A novel targeted/untargeted GC-Orbitrap metabolomics methodology applied to Candida albicans and Staphylococcus aureus biofilms

Introduction: Combined infections from Candida albicans and Staphylococcus aureus are a leading cause of death in the developed world. Evidence suggests that Candida enhances the virulence of Staphylococcus—hyphae penetrate through tissue barriers, while S. aureus tightly associates with the hyphae to obtain entry to the host organism. Indeed, in a biofilm state, C. albicans enhances the antimicrobial resistance characteristics of S. aureus. The association of these microorganisms is also associated with significantly increased morbidity and mortality. Due to this tight association we hypothesised that metabolic effects were also in evidence. Objectives: To explore the interaction, we used a novel GC-Orbitrap-based mass spectrometer, the Q Exactive GC, which combines the high peak capacity and chromatographic resolution of gas chromatography with the sub-ppm mass accuracy of an Orbitrap system. This allows the capability to leverage the widely available electron ionisation libraries for untargeted applications, along with expanding accurate mass libraries and targeted matches based around authentic standards. Methods: Optimised C. albicans and S. aureus mono- and co-cultured biofilms were analysed using the new instrument in addition to the fresh and spent bacterial growth media. Results: The targeted analysis experiment was based around 36 sugars and sugar phosphates, 22 amino acids and five organic acids. Untargeted analysis resulted in the detection of 465 features from fresh and spent medium and 405 from biofilm samples. Three significantly changing compounds that matched to high scoring library fragment patterns were chosen for validation. Conclusion: Evaluation of the results demonstrates that the Q Exactive GC is suitable for metabolomics analysis using a targeted/untargeted methodology. Many of the results were as expected: e.g. rapid consumption of glucose and fructose from the medium regardless of the cell type. Modulation of sugar-phosphate levels also suggest that the pentose phosphate pathway could be enhanced in the cells from co-cultured biofilms. Untargeted metabolomics results suggested significant production of cell-wall biosynthesis components and the consumption of non-proteinaceous amino-acids

Discovery of Emerging Disinfection by-products in Water using Gas Chromatography coupled with Orbitrap-based Mass Spectrometry

The disinfection of drinking water is required in order to protect consumers from potential waterborne infectious and parasitic pathogens. Water is commonly treated by adding chemical disinfectants, such as free chlorine, chloramines, chlorine dioxide, and ozone. However, although very effective in removing disease-causing microorganisms, these disinfectants can react with naturally occurring materials in the water and can form disinfection by-products (DBPs) which can be harmful to human health. In particular, compounds containing an iodo-group, i.e., iodinated DBPs (iodo-DBPs), may pose a greater health risk for the population exposed to them than their brominated and chlorinated analogues [1]. In recent years, several chemical classes of low molecular weight iodo-DBPs have been reported; however, many more may be still present in the unknown fraction (~50%) of halogenated material formed during disinfection treatments [2]. Therefore, complete characterization of iodo-DBPs present in DBP mixtures is crucial to further investigate their occurrence in disinfected waters and potential toxicity effects. The identification of emerging iodinated DBPs in water is difficult due to the complexity of this matrix and the low concentrations of these compounds. For this, analytical techniques with high resolving power, high mass accuracy and sensitivity are required. In this work, a novel gas chromatography (GC), coupled with high-resolution accurate mass Orbitrap mass spectrometer (the Thermo Scientific™ Q Exactive™ GC hybrid quadrupole-Orbitrap mass spectrometer), has been used for iodo-DBPs detection and accurate mass identification in chlorinated and chloraminated water samplesC. P. acknowledges support provided by the European Union 7th R&D Framework Programme (FP7/2007e2013) under grant agreement 274379 (Marie Curie IOF) and the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia and the COFUND programme of the Marie Curie Actions of the EU’s FP7 (2014 BP_B00064). This work has been financially supported by the Generalitat de Catalunya (Consolidated Research Groups “2014 SGR 418 - Water and Soil Quality Unit” and 392 2014 SGR 291 - ICRA) and by the European Union’s FP7 for research, technological development and demonstration under grant agreement nº 603437 (SOLUTIONS). The EU is not liable for any use that may be made of the information contained therein.Peer reviewe

Characterization of iodinated disinfection by-products in chlorinated and chloraminated waters using Orbitrap based gas chromatography-mass spectrometry

Recent developments in gas chromatography (GC)-mass spectrometry (MS) have opened up the possibility to use the high resolution-accurate mass (HRAM) Orbitrap mass analyzer to further characterize the volatile and semivolatile fractions of environmental samples. This work describes the utilization of GC Orbitrap MS technology to characterize iodine-containing disinfection by-products (iodo-DBPs) in chlorinated and chloraminated DBP mixture concentrates. These DBP mixtures were generated in lab-scale disinfection reactions using Llobregat river water and solutions containing Nordic Lake natural organic matter (NOM). The DBPs generated were concentrated using XAD resins, and extracts obtained were analyzed in full scan mode with the GC Orbitrap MS. Integration of high resolution accurate mass information and fragment rationalization allowed the characterization of up to 11 different iodo-DBPs in the water extracts analyzed, including one new iodo-DBP reported for the first time. Overall, formation of iodo-DBPs was enhanced during chloramination reactions. As expected, NOM characteristics and iodide and bromide content of the tested waters affected the amount and type of iodo-DBPs generated.C.P. acknowledges support provided by the European Union 7th R&D Framework Programme (FP7/2007-2013) under grant agreement 274379 (Marie Curie IOF) and the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia and the COFUND programme of the Marie Curie Actions of the EU’s FP7 (2014 BP_B00064). This work was financially supported by the Government of Catalonia (Consolidated Research Groups “2014 SGR 418 - Water and Soil Quality Unit” and “2014 SGR 291 – ICRA”) and by the EU’s FP7 for research, technological development, and demonstration (grant agreement no. 603437 - SOLUTIONS).Peer reviewe

Erratum to: Characterization of iodinated disinfection by-products in chlorinated and chloraminated waters using Orbitrap based gas chromatography-mass spectrometry

Recent developments in gas chromatography (GC)-mass spectrometry (MS) have opened up the possibility to use the high resolution-accurate mass (HRAM) Orbitrap mass analyzer to further characterize the volatile and semivolatile fractions of environmental samples. This work describes the utilization of GC Orbitrap MS technology to characterize iodine-containing disinfection by-products (iodo-DBPs) in chlorinated and chloraminated DBP mixture concentrates. These DBP mixtures were generated in lab-scale disinfection reactions using Llobregat river water and solutions containing Nordic Lake natural organic matter (NOM). The DBPs generated were concentrated using XAD resins, and extracts obtained were analyzed in full scan mode with the GC Orbitrap MS. Integration of high resolution accurate mass information and fragment rationalization allowed the characterization of up to 11 different iodo-DBPs in the water extracts analyzed, including one new iodo-DBP reported for the first time. Overall, formation of iodo-DBPs was enhanced during chloramination reactions. As expected, NOM characteristics and iodide and bromide content of the tested waters affected the amount and type of iodo-DBPs generated.Peer reviewe

GC×GC-(HR)TOFMS in colorectal cancer metabolomics

Colorectal cancer globally affects more than one million new persons each year, and kills more than 700.000. Nevertheless, its diagnosis is still largely based on invasive tissue sampling and gaps remain in the understanding of its pathogenesis, with complex combinations between lifestyle, genetics, epigenetics, chronic inflammation (IBD) and microbiota. Untargeted metabolomics is one of the approaches that can be used to solve these issues. To do so, an optimized and validated (NIST SRM 1950) comprehensive GC×GC-(HR)TOFMS method we developed was used, that also included an in-house QC system and data processing based on multiple statistical techniques. Practically, we analyzed serum samples from patients affected by colorectal cancer (CRC, n = 18) and by colorectal cancer in remission (R-CRC, n = 17), and samples from healthy patients matched for biases (HC, n = 19 and R-HC, n = 17). We highlighted candidate biomarkers able to discriminate between matched HC and CRC or R-CRC, which discrimination potential was assessed using supervised and unsupervised models, discriminant analysis and ROC curves. Overfitting was avoided by re-sampling and test validation testing. Annotation used full mass spectrum, linear retention indices and accuracte mass provided by state-of-the-art high-resolution (HR) time-of-flight mass spectrometry. Finally, we studied the main metabolic pathways altered in the disease, whether in active or in remission state. In addition, newly developed GC-MS Orbitrap was applied, using the same global method, to biological replicates to determine the capacity of this technology to perform in untargeted metabolomics as well as to validate the results previously obtained

Transient Release of Oxygenated Volatile Organic Compounds during Light-Dark Transitions in Grey Poplar Leaves

In this study, we investigated the prompt release of acetaldehyde and other oxygenated volatile organic compounds (VOCs) from leaves of Grey poplar [Populus x canescens (Aiton) Smith] following light-dark transitions. Mass scans utilizing the extremely fast and sensitive proton transfer reaction-mass spectrometry technique revealed the following temporal pattern after light-dark transitions: hexenal was emitted first, followed by acetaldehyde and other C(6)-VOCs. Under anoxic conditions, acetaldehyde was the only compound released after switching off the light. This clearly indicated that hexenal and other C(6)-VOCs were released from the lipoxygenase reaction taking place during light-dark transitions under aerobic conditions. Experiments with enzyme inhibitors that artificially increased cytosolic pyruvate demonstrated that the acetaldehyde burst after light-dark transition could not be explained by the recently suggested pyruvate overflow mechanism. The simulation of light fleck situations in the canopy by exposing leaves to alternating light-dark and dark-light transitions or fast changes from high to low photosynthetic photon flux density showed that this process is of minor importance for acetaldehyde emission into the Earth's atmosphere

Assessment of the Impact of Population Reduction on Grasslands with a New “Tool”: A Case Study on the “Mountainous Banat” Area of Romania

The landscapes and, implicitly, the surfaces of secondary grasslands in the mountain areas have been intensively modified and transformed by humans. In this context, this paper analyses the spatial and temporal changes of grassland surfaces following the impact of human population reduction. Thus, the study proposes the implementation of the Grassland Anthropic Impact Index (GAII) as a “measurement tool” to functionally link the two components, grassland surface and human population. The spatiotemporal analyses are based on Corine Land Cover data and demographic data, processed via Geographic Information Systems (GIS) methods and the Land Change Modeler (LCM) tool. The research shows that over a period of 28 years, the population, which was continuously decreasing, caused a series of transformations to the grasslands over an area of 33343 ha. The influence of the reduction in the number of inhabitants was also demonstrated by the direction of the changes produced in the grassland surfaces: in the better populated areas, the grasslands expanded over lands with other uses, and in the sparsely populated areas, they were abandoned. GAII values generally increase with the decrease of the population in the target area, meaning that for an inhabitant (potential user) a greater grassland surface is reported, resulting in a greater responsibility for the management of this resource on a space and time scale. Following the evaluation of the trend of the last 28 years, it was observed that the depopulation of mountain areas can be seen as a threat to grassland ecosystems, either through the transition to other categories of use, or through abandonment. The implications of these phenomena are much broader: they produce chain reactions and affect other components of the regional geosystem

Genetic Counseling and Management: The First Study to Report NIPT Findings in a Romanian Population

Background and Objectives: Non-invasive prenatal testing (NIPT) has been confirmed as the most accurate screening test for trisomies 21, 18, 13, sex chromosomes aneuploidies and several microdeletions. This study aimed to assess the accuracy of cell free DNA testing based on low-level whole-genome sequencing to screen for these chromosomal abnormalities and to evaluate the clinical performance of NIPT. Materials and Methods: 380 consecutive cases from a single genetic center, from Western Romania were included in this retrospective study. Cell-free nucleic acid extraction from maternal blood, DNA sequencing and analysis of sequenced regions were performed by BGI Hong Kong and Invitae USA to determine the risk of specific fetal chromosomal abnormalities. In high-risk cases the results were checked by direct analysis of fetal cells obtained by invasive methods: 6 chorionic villus sampling and 10 amniocenteses followed by combinations of QF-PCR, karyotyping and aCGH. Results: NIPT results indicated low risk in 95.76% of cases and high risk in 4.23%. Seven aneuploidies and one microdeletion were confirmed, the other results were found to be a false-positive. A gestational age of up to 22 weeks had no influence on fetal fraction. There were no significant differences in fetal fraction across the high and low risk groups. Conclusions: This is the first study in Romania to report the NIPT results. The confirmation rate was higher for autosomal aneuploidies compared to sex chromosome aneuploidies and microdeletions. All cases at risk for trisomy 21 were confirmed. Only one large fetal microdeletion detected by NIPT has been confirmed. False positive NIPT results, not confirmed by invasive methods, led to the decision to continue the pregnancy. The main limitation of the study is the small number of patients included. NIPT can be used as a screening method for all pregnancies, but in high-risk cases, an invasive confirmation test was performed