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Amino acid auxotrophies are prevalent among bacteria. They can govern ecological dynamics in microbial communities and indicate metabolic cross-feeding interactions among coexisting genotypes. Despite the ecological importance of auxotrophies, their distribution and impact on the diversity and function of the human gut microbiome remain poorly understood. This study performed the first systematic analysis of the distribution of amino acid auxotrophies in the human gut microbiome using a combined metabolomic, metagenomic, and metabolic modeling approach. Results showed that amino acid auxotrophies are ubiquitous in the colon microbiome, with tryptophan auxotrophy being the most common. Auxotrophy frequencies were found to be higher for those amino acids, that are also essential to the human host. Moreover, a higher overall abundance of auxotrophies was associated with greater microbiome diversity and stability, and the distribution of auxotrophs was found to be related to the human host’s metabolome, including trimethylamine oxide, small aromatic acids, and secondary bile acids. Thus, our results suggest that amino acid auxotrophies are important factors that contribute to microbiome ecology and host-microbiome metabolic interactions.

Insect biomass obtained from large-scale mass-rearing of insect larvae has gained considerable attention in recent years as an alternative and sustainable source of food and feed.

Occupational exposure to veterinary antibiotics in hen houses at poultry feeding farms was demonstrated by biomonitoring campaigns in the past. The objective of this study was to investigate pharmacokinetics of three uptake routes: dermal, oral and inhaled. In an open-label cross-over study, six healthy volunteers were exposed to single occupational relevant doses of enrofloxacin. Plasma and urine samples were analysed for enrofloxacin and ciprofloxacin. Physiologically based pharmacokinetic (PBPK) modelling based on bioanalysis data showed underestimation for the elimination rate in comparison to experimental data pointing towards a lack of sufficient ADME information and limitations of available physico-chemical properties of the parent drug. The data obtained in this study indicate that oral uptake with its various sources, e.g. airborne enrofloxacin, direct hand-mouth contact, is the major source for occupational exposure to enrofloxacin in hen houses. Dermal exposure was considered negligible.

Inhalation exposure to copper and its inorganic compounds can occur in various industrial sectors such as ore mining, production of electrical equipment etc. Toxic effects of copper and its oxides have been shown to be attributed to i. particle size and ii. dissolution/release of copper ions. The latter being dependend on the chemical composition of the copper particle and its size. Hence, in the context of occupational inhalation exposure to those compounds emphasis should not only be directed towards particle size but also to the copper species. We have developed an ICP-MS based method that allows quantitative distinction between copper oxides (CuxO) and metallic copper (mCu) upon sampling on mixed-cellulose ester (MCE) membrane filters. This approach is based on the different dissolution properties of CuxO and mCu in dilute acid. Systematic assessment of the approach in regard to filter load and extraction time have been conducted and successfully applied to field samples.

Ulcerating skin lesions are manifestations of human ISG15 deficiency, a type I interferonopathy. However, chronic inflammation may not be their exclusive cause. We describe two siblings with recurrent skin ulcers that healed with scar formation upon corticosteroid treatment. Both had a homozygous nonsense mutation in the ISG15 gene, leading to unstable ISG15 protein lacking the functional domain. We characterized ISG15(-/-) dermal fibroblasts, HaCaT keratinocytes, and human induced pluripotent stem cell-derived vascular endothelial cells. ISG15-deficient cells exhibited the expected hyperinflammatory phenotype, but also dysregulated expression of molecules critical for connective tissue and epidermis integrity, including reduced collagens and adhesion molecules, but increased matrix metalloproteinases. ISG15(-/-) fibroblasts exhibited elevated ROS levels and reduced ROS scavenger expression. As opposed to hyperinflammation, defective collagen and integrin synthesis was not rescued by conjugation-deficient ISG15. Cell migration was retarded in ISG15(-/-) fibroblasts and HaCaT keratinocytes, but normalized under ruxolitinib treatment. Desmosome density was reduced in an ISG15(-/-) 3D epidermis model. Additionally, there were loose architecture and reduced collagen and desmoglein expression, which could be reversed by treatment with ruxolitinib/doxycycline/TGF-beta 1. These results reveal critical roles of ISG15 in maintaining cell migration and epidermis and connective tissue homeostasis, whereby the latter likely requires its conjugation to yet unidentified targets.

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.

The identification of CSF biomarkers for bacterial meningitis can potentially improve diagnosis and understanding of pathogenesis, and the differentiation from viral CNS infections is of particular clinical importance. Considering that substantial changes in CSF metabolites in CNS infections have recently been demonstrated, we compared concentrations of 188 metabolites in CSF samples from patients with bacterial meningitis (n = 32), viral meningitis/encephalitis (n = 34), and noninflamed controls (n = 66). Metabolite reprogramming in bacterial meningitis was greatest among phosphatidylcholines, and concentrations of all 54 phosphatidylcholines were significantly (p = 1.2 × 10−25–1.5 × 10−4) higher than in controls. Indeed, all biomarkers for bacterial meningitis vs. viral meningitis/encephalitis with an AUC ≥ 0.86 (ROC curve analysis) were phosphatidylcholines. Four of the five most accurate (AUC ≥ 0.9) phosphatidylcholine biomarkers had higher sensitivity and negative predictive values than CSF lactate or cell count. Concentrations of the 10 most accurate phosphatidylcholine biomarkers were lower in meningitis due to opportunistic pathogens than in meningitis due to typical meningitis pathogens, and they correlated most strongly with parameters reflecting blood–CSF barrier dysfunction and CSF lactate (r = 0.73–0.82), less so with CSF cell count, and not with blood CRP. In contrast to the elevated phosphatidylcholine concentrations in CSF, serum concentrations remained relatively unchanged. Taken together, these results suggest that increased free CSF phosphatidylcholines are sensitive biomarkers for bacterial meningitis and do not merely reflect inflammation but are associated with local disease and a shift in CNS metabolism.

Background: Detection of early metabolic changes in critically-ill COVID-19 patients under invasive mechanical ventilation (IMV) at the intensive care unit (ICU) could predict recovery patterns and help in disease management. Methods: Targeted metabolomics of serum samples from 39 COVID-19 patients under IMV in ICU was performed within 48 hours of intubation and a week later. A generalized linear model (GLM) was used to identify, at both time points, metabolites and clinical traits that predict the length of stay (LOS) at ICU (short ≤14 days /long >14 days) as well as the duration under IMV. All models were initially trained on a set of randomly selected individuals and validated on the remaining individuals in the cohort. Further validation in recently published Metabolomics data of COVID-19 severity was performed. Results: A model based on hypoxanthine and betaine measured at first time point was best at predicting whether a patient is likely to experience a short or long stay at ICU (AUC=0.92). A further model based on kynurenine, 3-methylhistidine, Ornithine, p-Cresol sulfate and C24.0 sphingomyelin, measured one-week later, accurately predicted the duration of IMV (Pearson correlation=0.94). Both predictive models outperformed APACHE II scores and differentiated COVID-19 severity in published data. Conclusion: This study has identified specific metabolites that can predict in advance LOS and IMV, which could help in the management of COVID-19 cases at ICU.

Detection of early metabolic changes in critically-ill coronavirus disease 2019 (COVID-19) patients under invasive mechanical ventilation (IMV) at the intensive care unit (ICU) could predict recovery patterns and help in disease management. Targeted metabolomics of serum samples from 39 COVID-19 patients under IMV in ICU was performed within 48 h of intubation and a week later. A generalized linear model (GLM) was used to identify, at both time points, metabolites and clinical traits that predict the length of stay (LOS) at ICU (short ≤ 14 days/long >14 days) as well as the duration under IMV. All models were initially trained on a set of randomly selected individuals and validated on the remaining individuals in the cohort. Further validation in recently published metabolomics data of COVID-19 severity was performed. A model based on hypoxanthine and betaine measured at first time point was best at predicting whether a patient is likely to experience a short or long stay at ICU [area under curve (AUC) = 0.92]. A further model based on kynurenine, 3-methylhistidine, ornithine, p-cresol sulfate, and C24.0 sphingomyelin, measured 1 week later, accurately predicted the duration of IMV (Pearson correlation = 0.94). Both predictive models outperformed Acute Physiology and Chronic Health Evaluation II (APACHE II) scores and differentiated COVID-19 severity in published data. This study has identified specific metabolites that can predict in advance LOS and IMV, which could help in the management of COVID-19 cases at ICU. This project was funded by Qatar University's internal grant number QUHI-BRC-20/21-1. This publication was made possible by GSRA grant, ID# GSRA5-1-0602-18124, from the Qatar National Research Fund (a member of Qatar Foundation).

Systemic effects of advanced cancer impact on the heart leading to cardiac atrophy and functional impairment. Using a murine melanoma cancer model (B16F10 melanoma cells stably transduced with a Ganciclovir (GCV)-inducible suicide gene), the present study analysed the recovery potential of cancer-induced cardiomyopathy with or without use of doxorubicin (Dox). After Dox-free tumor elimination and recovery for 70 ± 5 days, cancer-induced morphologic, functional, metabolic and molecular changes were largely reversible in mice previously bearing tumors. Moreover, grip strength and cardiac response to angiotensin II-induced high blood pressure were comparable with healthy control mice. In turn, addition of Dox (12 mg/kg BW) to melanoma-bearing mice reduced survival in the acute phase compared to GCV-alone induced recovery, while long-term effects on cardiac morphologic and functional recovery were similar. However, Dox treatment was associated with permanent changes in the cardiac gene expression pattern, especially the circadian rhythm pathway associated with the DNA damage repair system. Thus, the heart can recover from cancer-induced damage after chemotherapy-free tumor elimination. In contrast, treatment with the cardiotoxic drug Dox induces, besides well-known adverse acute effects, long-term subclinical changes in the heart, especially of circadian clock genes. Since the circadian clock is known to impact on cardiac repair mechanisms, these changes may render the heart more sensitive to additional stress during lifetime, which, at least in part, could contribute to late cardiac toxicity. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00902-7.

Administration of pivalate has been demonstrated to be suitable for the induction of secondary carnitine deficiency (CD) in pigs, as model objects for humans. In order to comprehensively characterize the metabolic effects of secondary CD in the liver of pigs, the present study aimed to carry out comparative analysis of the hepatic transcriptome and hepatic and plasma metabolome of a total of 12 male 5-week-old pigs administered either pivalate (group PIV, n = 6) or vehicle (group CON, n = 6) for 28 days. Pigs of group PIV had approximately 40–60% lower concentrations of free carnitine and acetylcarnitine in plasma, liver and different skeletal muscles than pigs of group CON (p &lt, 0.05). Transcript profiling of the liver revealed 140 differentially expressed genes (DEGs) between group PIV and group CON (fold change &gt, 1.2 or &lt, −1.2, p-value &lt, 0.05). Biological process terms dealing with the innate immune response were found to be enriched with the DEGs (p &lt, 0.05). Using a targeted metabolomics approach for the simultaneous quantification of 630 metabolites, 9 liver metabolites and 18 plasma metabolites were identified to be different between group PIV and group CON (p &lt, 0.05). Considering the limited alterations of the hepatic transcriptome and of the liver and plasma metabolome, it can be concluded that pivalate-induced secondary CD is not associated with significant hepatic metabolism changes in pigs.

Aims: Peripartum cardiomyopathy (PPCM) is a heart disease affecting women during the last month of pregnancy or in the first months after delivery. The impact of the disease on mental health is largely unknown. Methods and results: Major mental disorders were assessed by a structured clinical interview in 40 patients with a confirmed PPCM diagnosis, and the data were compared with published prevalence in postpartum women. Circulating biomarkers associated with mental health, such as kynurenine, serotonin, and microRNA (miR)‐30e, were evaluated in PPCM and compared with matched healthy pregnancy‐matched postpartum controls (PP‐Ctrl). Major mental disorders were diagnosed in 65% (26/40) of the PPCM cohort. The prevalence for major depressive disorders was 4‐fold, for post‐traumatic stress disorder 14‐fold, and for panic disorder 6‐fold higher in PPCM patients compared with postpartum women without a PPCM diagnosis. Compared with PP‐Ctrl, PPCM patients displayed elevated levels of serum kynurenine (P < 0.01), reduced levels of serum serotonin (P < 0.05), and elevated levels of plasma miR‐30e (P < 0.05). Conclusions: The majority of PPCM patients in the present cohort displayed mental disorders with a higher prevalence of major depressive disorders, post‐traumatic stress disorder (PTBS), and panic disorder, compared with postpartum women without a PPCM diagnosis. This higher prevalence was associated with an impaired tryptophan metabolism and elevated levels of the depression‐associated miR‐30e, suggesting a potential predisposition for mental disorders at the time of PPCM diagnosis. Consequently, physicians should be aware of the increased risk for mental disorders in PPCM patients, and psychiatric assessment should be included in the diagnosis and management of PPCM patients.

Introduction: A breath test that provides information about the inflammatory phenotype of asthma patients could be a valuable tool in daily clinical practice. We analyzed breath in the adult arm of the ALLIANCE cohort to evaluate if breath VOC patterns differ between asthma phenotypes. Methods: We included 111 patients of three phenotypes: active smokers with asthma (n=29), non-smokers with severe asthma (n=36; ERS/ATS guidelines), and non-smokers with mild-to-moderate asthma (n=46). Patients inhaled pre-cleaned room air and exhaled into an aluminum reservoir tube. The breath was loaded onto two adsorption tubes for the analysis by GC-MS. 134 different VOCs were assessed. Results: The high comparability between simultaneously loaded adsorption tubes and the differences between actively smoking and non-smoking patients (44/134 VOCs with p Conclusion: Breath VOCs appear to be closer related to the clinical than the inflammatory asthma phenotype. One breath VOC might have a clinical relevance as it could support other readily accessible markers like blood eosinophils and exhaled NO to predict the level of eosinophilic airway inflammation.

Today, antibiotics are essential for effective treatment of infectious diseases both in human and veterinary medicine. The increasing development and distribution of antibiotic-resistant microorganisms are subject of concern. In some sectors of animal agriculture, such as poultry feeding farms, the application of antibiotics and hence occupational exposure is inevitable. In the past, numerous studies focussed on the occurrence of antibiotic-resistant bacteria in livestock farming, but little attention was paid to the employees. The exposure of workers to antibiotics was the focus of the study detailed in this article. Four biomonitoring campaigns monitoring systemic exposure of workers to antibiotics were run at two farms over four fattening periods. Urine samples of potentially affected employees were sampled and analysed for the antibiotics of interest by liquid chromatography–mass spectrometry. The highest antibiotic concentrations detected in urine samples exceeded the minimum inhibitory concentration of some bacteria strains. In some cases, the amount of antibiotics excreted over a time-period of 24 h indicated the exceedance of the tolerable daily intake.

Quantitative assessment of human exposure to semi-volatile organic compounds (SVOC), such as tricresyl phosphates (TCP) that may originate from engine oil contamination of the cabin air, during air travel is challenging due to the technical complexity of the air supply in commercial jet aircraft. Normal flight operations involve reduced air exchange before and during takeoff, which results in increased concentrations of potential cabin air pollutants. During cruise, normal ventilation rates (>20 h− 1) are reestablished and thus lower pollutant concentrations are also reestablished. This relationship between changes in ventilation rate and associated changes in pollutant concentrations during the departure phase is first described in the present study, although this effect was found by previous studies that investigated distinct flight phases. The perception of so-called "smell events" in cabin air does not necessarily indicate the presence of harmful contaminants and TCP-containing oil mist must be clearly distinguished thereof. Thus, aldehydes, VOCs, and organophosphates such as TCP were investigated. In this paper, the occurrence of TCP contamination in a bleed air free Boeing 787 (B787) aircraft is reported for the first time. The results presented here show that there are TCP sources other than bleed air from leaky engines. Furthermore, exceptional release behavior of TCP suggests that a more detailed classification for engine oil-triggered cabin air contamination (CAC) events is necessary. This study evaluated measurement data from 177 flights that were either commissioned by the EASA or conducted as part of studies with the support of Lufthansa, Condor, and British Airways.

The aerosol release during the professional application of two different isocyanate based two component spray systems was identified and the physicochemical properties of the released airborne aerosols were characterized. For this purpose, aerosol release fractions were measured using a mass balance method described by Schwarz and Koch. Besides the release of total aerosol mass special emphasis was directed to the content of free monomeric MDI (4,4′- and 2,4′-diphenylmethane diisocyanate) in three particle size fractions relevant for inhalation uptake: inhalable, thoracic, and respirable size fraction. Two products were investigated: a two component PUR (polyurethane) spray foam (Elastopor) and a polyurea spray coating (Elastocoat). The mass fraction of the applied products released with the overspray as inhalable aerosol is 6.3 × 10−4 (Elastopor) and 4.0 × 10−4 (Elastocoat). Of the released total overspray aerosol 75 or 80% were in the thoracic size range, and 26 or 47% in the respirable regime for the PUR spray foam or the polyurea spray coating, respectively. At the time point of release the content of monomeric MDI in the aerosol corresponds to the composition of the bulk product. However, analysis of air samples indicates that

INTRODUCTION: Chronic lung allograft dysfunction with its clinical correlative of bronchiolitis obliterans syndrome (BOS) remains the major limiting factor for long-term graft survival. Currently there are no established methods for the early diagnosis or prediction of BOS. To assess the feasibility of breath collection as a non-invasive tool and the potential of breath volatile organic compounds (VOC) for the early detection of BOS, we compared the breath VOC composition between transplant patients without and different stages of BOS. METHODS: 75 outpatients (25 BOS stage 0, 25 BOS stage 1 + 2, 25 BOS stage 3) after bilateral lung transplantation were included. Exclusion criteria were active smoking, oxygen therapy and acute infection. Patients inhaled room air through a VOC and sterile filter and exhaled into an aluminum reservoir tube. Breath was loaded directly onto Tenax((R)) TA adsorption tubes and was subsequently analyzed by gas-chromatography/mass-spectrometry. RESULTS: The three groups were age and gender matched, but differed with respect to time since transplantation, the spectrum of underlying disease, and treatment regimes. Relative to patients without BOS, BOS stage 3 patients showed a larger number of different VOCs, and more pronounced differences in the level of VOCs as compared to BOS stage 1 + 2 patients. Logistic regression analysis found no differences between controls and BOS 1 + 2, but four VOCs (heptane, isopropyl-myristate, ethyl-acetate, ionone) with a significant contribution to the discrimination between controls and BOS stage 3. A combination of these four VOCs separated these groups with an area under the curve of 0.87. CONCLUSION: Breath sample collection using our reservoir sampler in the clinical environment was feasible. Our results suggest that breath VOCs can discriminate severe BOS. However, convincing evidence for VOCs with a potential to detect early onset BOS is lacking.

In order to quantify the relative bioavailability of glycidol from glycidyl fatty acid esters in vivo, glycidyl palmitoyl ester and glycidol were orally applied to rats in equimolar doses. The time courses of the amounts of glycidol binding to hemoglobin as well as the excretion of 2,3-dihydroxypropyl mercapturic acids were determined. The results indicate that glycidol is released from the glycidyl ester by hydrolysis and rapidly distributed in the organism. In relation to glycidol, there was only a small timely delay in the binding to hemoglobin for the glycidol moiety released from the ester which may be certainly attributed to enzymatic hydrolysis. In both cases, however, an analogous plateau was observed representing similar amounts of hemoglobin binding. With regard to the urinary excretion of mercapturic acids, also similar amounts of dihydroxypropyl mercapturic acids could be detected. In an ADME test using a virtual double tag (3H, 14C) of glycidyl palmitoyl ester, a diverging isotope distribution was detected. The kinetics of the 14C-activity reflected the kinetics of free glycidol released after hydrolysis of the palmitoyl ester. In view of this experimental data obtained in rats, it is at present justified for the purpose of risk assessment to assume complete hydrolysis of the glycidyl ester in the gastrointestinal tract. Therefore, assessment of human exposure to glycidyl fatty acid ester should be regarded as an exposure to the same molar quantity of glycidol.

Background: The WHO estimated that air pollution is the 13th leading cause of mortality worldwide largely through excess cardiovascular mortality. But it is unknown whether air pollutants actually raise sympathetic activity. Therefore it was the primary aim of this study to test if ultrafine particle (UFP) exposure increases sympathetic nervous system activity in human subjects and if ozone-induced airway inflammation causes autonomic dysregulation. Here we report the results of the inflammatory response to these experimental exposures. Methods: 20 subjects underwent exposures to filtered air (FA), carbon black UFP (50μg/m³) and to UFP and 250ppb ozone (UFPO3) for 3h of intermittent exercise. The exposures were at least 50d apart and performed double blind, and in random order. Sputum was induced 3h after the exposure. Exhaled breath samples were collected onto Tenax adsorption tubes and analyzed by GC-MS and GC-APCI-MS. Results: The combined exposure caused a significant increase in the number of sputum cells, the number and the percentage of sputum neutrophils, and in sputum supernatant IL6 and MMP9. We also observed an increase in the level of serum CC16. A significant effect of UFP exposure could only be detected in the 14 subjects with Conclusion: We know from our previous study (Resp. Med. 2011) that exercise can induce a mild increase in sputum and systemic leukocytes. Only in subjects with a mild response to exercise the small inflammatory response to UFP alone could be detected. In combination with ozone the typical increase in sputum parameters was observed that is known from studies using the ozone challenge model. Sponsored by DFG (JO284/10-1) and by DZL.

Due to its high sensitivity, compact size and low cost ion mobility spectrometry (IMS) has the potential to become a point-of-care breath analyzer. Therefore, we developed a prototype of a compact, closed gas loop IMS with gas chromatographic (GC) pre-separation and high resolving power of R = 90. In this study, we evaluated the performance of this GC-IMS under clinical conditions in a COPD study to find correlations between VOCs (10 ppbv to 1 ppmv) and COPD. Furthermore, in order to investigate possible correlations between ultra-low concentrated breath VOCs (0.1 pptv to 1 ppbv) and COPD, a modified mass spectrometer (MS) with atmospheric pressure chemical ionization (APCI) and GC pre-separation (GC-APCI-MS) was used. The GC-IMS has been used in 58 subjects (21 smokers with moderate COPD, 12 ex-smokers with COPD, 16 healthy smokers and 9 non-smokers). GC-APCI-MS data were available for 94 subjects (21 smokers with moderate COPD, 25 ex-smokers with COPD, 25 healthy smokers and 23 non-smokers). For 44 subjects, a comparison between GC-IMS and GC-APCI-MS data could be performed. Due to service intervals, subject availability and corrupt data, patient numbers were different for GC-APCI-MS and GC-IMS measurements. Using GC-IMS, three VOCs have been found showing a significant difference between healthy controls and patients with COPD. In the GC-APCI-MS data, we only observed one distinctive VOC, which has been identified as 2-pentanone. This proof-of-principle study shows the potential of our high-resolution GC-IMS in the clinical environment. Due to different linear dynamic response ranges, the data of GC-IMS and GC-APCI-MS were only comparable to a limited extent.

In order for digital health offerings to be worthy of the investment, they must have an evidence base that demonstrates their contribution to improving health outcomes, their cost-effectiveness and have the ability to demonstrate improvements in quality of care. For telecommunications and digital service providers, this translates into requirements for scalability and performance and quality management. All these performance indicators are challenged by cost, quality, and availability of the delivered service.

There is increasing evidence that breath volatile organic compounds (VOC) have the potential to support the diagnosis and management of inflammatory diseases such as COPD. In this study we used a novel breath sampling device to search for COPD related VOCs. We included a large number of healthy controls and patients with mild to moderate COPD, recruited subjects at two different sites and carefully controlled for smoking. 222 subjects were recruited in Hannover and Marburg, and inhaled cleaned room air before exhaling into a stainless steel reservoir under exhalation flow control. Breath samples (2.5 l) were continuously drawn onto two Tenax(®) TA adsorption tubes and analyzed in Hannover using thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). Data of 134 identified VOCs from 190 subjects (52 healthy non-smokers, 52 COPD ex-smokers, 49 healthy smokers, 37 smokers with COPD) were included into the analysis. Active smokers could be clearly discriminated by higher values for combustion products and smoking related VOCs correlated with exhaled carbon monoxide (CO), indicating the validity of our data. Subjects from the study sites could be discriminated even after exclusion of cleaning related VOCs. Linear discriminant analysis correctly classified 89.4% of COPD patients in the non/ex-smoking group (cross validation (CV): 85.6%), and 82.6% of COPD patients in the actively smoking group (CV: 77.9%). We extensively characterized 134 breath VOCs and provide evidence for 14 COPD related VOCs of which 10 have not been reported before. Our results show that, for the utilization of breath VOCs for diagnosis and disease management of COPD, not only the known effects of smoking but also site specific differences need to be considered. We detected novel COPD related breath VOCs that now need to be tested in longitudinal studies for reproducibility, response to treatment and changes in disease severity.

The present study simulated large-scale indoor mold damage in order to test the efficiency of air sampling for the detection of microbial volatile organic compounds (MVOCs). To do this, a wallpaper damaged by condensation was stored in a climate chamber (representing a hypothetical test room of 40 m(3) volume) and was inoculated with 14 typical indoor fungal strains. The chamber ventilation conditions were adjusted to common values found in moldy homes, and the mold growth was allowed to continue to higher than average values. The MVOC content of the chamber air was analyzed daily for a period of 105 days using coupled gas chromatography/mass spectrometry (GC-MS). This procedure guarantees MVOC profiling without external factors such as outdoor air, building materials, furniture, and occupants. However, only nine MVOCs could be detected during the sampling period, which indicates that the very low concentrated MVOCs are hardly accessible, even under these favorable conditions. Furthermore, most of the MVOCs that were detected cannot be considered as reliable indicators of mold growth in indoor environments.

Microorganisms such as bacteria and molds produce an enormous variety of volatile metabolites. To determine whether typical microbial volatile metabolites can be used as indicator compounds for the detection of hidden mold in indoor environments, we examined 14 typical indoor fungal strains for their growth rates and their capability to produce volatile organic compounds (VOC) on standard clinical media and on agar medium made from building materials. Air samples from Headspace Chambers (HSC) were adsorbed daily on Tenax TA tubes and analyzed by thermal desorption gas chromatography and mass spectrometry. In parallel, metabolic activity was measured by determining oxygen demand, the microbial biomass was assessed by dry weighing. Profiling of the volatile metabolites showed that VOC production depended greatly on fungal strain, culture medium, biological activity, and time. The laboratory-derived maximum emission rates were extrapolated to approximate indoor air concentrations in a hypothetical mold-infested room. The extrapolated indoor air data suggest that most of the microbial-produced VOC concentrations were below the analytical detection limit for conventional indoor air analysis. Additionally, conducted indoor air analysis in mold homes confirmed these findings for the most part. The present findings raise doubts about the utility of indicator VOC for the detection of hidden mold growth in indoor environments. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

In a two-center collaboration study we collected breath samples of COPD patients and controls and evaluated the impact of site and shipment on VOC sample quality and composition. Tenax tubes were conditioned in Hannover and shipped to Marburg. Breath collection was performed using identical sampling devices (inhalation via A2 filter and exhalation (flow control) into a stainless steel reservoir), but different mouth pieces. 3L of breath were continuously drawn onto 2 adsorption tubes. For this analysis we used data of 10 non-smoking subjects and 10 smokers from each site. 65 commonly observed substances were chosen for single-ion analysis. The chromatograms showed major site specific VOC peaks which could not or only in traces be found in samples collected at the respective other site. Most of these VOCs could not be detected in the accompanied room air samples, excluding room air or shipment as potential sources. Among the VOCs that discriminated sites were 2-Butyl-1-octanol and 1-Phenoxy-2-propanon, the latter being known to be part of cleaning solutions. No significant differences could be observed between sites with respect to the levels of Benzene, which clearly discriminated active smokers from controls at both sites. We found site-specific VOCs which could be linked to different cleaning procedures. These VOCs did not show a negative impact on the analysis of breath-related VOCs, but need to be considered as site specific effects in multicenter-studies when using cross-reactive sensor devices (eNoses). The comparable level of benzene indicates that shipping of thermal-desorption tubes does not contaminate breath samples and that shipment between sites is feasible.

In this ongoing two-center collaboration study (Marburg, Hannover, German Center for Lung Research) we aim to include sufficiently high numbers of patients and controls (total 240, >70% completed) to identify specific COPD related VOC biomarker signals that are independent of the influence of study site. Tenax adsorption tubes were conditioned in Hannover and shipped to Marburg. Breath collection was performed using identical sampling devices (inhalation via A2 filter and exhalation (flow control) into a stainless steel reservoir), but different mouth pieces. 3L of breath were continuously drawn onto 2 adsorption tubes. Currently data for 53 commonly observed substances is available. Of these 13 VOCs were excluded due to significant differences between study sites. Including data of 72 samples from healthy non-smokers and COPD ex-smokers from both sites the stepwise inclusion of variables resulted in a model with 4 VOCs that discriminated 83.0% of subjects correctly. In the cross-validation process 77.8% of subjects were correctly identified. Including the 41 healthy smokers and smokers with COPD the model correctly identified only 68.3% of the subjects and the cross-validation value was 61.0%. Excluding site specific VOCs we were able to identify a VOC pattern of 4 substances that appear to be related to COPD. The model to discriminate COPD patients from controls showed a better performance in the non-smoking groups. Despite lower subject numbers in the actively smoking groups, this result emphasizes that it is important to carefully control for active smoking when searching for COPD related VOC patterns.

The objective of this project was to gain information on toxicokinetic behaviour and in vivo genotoxicity of alternariol (AOH) in NMRI mice. Therefore, commercially available AOH was purified using a preparative HPLC-method. Initial dose-range finding proved AOH to be nontoxic after single or repeated oral application of up to 2000 mg/kg (limit dose). Subsequently, an in vivo oral toxicokinetic study (OECD guideline 417) was performed with 200 and 1000 mg/kg radiolabelled AOH. The study revealed low systemic absorption, with about 90 % of the total dose excreted via faeces and up to 9 % via urine. Blood levels did not exceed 0.06 % of the administered dose during the first 24 h after administration. Thus, target organ toxicity would most likely be restricted to the gastrointestinal tract. Metabolism of AOH was then investigated in a toxicokinetic study with non-radiolabelled AOH. Three dosage schemes were used: 200 and 2000 mg/kg (single dose) and 3 x 2000 mg/kg (0, 24 and 45 h). Whole blood (LC-MS/MS analysis) and urine (GC-MS in SIM mode) were analysed for AOH and its hydroxy-metabolites. Four metabolites (8-hydroxy-AOH, 4-hydroxy-AOH, 10-hydroxy-AOH, 2-hydroxy-AOH) were detected and ID was confirmed by NMR and mass spectrometry. Results also pointed to low systemic absorption, but mean blood levels (0.5 µM AOH, 3 h after the last of three applications) was considered sufficient to justify performance of a combined bone marrow micronucleus test (OECD 474) and in vivo alkaline comet assay (stomach, gut, liver). Therefore, 3 x 2000 mg/kg AOH were applied in corn oil (at 0, 24, and 45 h) and animals were sacrificed 48 h after the first application. The micronucleus assay revealed no toxic or genotoxic effect of AOH in bone marrow and the comet assay with liver tissue also did not indicate systemic genotoxicicity.

The bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 is encoded by five genes (hoxEFUYH) which are transcribed as one unit. The transcription of the hox-operon is regulated by a promoter situated upstream of hoxE. The transcription start point was located at -168 by 5'Race. Several promoter probe vectors carrying different promoter fragments revealed two regions to be essential for the promoter activity. One is situated in the untranslated 5'leader region and the other is found -569 to -690 nucleotides upstream of the ATG. The region further upstream was shown to bind a protein. Even though an imperfect NtcA binding site was identified, NtcA did not bind to this region. The protein binding to the DNA was purified and found to be LexA by MALDI-TOF. The complete LexA and its DNA binding domain were overexpressed in Escherichia coli. Both were able to bind to two sites in the examined region in band-shift-assays. Accordingly, the hydrogenase activity of a LexA-depleted mutant was reduced. This is the first report on LexA acting not as a repressor but as a transcriptional activator. Furthermore, LexA is the first transcription factor identified so far for the expression of bidirectional hydrogenases in cyanobacteria.

More than 60 years after an illicit love affair had occurred between Erika H, wife of a Wehrmacht soldier, and a Polish slave worker during World War II, we could clarify the blood relationships of her daughter Uta. When Erika H had become pregnant both of the men could have fathered the child. Erika H was found guilty of fraternization and imprisoned at Ravensbruck concentration camp. She gave birth to Uta and died there in 1944. Uta survived the war as did Erika’s husband Gustav, who accepted Uta as his child. Blood samples from family members were taken and DNA extracted. A panel of 16 short tandem repeat (STR) loci were amplified and separated by capillary electrophoresis and the likelihoods calculated using the MLINK software. The combined genotypes yielded a cumulative likelihood ratio of over 200,000 against paternity of Gustav H. This case serves to illustrate the utility of STR profiles for complex deficiency kinship analysis.

The pulmonary effects of a finished toner were evaluated in intratracheal instillation and inhalation studies, using toners with external additives (titanium dioxide nanoparticles and amorphous silica nanoparticles). Rats received an intratracheal dose of 1 mg or 2 mg of toner and were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months. The toner induced pulmonary inflammation, as evidenced by a transient neutrophil response in the low-dose groups and persistent neutrophil infiltration in the high-dose groups. There were increased concentrations of heme oxygenase-1 (HO-1) as a marker of oxidative stress in the bronchoalveolar lavage fluid (BALF) and the lung. In a 90-day inhalation study, rats were exposed to well-dispersed toner (mean of MMAD: 3.76 µm). The three mass concentrations of toner were 1, 4 and 16 mg/m(3) for 13 weeks, and the rats were sacrificed at 6 days and 91 days after the end of the exposure period. The low and medium concentrations did not induce neutrophil infiltration in the lung of statistical significance, but the high concentration did, and, in addition, upon histopathological examination not only showed findings of inflammation but also of fibrosis in the lung. Taken together, the results of our studies suggest that toners with external additives lead to pulmonary inflammation and fibrosis at lung burdens suggest beyond the overload. The changes observed in the pulmonary responses in this inhalation study indicate that the high concentration (16 mg/m(3)) is an LOAEL and that the medium concentration (4 mg/m(3)) is an NOAEL.

Magnesium materials are of increasing interest in the development of biodegradable implants as they exhibit properties that make them promising candidates. However, the formation of gas cavities after implantation of magnesium alloys has been widely reported in the literature. The composition of the gas and the concentration of its components in these cavities are not known as only a few studies using non-specific techniques were done about 60 years ago. Currently many researchers assume that these cavities contain primarily hydrogen because it is a product of magnesium corrosion in aqueous media. In order to clearly answer this question we implanted rare earth-containing magnesium alloy disks in mice and determined the concentration of hydrogen gas for up to 10 days using an amperometric hydrogen sensor and mass spectrometric measurements. We were able to directly monitor the hydrogen concentration over a period of 10 days and show that the gas cavities contained only a low concentration of hydrogen gas, even shortly after formation of the cavities. This means that hydrogen must be exchanged very quickly after implantation. To confirm these results hydrogen gas was directly injected subcutaneously. Most of the hydrogen gas was found to exchange within 1 h after injection. Overall, our results disprove the common misbelief that these cavities mainly contain hydrogen and show how quickly this gas is exchanged with the surrounding tissue.

The present study simulated large-scale indoor mold damage in order to test the efficiency of air sampling for the detection of microbial volatile organic compounds (MVOCs). To do this, a wallpaper damaged by condensation was stored in a climate chamber (representing a hypothetical test room of 40 m(3) volume) and was inoculated with 14 typical indoor fungal strains. The chamber ventilation conditions were adjusted to common values found in moldy homes, and the mold growth was allowed to continue to higher than average values. The MVOC content of the chamber air was analyzed daily for a period of 105 days using coupled gas chromatography/mass spectrometry (GC-MS). This procedure guarantees MVOC profiling without external factors such as outdoor air, building materials, furniture, and occupants. However, only nine MVOCs could be detected during the sampling period, which indicates that the very low concentrated MVOCs are hardly accessible, even under these favorable conditions. Furthermore, most of the MVOCs that were detected cannot be considered as reliable indicators of mold growth in indoor environments.