Absolute limonene breath levels were measured using gas chromatography-mass spectrometry. Results: All subjects showed a >100-fold limonene spike in breath after administration compared to baseline. Limonene breath kinetics showed first-order decay in >90% of the participants, with higher bioavailability in the cirrhosis group. At the Youden index, baseline limonene levels showed classification performance with an area under the roc curve (AUROC) of 0.83 ± 0.012, sensitivity of 0.66 ± 0.09, and specificity of 0.83 ± 0.07. The best performing timepoint post-administration was 60 min, with an AUROC of 0.91, sensitivity of 0.83 ± 0.07, and specificity of 0.9 ± 0.06. In the cirrhosis group, limonene bioavailability showed a correlation with MELD and fibrosis indicators, and was associated with signs of portal hypertension. Conclusions: Dynamic limonene breath testing enhances diagnostic performance for cirrhosis compared to static testing. The correlation with disease severity suggests potential for monitoring therapeutic interventions. Given the non-invasive nature of breath collection, a dynamic limonene breath test could be implemented in primary care.

Breath Biopsy® to Identify Exhaled Volatile Organic Compounds Biomarkers for Liver Cirrhosis Detection.

Background and Aims: The prevalence of chronic liver disease in adults exceeds 30% in some countries and there is significant interest in developing tests and treatments to help control disease progression and reduce healthcare burden. Breath is a rich sampling matrix that offers non-invasive solutions suitable for early-stage detection and disease monitoring. Having previously investigated targeted analysis of a single biomarker, here we investigated a multiparametric approach to breath testing that would provide more robust and reliable results for clinical use. Methods: To identify candidate biomarkers we compared 46 breath samples from cirrhosis patients and 42 from controls. Collection and analysis used Breath Biopsy OMNI™, maximizing signal and contrast to background to provide high confidence biomarker detection based upon gas chromatography mass spectrometry (GC-MS). Blank samples were also analyzed to provide detailed information on background volatile organic compounds (VOCs) levels. Results: A set of 29 breath VOCs differed significantly between cirrhosis and controls. A classification model based on these VOCs had an area under the curve (AUC) of 0.95±0.04 in cross-validated test sets. The seven best performing VOCs were sufficient to maximize classification performance. A subset of 11 VOCs was correlated with blood metrics of liver function (bilirubin, albumin, prothrombin time) and separated patients by cirrhosis severity using principal component analysis. Conclusions: A set of seven VOCs consisting of previously reported and novel candidates show promise as a panel for liver disease detection and monitoring, showing correlation to disease severity and serum biomarkers at late stage.

Simultaneous analysis of plasma and CSF by NMR and hierarchical models fusion.

Because cerebrospinal fluid (CSF) is the biofluid which interacts most closely with the central nervous system, it holds promise as a reporter of neurological disease, for example multiple sclerosis (MScl). To characterize the metabolomics profile of neuroinflammatory aspects of this disease we studied an animal model of MScl-experimental autoimmune/allergic encephalomyelitis (EAE). Because CSF also exchanges metabolites with blood via the blood-brain barrier, malfunctions occurring in the CNS may be reflected in the biochemical composition of blood plasma. The combination of blood plasma and CSF provides more complete information about the disease. Both biofluids can be studied by use of NMR spectroscopy. It is then necessary to perform combined analysis of the two different datasets. Mid-level data fusion was therefore applied to blood plasma and CSF datasets. First, relevant information was extracted from each biofluid dataset by use of linear support vector machine recursive feature elimination. The selected variables from each dataset were concatenated for joint analysis by partial least squares discriminant analysis (PLS-DA). The combined metabolomics information from plasma and CSF enables more efficient and reliable discrimination of the onset of EAE. Second, we introduced hierarchical models fusion, in which previously developed PLS-DA models are hierarchically combined. We show that this approach enables neuroinflamed rats (even on the day of onset) to be distinguished from either healthy or peripherally inflamed rats. Moreover, progression of EAE can be investigated because the model separates the onset and peak of the disease.

Quantitative proteomics and metabolomics analysis of normal human cerebrospinal fluid samples.

The analysis of cerebrospinal fluid (CSF) is used in biomarker discovery studies for various neurodegenerative central nervous system (CNS) disorders. However, little is known about variation of CSF proteins and metabolites between patients without neurological disorders. A baseline for a large number of CSF compounds appears to be lacking. To analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples of patients undergoing routine, non-neurological surgical procedures, we determined the variation of various proteins and metabolites by multiple analytical platforms. A total of 126 common proteins were assessed for biological variations between individuals by ESI-Orbitrap. A large spread in inter-individual variation was observed (relative standard deviations [RSDs] ranged from 18 to 148%) for proteins with both high abundance and low abundance. Technical variation was between 15 and 30% for all 126 proteins. Metabolomics analysis was performed by means of GC-MS and nuclear magnetic resonance (NMR) imaging and amino acids were specifically analyzed by LC-MS/MS, resulting in the detection of more than 100 metabolites. The variation in the metabolome appears to be much more limited compared with the proteome: the observed RSDs ranged from 12 to 70%. Technical variation was less than 20% for almost all metabolites. Consequently, an understanding of the biological variation of proteins and metabolites in CSF of neurologically normal individuals appears to be essential for reliable interpretation of biomarker discovery studies for CNS disorders because such results may be influenced by natural inter-individual variations. Therefore, proteins and metabolites with high variation between individuals ought to be assessed with caution as candidate biomarkers because at least part of the difference observed between the diseased individuals and the controls will not be caused by the disease, but rather by the natural biological variation between individuals.

Reproducibility and reporting, the routes to progress in breath research-highlights from the Breath Biopsy Conference 2021.

With the continued presence of COVID-19 worldwide, it has been a challenge for the breath research community to progress with clinical studies and travel restrictions have also limited the opportunities to meet up, share ideas and celebrate the latest advances. The Breath Biopsy Conference 2021 offered the chance to catch up with the latest breath research and to share progress that researchers in the community have been able to make in these difficult times. Limited opportunities for clinical research have led many in the field to look more closely at different methods for breath collection and have contributed to the growing calls for consistent standards in how results are reported, shared and even how breath studies themselves are carried out. As such, standardization was a key theme for this year's event and featured prominently in the keynotes, discussions and throughout many of the presentations. With over 900 registrants, almost 400 live attendees and 16 speakers, the Breath Biopsy Conference continues to bring together breath research leaders from around the world. This article provides an overview of the highlights from this event.

Changes of breath volatile organic compounds in healthy volunteers following segmental and inhalation endotoxin challenge.

It is still unclear how airway inflammation affects the breath volatile organic compounds (VOCs) profile in exhaled air. We therefore analyzed breath following well-defined pulmonary endotoxin (lipopolysaccharide, LPS) challenges. Breath was collected from ten healthy non-smoking subjects at eight time points before and after segmental and whole lung LPS inhalation challenge. Four Tenax-TA® adsorption tubes were simultaneously loaded from an aluminum reservoir cylinder and independently analyzed by two research groups using gas chromatography-mass spectrometry. Airway inflammation was assessed in bronchoalveolar lavage (BAL) and in sputum after segmental and inhaled LPS challenge, respectively. Segmental LPS challenge significantly increased the median (interquartile range, IQR) percentage of neutrophils in BAL from 3.0 (4.2) % to 64.0 (7.3) %. The inhalation challenge increased sputum neutrophils from 33.9 (26.8) % to 78.3 (13.5) %. We observed increases in breath aldehydes at both time points after segmental and inhaled LPS challenge. These results were confirmed by an independent laboratory. The longitudinal breath analysis also revealed distinct VOC patterns related to environmental exposures, clinical procedures, and to metabolic changes after food intake. Changes in breath aldehydes suggest a relationship to LPS induced inflammation compatible with lipid peroxidation processes within the lung. Findings from our longitudinal data highlight the need for future studies to better consider the potential impact of the multiple VOCs from detergents, hygiene or lifestyle products a subject is continuously exposed to. We suspect that this very individual 'owncloud' exposure is contributing to an increased variability of breath aldehydes, which might limit a use as inflammatory markers in daily clinical practice.

Exhaled Breath Analysis for Investigating the Use of Inhaled Corticosteroids and Corticosteroid Responsiveness in Wheezing Preschool Children.

Exhaled breath analysis has great potential in diagnosing various respiratory and non-respiratory diseases. In this study, we investigated the influence of inhaled corticosteroids (ICS) on exhaled volatile organic compounds (VOCs) of wheezing preschool children. Furthermore, we assessed whether exhaled VOCs could predict a clinical steroid response in wheezing preschool children. We performed a crossover 8-week ICS trial, in which 147 children were included. Complete data were available for 89 children, of which 46 children were defined as steroid-responsive. Exhaled VOCs were measured by GC-tof-MS. Statistical analysis by means of Random Forest was used to investigate the effect of ICS on exhaled VOCs. A set of 20 VOCs could best discriminate between measurements before and after ICS treatment, with a sensitivity of 73% and specificity of 67% (area under ROC curve = 0.72). Most discriminative VOCs were branched C11H24, butanal, octanal, acetic acid and methylated pentane. Other VOCs predominantly included alkanes. Regularised multivariate analysis of variance (rMANOVA) was used to determine treatment response, which showed a significant effect between responders and non-responders (p < 0.01). These results show that ICS significantly altered the exhaled breath profiles of wheezing preschool children, irrespective of clinical treatment response. Furthermore, exhaled VOCs were capable of determining corticosteroid responsiveness in wheezing preschool children.

Systematic Review: Contribution of the Gut Microbiome to the Volatile Metabolic Fingerprint of Colorectal Neoplasia.

Colorectal cancer (CRC) has been associated with changes in volatile metabolic profiles in several human biological matrices. This enables its non-invasive detection, but the origin of these volatile organic compounds (VOCs) and their relation to the gut microbiome are not yet fully understood. This systematic review provides an overview of the current understanding of this topic. A systematic search using PubMed, Embase, Medline, Cochrane Library, and the Web of Science according to PRISMA guidelines resulted in seventy-one included studies. In addition, a systematic search was conducted that identified five systematic reviews from which CRC-associated gut microbiota data were extracted. The included studies analyzed VOCs in feces, urine, breath, blood, tissue, and saliva. Eight studies performed microbiota analysis in addition to VOC analysis. The most frequently reported dysregulations over all matrices included short-chain fatty acids, amino acids, proteolytic fermentation products, and products related to the tricarboxylic acid cycle and Warburg metabolism. Many of these dysregulations could be related to the shifts in CRC-associated microbiota, and thus the gut microbiota presumably contributes to the metabolic fingerprint of VOC in CRC. Future research involving VOCs analysis should include simultaneous gut microbiota analysis.