New perspectives on 'Breathomics': metabolomic profiling of non-volatile organic compounds in exhaled breath using DI-FT-ICR-MS.

Breath analysis offers tremendous potential for diagnostic approaches, since it allows for easy and non-invasive sample collection. "Breathomics" as one major research field comprehensively analyses the metabolomic profile of exhaled breath providing insights into various (patho)physiological processes. Recent research, however, primarily focuses on volatile compounds. This is the first study that evaluates the non-volatile organic compounds (nVOCs) in breath following an untargeted metabolomic approach. Herein, we developed an innovative method utilizing a filter-based device for metabolite extraction. Breath samples of 101 healthy volunteers (female n = 50) were analysed using DI-FT-ICR-MS and biostatistically evaluated. The characterisation of the non-volatile core breathome identified more than 1100 metabolites including various amino acids, organic and fatty acids and conjugates thereof, carbohydrates as well as diverse hydrophilic and lipophilic nVOCs. The data shows gender-specific differences in metabolic patterns with 570 significant metabolites. Male and female metabolomic profiles of breath were distinguished by a random forest approach with an out-of-bag error of 0.0099. Additionally, the study examines how oral contraceptives and various lifestyle factors, like alcohol consumption, affect the non-volatile breathome. In conclusion, the successful application of a filter-based device combined with metabolomics-analyses delineate a non-volatile breathprint laying the foundation for discovering clinical biomarkers in exhaled breath.

Reverse Mapping of Coarse Grained Polyglutamine Conformations from PRIME20 Sampling.

An inverse coarse-graining protocol is presented for generating and validating atomistic structures of large (bio-) molecules from conformations obtained via a coarse-grained sampling method. Specifically, the protocol is implemented and tested based on the (coarse-grained) PRIME20 protein model (P20/SAMC), and the resulting all-atom conformations are simulated using conventional biomolecular force fields. The phase space sampling at the coarse-grained level is performed with a stochastical approximation Monte Carlo approach. The method is applied to a series of polypeptides, specifically dimers of polyglutamine with varying chain length in aqueous solution. The majority (>70 %) of the conformations obtained from the coarse-grained peptide model can successfully be mapped back to atomistic structures that remain conformationally stable during 10 ns of molecular dynamics simulations. This work can be seen as the first step towards the overarching goal of improving our understanding of protein aggregation phenomena through simulation methods.

Reduction of Hydrogen Peroxide by Human Mitochondrial Amidoxime Reducing Component Enzymes.

The mitochondrial amidoxime reducing component (mARC) is a human molybdoenzyme known to catalyze the reduction of various N-oxygenated substrates. The physiological function of mARC enzymes, however, remains unknown. In this study, we examine the reduction of hydrogen peroxide (H2O2) by the human mARC1 and mARC2 enzymes. Furthermore, we demonstrate an increased sensitivity toward H2O2 for HEK-293T cells with an MTARC1 knockout, which implies a role of mARC enzymes in the cellular response to oxidative stress. H2O2 is a reactive oxygen species (ROS) formed in all living cells involved in many physiological processes. Furthermore, H2O2 constitutes the first mARC substrate without a nitrogen-oxygen bond, implying that mARC enzymes may have a substrate spectrum going beyond the previously examined N-oxygenated compounds.

Detection of SARS-CoV-2 RNA in exhaled breath and its potential for prevention measures.

Background: To propose infection prevention measures it is essential to understand the dynamics of SARS-CoV-2 shedding, particularly in asymptomatic patients. This report compares the viral load progression in exhaled breath (EB) with the symptom severity. We aim to evaluate the adequacy of symptom assessment regarding the infectivity level of individuals. Methods: We observed infected patients since their first positive test during hospitalization. EB samples were collected on days 1, 3, 5, 7, 10, 12 and 14 of hospitalization using a filter-based device. After extraction, viral loads were quantified with qRT-PCR. The infection trajectory was documented after symptom onset. Case Presentation and Discussion: A 34-year old patient showed mild symptoms, e.g. fever, cough, headache, muscle pain and loss of taste and smell across trajectory of infection (Case 1). The viral loads emitted via exhaling were nearly constant and ranged from 8.6 x 103 and 4.1 x 104 RNA copies per hour. After the infection, the patient developed a pneumonia. The second case of a 65-year old patient depicted an asymptomatic infection trajectory for 14 days after the first diagnosis (Case 2). Nevertheless, the patient exhaled up to 2 x 105 SARS-CoV-2 virus copies hourly, approximately 10 fold higher than measured for Case 1. Conclusion: Symptomatic and asymptomatic COVID-19 patients exhale distinctive amounts of SARS-CoV-2 not necessarily correlating with symptom severity. Particularly, asymptomatic patients might show higher EB viral shedding. Therefore, EB testing should be included in infection prevention measures as it has high potential to reveal the most infectious individuals regardless of their symptoms during infection.

Ultrafast Proton Transfer Pathways Mediated by Amphoteric Imidazole.

Imidazole, being an amphoteric molecule, can act both as an acid and as a base. This property enables imidazole, as an essential building block, to effectively facilitate proton transport in high-temperature proton exchange membrane fuel cells and in proton channel transmembrane proteins, enabling those systems to exhibit high energy conversion yields and optimal biological function. We explore the amphoteric properties of imidazole by following the proton transfer exchange reaction dynamics with the bifunctional photoacid 7-hydroxyquinoline (7HQ). We show with ultrafast ultraviolet-mid-infrared pump-probe spectroscopy how for imidazole, in contrast to expectations based on textbook knowledge of acid-base reactivity, the preferential reaction pathway is that of an initial proton transfer from 7HQ to imidazole, and only at a later stage a transfer from imidazole to 7HQ, completing the 7HQ tautomerization reaction. An assessment of the molecular distribution functions and first-principles calculations of proton transfer reaction barriers reveal the underlying reasons for our observations.

Combined physicochemical and functional assessment of pertuzumab integrity supports extended in-use stability.

Pertuzumab (Perjeta®) is a monoclonal antibody approved for the treatment of HER2-positive breast cancer. Before treatment, the concentrate must be diluted to obtain the ready-to-use infusion solution. Data on the storage stabilities of these preparations are lacking but important for all healthcare professionals in the area of outpatient chemotherapy. The aim of this study was to investigate the storage stability of the ready-to-use infusion bags and the concentrates from once-opened vials over a period of up to 42 days. For a comprehensive and unambiguous assessment of pertuzumab's integrity, a panel of orthogonal analytical methods was employed, including a newly established mass spectrometry-based peptide mapping procedure along with a reporter gene assay for monitoring cellular bioactivity. The herein presented data showed that the ready-to-use infusion solutions stored at 4 ± 2°C and at 20 ± 3°C without light protection, as well as the undiluted Perjeta® concentrates stored at 4 ± 2°C, were physicochemically stable and biologically active for 28 days. These results might eventually allow for infusion preparations in advance, thus improving the quality of patient care as well as the economic usage of pertuzumab.

Polycapillary-boosted instrument performance in the extreme ultraviolet regime for inverse photoemission spectroscopy: erratum.

We correct values and figures for the resolution of the spectrometer, as proposed in [Opt. Express25, 31840 (2017)10.1364/OE.25.031840OPEXFF1094-4087]. The new results take into account previously unknown, incoherent phase fluctuations, caused by the polycapillary lens (PCL), and estimate the realistic performance of the instrument.

SARS-CoV-2: Viral Loads of Exhaled Breath and Oronasopharyngeal Specimens in Hospitalized Patients with COVID-19.

BACKGROUND: SARS-CoV-2 seems mainly transmissible via respiratory droplets. We compared the time-dependent SARS-CoV-2 viral load in serial pharyngeal swab with exhaled breath (EB) samples of hospitalized COVID-19 patients. METHODS: In this prospective proof of concept study, we examined hospitalized patients who initially tested positive for SARS-CoV-2. Paired oronasopharyngeal swab and EB specimens were taken at different days of hospitalization. EB collection was performed through a simple, noninvasive method using an electret air filter-based device. SARS-CoV-2 RNA detection was determined with real-time quantitative reverse transcription polymerase chain reaction. RESULTS: Of 187 serial samples from 15 hospitalized patients, 87/87 oronasopharyngeal swabs and 70/100 EB specimens tested positive. Comparing the number of SARS-CoV-2 copies, the viral load of the oronasopharyngeal swabs was significantly higher (CI 99%, P<<0,001) than for EB samples. The mean viral load per swab was 7.97 × 106 (1.65 × 102-1.4 × 108), whereas EB samples showed 2.47 × 103 (7.19 × 101-2.94 × 104) copies per 20 times exhaling. Viral loads of paired oronasopharyngeal swab and EB samples showed no correlation. CONCLUSIONS: Assessing the infectiousness of COVID-19 patients merely through pharyngeal swabs might not be accurate. Exhaled breath could represent a more suitable matrix for evaluating infectiousness and might allow screening for superspreader individuals and widespread variants such as Delta.

Impact of SnF2 Addition on the Chemical and Electronic Surface Structure of CsSnBr3.

We report on the chemical and electronic structure of cesium tin bromide (CsSnBr3) and how it is impacted by the addition of 20 mol % tin fluoride (SnF2) to the precursor solution, using both surface-sensitive lab-based soft X-ray photoelectron spectroscopy (XPS) and near-surface bulk-sensitive synchrotron-based hard XPS (HAXPES). To determine the reproducibility and reliability of conclusions, several (nominally identically prepared) sample sets were investigated. The effects of deposition reproducibility, handling, and transport are found to cause significant changes in the measured properties of the films. Variations in the HAXPES-derived compositions between individual sample sets were observed, but in general, they confirm that the addition of 20 mol % SnF2 improves coverage of the titanium dioxide substrate by CsSnBr3 and decreases the oxidation of SnII to SnIV while also suppressing formation of secondary Br and Cs species. Furthermore, the (surface) composition is found to be Cs-deficient and Sn-rich compared to the nominal stoichiometry. The valence band (VB) shows a SnF2-induced redistribution of Sn 5s-derived density of states, reflecting the changing SnII/SnIV ratio. Notwithstanding some variability in the data, we conclude that SnF2 addition decreases the energy difference between the VB maximum of CsSnBr3 and the Fermi level, which we explain by defect chemistry considerations.

Drug Metabolism by the Mitochondrial Amidoxime Reducing Component (mARC): Rapid Assay and Identification of New Substrates.

For the development of new drugs, the investigation of their metabolism is of central importance. In the past, the focus was mostly on the consideration of established enzymes leading to oxidations such as cytochrome P450. However, reductive metabolism by the mARC enzyme system can play an important role in particular for nitrogen containing functional groups. A rapid test was established to give developers of new drugs in the preclinical stage the opportunity to test the metabolism by mARC. To demonstrate the relevance and validity of the new test system, known and potential substrates were applied to this new assay. All known substrates could be detected by the system. Furthermore, several new substrates were found including long-established drugs such as hydroxyurea and new compounds in development such as epacdadostat.

Alkali Postdeposition Treatment-Induced Changes of the Chemical and Electronic Structure of Cu(In,Ga)Se2 Thin-Film Solar Cell Absorbers: A First-Principle Perspective.

The effects of alkali postdeposition treatment (PDT) on the valence band structure of Cu(In,Ga)Se2 (CIGSe) thin-film solar cell absorbers are addressed from a first-principles perspective. In detail, experimentally derived hard X-ray photoelectron spectroscopy (HAXPES) data [ Handick , E. ; ACS Appl. Mater. Interfaces 2015 , 7 , 27414 - 27420 ] of the valence band structure of alkali-free and NaF/KF-PDT CIGSe are directly compared and fit by calculated density of states (DOS) of CuInSe2, its Cu-deficient counterpart CuIn5Se8, and different potentially formed secondary phases, such as KInSe2, InSe, and In2Se3. The DOSs are based on first-principles electronic structure calculations and weighted according to element-, symmetry-, and energy-dependent photoionization cross sections for the comparison to experimental data. The HAXPES spectra were recorded using photon energies ranging from 2 to 8 keV, allowing extraction of information from different sample depths. The analysis of the alkali-free CIGSe valence band structure reveals that it can best be described by a mixture of the DOS of CuInSe2 and CuIn5Se8, resulting in a stoichiometry slightly more Cu-rich than that of CuIn3Se5. The NaF/KF-PDT-induced changes in the HAXPES spectra for different alkali exposures are best reproduced by additional contributions from KInSe2, with some indications that the formation of a pronounced K-In-Se-type surface species might crucially depend on the amount of K available during PDT.

[Survival increases for all cancer diagnoses - but inequality persists]. / Överlevnaden ökar vid all cancer ­ men ojämlikheten kvarstår - Konceptet ¼rätt resurser med rätt åtgärd till rätt patient vid rätt tillfälle« måste få genomslag.

New data show a continuously increased five-year survival for almost all analyzed cancer diagnoses since 1990. It has to be emphasized that the figures are uncertain due to the limited number of patients. The variation is huge and the greatest improvements are seen not least among the three major tumor diseases (breast, colorectal and prostate cancer), where the society, industry and research bodies made the biggest investments over the years. The causes of improved survival can be sought in several areas, such as earlier detection and better treatments. In addition to survival estimates, it is also always of importance to consider aspects around patient related outcome, such as quality of life.

The antibiotic resistome and microbiota landscape of refugees from Syria, Iraq and Afghanistan in Germany.

BACKGROUND: Multidrug-resistant bacteria represent a substantial global burden for human health, potentially fuelled by migration waves: in 2015, 476,649 refugees applied for asylum in Germany mostly as a result of the Syrian crisis. In Arabic countries, multiresistant bacteria cause significant problems for healthcare systems. Currently, no data exist describing antibiotic resistances in healthy refugees. Here, we assess the microbial landscape and presence of antibiotic resistance genes (ARGs) in refugees and German controls. To achieve this, a systematic study was conducted in 500 consecutive refugees, mainly from Syria, Iraq, and Afghanistan and 100 German controls. Stool samples were subjected to PCR-based quantification of 42 most relevant ARGs, 16S ribosomal RNA gene sequencing-based microbiota analysis, and culture-based validation of multidrug-resistant microorganisms. RESULTS: The fecal microbiota of refugees is substantially different from that of resident Germans. Three categories of resistance profiles were found: (i) ARGs independent of geographic origin of individuals comprising BIL/LAT/CMA, ErmB, and mefE; (ii) vanB with a high prevalence in Germany; and (iii) ARGs showing substantially increased prevalences in refugees comprising CTX-M group 1, SHV, vanC1, OXA-1, and QnrB. The majority of refugees carried five or more ARGs while the majority of German controls carried three or less ARGs, although the observed ARGs occurred independent of signatures of potential pathogens. CONCLUSIONS: Our results, for the first time, assess antibiotic resistance genes in refugees and demonstrate a substantially increased prevalence for most resistances compared to German controls. The antibiotic resistome in refugees may thus require particular attention in the healthcare system of host countries.

Polycapillary-boosted instrument performance in the extreme ultraviolet regime for inverse photoemission spectroscopy.

A collimating polycapillary half lens, traditionally used in the medium and hard X-ray band, is operated at a photon energy of 36 eV for the first time. While the transmission still exceeds 50%, the measured and simulated spatial resolution and angular divergence approach 0.4 mm or less and at most 20 mrad, respectively. This unexpected, superior performance of the polycapillary optic in the extreme Ultraviolet could enable the design of an efficient, versatile and compact spectrometer for inverse photoemission spectroscopy (IPES): Its wavelength-dispersive component, a customized reflection zone plate, can maintain an energy resolution of 0.3 eV, whereas the sensitivity may be enhanced by more than one order of magnitude, compared to conventional spectrometers. Furthermore, the overall length of 0.9 m would allow for an eased alignment and evacuation. We see a significant potential for numerous polycapillary-based XUV / soft X-ray instruments in the future, in particular after further optimization for this long wavelength regime.

Evidence for Chemical and Electronic Nonuniformities in the Formation of the Interface of RbF-Treated Cu(In,Ga)Se2 with CdS.

We report on the initial stages of CdS buffer layer formation on Cu(In,Ga)Se2 (CIGSe) thin-film solar cell absorbers subjected to rubidium fluoride (RbF) postdeposition treatment (PDT). A detailed characterization of the CIGSe/CdS interface for different chemical bath deposition (CBD) times of the CdS layer is obtained from spatially resolved atomic and Kelvin probe force microscopy and laterally integrating X-ray spectroscopies. The observed spatial inhomogeneity in the interface's structural, chemical, and electronic properties of samples undergoing up to 3 min of CBD treatments is indicative of a complex interface formation including an incomplete coverage and/or nonuniform composition of the buffer layer. It is expected that this result impacts solar cell performance, in particular when reducing the CdS layer thickness (e.g., in an attempt to increase the collection in the ultraviolet wavelength region). Our work provides important findings on the absorber/buffer interface formation and reveals the underlying mechanism for limitations in the reduction of the CdS thickness, even when an alkali PDT is applied to the CIGSe absorber.

Formation of a K-In-Se Surface Species by NaF/KF Postdeposition Treatment of Cu(In,Ga)Se2 Thin-Film Solar Cell Absorbers.

A NaF/KF postdeposition treatment (PDT) has recently been employed to achieve new record efficiencies of Cu(In,Ga)Se2 (CIGSe) thin film solar cells. We have used a combination of depth-dependent soft and hard X-ray photoelectron spectroscopy as well as soft X-ray absorption and emission spectroscopy to gain detailed insight into the chemical structure of the CIGSe surface and how it is changed by different PDTs. Alkali-free CIGSe, NaF-PDT CIGSe, and NaF/KF-PDT CIGSe absorbers grown by low-temperature coevaporation have been interrogated. We find that the alkali-free and NaF-PDT CIGSe surfaces both display the well-known Cu-poor CIGSe chemical surface structure. The NaF/KF-PDT, however, leads to the formation of bilayer structure in which a K-In-Se species covers the CIGSe compound that in composition is identical to the chalcopyrite structure of the alkali-free and NaF-PDT absorber.

Physicochemical compatibility and emulsion stability of propofol with commonly used analgesics and sedatives in an intensive care unit.

OBJECTIVES: The purpose of this study was the determination of the physicochemical compatibility and emulsion stability of propofol with other sedatives and analgesics (clonidine hydrochloride, dexmedetomidine, 4-hydroxybutyric acid, (S)-ketamine, lormetazepam, midazolam hydrochloride, piritramide, remifentanil hydrochloride and sufentanil citrate) that are frequently administered together intravenously. METHODS: Drugs were mixed with propofol and stored without light protection at room temperature. Samples were taken at 10 points of time over 7 days. The physical stability and emulsion stability in particular were analysed by visual and microscopical inspection and by measurement of the pH value, zeta potential and globule size distribution. In addition, high-performance liquid chromatography and mass spectrometry were used to identify chemical incompatibilities. RESULTS: 4-Hydroxybutyric acid, midazolam hydrochloride, piritramide and remifentanil hydrochloride are physically incompatible when mixed with propofol. The reason for this is the development of an increased fraction of oil droplets >5 µm leading to a higher risk of emboli. Moreover, propofol is chemically incompatible with remifentanil. The sorption of propofol to the rubber stopper of the syringe was another detectable incompatibility. CONCLUSIONS: Propofol should not be administered with 4-hydroxybutyric acid, remifentanil hydrochloride, midazolam hydrochloride and piritramide through the same intravenous line. Based on the risk of sorption to the rubber material, propofol should be used with caution. A drug loss might occur that leads to an underdosing of the patient requiring a dose adjustment to avoid any adverse consequences. As a result of this study, the drug safety in intensive care units could be optimised.

The pivotal role of the mitochondrial amidoxime reducing component 2 in protecting human cells against apoptotic effects of the base analog N6-hydroxylaminopurine.

N-Hydroxylated nucleobases and nucleosides as N-hydroxylaminopurine (HAP) or N-hydroxyadenosine (HAPR) may be generated endogenously in the course of cell metabolism by cytochrome P450, by oxidative stress or by a deviating nucleotide biosynthesis. These compounds have shown to be toxic and mutagenic for procaryotic and eucaryotic cells. For DNA replication fidelity it is therefore of great importance that organisms exhibit effective mechanisms to remove such non-canonical base analogs from DNA precursor pools. In vitro, the molybdoenzymes mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) have shown to be capable of reducing N-hydroxylated base analogs and nucleoside analogs to the corresponding canonical nucleobases and nucleosides upon reconstitution with the electron transport proteins cytochrome b5 and NADH-cytochrome b5 reductase. By RNAi-mediated down-regulation of mARC in human cell lines the mARC-dependent N-reductive detoxication of HAP in cell metabolism could be demonstrated. For HAPR, on the other hand, the reduction to adenosine seems to be of less significance in the detoxication pathway of human cells as HAPR is primarily metabolized to inosine by direct dehydroxylamination catalyzed by adenosine deaminase. Furthermore, the effect of mARC knockdown on sensitivity of human cells to HAP was examined by flow cytometric quantification of apoptotic cell death and detection of poly (ADP-ribose) polymerase (PARP) cleavage. mARC2 was shown to protect HeLa cells against the apoptotic effects of the base analog, whereas the involvement of mARC1 in reductive detoxication of HAP does not seem to be pivotal.

Reduction of sulfamethoxazole hydroxylamine (SMX-HA) by the mitochondrial amidoxime reducing component (mARC).

Under high dose treatment with sulfamethoxazole (SMX)/trimethoprim (TMP), hypersensitivity reactions occur with a high incidence. The mechanism of this adverse drug reaction is not fully understood. Several steps in the toxification pathway of SMX were investigated. The aim of our study was to investigate the reduction of sulfamethoxazole hydroxylamine (SMX-HA) in this toxification pathway, which can possibly be catalyzed by the mARC-containing N-reductive enzyme system. Western blot analyses of subcellular fractions of porcine tissue were performed with antibodies against mARC-1, mARC-2, cytochrome b5 type B, and NADH cytochrome b5 reductase. Incubations of porcine and human subcellular tissue fractions and of the heterologously expressed human components of the N-reductive enzyme system were carried out with SMX-HA. mARC-1 and mARC-2 knockdown was performed in HEK-293 cells. Kinetic parameters of the heterologously expressed human protein variants V96L, A165T, M187 K, C246S, D247H, and M268I of mARC-1 and G244S and C245W of mARC-2 and N-reductive activity of 2SF, D14G, K16E, and T22A of cytochrome b5 type B were analyzed. Western blot analyses were consistent with the hypothesis that the mARC-containing N-reductive enzyme system might be involved in the reduction of SMX-HA. In agreement with these results, highest reduction rates were found in mitochondrial subcellular fractions of porcine tissue and in the outer membrane vesicle (OMV) of human liver tissue. Knockdown studies in HEK-293 cells demonstrated that mARC-1 and mARC-2 were capable of reducing SMX-HA in cell metabolism. Investigations with the heterologously expressed human mARC-2 protein showed a higher catalytic efficiency toward SMX-HA than mARC-1, but none of the investigated human protein variants showed statistically significant differences of its N-reductive activity and was therefore likely to participate in the pathogenesis of hypersensitivity reaction under treatment with SMX.

Functional characterization of protein variants encoded by nonsynonymous single nucleotide polymorphisms in MARC1 and MARC2 in healthy Caucasians.

Human molybdenum-containing enzyme mitochondrial amidoxime reducing component (mARC), cytochrome b5 type B, and NADH cytochrome b5 reductase form an N-reductive enzyme system that is capable of reducing N-hydroxylated compounds. Genetic variations are known, but their functional relevance is unclear. Our study aimed to investigate the incidence of nonsynonymous single nucleotide polymorphisms (SNPs) in the mARC genes in healthy Caucasian volunteers, to determine saturation of the protein variants with molybdenum cofactor (Moco), and to characterize the kinetic behavior of the protein variants by in vitro biotransformation studies. Genotype frequencies of six SNPs in the mARC genes (c.493A>G, c.560T>A, c.736T>A, and c.739G>C in MARC1; c.730G>A and c.735T>G in MARC2) were determined by pyrosequencing in a cohort of 340 healthy Caucasians. Protein variants were expressed in Escherichia coli. Saturation with Moco was determined by measurement of molybdenum by inductively coupled mass spectrometry. Steady state assays were performed with benzamidoxime. The six variants were of low frequency in this Caucasian population. Only one homozygous variant (c.493A; MARC1) was detected. All protein variants were able to bind Moco. Steady state assays showed statistically significant decreases of catalytic efficiency values for the mARC-2 wild type compared with the mARC-1 wild type (P < 0.05) and for two mARC-2 variants compared with the mARC-2 wild type (G244S, P < 0.05; C245W, P < 0.05). After simultaneous substitution of more than two amino acids in the mARC-1 protein, N-reductive activity was decreased 5-fold. One homozygous variant of MARC1 was detected in our sample. The encoded protein variant (A165T) showed no different kinetic parameters in the N-reduction of benzamidoxime.