Evaluation of peroxidative stress of cancer cells in vitro by real time quantification of volatile aldehydes in culture headspace

Rationale Peroxidation of lipids in cellular membranes results in the release of volatile organic compounds (VOCs), including saturated aldehydes. The real‐time quantification of trace VOCs produced by cancer cells during peroxidative stress presents a new challenge to non‐invasive clinical diagnostics, which as described here, we have met with some success. Methods A combination of selected ion flow tube mass spectrometry (SIFT‐MS), a technique that allows rapid, reliable quantification of VOCs in humid air and liquid headspace, and electrochemistry to generate reactive oxygen species (ROS) in vitro has been used. Thus, VOCs present in the headspace of CALU‐1 cancer cell line cultures exposed to ROS have been monitored and quantified in real time using SIFT‐MS. Results The CALU‐1 lung cancer cells were cultured in 3D collagen to mimic in vivo tissue. Real‐time SIFT‐MS analyses focused on the volatile aldehydes: propanal, butanal, pentanal, hexanal, heptanal and malondialdehyde (propanedial), that are expected to be products of cellular membrane peroxidation. All six aldehydes were identified in the culture headspace, each reaching peak concentrations during the time of exposure to ROS and eventually reducing as the reactants were depleted in the culture. Pentanal and hexanal were the most abundant, reaching concentrations of a few hundred parts‐per‐billion by volume, ppbv, in the culture headspace. Conclusions The results of these experiments demonstrate that peroxidation of cancer cells in vitro can be monitored and evaluated by direct real‐time analysis of the volatile aldehydes produced. The combination of adopted methodology potentially has value for the study of other types of VOCs that may be produced by cellular damage

Direct detection and quantification of malondialdehyde vapour in humid air using selected ion flow tube mass spectrometry supported by gas chromatography/mass spectrometry

RATIONALE: It has been proposed that malondialdehyde (MDA) reflects free oxygen-radical lipid peroxidation and can be useful as a biomarker to track this process. For the analysis of MDA molecules in humid air by selected ion flow tube mass spectrometry (SIFT-MS), the rate coefficients and the ion product distributions for the reactions of the SIFT-MS reagent ions with volatile MDA in the presence of water vapour are required. METHODS: The SIFT technique has been used to determine the rate coefficients and ion product distributions for the reactions of H3O(+), NO(+) and O2 (+•) with gas-phase MDA. In support of the SIFT-MS analysis of MDA, solid-phase microextraction, SPME, coupled with gas chromatography/mass spectrometry, GC/MS, has been used to confirm the identification of MDA. RESULTS: The primary product ions have been identified for the reactions of H3O(+), NO(+) and O2 (+•) with MDA and the formation of their hydrates formed in humid samples is described. The following combinations of reagent and the analyte ions (given as m/z values) have been adopted for SIFT-MS analyses of MDA in the gas phase: H3O(+): 109; NO(+): 89, 102; O2 (+•): 72, 90, 108, 126. The detection and quantification of MDA released by a cell culture by SIFT-MS are demonstrated. CONCLUSIONS: This detailed study has provided the kinetics data required for the SIFT-MS analysis of MDA in humid air, including exhaled breath and the headspace of liquid-phase biogenic media. The detection and quantification by SIFT-MS of MDA released by a cell culture are demonstrated. Copyright © 2015 John Wiley & Sons, Ltd

Sunflower Plants as Bioindicators of Environmental Pollution with Lead (II) Ions

In this study, the influence of lead (II) ions on sunflower growth and biochemistry was investigated from various points of view. Sunflower plants were treated with 0, 10, 50, 100 and/or 500 μM Pb-EDTA for eight days. We observed alterations in growth in all experimental groups compared with non-treated control plants. Further we determined total content of proteins by a Bradford protein assay. By the eighth day of the experiment, total protein contents in all treated plants were much lower compared to control. Particularly noticeable was the loss of approx. 8 μg/mL or 15 μg/mL in shoots or roots of plants treated with 100 mM Pb-EDTA. We also focused our attention on the activity of alanine transaminase (ALT), aspartate transaminase (AST) and urease. Activity of the enzymes increased with increasing length of the treatment and applied concentration of lead (II) ions. This increase corresponds well with a higher metabolic activity of treated plants. Contents of cysteine, reduced glutathione (GSH), oxidized glutathione (GSSG) and phytochelatin 2 (PC2) were determined by high performance liquid chromatography with electrochemical detection. Cysteine content declined in roots of plants with the increasing time of treatment of plants with Pb-EDTA and the concentration of toxic substance. Moreover, we observed ten times higher content of cysteine in roots in comparison with shoots. The observed reduction of cysteine content probably relates with its utilization for biosynthesis of GSH and phytochelatins, because the content of GSH and PC2 was similar in roots and shoots and increased with increased treatment time and concentration of Pb-EDTA. Moreover, we observed oxidative stress caused by Pb-EDTA in roots where the GSSG/GSH ratio was about 0.66. In shoots, the oxidative stress was less distinctive, with a GSSG/GSH ratio 0.14. We also estimated the rate of phytochelatin biosynthesis from the slope of linear equations plotted with data measured in the particular experimental group. The highest rate was detected in roots treated with 100 μM of Pb-EDTA. To determine heavy metal ions many analytical instruments can be used, however, most of them are only able to quantify total content of the metals. This problem can be overcome using laser induced breakdown spectroscopy, because it is able to provide a high spatial-distribution of metal ions in different types of materials, including plant tissues. Data obtained were used to assemble 3D maps of Pb and Mg distribution. Distribution of these elements is concentrated around main vascular bundle of leaf, which means around midrib

Quantitative analysis of volatile metabolites released in vitro by bacteria of the genus Stenotrophomonas for identification of breath biomarkers of respiratory infection in cystic fibrosis

The aim of the present study was to characterize the volatile metabolites produced by genotypically diverse strains of the Stenotrophomonas genus in order to evaluate their potential as biomarkers of lung infection by non-invasive breath analysis. Volatile organic compounds (VOCs) emitted from 15 clinical and five environmental strains belonging to different genogroups of Stenotrophomonas maltophilia (n = 18) and Stenotrophomonas rhizophila (n = 2) cultured in Mueller-Hinton Broth (MHB) liquid media were analysed by gas chromatography mass spectrometry (GC-MS) and selected ion flow tube mass spectrometry (SIFT-MS). Several VOCs were detected in high concentration, including ammonia, propanol, dimethyl disulphide propanol and dimethyl disulphide. The GC-MS measurements showed that all 15 clinical strains produced similar headspace VOCs compositions, and SIFT-MS quantification showed that the rates of production of the VOCs by the genotypically distinct strains were very similar. All in vitro cultures of both the Stenotrophomonas species were characterised by efficient production of two isomers of methyl butanol, which can be described by known biochemical pathways and which is absent in other pathogens, including Pseudomonas aeruginosa. These in-vitro data indicate that methyl butanol isomers may be exhaled breath biomarkers of S. maltophilia lung infection in patients with cystic fibrosis

Acinetobacter silvestris sp. nov. discovered in forest ecosystems in Czechia

We investigated a taxonomically novel group of the genus Acinetobacter, which included five strains isolated from soil and water samples collected in preserved forest areas in Czechia between 2013 and 2021. The whole-genome sequences of the strains were 3.1-3.2 Mb in size, with G+C contents of 38.0-38.2mol%. Core genome-based phylogenetic analysis showed that they formed a compact and deeply branched Glade within the genus. The genomic average nucleotide identity based on BLAST/digital DNA-DNA hybridization values for the novel strains were 99.2-99.6%/95.2-98.4%, whereas those between the novel strains and the type strains of the known Acinetobacter species reached <78%/<24%. The results of the genus-wide analysis of whole-cell MALDI-TOF mass spectra supported the sharp distinctness of the group. The five strains were non-glucose acidifying. nonhaemolytic, nonproteolytic and growing at 28 degrees C. but not at 32 degrees C; they assimilated acetate, benzoate, ethanol, L-histidine, 4-hydroxybenzoate, DL-lactate and malonate but not 4-aminobutyrate, L-aspartate or 2,3-butanediol; this phenotype is unique among the known Acinetobacter species. We conclude that the five strains represent a novel environmental species, for which the name Acinetobacter silvestris sp. nov. is proposed, with the type strain ANC 4999(T) (=CCM 920(T)=CCUG 7587(T)=CNCTC 8124(T))

Formation of nucleobases in a Miller-Urey reducing atmosphere

The Miller-Urey experiments pioneered modern research on the molecular origins of life, but their actual relevance in this field was later questioned because the gas mixture used in their research is considered too reducing with respect to the most accepted hypotheses for the conditions on primordial Earth. In particular, the production of only amino acids has been taken as evidence of the limited relevance of the results. Here, we report an experimental work, combined with state-of-the-art computational methods, in which both electric discharge and laser-driven plasma impact simulations were carried out in a reducing atmosphere containing NH 3 + CO. We show that RNA nucleobases are synthesized in these experiments, strongly supporting the possibility of the emergence of biologically relevant molecules in a reducing atmosphere. The reconstructed synthetic pathways indicate that small radicals and formamide play a crucial role, in agreement with a number of recent experimental and theoretical results. The following explorations showed that a broad array of amino acids could be synthesized, but there was no evidence that all of the fundamental molecules of the RNA genetic code could be produced alongside others in this type of experiment (2-5). Additionally, the significant persistence of reducing atmospheres in a geological timescale has been seriously debated (6). Finally, many scientists have claimed that this experiment is not related to early-Earth conditions and does not provide fundamental building blocks (i.e., nucleobases) important for the evolution of early life possibly based on RNA (7-13). In 2001, Saladino, Di Mauro, and coworkers During the past decade, comets (39), HCN hydrolysis (40), chemistry in interstellar space (41), reducing atmospheres (42), or ammonium formate dehydration (43) have been proposed as sources of formamide. However, in most cases, the exact chemistry of such systems has not been well explored either experimentally or theoretically. Moreover, the plausibility and relation to a prebiotic environment is also questioned. Using the large laser facility at the terrawatt Prague Asterix Laser System, we comprehensively explored asteroid shock wave impact plasma, in addition to electric discharge, in a simple reducing mixture of NH 3 + CO and H 2 O. The formamide molecule does not directly play the role of starting substrate, but it is rather a suspected intermediate of reactions leading from simple model prebiotic mixtures to biomolecules. The results are compared with similar experiments, in which formamide is the starting compound. The chemistry is also mapped using state-of-the-art ab initio molecular-dynamics simulations. We focused our effort on two environments relevant to prebiotic chemistry: (i) transformation of an atmosphere exposed a shock wave induced by an extraterrestrial body and the resulting impact Significance The study shows that Miller-Urey experiments produce RNA nucleobases in discharges and laser-driven plasma impact simulations carried out in a simple prototype of reducing atmosphere containing ammonia and carbon monoxide. We carried out a self-standing description of chemistry relevant to hypothesis of abiotic synthesis of RNA nucleobases related to early-Earth chemical evolution under reducing conditions. The research addresses the chemistry of simple-model reducing atmosphere (NH 3 + CO + H 2 O) and the role of formamide as an intermediate of nucleobase formation in Miller-Urey experiment. The explorations combine experiments performed using modern techniques of large, high-power shock wave plasma generation by hall terawatt lasers, electric discharges, and state-of-the-art ab initio free-energy calculations

Formation of nucleobases in a Miller-Urey reducing atmosphere

The Miller-Urey experiments pioneered modern research on the molecular origins of life, but their actual relevance in this field was later questioned because the gas mixture used in their research is considered too reducing with respect to the most accepted hypotheses for the conditions on primordial Earth. In particular, the production of only amino acids has been taken as evidence of the limited relevance of the results. Here, we report an experimental work, combined with state-of-the-art computational methods, in which both electric discharge and laser-driven plasma impact simulations were carried out in a reducing atmosphere containing NH 3 + CO. We show that RNA nucleobases are synthesized in these experiments, strongly supporting the possibility of the emergence of biologically relevant molecules in a reducing atmosphere. The reconstructed synthetic pathways indicate that small radicals and formamide play a crucial role, in agreement with a number of recent experimental and theoretical results. The following explorations showed that a broad array of amino acids could be synthesized, but there was no evidence that all of the fundamental molecules of the RNA genetic code could be produced alongside others in this type of experiment (2-5). Additionally, the significant persistence of reducing atmospheres in a geological timescale has been seriously debated (6). Finally, many scientists have claimed that this experiment is not related to early-Earth conditions and does not provide fundamental building blocks (i.e., nucleobases) important for the evolution of early life possibly based on RNA (7-13). In 2001, Saladino, Di Mauro, and coworkers During the past decade, comets (39), HCN hydrolysis (40), chemistry in interstellar space (41), reducing atmospheres (42), or ammonium formate dehydration (43) have been proposed as sources of formamide. However, in most cases, the exact chemistry of such systems has not been well explored either experimentally or theoretically. Moreover, the plausibility and relation to a prebiotic environment is also questioned. Using the large laser facility at the terrawatt Prague Asterix Laser System, we comprehensively explored asteroid shock wave impact plasma, in addition to electric discharge, in a simple reducing mixture of NH 3 + CO and H 2 O. The formamide molecule does not directly play the role of starting substrate, but it is rather a suspected intermediate of reactions leading from simple model prebiotic mixtures to biomolecules. The results are compared with similar experiments, in which formamide is the starting compound. The chemistry is also mapped using state-of-the-art ab initio molecular-dynamics simulations. We focused our effort on two environments relevant to prebiotic chemistry: (i) transformation of an atmosphere exposed a shock wave induced by an extraterrestrial body and the resulting impact Significance The study shows that Miller-Urey experiments produce RNA nucleobases in discharges and laser-driven plasma impact simulations carried out in a simple prototype of reducing atmosphere containing ammonia and carbon monoxide. We carried out a self-standing description of chemistry relevant to hypothesis of abiotic synthesis of RNA nucleobases related to early-Earth chemical evolution under reducing conditions. The research addresses the chemistry of simple-model reducing atmosphere (NH 3 + CO + H 2 O) and the role of formamide as an intermediate of nucleobase formation in Miller-Urey experiment. The explorations combine experiments performed using modern techniques of large, high-power shock wave plasma generation by hall terawatt lasers, electric discharges, and state-of-the-art ab initio free-energy calculations