Tandem Mass Spectrometry Analysis Research Articles

The distinct initiation sites and processing activities of TTLL4 and TTLL7 in glutamylation of brain tubulin

Mammalian brain tubulins undergo a reversible posttranslational modification-polyglutamylation-which attaches a secondary polyglutamate chain to the primary sequence of proteins. Loss of its erasers can disrupt polyglutamylation homeostasis and cause neurodegeneration. Tubulin tyrosine ligase like 4 (TTLL4) and TTLL7 were known to modify tubulins, both with preference for the β-isoform, but differently contribute to neurodegeneration. However, differences in their biochemical properties and functions remain largely unknown. Here, using an antibody-based method, we characterized the properties of a purified recombinant TTLL4 and confirmed its sole role as an initiator, unlike TTLL7, which both initiates and elongates the side chains. Unexpectedly, TTLL4 produced stronger glutamylation immunosignals for α-isoform than β-isoform in brain tubulins. Contrarily, the recombinant TTLL7 raised comparable glutamylation immunoreactivity for two isoforms. Given the site selectivity of the glutamylation antibody, we analyzed modification sites of two enzymes. Tandem mass spectrometry analysis revealed their incompatible site selectivity on synthetic peptides mimicking carboxyl termini of α1- and β2-tubulins and a recombinant tubulin. Particularly, in the recombinant α1A-tubulin, a novel region was found glutamylated by TTLL4 and TTLL7, that again at distinct sites. These results pinpoint different site specificities between two enzymes. Moreover, TTLL7 exhibits less efficiency to elongate microtubules premodified by TTLL4, suggesting possible regulation of TTLL7 elongation activity by TTLL4-initiated sites. Finally, we showed that kinesin behaves differentially on microtubules modified by two enzymes. This study underpins the different reactivity, site selectivity, and function of TTLL4 and TTLL7 on brain tubulins and sheds light on their distinct rolein vivo.

Structural and performance variation of PES/PVDF membranes after exposure to the pretreated feed water of CECs

In this study, the removal efficiency of chemicals of emerging concerns (CECs) was evaluated under exposure to various doses of UV/H2O2-based oxidation processes in combination with membrane filtration for three cleaning cycles. Polyethersulphone (PES) and polyvinylidene fluoride (PVDF) materials based membranes were used for this study. The chemical cleaning of the membranes was performed by immersion of the membranes into 1 N HCl followed by adding 3000 mg.L−1 NaOCl for 1hr. Degradation and filtration performance was evaluated using Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) and total organic carbon (TOC) analysis. Membrane fouling analysis for assessing the comparative performance of PES and PVDF membranes was determined by specific fouling and fouling indices evaluation. Membrane characterization results show that the alkynes and carbonyl group formation are due to dehydrofluorination and oxidation of PVDF and PES membranes under the attack of foulants and cleaning chemicals, which resulted in a reduction of fluoride percentage and an increase in sulfur percentage in the PVDF and PES membranes. A decrease in the hydrophilicity of the membranes in underexposed conditions was observed and is consistent with an increase in dose. Degradation results of CECs follow with the highest removal efficiency of chlortetracycline (CTC) followed by atenolol (ATL), acetaminophen (ACT), and caffeine (CAF) degradation due to attack on the aromatic ring and the carbonyl group of CECs by OH exposure. Membrane exposed at 3 mg.L-1 dose of UV/H2O2-based CECs shows minimum alteration with higher filtration efficiency and lower fouling, particularly in PES membranes.

Targeted metabolomic analysis in Parkinson’s disease brain frontal cortex and putamen with relation to cognitive impairment

We performed liquid chromatography tandem mass spectrometry analysis with the targeted metabolomic kit Biocrates MxP Quant 500, in human brain cortex (Brodmann area 9) and putamen, to reveal metabolic changes characteristic of Parkinson’s disease (PD) and PD-related cognitive decline. This case-control study involved 101 subjects (33 PD without dementia, 32 PD with dementia (cortex only), 36 controls). We found changes associated with PD, cognitive status, levodopa levels, and disease progression. The affected pathways include neurotransmitters, bile acids, homocysteine metabolism, amino acids, TCA cycle, polyamines, β-alanine metabolism, fatty acids, acylcarnitines, ceramides, phosphatidylcholines, and several microbiome-derived metabolites. Previously reported levodopa-related homocysteine accumulation in cortex still best explains the dementia status in PD, which can be modified by dietary supplementation. Further investigation is needed to reveal the exact mechanisms behind this pathological change.

Discrimination of Traditional Chinese Medicine Syndromes in Type 2 Diabetic Patients Based on Metabolomics-Proteomics Profiles.

The metabolomics-proteomics of sixty patients with T2DM were acquired by high-performance liquid chromatography (HPLC). In addition, some clinical features, containing total cholesterol (TC), triglycerides (TG), hemoglobin A1c (HbA1c), body mass index (BMI), and low-density lipoprotein (LDL) together with high-density lipoprotein (HDL), were determined via clinical detection strategies. Abundant metabolites and proteins, respectively, were identified with the analysis of liquid chromatography tandem mass spectrometry (LC-MS/MS). 22 differentially abundant metabolites and 15 differentially abundant proteins were determined. The analysis of bioinformatics suggested that the differentially abundant proteins were commonly associated with the renin-angiotensin system, vitamin digestion and absorption, hypertrophic cardiomyopathy, and so on. Furthermore, differentially abundant metabolites were amino acids and were associated with the biosynthesis of CoA and pantothenate, together with the metabolisms of phenylalanine, beta-alanine, proline, and arginine. Combination analysis revealed that the vitamin metabolism pathway was predominantly affected. DHS syndrome can be separated by certain metabolic-proteomic differences, and metabolism is particularly prominent, especially in vitamin digestion and absorption. From the molecular level, we provide preliminary data for the extensive application of TCM in the study of T2DM, and at the same time benefited in a sense diagnosis and treatment of T2DM.

Fluralaner systemic treatment of chickens results in mortality in Triatoma gerstaeckeri, vector of the agent of Chagas disease

BackgroundChagas disease remains a persistent vector-borne neglected tropical disease throughout the Americas and threatens both human and animal health. Diverse control methods have been used to target triatomine vector populations, with household insecticides being the most common. As an alternative to environmental sprays, host-targeted systemic insecticides (or endectocides) allow for application of chemicals to vertebrate hosts, resulting in toxic blood meals for arthropods (xenointoxication). In this study, we evaluated three systemic insecticide products for their ability to kill triatomines.Methods Chickens were fed the insecticides orally, following which triatomines were allowed to feed on the treated chickens. The insecticide products tested included: Safe-Guard® Aquasol (fenbendazole), Ivomec® Pour-On (ivermectin) and Bravecto® (fluralaner). Triatoma gerstaeckeri nymphs were allowed to feed on insecticide-live birds at 0, 3, 7, 14, 28 and 56 days post-treatment. The survival and feeding status of the T. gerstaeckeri insects were recorded and analyzed using Kaplan–Meier curves and logistic regression.ResultsFeeding on fluralaner-treated chickens resulted 50–100% mortality in T. gerstaeckeri over the first 14 days post-treatment but not later; in contrast, all insects that fed on fenbendazole- and ivermectin-treated chickens survived. Liquid chromatography tandem mass spectrometry (LC-QQQ) analysis, used to detect the concentration of fluralaner and fenbendazole in chicken plasma, revealed the presence of fluralaner in plasma at 3, 7, and 14 days post-treatment but not later, with the highest concentrations found at 3 and 7 days post-treatment. However, fenbendazole concentration was below the limit of detection at all time points.ConclusionsXenointoxication using fluralaner in poultry is a potential new tool for integrated vector control to reduce risk of Chagas disease.Graphical

Perturbations in gastrointestinal tract microbiota composition and function in individuals with yellow-greasy tongue coating

ObjectiveTo study the composition and function of tongue coating (TC) and gastrointestinal tract (GIT) microbiota in participants with yellow-greasy tongue coating (YGTC), and to explore the representative metabolite markers and pathways in this group. MethodsSubjects with YGTC or thin-white tongue coating (TWTC) were recruited from December 1, 2021 to October 30, 2022, and the TC and fecal samples were collected. Samples were subjected to both whole-genome shotgun (WGS), and 16S rRNA gene sequencing. The α-diversity analysis, principal component analysis (PCA), and Spearman correlation analysis were performed for two groups. Ultra-performance liquid chromatography combined with tandem mass spectrometry (UPLC–MS/MS) analysis was used to analyze metabolomics and enrichment of metabolic pathways. ResultsThe results revealed 20 YGTC participates and 19 TWTC participates. At the genus level, the dominant bacterial species of TC flora and intestinal flora in the two groups were roughly the same, but the relative kurtosis difference was marked, and the abundance of potentially pathogenic bacteria in TC and fecal samples of YGTC subjects was higher. There were 9 down-regulated microorganisms in the TC samples, 26 down-regulated microorganisms, and 6 up-regulated microorganisms in YGTC subjects. The α-diversity analysis indicated that the Chao and abundance-based coverage estimator (ACE) indices of TC bacteria in the YGTC subjects showed a decreasing trend, but the difference was not statistically significant (P > 0.05). The α-diversity of fecal samples and the Chao and ACE indices decreased significantly (P < 0.05). PCA showed that the microflora structure of TC and fecal samples were significantly different between the two groups. Spearman correlation analysis showed that there was no correlation between TC and fecal microorganisms at phyla and genus levels in the same subjects (P > 0.05). The metabolomics results demonstrated that fumarate reductase, V/A ATPase, and phosphatidylethanolamine were increased, and glycerate-3p, UDP-glucose, and quinone oxidoreductase metabolites were decreased in YGTC TC samples. Inosine monophosphate (IMP), uridine monophosphate (UMP), and gamma-aminobutyric acid (GABA) were increased in YGTC fecal samples, while the contents of ribo-5P, histidine, biotin, and cobalamin were decreased. Metabolic pathway analysis indicated that the abundance of the TC and fecal samples of the YGTC subjects was relatively low in various metabolic pathways, including amino acid metabolism, carbohydrate metabolism, nitrogen metabolism, and energy metabolism. ConclusionStructural and functional changes in TC and GIT microbiota or metabolite markers could be potential biological bases of YGTC formation.

Determination of hydrazine in air by liquid chromatography/tandem mass spectrometry combined with precolumn derivatization.

In this study, we developed a determination method for hydrazine in the air. Hydrazine was derivatized with p-dimethyl amino benzaldehyde (DBA) to yield p-dimethylaminobenzalazine, which was subjected to liquid chromatography-electrospray tandem mass spectrometry (LC/MS/MS) analysis. The derivative exhibited good sensitivity in the LC/MS/MS analysis, and its instrument detection limit and instrument quantification limit were 0.003 and 0.008ng/mL, respectively. The air sample was collected using an air sampler equipped with a peristaltic pump at 0.2L/min for 8h. We demonstrated that a silica cartridge impregnated with DBA and 1,2-bis(4-pyridyl) ethylene can collect hydrazine in the air stably. The mean recovery rates in outdoor and indoor locations were 97.6% and 92.4%, respectively. Further, the method detection and quantification limits were 0.1 and 0.4ng/m3, respectively. The proposed method does not require any pretreatment and/or concentration step, enabling high-throughput analyses.

Investigations of Antibacterial, Antioxidant, and Antidiabetic Potential of Extract and Its Active Fractions from the Leaves of Horsfieldia spicata (Roxb.) J. Sinclair.

This study was undertaken to analyse the potential bioactivities including antibacterial, antioxidant and antidiabetic derived from the methanolic extract and the column chromatography ethyl acetate fraction (AcOEt Fr) of Horsfieldia spicata leaves. Methanolic extract and 4 other fractions was calculated for total phenol and flavonoid contents along with tested for antibacterial, antioxidant and antidiabetic properties. Interestingly, the AcOEt Fr had the highest value for total flavonoid content and the best antioxidant, and antidiabetic activities. Therefore, the AcOEt Fr was further separated using column chromatography technique for obtaining 9 selected fractions namely fraction 1 (F1) - fraction 9 (F9) which were further tested. The results showed that the AcOEt column chromatography fractions namely F2, F3, F4 and F6 had the best clear inhibition antibacterial value against all bacterial tested. In addition, these fractions also exhibited better Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentrations (MBC) values than others. Antioxidant, 2,2-diphenylpicrylhydrazyl (DPPH) assayed indicated that AcOEt Fr had the strongest IC50 value of 47.30 μg/mL. Further, F4 column chromatography fraction showed the best inhibition against α-Glucosidase enzyme related to antidiabetic activity with an IC50 value of 6.11 μg/mL. Liquid chromatography tandem-mass spectrometry (LC/MS/MS) analysis identified that F4 derived from AcOEt fraction had several compounds belonging to the flavonoid and phenolics such as 3',5-dihydroxy-7,4'-dimethoxyflavone, 5,7-dihydroxy-3-(4'-hydroxybenzyl)chromone, and Kadsurenin I.

Direct analysis in real time tandem mass spectrometry method for the rapid screening of 11 NPS in blood and urine.

With the continuous renewal of new psychoactive substances (NPS), the abuse of NPS has seriously harmed social security and public safety. The number of deaths from the abuse of NPS is increasing year by year. Therefore, it is urgent to develop an effective method for detecting NPS. Direct analysis in real time-tandem mass spectrometry (DART-MS/MS) was used to detect 11 NPS in blood and urine. The temperature of ion source was optimized and set to 400°C. The mixture solvent of acetonitrile/methanol (4:1, v/v) was used as precipitant. SKF-525 was selected as the internal standard for quantification. After pretreatment of the analytes in blood or urine, the supernatant was prepared for instrumental analysis. The results indicated that the correlation coefficients (r2 ) of all analytes were ranging from 0.99 to 1 in the linear range. The recoveries of 11 analytes at three spiked levels ranged between 83.4% and 110.4% in blood and between 81.7% and 108.5% in urine. The matrix effects of 11 analytes ranged between 79.5% and 109.5% in blood and between 85.0% and 109.4% in urine. The relative standard deviations (RSDs) of intra-day, inter-day precisions and repeatability were lower than 12.4%, 14.1%, and 14.3% in blood and lower than 11.4%, 13.9%, and 14.3% in urine, respectively. The method established for the detection of 11 NPS could meet the needs of rapid screening of NPS samples. The DART-MS/MS method has the advantages of being efficient, fast, and green. Therefore, it may become a promising technology for the detection of NPS in the future.

Interaction studies of Serendipita indica and Zhihengliuella sp. ISTPL4 and their synergistic role in growth promotion in rice.

Rapid urbanization and globalization demand increasing agricultural productivity. Soil nutrient supply capacity is continuously decreasing due to soil erosion, degradation, salt deposition, undesired element, metal deposition, water scarcity, and an uneven nutrient delivery system. Rice cultivation requires a large amount of water which is becoming detrimental due to these activities. There is a need to increase its productivity. Microbial inoculants are becoming increasingly important in achieving sustainable agricultural production systems. The current study was conducted to investigate the interaction between the root endophytic fungus Serendipita indica (S. indica) and the actinobacterium Zhihengliuella sp. ISTPL4 (Z. sp. ISTPL4) and their synergistic effects on the growth of rice (Oryza sativa L). Both S. indica and Z. sp. ISTPL4 showed positive interactions. Growth of S. indica was observed at different days after Z. sp. ISTPL4 inoculation, and stimulated growth of S. indica was observed when Z. sp. ISTPL4 was inoculated at 5 dafi (days after fungal inoculation). Z. sp. ISTPL4 promoted the growth of S. indica as it increased spore germination. Furthermore, confocal and scanning electron microscopy (SEM) analyses showed a 27% increase in the spore size of S. indica in the presence of Z. sp. ISTPL4. In a liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis increased production of alanine and glutamic acid was observed in their sequential co-culture as compared with individual cultures. Sequential inoculation of S. indica and Z. sp. ISTPL4 significantly increased the biochemical and physical characteristics of rice as compared with their individual inoculum. Biochemical parameters such as chlorophyll content, total soluble sugar, and flavonoid content in the rice increased by up to 57%, 47%, and 39%, respectively, in the presence of the combined inoculum of S. indica and Z. sp. ISTPL4. This will be the first study, to the best of our knowledge, which shows the fungus and actinobacterium interaction and their synergistic roles in the growth promotion of rice. Furthermore, this novel combination can also be used to boost the growth of other crops to increase the agricultural yield.

HSPA5 Promotes Attachment and Internalization of Porcine Epidemic Diarrhea Virus through Interaction with the Spike Protein and the Endo-/Lysosomal Pathway.

Porcine epidemic diarrhea virus (PEDV) has caused huge economic losses to the global pig industry. The swine enteric coronavirus spike (S) protein recognizes various cell surface molecules to regulate viral infection. In this study, we identified 211 host membrane proteins related to the S1 protein by pulldown combined with liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis. Among these, heat shock protein family A member 5 (HSPA5) was identified through screening as having a specific interaction with the PEDV S protein, and positive regulation of PEDV infection was validated by knockdown and overexpression tests. Further studies verified the role of HSPA5 in viral attachment and internalization. In addition, we found that HSPA5 interacts with S proteins through its nucleotide-binding structural domain (NBD) and that polyclonal antibodies can block viral infection. In detail, HSPA5 was found to be involved in viral trafficking via the endo-/lysosomal pathway. Inhibition of HSPA5 activity during internalization would reduce the subcellular colocalization of PEDV with lysosomes in the endo-/lysosomal pathway. Together, these findings show that HSPA5 is a novel PEDV potential target for the creation of therapeutic drugs. IMPORTANCE PEDV infection causes severe piglet mortality and threatens the global pig industry. However, the complex invasion mechanism of PEDV makes its prevention and control difficult. Here, we determined that HSPA5 is a novel target for PEDV which interacts with its S protein and is involved in viral attachment and internalization, influencing its transport via the endo-/lysosomal pathway. Our work extends knowledge about the relationship between the PEDV S and host proteins and provides a new therapeutic target against PEDV infection.

Chemical profiling and quantitative analysis on the aqueous extract of Pimpinella anisum fruit by liquid chromatography-tandem mass spectrometry.

Anise fruit (Aniseed) has been used for many years as a traditional medicine in various countries throughout the world; however, the chemical material basis of Aniseed water extract (AWE) has not been examined in detail, limiting our understanding of its pharmacological mechanism and methods for practical quality control. A high-efficiency and high-sensitivity LC-triple time-of-flight tandem mass spectrometry (MS/MS) analysis method using data processing method combined with product ion and neutral loss filtering for systematic screening and identification of the constituents of AWE was established. A quantitative method was established by using LC-MS/MS with multiple reaction monitoring for 10 min to determine the concentration of 17 representative constituents. A total of 89 compounds were discovered in AWE, of which 31 were confirmed by the reference standards, while 24 were found in Aniseed for the first time. The qualification analysis results showed that chlorogenic acids and luteolin derivatives were the major compounds. The linearity, sensitivity, precision, stability, repeatability, and accuracy of the method were verified, which demonstrated that the method could meet the requirements for quantification. This work contributes to a better understanding of the chemical material basis of Aniseed and assists in the development of effective analytical methods for natural medicines.

Integrated Metabolomic and Transcriptomic Analysis Reveals Potential Gut-Liver Crosstalks in the Lipogenesis of Chicken.

Growing evidence has shown the involvement of the gut-liver axis in lipogenesis and fat deposition. However, how the gut crosstalk with the liver and the potential role of gut-liver crosstalk in the lipogenesis of chicken remains largely unknown. In this study, to identify gut-liver crosstalks involved in regulating the lipogenesis of chicken, we first established an HFD-induced obese chicken model. Using this model, we detected the changes in the metabolic profiles of the cecum and liver in response to the HFD-induced excessive lipogenesis using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The changes in the gene expression profiles of the liver were examined by RNA sequencing. The potential gut-liver crosstalks were identified by the correlation analysis of key metabolites and genes. The results showed that a total of 113 and 73 differentially abundant metabolites (DAMs) between NFD and HFD groups were identified in the chicken cecum and liver, respectively. Eleven DAMs overlayed between the two comparisons, in which ten DAMs showed consistent abundance trends in the cecum and liver after HFD feeding, suggesting their potential as signaling molecules between the gut and liver. RNA sequencing identified 271 differentially expressed genes (DEGs) in the liver of chickens fed with NFD vs. HFD. Thirty-five DEGs were involved in the lipid metabolic process, which might be candidate genes regulating the lipogenesis of chicken. Correlation analysis indicated that 5-hydroxyisourate, alpha-linolenic acid, bovinic acid, linoleic acid, and trans-2-octenoic acid might be transported from gut to liver, and thereby up-regulate the expression of ACSS2, PCSK9, and CYP2C18 and down-regulate one or more genes of CDS1, ST8SIA6, LOC415787, MOGAT1, PLIN1, LOC423719, and EDN2 in the liver to enhance the lipogenesis of chicken. Moreover, taurocholic acid might be transported from the gut to the liver and contribute to HFD-induced lipogenesis by regulating the expression of ACACA, FASN, AACS, and LPL in the liver. Our findings contribute to a better understanding of gut-liver crosstalks and their potential roles in regulating chicken lipogenesis.

LASP1, CERS6, and Actin Form a Ternary Complex That Promotes Cancer Cell Migration.

CERS6 is associated with metastasis and poor prognosis in non-small cell lung cancer (NSCLC) patients through d18:1/C16:0 ceramide (C16 ceramide)-mediated cell migration, though the detailed mechanism has not been elucidated. In the present study, examinations including co-immunoprecipitation, liquid chromatography, and tandem mass spectrometry analysis were performed to identify a novel binding partner of CERS6. Among the examined candidates, LASP1 was a top-ranked binding partner, with the LIM domain possibly required for direct interaction. In accord with those findings, CERS6 and LASP1 were found to co-localize on lamellipodia in several lung cancer cell lines. Furthermore, silencing of CERS6 and/or LASP1 significantly suppressed cell migration and lamellipodia formation, whereas ectopic addition of C16 ceramide partially rescued those phenotypes. Both LASP1 and CERS6 showed co-immunoprecipitation with actin, with those interactions markedly reduced when the LASP1-CERS6 complex was abolished. Based on these findings, it is proposed that LASP1-CERS6 interaction promotes cancer cell migration.

Simultaneous determination of multiple mycotoxins in corn and wheat by high efficiency extraction and purification based on polydopamine and ionic liquid bifunctional nanofiber mat

Due to the universality and harmfulness of mycotoxin co-contamination in cereals, it is of great significance to simultaneously monitor various mycotoxins co-polluted to ensure food safety and public health. In this work, a nanofiber mat modified by polydopamine and ionic liquid (PDA-IL-NFsM) was prepared and utilized as a solid-phase extraction (SPE) adsorbent for the simultaneous quantitative detection of multiple mycotoxins in corn and wheat. The PDA-IL-NFsM can form multiple retention mechanisms with the targets through hydrogen bond, π-π interaction, electrostatic or hydrophobic interaction, it shows favorable simultaneous adsorption performance (adsorption efficiency mostly higher than 88.27%) for fifteen mycotoxins in seven classes. Moreover, it can significantly reduce the matrix effect (lower than −13.69%), showing a good purification effect on the sample matrix. Based on the superior performance of PDA-IL-NFsM, a simple sample preparation method was established. The sample extract is simply diluted with water for SPE, and the eluent can be directly collected for ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis. The detection limit can reach 0.04–4.21 μg kg−1, the recovery was 80.09%–113.01%, and the relative standard deviations of intra-day and inter-day precision were 2.80%–14.81% and 0.68%–13.80% respectively. The results show that the proposed method has good sensitivity, accuracy and precision, and has practical application potential.

In situ biomonitoring using caged lumpfish (Cyclopterus lumpus) eggs reveal plastic and rubber associated chemicals in a harbour area in Central Norway

ABSTRACT Plastics- and rubber-derived chemicals are given increasing focus due to their migration into the environment and potential for causing detrimental effects. The current study demonstrates the use of a novel biomonitoring platform using caged fertilized eggs of lumpfish (Cyclopterus lumpus) in combination with gas chromatography tandem mass spectrometry analysis of a selection of target chemicals extracted from the lumpfish eggs after deployment. A monitoring campaign in the Trondheim harbor and off the coast of Trøndelag in Norway was executed using the described system. Here we found accumulation of UV stabilizers (benzophenone and benzothiazoles), plasticizers (n-butylbenzenesulfonamide), reagents, and polymer synthesis precursors (bisphenol A, acetophenone, phthalide, and phthalimide) in deployed eggs. Several of the compounds were detected in concentrations above previously quantified legacy contaminants in the same study areas.

Metabolite Profiling and Transcriptome Analysis Explain the Difference in Accumulation of Bioactive Constituents in Taxilli Herba from Two Hosts.

Taxilli Herba (TH) is a semi-parasitic herb and the host is a key factor affecting its quality. Flavonoids are the main bioactive constituents in TH. However, studies on the difference in accumulation of flavonoids in TH from different hosts are vacant. In this study, integrated transcriptomic and metabolomic analyses were performed on TH from Morus alba L. (SS) and Liquidambar formosana Hance (FXS) to investigate the relationship between the regulation of gene expression and the accumulation of bioactive constituents. The results showed that a total of 3319 differentially expressed genes (DEGs) were screened in transcriptomic analysis, including 1726 up-regulated genes and 1547 down-regulated genes. In addition, 81 compounds were identified using ultra-fast performance liquid chromatography coupled with triple quadrupole-time of flight ion trap tandem mass spectrometry (UFLC-Triple TOF-MS/MS) analysis, and the relative contents of flavonol aglycones and glycosides were higher in TH from SS group than those from the FXS group. A putative biosynthesis network of flavonoids was created, combined with structural genes, and the expression patterns of genes were mostly consistent with the variation of bioactive constituents. It was noteworthy that the UDP-glycosyltransferase genes might participate in downstream flavonoid glycosides synthesis. The findings of this work will provide a new way to understand the quality formation of TH from the aspects of metabolite changes and molecular mechanism.

Liquid Chromatography–Tandem Mass Spectrometry Analysis of Aminoglycosides in Foods Using an Ethylene-Bridged Hybrid Zwitterionic Stationary Phase and Hydrophilic–Lipophilic-Balanced Solid-Phase Extraction Cartridges

This work aimed to develop an analytical method for the screening of multiple aminoglycoside residues in foods of animal origin using an ethylene-bridged hybrid (BEH) particle-based sulfoalkylbetaine stationary phase. The effects of chromatographic conditions on the separation of 17 aminoglycosides have been systematically investigated. Sample preparation and mass spectrometry detection have also been investigated and optimized. In contrast to high buffer concentrations in the mobile phase required for silica-based sulfoalkylbetaine stationary phases, a moderate buffer concentration (20 mM) provided the optimal separation of 17 aminoglycosides with the BEH sulfoalkylbetaine stationary phase. The developed method has been evaluated in milk, beef, pork, liver, and honey samples with good performance for retention, selectivity, sensitivity, linearity, precision, and accuracy. The majority of the limit of quantitation estimated with the matrix was less than 25 μg/kg. The overall accuracy across five matrices was in the range from 96 to 111%, with standard deviations of less than 19%.

UHPLC-Quadrupole-Exactive-Orbitrap-MS/MS-based putative identification for Eucommiae Folium (Duzhongye) and its quality-marker candidate for pharmacopeia.

Eucommiae Folium (Duzhongye) is a traditional Chinese medicine with a long history of use in China. However, its quality-marker (Q-marker) in Chinese Pharmacopoeia is poorly defined nowadays. The study therefore conducted an ultra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap tandem mass spectrometry (UHPLC-Quadrupole-Exactive-Orbitrap MS/MS) analysis to obtain accurate data. The obtained data were then compared with authentic standards library using Xcalibur 4.1 software package and TraceFinder General Quan. Through the comparison, the study has putatively identified 26 bioactive compounds, which include 17 flavonoid derivatives (catechin, quercetin 3-gentiobioside, quercetin 3-O-β-D-glucose-7-O-β-D-gentiobioside, taxifolin, myricetin 3-O-galactoside, myricitrin, hyperoside, rutin, isoquercitrin, quercetin 3-O-β-xylopyranoside, quercitrin, isorhamnetin 3-O-β-D-glucoside, quercetin, kaempferol, S-eriodictyol, S-naringenin, and phloridzin), four caffeoylquinic acids (neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C), two alkaloids (vincamine and jervine), one lignan (pinoresinol), one xanthone (cowaxanthone B), and one steroid (cholesteryl acetate). Of these, flavonoid isoquercitrin is recommended as the new and additional pharmacopoeia Q-marker candidate, which can not only overcome the unreliability of old Q-marker but also recognize the possible counterfeit. This article is protected by copyright. All rights reserved.

18 Fecal microbial metabolite profiles are unaltered by laminitis history

Microbial metabolites of the equine gastrointestinal tract may confer information about health and susceptibility to disease. Laminitis is often associated with metabolic disturbances, but little is known of its impact on functional microbial metabolism. A pilot study compared metabolomic profiles of feces collected from 55 native ponies, comprising n = 15 pairs of previously laminitic (PL) and non-laminitic (NL) ponies matched for location, diet, and management, and n = 25 ponies suspected of having an active episode of laminitis (SL) but without clinical diagnosis. Targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was used. Univariate analyses (ANOVA with Fisher's least significant difference comparisons of PL, NL and SL groups) was performed with significance set at P < 0.05 following Benjamini-Hochberg correction. Principal component analysis (PCA) wasapplied to visualize clustering of individuals. Analysis was performed using the MetaboAnalyst 5.0 web-based interface. Of a panel of 167 metabolites, 104 were identified in the feces, however, none were significantly different between the laminitis status groups. Assessment of the PCA scores plot revealed no clustering of individuals by metabolic profile based on laminitis status. The similar fecal metabolite profile of previously laminitic and non-laminitic ponies indicates that laminitis may not result in significant long-term, alteration of microbial metabolism. Future work to combine these data with that of microbial abundance and species diversity in the feces may reveal further insights into the functional microbial metabolism in laminitic and healthy animals.