Mining for Active Molecules in Probiotic Supernatant by Combining Non-Targeted Metabolomics and Immunoregulation Testing

Chronic respiratory diseases such as asthma are highly prevalent in industrialized countries. As cases are expected to rise, there is a growing demand for alternative therapies. Our recent research on the potential benefits of probiotics suggests that they could prevent and reduce the symptoms of many diseases by modulating the host immune system with secreted metabolites. This article presents the first steps of the research that led us to identify the immunoregulatory bioactivity of the amino acid d-Trp reported in our previous study. Here we analyzed the cell culture metabolic footprinting of 25 commercially available probiotic strains to associate metabolic pathway activity information with their respective immune modulatory activity observed in vitro. Crude probiotic supernatant samples were processed in three different ways prior to untargeted analysis in positive and negative ionization mode by direct infusion ESI-FT-ICR-MS: protein precipitation and solid phase extraction (SPE) using HLB and CN-E sorbent cartridges. The data obtained were submitted to multivariate statistical analyses to distinguish supernatant samples into the bioactive and non-bioactive group. Pathway analysis using discriminant molecular features showed an overrepresentation of the tryptophan metabolic pathway for the bioactive supernatant class, suggesting that molecules taking part in that pathway may be involved in the immunomodulatory activity observed in vitro. This work showcases the potential of metabolomics to drive product development and novel bioactive compound discovery out of complex biological samples in a top-down manner.

The Influences of Perfluoroalkyl Substances on the Rheumatoid Arthritis Clinic

Objective. Perfluoroalkyl substances (PFASs) are a class of synthetic organic fluorine chemicals, which have been mass-produced and widely used in the past 60 years, and also have been shown to be one of the major pollutants affecting human health. The impact of fluoride on the development of RA is unclear. The objective of this study was to assess the relationship between perfluoroalkyl substances and the clinic of RA. Methods. A cohort of 155 patients with RA in Second Affiliated Hospital of Zhejiang University School of Medicine were investigated. Demographic and clinical data of these patients were recorded. The concentration of perfluoroalkyl substances were measured by ultra-performance liquid chromatography system (ACQUITY, UPLC) coupled to a tandem mass spectrometer that operated in negative ionization mode. The correlations between the clinical data and the concentration of perfluoroalkyl substances were analyzed.Results. There were 43 male patients and 112 female patients in the cohort. Some of perfluoroalkyl substances (PFOA, PFNA, PFTrA, PFOS) were correlated negatively with the Body Mass Index (BMI); some of them (PFOA, PFNA, PFTrA, PFOS, 8:2Cl-PFESA) were correlated positively with the Disease Activity Score 28 (DAS28); two (PFOA, PFOS) of them were correlated positively with the white blood cell count, and one (PFUnA) of them was correlated negatively with the hemoglobin; two (PFDA, PFUnA) of them were correlated negatively with the presence of interstitial lung disease. Conclusion. These data suggest that exposure to perfluoroalkyl substances may promote the disease activity of rheumatoid arthritis and the visceral lesions.

From mass spectral features to molecules in molecular networks: a novel workflow for untargeted metabolomics.

In the context of untargeted metabolomics, molecular networking is a popular and efficient tool which organizes and simplifies mass spectrometry fragmentation data (LC-MS/MS), by clustering ions based on a cosine similarity score. However, the nature of the ion species is rarely taken into account, causing redundancy as a single compound may be present in different forms throughout the network. Taking advantage of the presence of such redundant ions, we developed a new method named MolNotator. Using the different ion species produced by a molecule during ionization (adducts, dimers, trimers, in-source fragments), a predicted molecule node (or neutral node) is created by triangulation, and ultimately computing the associated molecule calculated mass. These neutral nodes provide researchers with several advantages. Firstly, each molecule is then represented in its ionization context, connected to all produced ions and indirectly to some coeluted compounds, thereby also highlighting unexpected widely present adduct species. Secondly, the predicted neutrals serve as anchors to merge the complementary positive and negative ionization modes into a single network. Lastly, the dereplication is improved by the use of all available ions connected to the neutral nodes, and the computed molecular masses can be used for exact mass dereplication. MolNotator is available as a Python library and was validated using the lichen database spectra acquired on an Orbitrap, computing neutral molecules for >90% of the 156 molecules in the dataset. By focusing on actual molecules instead of ions, MolNotator greatly facilitates the selection of molecules of interest.

On the slow ionization waves forming the breakdown in a long capillary tube with helium at low pressure

The results of studies of an electrical breakdown leading to the glow discharge ignition in a long capillary quartz tube are presented. Under such conditions, the breakdown completion is preceded by the development of direct, backward, and counter slow ionization waves traveling in the tube. The initiation of the waves was created in helium at low pressure (P=10 Torr) by the high-voltage pulses of positive and negative polarity with amplitude of several kilovolts. In the beginning, the regime without the breakdown completion in the tube was studied. In this regime, the propagation of only direct positive and direct negative ionization waves happens. The research on dynamics of the direct, backward, and counter positive and negative waves followed by a complete breakdown was done as well. The influence of the pre-existing plasma on the ionization waves propagation was also studied. The plasma was created in advance by low-current glow discharge being formed in the tube. The instant images of ionization waves were correlated with the electrical currents formed by the waves, that is, with the displacement current through the dielectric wall and the conductive current through the plasma column. In the experiments, the fine-sectioned electrode wrapped around the lateral tube surface was used. The usage of such electrode allowed one to study the dynamics of the surface charge deposition and deletion happening during the direct and backward wave propagation, respectively. Finally, a strong difference in the spatial structure and velocity of positive and negative direct waves traveling through non-ionized gas was revealed. Contrary, both the positive and negative backward waves traveling through the plasma formed by previous direct waves have the parameters close to each other.

Untargeted metabolomics of COVID-19 patient serum reveals potential prognostic markers of both severity and outcome

Introduction The diagnosis of COVID-19 is normally based on the qualitative detection of viral nucleic acid sequences. Properties of the host response are not measured but are key in determining outcome. Although metabolic profiles are well suited to capture host state, most metabolomics studies are either underpowered, measure only a restricted subset of metabolites, compare infected individuals against uninfected control cohorts that are not suitably matched, or do not provide a compact predictive model. Objectives Here we provide a well-powered, untargeted metabolomics assessment of 120 COVID-19 patient samples acquired at hospital admission. The study aims to predict the patient’s infection severity (i.e., mild or severe) and potential outcome (i.e., discharged or deceased). Methods High resolution untargeted UHPLC-MS/MS analysis was performed on patient serum using both positive and negative ionization modes. A subset of 20 intermediary metabolites predictive of severity or outcome were selected based on univariate statistical significance and a multiple predictor Bayesian logistic regression model was created. Results The predictors were selected for their relevant biological function and include deoxycytidine and ureidopropionate (indirectly reflecting viral load), kynurenine (reflecting host inflammatory response), and multiple short chain acylcarnitines (energy metabolism) among others. Currently, this approach predicts outcome and severity with a Monte Carlo cross validated area under the ROC curve of 0.792 (SD 0.09) and 0.793 (SD 0.08), respectively. A blind validation study on an additional 90 patients predicted outcome and severity at ROC AUC of 0.83 (CI 0.74–0.91) and 0.76 (CI 0.67–0.86). Conclusion Prognostic tests based on the markers discussed in this paper could allow improvement in the planning of COVID-19 patient treatment.

High-Resolution UPLC-MS Profiling of Anthocyanins and Flavonols of Red Cabbage (Brassica oleracea L. var. capitata f. rubra DC.) Cultivated in Egypt and Evaluation of Their Biological Activity

In this paper, biological investigations and a high-resolution UPLC-PDA-ESI-qTOF-HRMS technique were employed for Brassica oleracea L. var. capitata f. rubra DC. (red cabbage) of the family Brassicaceae (Cruciferae), cultivated in Egypt, for the first time. The positive ionization mode is usually performed to identify anthocyanins. However, this technique cannot differentiate between anthocyanins and corresponding non-anthocyanin polyphenols. Thus, the negative ionization mode was also used, as it provided a series of characteristic ions for the MS analysis of anthocyanins. This helped in identifying five kaempferol derivatives for the first time in red cabbage, as well as nine—previously reported—anthocyanins. For the biological investigations, the acidified methanolic extract of fresh leaves and the methanolic extract of air-dried powdered leaves were examined for their antioxidant, antimicrobial, and anticancer activities. The freshly prepared phenolic extract was proven to be more biologically potent. Statistical significance was determined for its anticancer activity in comparison with standard doxorubicin.

Simultaneous determination of three naphthoquinones from Arnebia euchroma (Royle) Johnst in beagle plasma by UPLC-MS/MS and application for pharmacokinetics study

A rapid, simple and efficient ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was established to simultaneous determination of shikonin, isobutyryl shikonin, β, βʹ-dimethylacryl alkanin in beagle plasma and evaluated by using esculetin as internal standard. The electrospray ionization (ESI) source was operated in negative ionization mode. Multi-reaction monitoring (MRM) was used to quantitatively analyzed shikonin m/z 287.0 → 217.9, isobutyryl shikonin m/z 357.0 → 268.9, β, βʹ-dimethylacryl alkanin m/z 370.0 → 270.1 and esculetin m/z 177.0 → 89.0, respectively. The method was validated for selectivity, linearity, lower limit of quantification, precision, accuracy, recovery, matrix effect and stability. All validation parameters met the acceptance criteria according to regulatory guidelines. This method was successfully applied for the pharmacokinetic study of shikonin, isobutyryl shikonin, β, βʹ-dimethylacryl alkanin in beagle dogs plasma after oral administration of A. euchroma extract.

Features of a negative ionization wave development preceding the full breakdown in a long capillary tube wrapped with a continuous or fine-sectioned grounded electrode

The experimental results on the study of negative ionization wave propagating along a long capillary tube are presented. The ionization wave was initiated by high-voltage pulse of negative polarity. The propagation of this surface ionization wave precedes and influences the establishment of complete electric breakdown within the tube. The spreading of this wave is accompanied by the surface charge deposition. The usage of the fine-sectioned outer electrode allows one to find out the general features of a negative ionization wave. The main of them is the tight correlation between local currents determining the formation of local surface charge and visual discharge images taken by the fast camera characterizing the pace of the ionization wave propagation.

Conversion of Organic Dyes into Pigments: Extraction of Flavonoids from Blackberries (Rubus ulmifolius) and Stabilization

The blackberry’s color is composed mainly of natural dyes called anthocyanins. Their color is red–purple, and they can be used as a natural colorant. Anthocyanins are flavonoids, which are products of plants, and their colors range from orange and red to various shades of blue, purple and green, according to pH. In this study, the chemical composition of an extract obtained from blackberries was defined by LC-ESI/LTQOrbitrap/MS in positive and negative ionization mode. Furthermore, we investigated the adsorption process of blackberry extract using several inorganic fillers, such as metakaolin, silica, Lipari pumice, white pozzolan and alumina. The pigments exhibit different colors as a function of their interactions with the fillers. The analysis of the absorption data allowed the estimation of the maximum adsorbing capacity of each individual filler tested. Through thermogravimetric measurements (TGA), the thermal stability and the real adsorption of the organic extract were determined.