Pursuing breath research in unprecedented circumstances-report from the Breath Biopsy Conference 2020.

The global outbreak of Sars-CoV-2 commencing early in 2020 had a dramatic impact on breath research, imposing abrupt restrictions but also presenting unforeseen opportunities. Taking place against the background of the COVID-19 pandemic, the 2020 Breath Biopsy Conference provided the breath research community with a platform to showcase and discuss the latest findings, including COVID-19 related research. As with most conferences under the present circumstance, it differed from its predecessor meetings by shifting to a virtual format, but retained its broad scope and interactive nature. The conference centred on four key themes, featuring applications of volatile organic compounds, breath biomarkers for liver disease, study design and data analytics, and, notably this year, breath-based endeavours to detect COVID-19 infection. This meeting report summarizes the events of the conference and spotlights selected contributions.

Driving progress in exhaled breath biomarkers: Breath Biopsy Conference 2019.

November 2019 saw Cambridge, UK play host to the second Breath Biopsy Conference, a community-focused event aimed at sharing and supporting advancements in the collection and analysis of volatile organic compounds in exhaled breath. The event expanded upon the previous year's format, spanning two days and concluding with an expert panel discussion. Presentations covered detection, monitoring and precision medicine studies examining diseases including asthma, cirrhosis, cancer and tuberculosis. The meeting attracted representatives from diverse backgrounds, such as metabolomics, artificial intelligence, clinical research and chemical analysis. This meeting report offers an overview of what was presented and discussed during the conference.

Towards standardization in the analysis of breath gas volatiles.

Despite growing interest and considerable progress in breath research over the last decade, standardized practices for the sampling and analysis of breath gas volatiles remain elusive. The primary reasons for this are (a) the rich chemical diversity of exhaled breath that covers an extensive range of volatile organic compounds at highly varied concentrations, (b) the vast disparity in the analytical tools employed, (c) diverse study goals and (d) the presence of (unidentified) confounders. These aspects place stringent but divergent demands on sampling and analysis: each analytical tool, target compound and concentration range requires its own specific protocol and in many cases the latter two are not even known a priori. The ongoing rapid developments and constant discoveries in the field of breath research and the lack of established best practices in breath gas sampling and analysis currently preclude an acceptable overall standardization of these methods. This paper addresses these manifold issues and suggests a framework that separately considers individual stages of sampling and analysis with a view to establishing standardization in the analysis of breath gas volatiles to suit different target compounds and analytical technologies.

First observation of a potential non-invasive breath gas biomarker for kidney function.

We report on the search for low molecular weight molecules-possibly accumulated in the bloodstream and body-in the exhaled breath of uremic patients with kidney malfunction. We performed non-invasive analysis of the breath gas of 96 patients shortly before and several times after kidney transplantation using proton-transfer-reaction mass spectrometry (PTR-MS), a very sensitive technique for detecting trace amounts of volatile organic compounds. A total of 642 individual breath analyses which included at least 41 different chemical components were carried out. Correlation analysis revealed one particular breath component with a molecular mass of 114 u (unified atomic mass units) that clearly correlated with blood serum creatinine, which is the currently accepted marker for assessing the function of the kidney. In particular, daily urine production showed good correlation with the identified breath marker. An independent set of seven samples taken from three patients at the onset of dialysis and three controls with normal kidney function confirmed a significant difference in concentration between patients and controls for a compound with a molecular mass of 114.1035 u using high mass resolving proton-transfer-reaction time-of-flight mass spectrometry (PTR-TOF-MS). A chemical composition of C7H14O was derived for the respective component. Fragmentation experiments on the same samples using proton-transfer-reaction triple-quadrupole tandem mass spectrometry (PTR-QqQ-MS) suggested that this breath marker is a C7-ketone or a branched C7-aldehyde. Non-invasive real-time monitoring of the kidney function via this breath marker could be a possible future procedure in the clinical setting.

Buffered end-tidal (BET) sampling-a novel method for real-time breath-gas analysis.

We present a novel method for real-time breath-gas analysis using mass-spectrometric techniques: buffered end-tidal (BET) on-line sampling. BET has several advantages over conventional direct on-line sampling where the subject inhales and exhales through a sampling tube. In our approach, a single exhalation is administered through a tailored tube in which the end-tidal fraction of the breath-gas sample is buffered. This increases sampling time by an order of magnitude to several seconds, improving signal quality and reducing the total measurement time per test subject. Furthermore, only one exhalation per minute is required for sampling and the test subject can otherwise maintain a normal breathing pattern, thereby reducing the risk of hyperventilation. To validate our new BET sampling method we conducted comparative measurements with direct on-line sampling using proton-transfer-reaction mass spectrometry. We find excellent agreement in measured acetone and acetonitrile concentrations. High variability observed in breath-by-breath isoprene concentrations is attributed to differences in exhalation depth and influences of hyperventilation on end-tidal concentrations.

On the use of Tedlar® bags for breath-gas sampling and analysis.

The storage capability of Tedlar® bags for gaseous compounds was assessed using on-line proton-transfer-reaction mass spectrometry (PTR-MS). Sample bags were filled with a mixture of volatile organic compounds (VOCs) at known quantities in the ppbv range. The test gas included alcohol, nitrile, aldehyde, ketone, terpene and aromatic compounds. PTR-MS enabled frequent bag-direct measurements of compound abundances over a 70 h storage period. Concentrations of all compounds decreased with bag storage time, with compound-specific decay rates. The most rapid decline in concentration levels was seen for water vapour in the bag, i.e. sample humidity. Such a decrease is particularly relevant for breath-gas samples, where water vapour content is high. Compound losses were attributed to a combination of adsorption to and diffusion through the bag walls. Storage property observations suggest that sample analyses made within 10 h of sampling offer adequate sample authenticity replication. Based on observations, an appropriate bag-cleaning procedure was established and assessed. Results indicated that acceptable bag cleanliness for breath-gas sampling is achievable.

Skeletal muscle stem cells express anti-apoptotic ErbB receptors during activation from quiescence.

To be effective for tissue repair, satellite cells (the stem cells of adult muscle) must survive the initial activation from quiescence. Using an in vitro model of satellite cell activation, we show that erbB1, erbB2 and erbB3, members of the EGF receptor tyrosine kinase family, appear on satellite cells within 6 h of activation. We show that signalling via erbB2 provides an anti-apoptotic survival mechanism for satellite cells during the first 24 h, as they progress to a proliferative state. Inhibition of erbB2 signalling with AG825 reduced satellite cell numbers, concomitant with elevated caspase-8 activation and TUNEL labelling of apoptotic satellite cells. In serum-free conditions, satellite cell apoptosis could be largely prevented by a mixture of erbB1, erbB3 and erbB4 ligand growth factors, but not by neuregulin alone (erbB3/erbB4 ligand). Furthermore, using inhibitors specific to discrete intracellular signalling pathways, we identify MEK as a pro-apoptotic mediator, and the erbB-regulated factor STAT3 as an anti-apoptotic mediator during satellite cell activation. These results implicate erbB2 signalling in the preservation of a full compliment of satellite cells as they activate in the context of a damaged muscle.

Pax7 distribution in human skeletal muscle biopsies and myogenic tissue cultures.

Demonstration of the importance of the paired box transcription factor Pax7 for the murine myosatellite cell population, with persistent expression in mature skeletal muscle, prompted us to investigate the distribution of Pax7 protein in biopsy samples of normal and pathological human skeletal limb muscle. Immunostaining for M-cadherin, an adhesion molecule present at the interface between myofibre and satellite cell, and the characteristic position adjacent to the muscle fibre and beneath the fibre's basement membrane were used to identify satellite cells. Anti-Pax7 reactivity was found in the majority of satellite cells but a small population was Pax7 negative. Neither could we identify Pax7-positive nuclei in freshly regenerating myotubes or in presumed myoblasts in these biopsies. Similarly, in myogenic cell cultures derived from the explantation of human foetal muscle Pax7 expression was low or undetectable at the proliferative myoblast stage but it became prominent in an increasing proportion of mononucleate cells after the induction of differentiation. This expression was, however, restricted to mononucleate cells; it did not persist into the differentiation stage of newly formed multinucleate myotubes. Despite this, in the biopsy samples, we occasionally found Pax7-positive nuclei in muscle fibres that seemed to be undergoing degenerative changes. Most of these were found to be the nuclei of cells engaged in focal regenerative processes, but Pax7 re-expression by myonuclei "in distress" cannot be ruled out entirely.

Myogenic cell proliferation and generation of a reversible tumorigenic phenotype are triggered by preirradiation of the recipient site.

Environmental influences have profound yet reversible effects on the behavior of resident cells. Earlier data have indicated that the amount of muscle formed from implanted myogenic cells is greatly augmented by prior irradiation (18 Gy) of the host mouse muscle. Here we confirm this phenomenon, showing that it varies between host mouse strains. However, it is unclear whether it is due to secretion of proliferative factors or reduction of antiproliferative agents. To investigate this further, we have exploited the observation that the immortal myogenic C2 C12 cell line forms tumors far more rapidly in irradiated than in nonirradiated host muscle. We show that the effect of preirradiation on tumor formation is persistent and dose dependent. However, C2 C12 cells are not irreversibly compelled to form undifferentiated tumor cells by the irradiated muscle environment and are still capable of forming large amounts of muscle when reimplanted into a nonirradiated muscle. In a clonal analysis of this effect, we discovered that C2 C12 cells have a bimodal propensity to form tumors; some clones form no tumors even after extensive periods in irradiated graft sites, whereas others rapidly form extensive tumors. This illustrates the subtle interplay between the phenotype of implanted cells and the factors in the muscle environment.