Hypoxia and lactate influence VOC production in A549 lung cancer cells.

Introduction: Cancer cells emit characteristic volatile organic compounds (VOCs), which are potentially generated from ROS-based lipid peroxidation of polyunsaturated fatty acids. The metabolism of such VOCs and their regulation remain to be fully investigated. In fact, the enzymes involved in the synthesis of these VOCs have not been described yet. Methods: In this study, we firstly conducted in vitro enzyme assays and demonstrated that recombinant alcohol dehydrogenase (ADH) converted Trans 2-hexenal into Trans 2-hexenol. The latter has previously been reported as a cancer VOC. To study VOC metabolism, 14 different culture conditions were compared in view of Trans 2-hexenol production. Results and discussion: The data indicate that hypoxia and the addition of lactate positively influenced Trans 2-hexenol production in A549 cancer cells. The RNAseq data suggested certain gene expressions in the VOC pathway and in lactate signaling, parallel to VOC production. This implies that hypoxia and lactate signaling with a VOC production can be characteristic for cancer in vitro.

Role of Light and Plant Hormones in Stem Parasitic Plant (Cuscuta and Cassytha) Twining and Haustoria Induction.

Cuscuta and Cassytha are two distinct stem parasitic plant genera developing haustoria at their stem. The initial step to parasitization is twining onto the host plant. Although twining is the critical first step, less attention has been paid to this aspect in stem haustoria parasitic plant studies. As tendril coiling is also controlled by light and plant hormones, we investigated the role of light (blue, red and far-red) and hormones (auxin, brassinolide, cytokinin) in twining of stem parasitic plants (Cuscuta japonica and Cassytha filiformis). In general, both Cuscuta and Cassytha showed similar behavior to light cues. The data show that blue light is essential for twining, and a lower far-red/red light (FR/R) ratio is important for subsequent haustoria induction. Regarding plant hormones, seedlings with solely auxin or cytokinin (iP) under blue light showed not only twining but also haustoria induction, demonstrating that auxin and iP appear to be especially important for induction. Seedlings with solely brassinolide showed no positive influence, but brassinolide together with iP caused twining even under dark conditions. This points to the presence of cross-talk between brassinolide and cytokinin for twining.

Non-linear regression analysis for estimating the intraoperative motor evoked potential recovery time after bolus neuromuscular blockade.

The recovery time of the motor evoked potential (MEP) amplitude following a neuromuscular blockade (NMB) during surgery is useful for interpreting low-amplitude waveforms or selecting the baseline waveform. In this study, the MEP data of 195 orthopedic cases who received a bolus dose of rocuronium at the beginning of surgery, between June 2009 and January 2016 were used. A non-linear regression analysis was applied to MEP amplitude data of multiple patients. The time taken for 90% of the maximum-amplitude recovery was estimated from the identified time series model. The 90% amplitude recovery time was 88.6 min in the pharmacological model and 89.4 min in the logistic model. These results were included in the 95% confidence interval of the previous studies. Although MEP amplitude is relatively unstable because of anesthesia, the averaged time series model of MEP amplitude can be estimated by using a large number of data.

Elucidation of Biochemical Pathways Underlying VOCs Production in A549 Cells.

Cellular volatile organic compounds (VOCs) are unique compounds whose metabolic pathways remain enigmatic. To elucidate their metabolism, we investigated the VOCs of lung cancer A549 and 2 non-cancer lung cells (HLB; HBEpC). Neutral sugars and lactate in the medium were measured by colorimetric assay. VOCs were enriched by monotrap and profiled by GC-MS. To investigate the enzymes that change VOC metabolism in cells, we conducted ALDH activity assays and qPCR. ROS (reactive oxygen species) assays were conducted to assess oxidation stress. The colorimetric assay showed that especially A549 and HLB took up sugars from the medium and rapidly secreted lactate into the medium. The VOC profile (GC-MS) revealed a trans-2-hexenol increase, especially in A549 lung cancer cells. This is a novel lipid peroxidation product from animal cells. Based on the absolute quantification data, trans-2-hexenol increased in parallel with number of A549 cancer cells incubated. The qPCR data implies that ADH1c potentially plays an important role in the conversion into trans-2-hexenol.

Urinary Pharmacokinetic and Pharmacodynamic Profiles of Fosfomycin against Extended-Spectrum ß-Lactamase-Producing Escherichia coli with Canine Ex Vivo Modeling: A Pilot Study.

Fosfomycin is a candidate drug for extended-spectrum ß-lactamase (ESBL)-producing bacteria, but its efficacy is yet to be investigated in dogs. This study investigated the urinary pharmacokinetic/pharmacodynamic (PK/PD) profile of fosfomycin orally administered at 80 mg/kg to six healthy dogs to assess its efficacy for canine urinary tract infections (UTIs) caused by ESBL-producing bacteria. Four strains of ESBL-producing Escherichia coli (ESBL-EC) characterized by fosfomycin minimum inhibitory concentrations (MICs) of 0.5, 1, 2, and 32 µg/mL were used. Urine samples for the measurement of urinary drug concentrations and urinary bactericidal titers (UBTs) were obtained after drug administration. The urinary concentrations (µg/mL, mean ± SE) were 1348.2 ± 163.5, 1191.6 ± 260.2, and 661.1 ± 190.4 at 0-4, 4-8, and 8-12 h, respectively, after drug administration. The mean urinary area under the curve during the test period (AUC0-12) of fosfomycin was estimated to be 12,803.8 µg·h/mL. The UBTs for all tested strains fluctuated closely with urine concentration during the test period (r = 0.944-1.000), and the area under the UBT-versus-time curve correlated with the urinary AUC/MIC of each strain (r = 0.991). According to the optimal urinary PK/PD target value, fosfomycin at 80 mg/kg twice daily may be suitable for the treatment of canine UTIs caused by ESBL-EC presenting MIC ≤ 128 µg/mL.

Rapid profiling method for mammalian feces short chain fatty acids by GC-MS.

Short chain fatty acids (SCFAs) are key feces metabolites generated by gut bacteria fermentation. Despite the importance of profiling feces SCFAs, technical difficulties in analysis remain due to their volatility and hydrophilicity. We improve previous protocols to profile SCFAs and optimize the metabolite profiling platform for mammalian feces samples. In this study, we investigated feces as biological samples using gas chromatography-mass spectrometry (GC-MS). Isobutyl chloroformate was used for a derivatization in aqueous solution without drying out the samples. Ultimately, we envisage being able to determine the way in which gut bacteria fermentation influences host gut condition by using our rapid metabolite profiling methods.

Application of GC/MS Soft Ionization for Isomeric Biological Compound Analysis.

Isomers are compounds with the same molecular formula. Many different types of isomers are ubiquitous and play important roles in living organisms. Despite their early discovery, the actual analysis of isomers has been tricky and has confounded researchers. Using mass spectrometry (MS) to distinguish or identify isomers is an emergent topic and challenge for analytical chemists. We review some techniques for analyzing isomers with emphasis on MS, e.g., the roles of ion reaction, hydrogen-deuterium exchange, ion mobility mass spectrometry, ion spectroscopy, and energy change in producing isomer-specific fragments. In particular, soft ionization for gas chromatography-mass spectrometry (GC-MS) is a focus in this review. Awareness of the advantages and technical problems of these techniques would inspire innovation in future approaches.

Analysis of Metabolites in Stem Parasitic Plant Interactions: Interaction of Cuscuta-Momordica versus Cassytha-Ipomoea.

Cuscuta and Cassytha are two well-known stem parasitic plant genera with reduced leaves and roots, inducing haustoria in their stems. Their similar appearance in the field has been recognized, but few comparative studies on their respective plant interactions are available. To compare their interactions, we conducted a metabolite analysis of both the Cassytha-Ipomoea and the Cuscuta-Momordica interaction. We investigated the energy charge of the metabolites by UFLC (ultra-high performance liquid chromatography), and conducted GC-MS (gas chromatography-mass spectrometry) analysis for polar metabolites (e.g., saccharides, polyols) and steroids. The energy charge after parasitization changed considerably in Cassytha but not in Cusucta. Cuscuta changed its steroid pattern during the plant interaction, whereas Cassytha did not. In the polar metabolite analysis, the laminaribiose increase after parasitization was conspicuous in Cuscuta, but not in Cassytha. This metabolite profile difference points to different lifestyles and parasitic strategies.

Biodiesel and poly-unsaturated fatty acids production from algae and crop plants - a rapid and comprehensive workflow for lipid analysis.

Fatty acid methyl ester analysis (FAME) by gas chromatography coupled to mass spectrometry (GC-MS) is a widely used technique in biodiesel/bioproduct (e.g. poly-unsaturated fatty acids, PUFA) research but typically does not allow distinguishing between bound and free fatty acids. To understand and optimize biosynthetic pathways, however, the origin of the fatty acid is an important information. Furthermore the annotation of PUFAs is compromised in classical GC-EI-MS because the precursor molecular ion is missing. In the present protocol an alkaline methyl esterification step with TMS derivatization enabling the simultaneous analysis of bound and free fatty acids but also further lipids such as sterols in one GC-MS chromatogram is combined. This protocol is applied to different lipid extracts from single cell algae to higher plants: Chlorella vulgaris, Chlamydomonas reinhardtii, Coffea arabica, Pisum sativum and Cuscuta japonica. Further, field ionization (GC-FI-MS) is introduced for a better annotation of fatty acids and exact determination of the number of double bonds in PUFAs. The proposed workflow provides a convenient strategy to analyze algae and other plant crop systems with respect to their capacity for third generation biodiesel and high-quality bioproducts for nutrition such as PUFAs.

Fragmentation patterns of methyloxime-trimethylsilyl derivatives of constitutive mono- and disaccharide isomers analyzed by gas chromatography/field ionization mass spectrometry.

RATIONALE: In saccharide analysis by gas chromatography/mass spectrometry (GC/MS), electron ionization (EI) is used almost exclusively, whereas other gentler methods of ionization are rarely used. Field ionization (FI) is recognized as a GC/MS ionization method that causes fewer fragment ions, but only few studies are available on its use in saccharide analysis. METHODS: To evaluate the usefulness of FI in profiling isomeric saccharides by GC/MS and to explore its potential application in metabolome analysis, we compared EI, chemical ionization (CI), and FI spectral patterns of consecutive mono- and disaccharides derivatized with methoxamine-HCl and N-methyl-N-(trimethylsilyl)trifluoroacetamide. RESULTS: FI produced molecular ions and fragment ions characteristic of constitutive isomeric disaccharides. All of the derivatized saccharides that originally had free anomeric OH showed methyloxime-moiety fragment ions, attributable to the cleavage between C2 and C3. Some fragment ions in FI were indicative of the position of dihexose linkages. Although EI with lowered voltage (18 V, 130 °C) produced fewer fragment ions than conventional EI (70 V, 250 °C) did, fragmentation patterns were different from those of FI. CONCLUSIONS: Our data show that FI is useful for distinguishing isomeric saccharides in qualitative analyses.

A bioinformatics approach to distinguish plant parasite and host transcriptomes in interface tissue by classifying RNA-Seq reads.

BACKGROUND: The genus Cuscuta is a group of parasitic plants that are distributed world-wide. The process of parasitization starts with a Cuscuta plant coiling around the host stem. The parasite's haustorial organs then establish a vascular connection allowing for access to the phloem content. The host and the parasite form new cellular connections, suggesting coordination of developmental and biochemical processes. Simultaneous monitoring of gene expression in the parasite's and host's tissues may shed light on the complex events occurring between the parasitic and host cells and may help to overcome experimental limitations (i.e. how to separate host tissue from Cuscuta tissue at the haustorial connection). A novel approach is to use bioinformatic analysis to classify sequencing reads as either belonging to the host or to the parasite and to characterize the expression patterns. Owing to the lack of a comprehensive genomic dataset from Cuscuta spp., such a classification has not been performed previously. RESULTS: We first classified RNA-Seq reads from an interface region between the non-model parasitic plant Cuscuta japonica and the non-model host plant Impatiens balsamina. Without established reference sequences, we classified reads as originating from either of the plants by stepwise similarity search against de novo assembled transcript sets of C. japonica and I. balsamina, unigene sets of the same genus, and cDNA sequences of the same family. We then assembled de novo transcriptomes from the classified read sets. We assessed the quality of the classification by mapping reads to contigs of both plants, achieving a misclassification rate low enough (0.22-0.39%) to be used reliably for differential gene expression analysis. Finally, we applied our read classification method to RNA-Seq data from the interface between the non-model parasitic plant C. japonica and the model host plant Glycine max. Analysis of gene expression profiles at 5 parasitizing stages revealed differentially expressed genes from both C. japonica and G. max, and uncovered the coordination of cellular processes between the two plants. CONCLUSIONS: We demonstrated that reliable identification of differentially expressed transcripts in undissected interface region of the parasite-host association is feasible and informative with respect to differential-expression patterns.

Exploration of polar lipid accumulation profiles in Euglena gracilis using LipidBlast, an MS/MS spectral library constructed in silico.

A rapid protocol for polar lipid profiling was applied to Euglena gracilis lipid metabolism by LipidBlast, an MS/MS spectral similarity search tool. The similarity search results suggested anoxia-induced polar lipid metabolism in Euglena characterized by the accumulation of differential lipid classes, carbon chain lengths, and unsaturated bond numbers. The informatics-supported MS spectral search provides an alternative option for global lipid profiling studies.

Boron nitride as desalting material in combination with phosphopeptide enrichment in shotgun proteomics.

Hydrophilic peptides in shotgun proteomics have been shown to be problematic in conventional chromatography. Typically, C18 solid phase extraction or peptide traps are used for desalting the sample prior to mass spectrometry analysis, but the capacity to retain hydrophilic peptides is not very high, causing a bias toward more hydrophobic peptides. This is particularly problematic in phosphoproteomic studies. We tested the compatibility of commercially available boron nitride as a novel material for peptide desalting. Boron nitride can be used to recover a wide range of peptides with different physicochemical properties comparable to combined C18 and graphite carbon material.

System-level network analysis of nitrogen starvation and recovery in Chlamydomonas reinhardtii reveals potential new targets for increased lipid accumulation.

BACKGROUND: Nitrogen starvation is known to cause drastic alterations in physiology and metabolism leading to the accumulation of lipid bodies in many microalgae, and it thus presents an important alternative for biofuel production. However, despite the importance of this process, the molecular mechanisms that mediate the metabolic remodeling induced by N starvation and especially by stress recovery are still poorly understood, and new candidates for bioengineering are needed to make this process useful for biofuel production. RESULTS: We have studied the molecular changes involved in the adaptive mechanisms to N starvation and full recovery of the vegetative cells in the microalga Chlamydomonas reinhardtii during a four-day time course. High throughput mass spectrometry was employed to integrate the proteome and the metabolome with physiological changes. N starvation led to an accumulation of oil bodies and reduced Fv/Fm.. Distinct enzymes potentially participating in the carbon-concentrating mechanism (CAH7, CAH8, PEPC1) are strongly accumulated. The membrane composition is changed, as indicated by quantitative lipid profiles. A reprogramming of protein biosynthesis was observed by increased levels of cytosolic ribosomes, while chloroplastidic were dramatically reduced. Readdition of N led to, the identification of early responsive proteins mediating stress recovery, indicating their key role in regaining and sustaining normal vegetative growth. Analysis of the data with multivariate correlation analysis, Granger causality, and sparse partial least square (sPLS) provided a functional network perspective of the molecular processes. Cell growth and N metabolism were clearly linked by the branched chain amino acids, suggesting an important role in this stress. Lipid accumulation was also tightly correlated to the COP II protein, involved in vesicle and lysosome coating, and a major lipid droplet protein. This protein, together with other key proteins mediating signal transduction and adaption (BRI1, snRKs), constitute a series of new metabolic and regulatory targets. CONCLUSIONS: This work not only provides new insights and corrects previous models by analyzing a complex dataset, but also increases our biochemical understanding of the adaptive mechanisms to N starvation in Chlamydomonas, pointing to new bioengineering targets for increased lipid accumulation, a key step for a sustainable and profitable microalgae-based biofuel production.

Systemic cold stress adaptation of Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is one of the most important model organisms nowadays phylogenetically situated between higher plants and animals (Merchant et al. 2007). Stress adaptation of this unicellular model algae is in the focus because of its relevance to biomass and biofuel production. Here, we have studied cold stress adaptation of C. reinhardtii hitherto not described for this algae whereas intensively studied in higher plants. Toward this goal, high throughput mass spectrometry was employed to integrate proteome, metabolome, physiological and cell-morphological changes during a time-course from 0 to 120 h. These data were complemented with RT-qPCR for target genes involved in central metabolism, signaling, and lipid biosynthesis. Using this approach dynamics in central metabolism were linked to cold-stress dependent sugar and autophagy pathways as well as novel genes in C. reinhardtii such as CKIN1, CKIN2 and a hitherto functionally not annotated protein named CKIN3. Cold stress affected extensively the physiology and the organization of the cell. Gluconeogenesis and starch biosynthesis pathways are activated leading to a pronounced starch and sugar accumulation. Quantitative lipid profiles indicate a sharp decrease in the lipophilic fraction and an increase in polyunsaturated fatty acids suggesting this as a mechanism of maintaining membrane fluidity. The proteome is completely remodeled during cold stress: specific candidates of the ribosome and the spliceosome indicate altered biosynthesis and degradation of proteins important for adaptation to low temperatures. Specific proteasome degradation may be mediated by the observed cold-specific changes in the ubiquitinylation system. Sparse partial least squares regression analysis was applied for protein correlation network analysis using proteins as predictors and Fv/Fm, FW, total lipids, and starch as responses. We applied also Granger causality analysis and revealed correlations between proteins and metabolites otherwise not detectable. Twenty percent of the proteins responsive to cold are uncharacterized proteins. This presents a considerable resource for new discoveries in cold stress biology in alga and plants.

A novel selective stimulus presentation for P300 speller.

The P300 speller is one of the brain-computer interfaces, allowing users to spell letters just by thoughts. Due to the low signal-to-noise ratio of the P300, however, stimuli are repeatedly presented so that EEG signals can be averaged, which improves the accuracy but degrades the speed. The authors have proposed to discontinue the stimulus presentation adaptively to the P300 response and have shown its superiority in the performance over the standard way that presents a prefixed number of stimuli. In addition to this adaptive stimulus termination, this paper proposes to select stimuli to be presented to avoid presenting redundant stimuli. Both off-line and on-line experiments show that the proposed method is more effective than our conventional method.

Comparison of aragonitic molluscan shell proteins.

Acidic macromolecules, as a nucleation factor for mollusc shell formation, are a major focus of research. It remains unclear, however, whether acidic macromolecules are present only in calcified shell organic matrices, and which acidic macromolecules are crucial for the nucleation process by binding to chitin as structural components. To clarify these questions, we applied 2D gel electrophoresis and amino acid analysis to soluble shell organic matrices from nacre shell, non-nacre aragonitic shell and non-calcified squid shells. The 2D gel electrophoresis results showed that the acidity of soluble proteins differs even between nacre shells, and some nacre (Haliotis gigantea) showed a basic protein migration pattern. Non-calcified shells also contained some moderately acidic proteins. The results did not support the correlation between the acidity of soluble shell proteins and shell structure.

Application of support vector machines to reliability-based automatic repeat request for Brain-Computer Interfaces.

A Brain-Computer Interface (BCI) is a system that could enable patients like those with Amyotrophic Lateral Sclerosis to control some equipment and to communicate with other people, and has been anticipated to be achieved. One of the problems in BCI research is a trade-off between speed and accuracy, and it is practically important to adjust those two performance measures effectively. So far the authors have considered BCIs as communications between users and computers, and have proposed an error control method, Reliability-Based Automatic Repeat reQuest (RB-ARQ). It has been shown that, with Linear Discriminant Analysis (LDA) as a classifier, RB-ARQ is more effective than other error control methods. In this paper, Support Vector Machines (SVMs), one of the most popular classifiers, are applied to RB-ARQ. A quantitative comparison showed that there was no significant difference between LDA and SVM. Also, it was demonstrated that RB-ARQ improved the accuracy from the one acquired by the top ranked methods in the BCI competition to 100 percents, with less loss of the speed.

Molluscan shell evolution with review of shell calcification hypothesis.

Biomineralization research on mollusc shells has mostly focused on nacre formation. Chitin, silk fibroin protein, and acidic macromolecules are important components for shell formation. Although the principle concept behind shell calcification was developed many years ago, the individual components have not been well scrutinized. Besides that, Mollusca are the second largest invertebrate phylum, but comprehensive biochemical research involving a comparison of different taxa is still rare. This study reconsiders the above three components with adding some biochemical data of aculiferans. The presence of chitin in polyplacophorans sclerites was confirmed by IR and pyrolysis GC/MS. DMMB staining data inferred that sulphated groups present in aplacophoran cuticle but not in polyplacophorans cuticle. These insight suggested importance of comparison between acuriferans and conchiferans.

Pyrolysis GC/MS and IR spectroscopy in chitin analysis of molluscan shells.

Chitin is an insoluble component in the shells of several molluscan species. It is thought to play important roles, in biomineralization and shell structure. To date, however, reports are scarce and sometimes contradictory, and suffer from methodological problems. Only in a single cephalopod species has the chitin been identified as beta-chitin. We present data on chitin occurrence in 22 species of shell-bearing Mollusca (Conchifera) and Polyplacophora, including the first evidence for scaphopods, based on pyrolysis gas chromatography, mass spectrometry (GC-MS), and infrared spectroscopy (IR). Pyrolysis GC-MS detected chitin in every tested member of the Conchifera. IR spectroscopy before and after chitinase treatment revealed at least three distinct patterns of peak changes. The contents of the insoluble shell organics included not only chitin and proteins, but also insoluble polysaccharides, e.g., glucan. We conclude that chitin was present in the last common ancestor of the Conchifera and that its abundance in the shell matrix depends on the differentiation of the shell.