Liquid Chromatography-tandem Mass Spectrometry Research Articles

Positive Intervention of Distinct Peptides in Clostridioides difficile Infection in a Mouse Model

Clostridioides difficile infection (CDI) is a common healthcare-associated infection and the leading cause of gastroenteritis-related deaths worldwide. To investigate the effects of peptide composition of different protein products on CDI, we analyzed and compared the peptide sequences and compositions from Engraulis japonicus and Glycine max using Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS). An animal model of CDI was also established to investigate the potential therapeutic effects of these peptides in vivo. The peptide compositions of E. japonicus and G. max differed, with only 11% of the peptide sequences being identical. Oral administration of the tested peptides could reduce intestinal inflammation, repair the intestinal barrier, increase the proportion of beneficial bacteria, and reduce the proportion of harmful bacteria, providing a therapeutic effect against CDI. However, the peptides may differ considerably in some aspects. E. japonicus peptides were superior to G. max peptides in promoting colon epithelial cell proliferation and repairing tight intestinal cell junctions. Interestingly, the two sources of peptides have different effects on the cecal microbiome. E. japonicus peptides can effectively restore the diversity and richness of intestinal microbiota, while G. max peptides have poor regulatory effects on the intestinal microbiota structure. Overall, E. japonicus peptides showed better results than G. max peptides in treating CDI. This study supports the potential treatment of CDI with natural peptides and promotes the development of specialty foods for CDI enteritis. Clostridioides difficile infection (CDI) is a common healthcare-associated infection and the leading cause of gastroenteritis-related deaths worldwide. To investigate the effects of peptide composition of different protein products on CDI, we analyzed and compared the peptide sequences and compositions from Engraulis japonicus and Glycine max using Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS). An animal model of CDI was also established to investigate the potential therapeutic effects of these peptides in vivo. The peptide compositions of E. japonicus and G. max differed, with only 11% of the peptide sequences being identical. Oral administration of the tested peptides could reduce intestinal inflammation, repair the intestinal barrier, increase the proportion of beneficial bacteria, and reduce the proportion of harmful bacteria, providing a therapeutic effect against CDI. However, the peptides may differ considerably in some aspects. E. japonicus peptides were superior to G. max peptides in promoting colon epithelial cell proliferation and repairing tight intestinal cell junctions. Interestingly, the two sources of peptides have different effects on the cecal microbiome. E. japonicus peptides can effectively restore the diversity and richness of intestinal microbiota, while G. max peptides have poor regulatory effects on the intestinal microbiota structure.

Influence of Pesticide Application Method, Timing, and Rate on Contamination of Nectar with Systemic and Nonsystemic Pesticides.

Exposure to pesticides is one potential factor contributing to the recent loss of pollinators and pollinator diversity. Few studies have specifically focused on the relationship between pesticide management during ornamental plant production and contamination of nectar. We evaluated contamination of nectar in Salvia 'Indigo Spires' (Salvia longispicata M. Martens & Galeotti × S. farinacea Benth.) associated with applications of the systemic insecticide thiamethoxam, and the nonsystemic fungicides boscalid and pyraclostrobin. Applications were made at the labeled rates for the commercially available products, and we compared the influence of application method (drench vs. spray), timing (relative to flowering), and rate (low vs. high) for each pesticide. Nectar was sampled using 50-µL microcapillary tubes and analyzed by liquid chromatography-tandem mass spectrometry. The results indicate that concentrations from the spray application resulted in the least contamination of nectar with the systemic thiamethoxam, with lower concentrations occurring when thiamethoxam was applied before blooming at the lowest rate. Concentrations of thiamethoxam and its metabolite clothianidin were detected in nectar in all treatments (regardless of the method, timing, or rate of application), and ranged from 3.6 ± 0.5 ng/mL (spray-applied before blooming, low rate) to 1720.0 ± 80.9 ng/mL (drench-applied after blooming, high rate). Residues of clothianidin in nectar ranged from below quantification limits (spray-applied before blooming, low rate) to 81.2 ± 4.6 ng/mL (drench-applied after blooming, high rate). Drench applications resulted in the highest levels of nectar contamination with thiamethoxam, and exceeded published median lethal concentrations (LC50s/median lethal doses for native bees and/or honeybees in all cases). Spray treatments resulted in nectar concentrations exceeding published LC50s for some bee species. In comparison, all nonsystemic treatments resulted in concentrations much lower than the published no-observable-effect doses and sublethal toxicity values, indicating low risks of toxicity. Environ Toxicol Chem 2024;001:1-12. © 2024 SETAC.

Exposure of Cattle Breeding Herds to Naturally Co-Contaminated Zearalenone and Deoxynivalenol: The Relevance of a Urinary Mycotoxin Monitoring System for Herd Health and Food Safety

The widespread presence of Fusarium mycotoxins in animal feed is a global issue, not only for the health of livestock but also for ensure the safety of food as an end product. High concentrations of zearalenone (ZEN) and deoxynivalenol (DON) have been detected in the diets of Japanese Black (JB) and Holstein Friesian (HF) breeding herds. Consequently, we monitored serum biochemical parameters over a long time in both herds, focusing on anti-Müllerian hormone (AMH) levels and acute-phase inflammation. Additionally, urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) and progesterone levels were measured in the HF herd. The JB herd, a ZEN-dominant model with low DON contamination, demonstrated ZEN levels that exceeded the Japanese limit in the purchased total mixed rations (TMR). Conversely, the HF herd, which primary consumes DON-dominant feed with low ZEN contamination, had high DON levels in the dent corn silage. Specifically, the JB herd’s TMR contained 1.79 mg/kg ZEN and 0.58 mg/kg DON, whereas the HF herd’s silage had 15.3 mg/kg DON (dried sample) and 0.1 mg/kg ZEN. Enzyme-linked immunoassay were used to measure urinary ZEN-DON levels following confirmation through liquid chromatography-tandem mass spectrometry. Urinary ZEN-DON levels measured were significantly correlated (p < 0.05, r > 0.6) in both herds. In the HF herd, AMH levels increased (p = 0.01) and serum amyloid A (SAA) levels decreased (p = 0.02) when contaminated and at the end of the monitoring period. Additionally, urinary ZEN and DON levels were significantly correlated with SAA levels (ZEN: p = 0.00, r = 0.46; DON: p = 0.03, r = 0.33), with an increase in ZEN and DON levels resulting in higher SAA levels. The JB herd showed no significant differences. Additionally, in the HF herd, 8-OHdG/Cre levels increased significantly during major contamination periods (p < 0.05). Clinical data from the HF herd indicated an increase in mastitis cases and treatment rates during periods of major contamination. Abortion rates in the HF herd decreased from 22.9% (before monitoring) to 8.9% (during the high contamination period) and finally to 1% (at the end of the monitoring period), with corresponding increases in progesterone levels. ZEN-DON contamination adversely affects breeding cattle’s productivity, reproductive performance, and health. Therefore, monitoring urinary ZEN-DON is valuable for detecting contaminants and ensuring the safety of food products.

Metabolomics and quantitative analysis to determine differences in the geographical origins and species of Chinese dragon’s blood

ObjectiveThe aim of this study was to comprehensively analyze the differences in Chinese dragon’s blood (CDB), specifically Dracaena cochinchinensis and Dracaena cambodiana, from different geographical origins.MethodsMetabolomic analysis of CDB was performed by liquid chromatography-tandem mass spectrometry (LC–MS/MS). A reliable ultrahigh-performance liquid chromatography method with a photodiode array detector (UHPLC-PDA) was developed and applied for the quantitative analysis of 12 phenolic compounds in 51 batches of samples.ResultsA total of 1394 metabolites were detected, of which 467 were identified as differentially accumulated metabolites. Multivariate analysis revealed that both origin and species had an effect on the composition of CDB, with greater variation between species. 19 phenolic compounds were selected as quality markers to distinguish D. cochinchinensis (Hdsp) from D. cambodiana (Hdca), and oppositin and spinoflavanone a were identified as quality markers to discriminate D. cochinchinensis samples from Hainan (Hdsp) and Guangxi Provinces (Gdc). Quantitative analysis indicated that four phenolic compounds, including loureirin D, 4H-1-benzopyran-4-one,2,3-dihydro-3,5,7-trihydroxy-3-[(4-methoxyphenyl)methyl]-,(R)-, loureirin B, and pterostilbene, showed significant differences between Gdc and Hdsp. Additionally, five phenolic compounds, namely resveratrol, loureirin D, pinostilbene, 4H-1-benzopyran-4-one,2,3-dihydro-3,5,7-trihydroxy-3-[(4-methoxyphenyl)methyl]-, (R)-, and loureirin B, exhibited significant differences between Hdsp and Hdca.ConclusionThere are significant differences in the quality of CDB from different geographical origins and species, which lays the foundation for the in-depth development and utilization of different sources of CDB.

Analysis of the gut microbiota and fecal metabolites in people living with HIV.

The gut microbiome has a pivotal function in human immunodeficiency virus (HIV). However, the associated alterations in the gut microbiome-host interaction are unknown. Herein, we aimed to investigate the gut microbiota and fecal metabolites in people living with HIV (PLWH). We collected stool samples from 70 PLWH and 34 healthy controls (HCs) and carried out 16S rRNA gene sequencing and analyzed the metabolites using liquid chromatography-mass spectrometry. Firmicutes, Proteobacteria, Actinobacteriota, and Bacteroidota were the most abundant phyla in both groups. Among genera, the level of Escherichia-Shigella was upregulated significantly in the PLWH group, whereas in the HC group, Bacteroides spp. were upregulated. Prediction of microbial function indicated significant reductions in alanine, aspartate, glutamate, and histidine metabolism. Furthermore, a comparison of the fecal metabolites between the HC and PLWH groups identified 38 differentially abundant metabolites in four differentially enriched human metabolic pathways. According to Spearman correlation analysis, there are close relationships between four differentially abundant microbiota members and five differentially abundant fecal metabolites, which might influence particular human metabolic pathways. Our findings provide a basis for further experimental investigation of the contribution of the gut microbiota and its associated metabolites to HIV/AIDS, providing a novel perspective for the further study of HIV/AIDS.IMPORTANCEGrowing evidence demonstrates that the gut microbiota is associated with HIV. This study investigated changes in the gut microbiota and fecal metabolites in PLWH. We identified 38 differentially abundant metabolites in four differentially enriched human metabolic pathways. Moreover, close relationships were noted between the four differentially abundant microbiota members and five differentially abundant fecal metabolites, which might influence particular human metabolic pathways. Thus, to benefit PLWH, potential pathobionts could be reduced (e.g., g_Enterococcus); probiotics could be increased (e.g., g_Faecalibacterium and g_Agathobacter); or certain metabolites (e.g., N-acetyl-L-phenylalanine and trehalose) could be reduced by changes in diet or the use of nutritional supplements. Our results provide insights into the interaction between the gut microbiota and the host, identifying possible targets that might be beneficial for PLWH.

Analysis of riociguat and desmethyl riociguat by UPLC-MS/MS and its interaction with quercetin

Riociguat, an orally soluble guanylate cyclase (sGC)-promoting drug, is mainly used in the clinical treatment of pulmonary hypertension (PH). In this study, a novel ultra-performance liquid chromatography-tandem mass spectrometry method was developed to quantify the concentrations of riociguat and its metabolite (M1) in plasma. The precision, stability, accuracy, matrix effect, and recovery of the methodology were satisfactory. Quercetin, a well-recognized compound, functions as a novel anticancer agent with the potential to alleviate symptoms of PH. Therefore, the potential interaction between quercetin and riociguat was investigated in this study. The levels of riociguat and M1 in rat plasma were measured using the method developed in this study to evaluate the interactions between riociguat and quercetin in rats. The results revealed that quercetin significantly inhibited riociguat and M1 metabolism with increased systemic exposure.

Identification of amino acids metabolomic profiling in human plasma distinguishes lupus nephritis from systemic lupus erythematosus

Lupus nephritis (LN) is an immunoinflammatory glomerulonephritis associated with renal involvement in systemic lupus erythematosus (SLE). Given the close relationship between plasma amino acids (AAs) and renal function, this study aimed to elucidate the plasma AA profiles in LN patients and identify key AAs and diagnostic patterns that distinguish LN patients from those with SLE and healthy controls. Participants were categorized into three groups: normal controls (NC), SLE, and LN. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed to quantify AA levels in human plasma. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were utilized to identify key AAs. The diagnostic capacity of the models was assessed using receiver operating characteristic (ROC) curve analysis and area under the ROC curve (AUC) values. Significant alterations in plasma AA profiles were observed in LN patients compared to the SLE and NC groups. The OPLS-DA model effectively separated LN patients from the SLE and NC groups. A joint model using histidine (His), lysine (Lys), and tryptophan (Trp) demonstrated exceptional diagnostic performance, achieving an AUC of 1.0 with 100% sensitivity, specificity, and accuracy in predicting LN. Another joint model comprising arginine (Arg), valine (Val), and Trp also exhibited robust predictive performance, with an AUC of 0.998, sensitivity of 93.80%, specificity of 100%, and accuracy of 95.78% in distinguishing between SLE and LN. The joint forecasting models showed excellent predictive capabilities in identifying LN and categorizing lupus disease status. This approach provides a novel perspective for the early identification, prevention, treatment, and management of LN based on variations in plasma AA levels.

SGLT2 inhibitors ameliorate NAFLD in mice via downregulating PFKFB3, suppressing glycolysis and modulating macrophage polarization.

Sodium-glucose co-transporter 2 (SGLT2) inhibitor (SGLT2i) is a novel class of anti-diabetic drug, which has displayed a promising benefit for non-alcoholic fatty liver disease (NAFLD). In this study, we investigated the protective effects of SGLT2i against NAFLD and the underlying mechanisms. The db/db mice and western diet-induced NAFLD mice were treated with dapagliflozin (1 mg·kg-1·d-1, i.g.) or canagliflozin (10 mg·kg-1·d-1, i.g.) for 8 weeks. We showed that the SGLT2i significantly improved NAFLD-associated metabolic indexes, and attenuated hepatic steatosis and fibrosis. Notably, SGLT2i reduced the levels of pro-inflammatory cytokines and chemokines, downregulated M1 macrophage marker expression and upregulated M2 macrophage marker expression in liver tissues. In cultured mouse bone marrow-derived macrophages and human peripheral blood mononuclear cell-derived macrophages, the SGLT2i (10, 20 and 40 μmol/L) significantly promoted macrophage polarization from M1 to M2 phenotype. RNA sequencing, Seahorse analysis and liquid chromatography-tandem mass spectrometry analysis revealed that the SGLT2i suppressed glycolysis and triggered metabolic reprogramming in macrophages. By using genetic manipulation and pharmacological inhibition, we identified that the SGLT2i targeted PFKFB3, a key enzyme of glycolysis, to modulate the macrophage polarization of M1 to M2 phenotype. Using a co-culture of macrophages with hepatocytes, we demonstrated that the SGLT2i inhibited lipogenesis in hepatocytes via crosstalk with macrophages. In conclusion, this study highlights a potential therapeutic application for repurposing SGLT2i and identifying a potential target PFKFB3 for NAFLD treatment.

Novel Metabolites Associated with Decreased GFR in Finnish Men: A 12-Year Follow-Up of the METSIM Cohort

Identification of the individuals having impaired kidney function is essential in preventing the complications of this disease. We measured 1009 metabolites at the baseline study in 10,159 Finnish men of the METSIM cohort and associated the metabolites with an estimated glomerular filtration rate (eGFR). A total of 7090 men participated in the 12-year follow-up study. Non-targeted metabolomics profiling was performed at Metabolon, Inc. (Morrisville, NC, USA) on EDTA plasma samples obtained after overnight fasting. We applied liquid chromatography mass spectrometry (LC-MS/MS) to identify the metabolites (the Metabolon DiscoveryHD4 platform). We performed association analyses between the eGFR and metabolites using linear regression adjusted for confounding factors. We found 108 metabolites significantly associated with a decrease in eGFR, and 28 of them were novel, including 12 amino acids, 8 xenobiotics, 5 lipids, 1 nucleotide, 1 peptide, and 1 partially characterized molecule. The most significant associations were with five amino acids, N-acetylmethionine, N-acetylvaline, gamma-carboxyglutamate, 3-methylglutaryl-carnitine, and pro-line. We identified 28 novel metabolites associated with decreased eGFR in the 12-year follow-up study of the METSIM cohort. These findings provide novel insights into the role of metabolites and metabolic pathways involved in the decline of kidney function.

Polyamine Pathway Inhibitor DENSPM Suppresses Lipid Metabolism in Pheochromocytoma Cell Line

Pheochromocytomas (PCCs) are tumors arising from chromaffin cells in the adrenal medulla, and paragangliomas (PGLs) are tumors derived from extra-adrenal sympathetic or parasympathetic paraganglia; these tumors are collectively referred to as PPGL cancer. Treatment for PPGL primarily involves surgical removal of the tumor, and only limited options are available for treatment of the disease once it becomes metastatic. Human carriers of the heterozygous mutations in the succinate dehydrogenase subunit B (SDHB) gene are susceptible to the development of PPGL. A physiologically relevant PCC patient-derived cell line hPheo1 was developed, and SDHB_KD cells carrying a stable short hairpin knockdown of SDHB were derived from it. An untargeted metabolomic approach uncovered an overactive polyamine pathway in the SDHB_KD cells that was subsequently fully validated in a large set of human SDHB-mutant PPGL tumor samples. We previously reported that treatment with the polyamine metabolism inhibitor N1,N11-diethylnorspermine (DENSPM) drastically inhibited growth of these PCC-derived cells in culture as well as in xenograft mouse models. Here we explored the mechanisms underlying DENSPM action in hPheo1 and SDHB_KD cells. Specifically, by performing an RNAseq analysis, we have identified gene expression changes associated with DENSPM treatment that broadly interfere with all aspects of lipid metabolism, including fatty acid (FA) synthesis, desaturation, and import/uptake. Furthermore, by performing an untargeted lipidomic liquid chromatography–mass spectrometry (LC/MS)-based analysis we uncovered specific groups of lipids that are dramatically reduced as a result of DENSPM treatment. Specifically, the bulk of plasmanyl ether lipid species that have been recently reported as the major determinants of cancer cell fate are notably decreased. In summary, this work suggests an intersection between active polyamine and lipid pathways in PCC cells.

Tear Proteomics in Children and Adolescents with Type 1 Diabetes: A Promising Approach to Biomarker Identification of Diabetes Pathogenesis and Complications

The aim of the current study was to investigate the tear proteome in children and adolescents with type 1 diabetes (T1D) compared to healthy controls, and to identify differences in the tear proteome of children with T1D depending on different characteristics of the disease. Fifty-six children with T1D at least one year after diagnosis, aged 6–17 years old, and fifty-six healthy age- and sex-matched controls were enrolled in this cross-sectional study. The proteomic analysis was based on liquid chromatography tandem mass spectrometry (LC-MS/MS) enabling the identification and quantification of the protein content via Data-Independent Acquisition by Neural Networks (DIA-NN). Data are available via ProteomeXchange with the identifier PXD052994. In total, 3302 proteins were identified from tear samples. Two hundred thirty-nine tear proteins were differentially expressed in children with T1D compared to healthy controls. Most of them were involved in the immune response, tissue homeostasis and inflammation. The presence of diabetic ketoacidosis at diagnosis and the level of glycemic control of children with T1D influenced the tear proteome. Tear proteomics analysis revealed a different proteome pattern in children with T1D compared to healthy controls offering insights on deregulated biological processes underlying the pathogenesis of T1D. Differences within the T1D group could unravel biomarkers for early detection of long-term complications of T1D.

EsxA, a type VII secretion system-dependent effector, reveals a novel function in the sporulation of Bacillus cereus ATCC14579

BackgroundBacillus cereus is a Gram-positive, spore-forming bacterium that produces a spectrum of effectors integral to bacterial niche adaptation and the development of various infections. Among those is EsxA, whose secretion depends on the EssC component of the type VII secretion system (T7SS). EsxA’s roles within the bacterial cell are poorly understood, although postulations indicate that it may be involved in sporulation. However, the T7SS repertoire in B. cereus has not been reported, and its functions are unestablished.MethodsWe used the type strain, B. cereus ATCC14579, to generate ΔessC mutant through homologous recombination using the homing endonuclease I-SceI mediated markerless gene replacement. Comparatively, we analyzed the culture supernatant of type strain and the ΔessC mutant through Liquid chromatography-tandem mass spectrometry (LC-MS/MS). We further generated T7SSb-specific gene mutations to explore the housekeeping roles of the T7SSb-dependent effectors. The sporulation process of B. cereus ATCC14579 and its mutants was observed microscopically through the classic Schaeffer-Fulton staining method. The spore viability of each strain in this study was established by enumerating the colony-forming units on LB agar.ResultsThrough LC-MS/MS, we identified a pair of nearly identical (94%) effector proteins named EsxA belonging to the sagEsxA-like subfamily of the WXG100 protein superfamily in the culture supernatant of the wild type and none in the ΔessC mutant. Homology analysis of the T7SSb gene cluster among B. cereus strains revealed diversity from the 3’ end of essC, encoding additional substrates. Deletions in esxA1 and esxA2 neither altered cellular morphology nor growth rate, but the ΔesxA1ΔesxA2 deletion resulted in significantly fewer viable spores and an overall slower sporulation process. Within 24 h culture, more than 80% of wild-type cells formed endospores compared to less than 5% in the ΔesxA1ΔesxA2 mutant. The maximum spore ratios for the wild type and ΔesxA1ΔesxA2 were 0.96 and 0.72, respectively. Altogether, these results indicated that EsxA1 and EsxA2 work cooperatively and are required for sporulation in B. cereus ATCC14567.ConclusionB. cereus ATCC14579 possesses two nearly identical T7SSb-dependent effectors belonging to the sagEsxA-like proteins. Simultaneous deletion of genes encoding these effectors significantly delayed and reduced sporulation, a novel finding for EsxA.

Faster ciguatoxin extraction methods for toxicity screening

Ciguatera poisoning (CP) is a severe global public health problem caused by the consumption of seafood products contaminated with ciguatoxins (CTXs). The growing demand for seafood products requires high-throughput testing for CTX-susceptible seafood, however complex extraction and slow cleanup methods inhibit this goal. Herein, several methods for extracting CTXs from fish tissue were established and compared; these methods are sensitive, specific, and valid while achieving higher sample extraction throughput than currently established protocols. The trial fish material was generated from multiple species, with different physical conditions (wet and freeze-dried tissue), and naturally contaminated with various CTXs (i.e., CTX-1B, CTX-3C, and C-CTX-1), thus ensuring these methods are robust and broadly applicable. The extraction methods used were based on mechanical maceration with acetone or methanol or enzymatic digestion followed by acetone and ethyl acetate extraction. Crude extracts were investigated for CTX-like toxicity using an in vitro mouse neuroblastoma (N2a) cell-based assay (CBA). Among the three methods, there was no significant difference in toxin estimates (p = 0.219, two-way ANOVA), indicating their interchangeability. For speed (> 16 samples/day), accuracy (100%), and CTX analog retention confirmation by liquid chromatography-tandem mass spectrometry (LC‒MS/MS), the preferred extraction methods were both methanol and enzyme-based. All extraction methods post hoc confirmation of CTX analogs successfully met international seafood market-based CTX contaminant guidance. These methods can drastically increase global CTX screening capabilities and subsequently relieve sample processing bottlenecks, inhibiting environmental and human health-based CTX analysis.

Residual levels of pesticides in filet green beans from Souss-Massa, Morocco.

A total of 30 samples of filet green beans (Phaseolus vulgaris) from various popular markets in the Souss-Massa region (Morocco) were analyzed during 2021 for the presence of pesticide residues. We performed a QuEChERS extraction (Quick, Easy, Cheap, Effective, Rugged, and Safe) combined with Liquid Chromatography-Mass Spectrometry (LC-MS/MS) and Gas Chromatography-Mass Spectrometry (GC-MS/MS). The method was validated in accordance with SANTE 11312/2021 guidelines. The results of this survey indicated that 8 out of 30 green bean samples are positives (0.015 to 0.112mg/kg). Azoxystrobin was the most frequently detected pesticide, occurring in 5 samples. For authorized pesticides, none of the samples exceeded the European Maximum residue level (EU MRL) in green beans, but residue of fipronil-unauthorized substance was detected in one sample at 0.027mg/kg. Hence, it is important to increase inspections for locally marketed fresh green beans and align agricultural practices with regulatory requirements.

An interactive R-based custom quantification program for semi-quantitative analysis of triacylglycerols in bovine milk.

The R programming language, RStudio, and open-source software solutions for analysis of liquid chromatography-mass spectrometry (LC-MS) data have been used with user-written R-based custom quantification programs (CQP) for semi-quantification of triacylglycerols (TAGs) in bovine milk lipid extracts. Using the peak-finding capabilities of the package "xcms" in RStudio, peaks were integrated, and retention times aligned, normalized, and then used for semi-quantitative analysis of a custom set of four extraction internal standards (EISs) and 29 TAG regioisomers using the choice of four analytical internal standards (AISs). Alternating stereospecific numbering (sn) 1,3 TAG regioisomers (standards 1, 3, and 5 of six calibration standards) and sn-1,2 TAG regioisomers (standards 2, 4, and 6 of six standards) were used to make a set of six calibration standards, which were used for quantification using a linear fit model, polynomial fit model, power fit model, level-bracketed linear fit, replicate-bracketed polynomial fit, replicate-bracketed power fit, and replicate- and level-bracketed linear fit and response factors. For example, the linear fit for EIS1 gave an unacceptable coefficient of determination (CoD), r2 = 0.9616, whereas the polynomial fit gave r2 = 0.9908 and the power fit gave r2 = 0.9928, while the double-bracketed linear fit gave CoDs of r2 = 0.9960, 0.9848, and 0.9781 for the three brackets, yet gave the least % difference to known calibration concentrations. For unparalleled transparency, the CQP produced webpages that allowed every step in the data processing and quantification sequence to be verified and reproduced, and contained interactive figures. The data are publicly available using a digital object identifier (DOI). The R code can be downloaded and used with the downloadable data to reproduce the results, to modify the code and further customize the results, or to copy and paste and adapt the code to other quantification applications.

Gibberellin 2-oxidase 1(CsGA2ox1) involved gibberellin biosynthesis regulates sprouting time in camellia sinensis

BackgroundTea is an important cash crop and buds are its main product. To elucidate the molecular mechanism of the sprouting time of tea plants, ‘Yuchunzao’, which was an early sprouting tea cultivar, was studied. ‘Echa 1’, sprout one week later than ‘Yuchunzao’ in spring, was used as the control.ResultsA total of 26 hormonal compounds and its derivatives in tea plants were qualified by using Ultra Performance Liquid Chromatography-Tandem mass spectrometry (UPLC-MS/MS). The result showed that GA20, GA3 and ICA were significantly different in ‘Yuchunzao’ than in ‘Echa 1’, with GA20 and GA3 up-regulated and ICA down-regulated. Based on the Illumina platform, transcriptome analysis revealed a total of 5,395 differentially expressed genes (DEGs). A diterpenoid biosynthesis related gene, gibberellin 2-oxidase 1 (CsGA2ox1), was downregulated in ‘Yuchunzao’ compared to ‘Echa 1’. CsGA2ox1 regulate the transformation of GA different forms in plants. The relative expression of CsGA2ox1 showed an adverse trend with the content of GA20 and GA3. Our results suggest that down regulation of CsGA2ox1 resulted in the accumulation of GA3 and GA20, and then promoted sprout of ‘Yuchunzao’.ConclusionThis study provides theoretical basis of tea plants sprout and guides the tea breeding in practice.

Comparative LC–MS-based metabolite profiling, antioxidant, and antibacterial properties of Bunium bulbocastanum tubers from two regions in Algeria

Traditional herbalists have been relied on for many years by Algerians to cure a wide range of diseases. Regardless of their nutritional values, mushrooms have chemical properties that make them attractive, beneficial, and more likely to be studied by researchers, according to ethnobotanical literature on traditional phytotherapy. Among all the edible mushrooms, tubers are a type of fungus that are traditionally used in fine dining and have garnered attention recently because of their many therapeutic applications. This research delves into a meticulous analysis of bioactive constituents in Bunium bulbocastanum tubers, sourced from Mostaganem and Relizane regions, with a keen focus on polyphenols, flavonoids, and condensed tannins. The quantification of total phenolic content was executed through the Folin-Ciocalteu assay, while flavonoids were assessed via the aluminum chloride colorimetric method. In addition, condensed tannins were evaluated in this study. Antioxidant capacities were scrutinized employing the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Microbial inhibition studies were conducted against five benchmark bacterial strains, utilizing the agar disc diffusion technique. Furthermore, a comprehensive liquid chromatography–mass spectrometry (LC–MS) analysis was performed to identify and quantify bioactive compounds. The findings underscore that the Mostaganem extracts were particularly rich in polyphenols (11.65 mg GAE/g of extract) and tannins (1.30 mg CE/g of extract), while the Relizane extracts boasted significant flavonoid concentrations (9.421 mg QE/g of extract). Notably, 4-methylguaiacol (82.04 mg/L), caffeic acid dimethyl ether (27.76 mg/L), syringic acid (20.48 mg/L), and naringenin (16.05 mg/L) emerged as the predominant volatile compounds. Compositional investigation of the extracts by LC–MS confirmed the presence of various compounds that were linked to the bioactivities exhibited by B. bulbocastanum tubers. These findings demonstrate the effective antibacterial and antioxidant properties of B. bulbocastanum tubers, indicating their potential use in pharmaceutical and nutraceutical applications.

Multianalyte Approach-Including Automated Preparation of Calibrators-for Validated Quantification of 82 Drugs in Whole Blood by Liquid Chromatography-Tandem Mass Spectrometry.

Bioanalysis, such as the quantification of drugs in different matrices, is of great importance in forensic toxicology. Nowadays, mainly so-called multianalyte approaches are used given their increased speed and effectiveness. However, such multianalyte procedures can be difficult to develop and maintain with sufficient robustness in the laboratory. One aspect of this is the tedious, manual preparation of spiking solutions containing such a great number of analytes. Therefore, the current study aimed to develop and validate a fast, simple, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 82 classic drugs and to evaluate an alternative autosampler-assisted automated approach for the preparation of spiking solutions. Simple protein precipitation of 200-μL whole blood was used followed by analysis by reversed-phase LC-MS/MS in advanced scheduled multiple reaction monitoring (MRM) mode. The method was fully validated according to international guidelines, including selectivity, recovery, matrix effects, linearity, bias/imprecision, processed-sample stability, and limits. Validation criteria were fulfilled for all analytes except for buprenorphine and five benzodiazepines. In the context of a multianalyte procedure, a (multipurpose) autosampler-assisted automatic preparation of calibrator spiking solutions proved comparable to manual preparation. Thus, automated preparation can overcome the frequently performed manual, time-consuming, and error-prone steps of multianalyte approaches and still allow for customized calibration ranges. Since its introduction, more than 8000 cases have been measured with the presented method, and 35 proficiency tests have been passed.

The Influence of Barley Proteome on Hop Bitter Acid Yield during Brewing.

A persistent challenge in brewing is the efficient utilization of hop bitter acids, with about 50% of these compounds precipitating with trub during wort boiling. This study aims to uncover the correlation between the barley cultivar proteome and hop bitter acid utilization during wort boiling. Therefore, comparative experiments were conducted using two cultivars, Liga and Solist, with varying proteomes to identify specific proteins' role in hop bitter acids precipitation. High-performance liquid chromatography (HPLC) was used to measure hop bitter acid content, while liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify and identify proteins. The 107 protein groups, particularly enzymes linked to barley metabolic defense mechanisms, exhibited significant differences between the two cultivars. Results revealed significantly lower α- and iso-α-acid content in wort produced from the barley cultivar Liga. This study highlights the critical role of the barley proteome in optimizing process efficiency by enhancing hop utilization through barley cultivar selection.

Integrated transcriptomic and metabolic analyses reveal the early response mechanism of Pinus tabulaeformis to pine wood nematodes

Pine wilt disease (PWD) is a devastating disease of pine trees caused by the pine wood nematode (Bursapherenchus xylophilus, PWN). To study how Pinus tabulaeformis responds to PWD infection, we collected 3-year-old P. tabulaeformis seedlings at 2 days, 5 days, and 8 days after being infected with B. xylophilus. We identified genes and metabolites early responding to infection using transcriptome and metabolomic data obtained by high-throughput mRNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assays, respectively. The following results were obtained: (1) After inoculation with PWN, the average number of days taken for 3-year-old P. tabulaeformis seedlings to develop symptoms was 8 days. (2) Combined transcriptome and metabolome analysis revealed that phenylpropanoid biosynthesis and flavonoid biosynthesis are critically important pathways for P. tabulaeformis to respond to PWD. (3) The response of P. tabulaeformis to stress was mainly through positive regulation of gene expression, including some key genes related to plant hormones or transcription factors that have been widely studied. Genes related to pathways such as photosynthesis, plant-pathogen interactions, and DNA replication were downregulated. (4) Terpenoid biosynthesis genes involved during the development of pine wilt disease. This study demonstrated the defence and pathogenic mechanisms of P. tabulaeformis against PWD, providing a reference for the early diagnosis of PWD.