High-resolution Electrospray Mass Spectrometry Research Articles

Efficient alcoholysis of total saponins in D. zingiberensis to diosgenin using ionic liquid-grafted MOF superacids

Diosgenin is an indispensable intermediate in the production of various steroid drugs. Conventional hydrolysis of sulfuric acid produces large amounts of acidic wastewater and has always faced stringent restrictions from environmental requirements. Here, UiO-66-NH2 and its derivative of UiO-66-NH2-AT (AT = 3-amino-1,2,4-triazole) were synthesized, and these two compounds were further grafted with high-concentration CF3SO3H molecules to obtain the MOF-based solid superacids, namely UiO-66-NH2-[SO3CF3] and UiO-66-NH2-AT-[SO3CF3], respectively. They have been applied to the alcoholysis of total saponins to extract diosgenin, and the alcoholysis conditions, including the solid acid dosage, extraction temperature and extraction time, have been optimized by single-factor experiments. Subsequently, the alcoholysis conditions of UiO-66-NH2-AT-[SO3CF3] with more prominent catalytic performance and cycle stability were further optimized by the response surface method, and under the optimum conditions, the diosgenin yield reached 20.8 %, which was close to the predicted value of 21.3 % and was superior to the conventional hydrolysis of sulfuric acid and our previously reported solid acids. Additionally, the alcoholysis pathways and alcoholysis mechanism of the total saponins catalyzed by UiO-66-NH2-AT-[SO3CF3] have been proposed by the analysis of high-resolution electrospray mass spectrometry.

Stereochemistry of aminoacylated cardiolipins and phosphatidylglycerols from bacteria.

Hydrophilic interaction liquid chromatography (HILIC) connected with electrospray high-resolution tandem mass spectrometry (MS) was used for the analysis of unusual amino acid (AA) substituted phosphatidylglycerols (PG) and cardiolipins (CL) in mesophilic and thermophilic bacteria. Individual peaks from the lipid class separation by HILIC were isolated and hydrolyzed to determine the absolute configuration of the aminoacyl side chain. The configuration of the aminoacyl side chain was assigned by indirect liquid chromatography (LC) enantiomer separation after the hydrolysis of the aminoacylated (aminoacyl) lipids using N-(4-nitrophenoxycarbonyl)-l-phenylalanine 2-methoxyethyl ester as chiral derivatizing agent and reversed phase LC-MS for analysis. When two chromatographic methods were combined, less common AAs, such as d-allo-Ile and d-allo-Thr, were identified. The taxonomic classification of bacteria showed that bacteria of the family Bacillaceae (Bacillus and Geobacillus) produce branched-chain AAs, that is, d-allo-Ile, d-Ile, and d-Leu. These AAs were present only in the genera Bacillus and Geobacillus and not in Alicyclobacillus acidoterrestris (family Alicyclobacillaceae). On the contrary, hydroxy AAs, that is, l- and d-Thr, and l- and d-allo-Thr, were identified as aminoacyl-PG and aminoacyl-CL in A. acidoterrestris and were not present in the genera Bacillus and Geobacillus. Therefore, the complete analysis made it possible to identify the stereochemistry of AAs in aminoacyl PGs and CLs and use this fact for chemotaxonomy.

Analysis of glycosylated cardiolipins from thermophilic bacteria using GC–MS and LC–ESI–MS/MS methods

Unusual glucose-substituted cardiolipins (Glcx-CLs) in three genera of thermophilic bacteria, having more than one glycosidically linked glucose to the hydroxyl of the central glycerol of Glcx-CLs were identified for the first time in thermophilic bacteria of the genera Geobacillus, Meiothermus, and Thermus. The number of glucoses reached up to five units. The structure of glycosidically linked oligosaccharides was determined based on shotgun analysis MS (electrospray high-resolution tandem mass spectrometry), partially methylated alditol acetates were identified by GC–MS, both electron ionization (EI) and positive chemical ionization (PCI), hydrophilic interaction liquid chromatography (HILIC) separation and identification of CLs glycosides by high resolution MS–ESI, and digestion by specific glycosidases.

Mobile phase and column chemistry selection for high sensitivity non-targeted LC/ESI/HRMS screening of water

Systematic selection of mobile phase and column chemistry type can be critical for achieving optimal chromatographic separation, high sensitivity, and low detection limits in liquid chromatography electrospray high resolution mass spectrometry (LC/MS). However, the selection process is challenging for non-targeted screening where the compounds of interest are not preselected nor available for method optimization. To provide general guidance, twenty different mobile phase compositions and four columns were compared for the analysis of 78 compounds with a wide range of physicochemical properties (logP range from −1.46 to 5.48), and analyte sensitivity was compared between methods. The pH, additive type, column, and organic modifier had significant effects on the analyte response factors, and acidic mobile phases (e.g. 0.1% formic acid) yielded highest sensitivity. In some cases, the effect was attributable to the difference in organic modifier content at the time of elution, depending on the mobile phase and column chemistry. Based on these findings, 0.1% formic acid, 0.1% ammonia and 5.0 mM ammonium fluoride were further evaluated for their performance in non-targeted LC/ESI/HRMS analysis of wastewater treatment plan influent and effluent, using a data dependent MS2 acquisition and two different data processing workflows (MS-DIAL, patRoon 2.1) to compare number of detected features and sensitivity. Both data-processing workflows indicated that 0.1% formic acid yielded the highest number of features in full scan spectrum (MS1), as well as the highest number of features that triggered fragmentation spectra (MS2) when dynamic exclusion was used.

Elimination of the Ion–Molecule Association in Electrospray High-Resolution Mass Spectrometry Analyses for Petroleum Fractions

Electrospray ionization (ESI) coupled with high-resolution mass spectrometry (HRMS) is rapidly developing into a powerful tool for the molecular characterization of petroleum fractions. However, the occurrence of ion–molecule aggregates seriously affects the interpretation of the mass spectrometry results. Ion–molecule aggregates are not inherent compounds in petroleum but they are produced during the analysis of petroleum samples by high-resolution mass spectrometry. Also, the classes of aggregates are the same as many compounds in petroleum. Therefore, the removal of these species is crucial, and their analysis from methods of dissociation and mechanism points of view is essential for this purpose. In this study, aggregates of a vacuum gas oil fraction were analyzed and the types were defined. The compositions of the aggregates were characterized by changing operating conditions. Several factors affecting the aggregates were evaluated by a parameter of dissociation efficiency. By establishing the operating methods, the residual content of the aggregates can be controlled by less than 1% of the total ions. The results are instructive for the molecular characterization of petroleum fractions by using mass spectrometry with ESI ionization.

Cytotoxic Metabolites from Calophyllum tacamahaca Willd.: Isolation and Detection through Feature-Based Molecular Networking

Isocaloteysmannic acid (1), a new chromanone, was isolated from the leaf extract of the medicinal species Calophyllum tacamahaca Willd. along with 13 known metabolites belonging to the families of biflavonoids (2), xanthones (3-5, 10), coumarins (6-8) and triterpenes (9, 11-14). The structure of the new compound was characterized based on nuclear magnetic resonance (NMR), high-resolution electrospray mass spectrometry (HRESIMS), ultraviolet (UV) and infrared (IR) data. Its absolute configuration was assigned through electronic circular dichroism (ECD) measurements. Compound (1) showed a moderate cytotoxicity against HepG2 and HT29 cell lines, with IC50 values of 19.65 and 25.68 µg/mL, respectively, according to the Red Dye method. Compounds 7, 8 and 10-13 exhibited a potent cytotoxic activity, with IC50 values ranging from 2.44 to 15.38 µg/mL, against one or both cell lines. A feature-based molecular networking (FBMN) approach led to the detection of a large amount of xanthones in the leaves extract, and particularly analogues of the cytotoxic isolated xanthone pyranojacareubin (10).

Evidences of the non-stoichiometry and control of the composition of the cluster-based solid solution K6[Re6-xMoxS8(CN)5] (x=2.75–3.63)

In this work, we investigated a high-temperature reaction between ReS2, MoS2 and KCN resulting in the formation of the cluster-based K6[Re6-xMoxS8(CN)5] solid solution. The crystal structure is based on chains of [Re6-xMoxS8(CN)6] cluster units. It was found that both the sintering temperature of starting sulfides and the reaction temperature with KCN have an influence on the composition of the product allowing x to be varied within the range x ​= ​2.75–3.63. The molar ration of ReS2:MoS2:KCN loaded composition is fixed to 3:3:14 in all experiments. Energy dispersive spectroscopy (EDS), powder X-ray diffraction (PXRD) and high-resolution electrospray (ESI) mass spectrometry were used to characterize the solid solution K6[Re6-xMoxS8(CN)5]. It was shown by mass spectrometry that the solid solution with fractional x contains anions based on the {Re4Mo2S8}, {Re3Mo3S8} and {Re2Mo4S8} cluster cores in different proportions depending on the above-mentioned factors. Particularly, the rise of the temperature of the synthesis or temperature of sintering of metal sulfides led to increase of the rhenium content in the solid solution.

Effects of a Volatile Organic Compound Filter on Breath Profiles Measured by Secondary Electrospray High-Resolution Mass Spectrometry.

Environmental volatile organic compounds (VOCs) from the ambient air potentially influence on-line breath analysis measurements by secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS). The aim of this study was to investigate how inhaling through a VOC filter affects the detected breath profiles and whether it is feasible to integrate such filters into routine measurements. A total of 24 adult participants performed paired breath analysis measurements with and without the use of an activated carbon filter for inspiration. Concordance correlation coefficients (CCCs) and the Bland−Altman analysis were used to assess the agreement between the two methods. Additionally, the effect on a selection of known metabolites and contaminants was analyzed. Out of all the detected features, 78.3% showed at least a moderate agreement before and after filter usage (CCC > 0.9). The decrease in agreement of the remaining m/z features was mostly associated with reduced signal intensities after filter usage. Although a moderate-to-substantial concordance was found for almost 80% of the m/z features, the filter still had an effect by decreasing signal intensities, not only for contaminants, but also for some of the studied metabolites. Operationally, the use of the filter complicated and slowed down the conductance of measurements, limiting its applicability in clinical studies.

Isotopologue pattern based data mining for selenium species from HILIC-ESI-Orbitrap-MS-derived spectra.

Automated and specific picking of selenium-containing molecular entities has not been an obvious option for software tools associated with electrospray high-resolution mass spectrometry (MS). In our study, a comprehensive pattern matching approach based on intra-isotopologue distance and isotopologue ratio data was critically evaluated in terms of reproducibility and selenium isotope selection on three samples, including selenized Torula yeast and the selenium hyperaccumulator plant Cardamine violifolia. Hydrophilic interaction liquid chromatography was applied to provide a one-step separation for water soluble metabolites to put an end to the need for either orthogonal setups or poor retention on reversed phase chromatography. Assistance from inductively coupled plasma-MS was taken only for chromatographic verification purposes, and the involvement of absolute mass defect (MD) data in selenometabolite-specific screening was assessed by multivariate statistical tools. High focus was placed on screening efficiency and on the validation of discovered selenized molecules to avoid reporting of artefacts. From the >1000 molecular entries detected, selenium-containing molecules were picked up with a recovery rate of >88% and a false positive rate of <10%. Isotop(ologu)e pairs of 78Se-80Se and 80Se-82Se proved to be the most performant in the detection. On the basis of accurate mass information and hypothetical deamination processes, elemental composition could be proposed for 72 species out of the 75 selenium species encountered without taking into account selenocompound databases. Absolute MD data were used to significantly differentiate a potentially sample-specific subgroup of false positive molecular entities from non-selenized and selenized entities.

Neurotrophic and Immunomodulatory Lanostane Triterpenoids from Wood-Inhabiting Basidiomycota.

Neurotrophins such as nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) play important roles in the central nervous system. They are potential therapeutic drugs for the treatment of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. In this study, we investigated the neurotrophic properties of triterpenes isolated from fruiting bodies of Laetiporus sulphureus and a mycelial culture of Antrodia sp. MUCL 56049. The structures of the isolated compounds were elucidated based on nuclear magnetic resonance (NMR) spectroscopy in combination with high-resolution electrospray mass spectrometry (HR-ESIMS). The secondary metabolites were tested for neurotrophin (ngf and bdnf) expression levels on human astrocytoma 1321N1 cells. Neurite outgrowth activity using rat pheochromocytoma (PC-12) cells was also determined. Twelve triterpenoids were isolated, of which several potently stimulated the expression of neurotrophic factors, namely, ngf (sulphurenic acid, 15α-dehydroxytrametenolic acid, fomefficinic acid D, and 16α-hydroxyeburicoic acid) and bdnf (sulphurenic acid and 15α-dehydroxytrametenolic acid), respectively. The triterpenes also potentiated ngf-induced neurite outgrowth in PC-12 cells. This is, to the best of our knowledge, the first report on the compound class of lanostanes in direct relation to bdnf and ngf enhancement. These compounds are widespread in medicinal mushrooms; hence, they appear promising as a starting point for the development of drugs and mycopharmaceuticals to combat neurodegenerative diseases. Interestingly, they do not show any pronounced cytotoxicity and may, therefore, be better suited for therapy than many other neurotrophic compounds that were previously reported.

Isolation and Identification of Secondary Metabolites Produced by Phytopathogenic Fungus Corynespora cassiicola from Hevea brasiliensis

The secondary metabolites of the phytopathogenic fungus Corynespora cassiicola CC01 from Hevea brasiliensis were investigated. As a result, two new compounds, 5-acetyl-7-hydroxy-6- methoxybenzofuran-2(3H)-one (1) and (S)-2-(2,3-dihydrofuro [3,2-c]pyridin-2-yl)propan-2-ol (2), together with seven known compounds, 4,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one (3), 3,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one (4), curvulin acid (5), 2-methyl-5-carboxymethyl- 7-hydroxychromone (6), tyrosol (7), p-hydroxybenzoic acid (8) and cerevisterol (9), were isolated from the fermentation extract by comprehensive silica gel, reverse phase silica gel, Sephadex-LH20 column chromatography and high-performance liquid chromatography (HPLC). The structures of these compounds were identified by using high-resolution electrospray mass spectrometry (HRESIMS), nuclear magnetic resonance spectroscopy (NMR), optical rotation, ultraviolet and infrared spectroscopy techniques and a comparison of NMR data with those reported in the literature. Compounds 1 and 2 were new compounds, and compounds 3-9 were discovered from this phytopathogenic fungus for the first time. Compounds 1-9 were tested for phytotoxicity against the fresh tender leaf of Hevea brasiliensis, and the results show that none of them were phytotoxic. Additionally, these compounds were subjected to an antimicrobial assay against three bacteria (E. coli, methicillin-resistant Staphylococcus aureus and Micrococcus luteus), but they showed no activity.

Alkali metal-linked triangular building blocks assemble a high-nucleation lanthanoid cluster based on single-molecule magnets

SummaryThe metallic central magnetic axes in high-nucleation clusters with complex structural connections tend to be disorganized and cancel each other out. Therefore, high-nucleation clusters cannot easily exhibit single-molecule magnets (SMMs) behaviors. Herein, we select a triple-core building block (Dy3K2, 1) and use linked diamagnetic alkali metal to form an open, spherical, high-nucleation cluster Dy12Na6 (3) with SMM behavior. Furthermore, by changing the reaction conditions, Dy6K2 (2) formed by linking two Dy3 by K(I) is obtained. High-resolution electrospray mass spectrometry of clusters 1–3 effectively captures the building block Dy3, and clusters 1 and 3 and Dy3 have high stability even with the increase in ion source energy. To the best of our knowledge, this is the first time that an SMM based on a high-nucleation cluster has been obtained by connecting magnetic primitives via diamagnetic metal ions. Dy12K6 is currently the highest nuclear ns-4f heterometallic SMM.

Metabolomic changes in lactating multiparous naturally MAP-infected Holstein-Friesian dairy cows suggest changes in mitochondrial energy pathways

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of Johne's Disease, a chronic intestinal infection of ruminants. Infected cows begin shedding MAP within the asymptomatic, subclinical stage of infection before clinical signs, such as weight loss, diarrhoea and reduced milk yields develop within the clinical stages of disease. Herein, we examine the milk metabolomic profiles of naturally MAP-infected Holstein-Friesian cows. The study used biobanked milk samples which were collected 73.4 ± 3.79 (early lactation) and 143 ± 3.79 (mean ± SE) (mid-lactation) days post-calving from 5 MAP-infected and 5 control multiparous cows. The milk metabolome was assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) for sensitive, non-targeted metabolite fingerprinting. Metabolite fingerprinting assessments using partial least squares discriminate analyses (PLS-DA) indicated that lactation stage was a larger source of variation than MAP status. Examining each lactation stage separately for changes associated to MAP-infection status identified 45 metabolites, 33 in early lactation and 12 in mid-lactation, but only 6 metabolites were targeted in both stages of lactation. Pathway enrichment analysis suggested that MAP affected the malate-aspartate shuffle during early lactation. Pearson's correlation analysis indicated relationships between milk lactose concentrations in mid-lactation and 6 metabolites that were tentatively linked to MAP-infection status. The targeted metabolites were suggestive of wider changes in the bioenergetic metabolism that appear to be an acceleration of the effects of progressing lactation in healthy cows. Additionally, milk lactose concentrations suggest that MAP reduces the availability of lactose derivatives.

Spectral binning as an approach to post-acquisition processing of high resolution FIE-MS metabolome fingerprinting data

IntroductionFlow infusion electrospray high resolution mass spectrometry (FIE-HRMS) fingerprinting produces complex, high dimensional data sets which require specialist in-silico software tools to process the data prior to analysis.ObjectivesPresent spectral binning as a pragmatic approach to post-acquisition procession of FIE-HRMS metabolome fingerprinting data.MethodsA spectral binning approach was developed that included the elimination of single scan m/z events, the binning of spectra and the averaging of spectra across the infusion profile. The modal accurate m/z was then extracted for each bin. This approach was assessed using four different biological matrices and a mix of 31 known chemical standards analysed by FIE-HRMS using an Exactive Orbitrap. Bin purity and centrality metrics were developed to objectively assess the distribution and position of accurate m/z within an individual bin respectively.ResultsThe optimal spectral binning width was found to be 0.01 amu. 80.8% of the extracted accurate m/z matched to predicted ionisation products of the chemical standards mix were found to have an error of below 3 ppm. The open-source R package binneR was developed as a user friendly implementation of the approach. This was able to process 100 data files using 4 Central Processing Units (CPU) workers in only 55 seconds with a maximum memory usage of 1.36 GB.ConclusionSpectral binning is a fast and robust method for the post-acquisition processing of FIE-HRMS data. The open-source R package binneR allows users to efficiently process data from FIE-HRMS experiments with the resources available on a standard desktop computer.

Untargeted metabolomic analysis of thoracic blood from badgers indicate changes linked to infection with bovine tuberculosis (Mycobacterium bovis): a pilot study

IntroductionMycobacterium bovis, the causative agent of bovine tuberculosis (bTB) in cattle, represents a major disease burden to UK cattle farming, with considerable costs associated with its control. The European badger (Meles meles) is a known wildlife reservoir for bTB and better knowledge of the epidemiology of bTB through testing wildlife is required for disease control. Current tests available for the diagnosis of bTB in badgers are limited by cost, processing time or sensitivities.Materials and MethodsWe assessed the ability of flow infusion electrospray—high-resolution mass spectrometry (FIE-HRMS) to determine potential differences between infected and non-infected badgers based on thoracic blood samples obtained from badgers found dead in Wales. Thoracic blood samples were autoclaved for handling in a containment level 2 (CL2) hazard laboratory.ResultsHere we show the major differences associated with with M. bovis infection were changes to folate, pyrimidine, histidine, glycerophospholipid and phosphonate metabolism.ConclusionsOur studies have indicated differences in the metabolomic signature of badgers found dead in relation to their infection status, suggesting metabolomics could hold potential for developing novel diagnostics for bTB in badgers. As well as highlighting a potential way to handle samples containing a highly pathogenic agent at CL2 for metabolomics studies.

Giant Crown-Shaped Dy34 Nanocluster with High Acid-Base Stability Assembled by an out-to-in Growth Mechanism.

Lanthanoid metal ions have large ionic radii, complex coordination modes, and easy distortion of coordination spheres, but the design and synthesis of high-nucleation lanthanoid clusters with high stability in solution (especially aqueous solution) are challenging. Herein, a diacylhydrazone ligand (H2L1) with multidentate chelating coordination sites was used to react with Dy(OAc)3·4H2O under solvothermal conditions to obtain an example of a 34-nucleus crown-shaped dysprosium cluster [Dy34(L)8(μ2-OH)(μ3-OH)21(μ3-O)14(OAc)31(OCH3)2(H2O)15](OAc)3 (1). Structural analysis showed that the bisacylhydrazone ligand H2L1 with polydentate chelate coordination sites could rapidly capture DyIII ions, thereby forming 34-nucleus crown-shaped dysprosium cluster 1 following the out-to-in growth mechanism. Cluster 1 remained stable after immersion in solutions with different pH values (3-14) for 24 h. To the best of the authors' knowledge, high-nucleation lanthanoid clusters with excellent strong acid and base stability and water stability are very rare. Meanwhile, high-resolution electrospray mass spectrometry molecular ion peaks produced by cluster 1 were captured, which proved to be stable also in organic solvents. Magnetic research showed that cluster 1 exhibited frequency-dependent behavior. This work provides a new idea for designing and synthesizing high-nucleation lanthanoid clusters with high stability.

Defining Fatty Acid Changes Linked to Rumen Development, Weaning and Growth in Holstein-Friesian Heifers.

After birth, as effectively monogastric animals, calves undergo substantial physiological changes to become ruminants by 3 months of age and reach sexual maturity at approximately 15 months of age. Herein, we assess longitudinal metabolomic changes in Holstein-Friesian (HF) heifers from birth until sexual maturity during this developmental process. Sera from 20 healthy, HF heifers were sampled biweekly from 2 weeks of age until 13 months of age and then monthly until 19 months of age. Sera were assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high-throughput, sensitive, non-targeted metabolite fingerprinting. Partial least squares discriminant analysis (PLS-DA) and unsupervised hierarchical clustering analysis (HCA) of the derived metabolomes indicated changes detectable in heifers’ sera over time. Time series analyses identified 30 metabolites that could be related to rumen development and weaning at ~3 months of age. Further time series analysis identified 40 metabolites that could be correlated with growth. These findings highlight the role of acetic acid and 3-phenylpropionate (3-PP) in rumen development and growth, suggest that weaning induces elevated levels of fatty acyls in response to a post-weaning stress-induced innate immune response and demonstrate the utilization of fatty acyls in growth. The identified metabolites offer serum metabolites which could inform the nutrition and healthy development of heifers.

Isolation and Characterisation of Quercitrin as a Potent Anti-Sickle Cell Anaemia Agent from Alchornea cordifolia.

Alchornea cordifolia Müll. Arg. (commonly known as Christmas Bush) has been used traditionally in Africa to treat sickle cell anaemia (a recessive disease, arising from the S haemoglobin (Hb) allele), but the active compounds are yet to be identified. Herein, we describe the use of sequential fractionation coupled with in vitro anti-sickling assays to purify the active component. Sickling was induced in HbSS genotype blood samples using sodium metabisulphite (Na2S2O5) or through incubation in 100% N2. Methanol extracts of A. cordifolia leaves and its sub-fractions showed >70% suppression of HbSS erythrocyte sickling. The purified compound demonstrated a 87.2 ± 2.39% significant anti-sickling activity and 93.1 ± 2.69% erythrocyte sickling-inhibition at 0.4 mg/mL. Nuclear magnetic resonance (NMR) spectra and high-resolution mass spectroscopy identified it as quercitrin (quercetin 3-rhamnoside). Purified quercitrin also inhibited the polymerisation of isolated HbS and stabilized sickle erythrocytes membranes. Metabolomic comparisons of blood samples using flow-infusion electrospray-high resolution mass spectrometry indicated that quercitrin could convert HbSS erythrocyte metabolomes to be like HbAA. Sickling was associated with changes in antioxidants, anaerobic bioenergy, and arachidonic acid metabolism, all of which were reversed by quercitrin. The findings described could inform efforts directed to the development of an anti-sickling drug or quality control assessments of A. cordifolia preparations.

Metabolomic changes in Mycobacterium avium subsp. paratuberculosis (MAP) challenged Holstein\u2013Friesian cattle highlight the role of serum amino acids as indicators of immune system activation

IntroductionParatuberculosis, commonly known as Johne’s disease, is a chronic granulomatous infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Clinical signs, including reduced milk yields, weight loss and diarrhoea, are typically absent until 2 to 6 years post exposure.ObjectivesTo identify metabolomic changes profiles of MAP challenged Holstein–Friesian (HF) cattle and correlate identified metabolites to haematological and immunological parameters.MethodsAt approximately 6 weeks of age, calves (n = 9) were challenged with 3.8 × 109 cells of MAP (clinical isolate CIT003) on 2 consecutive days. Additional unchallenged calves (n = 9) formed the control group. The study used biobanked serum from cattle sampled periodically from 3- to 33-months post challenge. The assessment of sera using flow infusion electrospray high resolution mass spectrometry (FIE-HRMS) for high throughput, sensitive, non-targeted metabolite fingerprinting highlighted differences in metabolite levels between the two groups.ResultsIn total, 25 metabolites which were differentially accumulated in MAP challenged cattle were identified, including 20 which displayed correlation to haematology parameters, particularly monocyte levels.ConclusionThe targeted metabolites suggest shifts in amino acid metabolism that could reflect immune system activation linked to MAP and as well as differences in phosphocholine levels which could reflect activation of the Th1 (tending towards pro-inflammatory) immune response. If verified by future work, selected metabolites could be used as biomarkers to diagnose and manage MAP infected cattle.

Anti-Inflammatory Effect of Three Isolated Compounds of Physalis alkekengi var. franchetii (PAF) in Lipopolysaccharide-Activated RAW 264.7 Cells.

(1) Background: Three isolated compounds from Physalis alkekengi var. franchetii (PAF) have been investigated to possess a variety of biological activities. Their structures were elucidated by spectroscopic analysis (Ultraviolet (UV), High-resolution electrospray mass spectrometry (HR-ESI-Ms), and their anti-inflammatory effects were evaluated in vitro; (2) Methods: To investigate the mechanisms of action of PAF extracts and their isolated compounds, their anti-inflammatory effects were assessed in RAW 264.7 macrophages stimulated by lipopolysaccharide (LPS). RAW 264.7 cells were treated with different concentrations of Physalis alkekengi var. franchetii three isolated compounds of PAF for 30 min prior to stimulation with or without LPS for the indicated times. The inflammatory cytokines, interleukin (IL)-1β and tumor necrosis factor (TNF)-α were determined using reverse transcription-polymerase chain (RT-PCR); (3) Results Treatment of RAW 264.7 cells with LPS alone resulted in significant increases in inflammatory cytokine production as compared to the control group (p < 0.001). However, with the treatment of isophysalin B 100 μg/mL, there was a significant decrease in the mRNA expression levels of TNF-α in LPS-stimulated raw 264.7 cells (p < 0.001). With treatment of physalin 1–100 μg/mL, there was a markedly decrease in the mRNA expression levels of TNF-α in LPS stimulated raw 264.7 (p < 0.05). Moreover, TNF-α mRNA (p < 0.05) and IL-1β mRNA (p < 0.001) mRNA levels were significantly suppressed after treatment with 3′,7-dimethylquercetin in LPS stimulated Raw 264.7 cells; (4) Conclusions: These findings suggest that three isolated compounds from can suppress inflammatory responses in LPS stimulated macrophage.