Ultra-performance Liquid Chromatography-tandem Mass Spectrometry Research Articles

Huangqin tang alleviates colitis-associated colorectal cancer via amino acids homeostasisand PI3K/AKT/mtor pathway modulation

Ethnopharmacological relevanceHuangqin Tang (HQT), a traditional Chinese medicine formula, is commonly used in clinical practice for the treatment of inflammatory bowel diseases. It has been reported that HQT exerts antitumor effects on colitis-associated colorectal cancer (CAC). However, the mechanism by which HQT interferes with the inflammation-to-cancer transformation remains unclear. Aims of the studyThe purpose of this study was to dynamically evaluate the efficacy of HQT in alleviating or delaying CAC and to reveal the underlying mechanism. MethodsWe established a mouse model of CAC using azoxymethane combined with 1.5% dextran sodium sulphate. The efficacy of HQT was evaluated based on pathological sections and serum biochemical indices. Subsequently, amino acids (AAs) metabolism analyses were performed using ultra-performance liquid chromatography-tandem mass spectrometry, and the phosphatidylinositol 3 kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR) pathway was detected by western blotting. ResultsThe data demonstrated that HQT could alleviate the development of CAC in the animal model. HQT effectively reduced the inflammatory response, particularly interleukin-6 (IL-6), in the inflammation induction stage, as well as in the stages of proliferation initiation and tumorigenesis. During the proliferation initiation and tumorigenesis stages, immunohistochemistry staining showed that the expression of the proliferation marker Ki67 was reduced, while apoptosis was increased in the HQT group. Accordingly, HQT substantially decreased the levels of specific AAs in the colon with CAC, including glutamic acid, glutamine, arginine, and isoleucine. Furthermore, HQT significantly inhibited the activated PI3K/AKT/mTOR pathway, which may contribute to suppression of cell proliferation and enhancement of apoptosis. ConclusionHQT is effective in alleviating and delaying the colon “inflammation-to-cancer”. The mechanism of action may involve HQT maintained AAs metabolism homeostasis and regulated PI3K/AKT/mTOR pathway, so as to maintain the balance between proliferation and apoptosis, and then interfere in the occurrence and development of CAC.

Targeting N4-acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2-mediated HMGB2 mRNA translation.

N4-acetylcytidine (ac4C) represents a novel messenger RNA (mRNA) modification, and its associated acetyltransferase N-acetyltransferase 10 (NAT10) plays a crucial role in the initiation and progression of tumors by regulating mRNA functionality. However, its role in hepatocellular carcinoma (HCC) development and prognosis is largely unknown. This study aimed to elucidate the role of NAT10-mediated ac4C in HCC progression and provide a promising therapeutic approach. The ac4C levels were evaluated by dot blot and ultra-performance liquid chromatography-tandem mass spectrometry with harvested HCC tissues. The expression of NAT10 was investigated using quantitative real-time polymerase chain reaction, western blotting, and immunohistochemical staining across 91 cohorts of HCC patients. To explore the underlying mechanisms of NAT10-ac4C in HCC, we employed a comprehensive approach integrating acetylated RNA immunoprecipitation and sequencing, RNA sequencing and ribosome profiling analyses, along with RNA immunoprecipitation, RNA pull-down, mass spectrometry, and site-specific mutation analyses. The drug affinity responsive targets stability, cellular thermal shift assay, and surface plasmon resonance assays were performed to assess the specific binding of NAT10 and Panobinostat. Furthermore, the efficacy of targeting NAT10-ac4C for HCC treatment was elucidated through in vitro experiments using HCC cells and in vivo HCC mouse models. Our investigation revealed a significant increase in both the ac4C RNA level and NAT10 expression in HCC. Notably, elevated NAT10 expression was associated with poor outcomes in HCC patients. Functionally, silencing NAT10 suppressed HCC proliferation and metastasis in vitro and in vivo. Mechanistically, NAT10 stimulates the ac4C modification within the coding sequence (CDS) of high mobility group protein B2 (HMGB2), which subsequently enhances HMGB2 translation by facilitating eukaryotic elongation factor 2 (eEF2) binding to the ac4C sites on HMGB2 mRNA's CDS. Additionally, high-throughput compound library screening revealed Panobinostat as a potent inhibitor of NAT10-mediated ac4C modification. This inhibition significantly attenuated HCC growth and metastasis in both in vitro experiments using HCC cells and in vivo HCC mouse models. Our study identified a novel oncogenic epi-transcriptome axis involving NAT10-ac4C/eEF2-HMGB2, which plays a pivotal role in regulating HCC growth and metastasis. The drug Panobinostat validates the therapeutic potential of targeting this axis for HCC treatment.

Is Dosage Adjustment Based on Age Necessary for Intravenous Lidocaine in Patients Undergoing General Anesthesia: A Prospective Multi-Arm Comparative Study

It remains unclear whether dosage adjustment of intravenous lidocaine is necessary during general anesthesia for elderly patients over 75 years old. This study aimed to investigate the effects of age on the pharmacokinetics (PK) and safety of intravenous lidocaine in patients undergoing general anesthesia. A total of 599 plasma samples were collected from 76 general anesthesia patients across three age groups: 18–64, 65–74, and ≥ 75 years. Lidocaine was administered intravenously at a dose of 1.5 mg/kg for the 18–64 and 65–74 years groups, while the dose was adjusted to 1.0 mg/kg for the ≥ 75 years group. The plasma concentrations of lidocaine and its active metabolites were measured using a validated ultra-performance liquid chromatography-tandem mass spectrometry assay, and the data were analyzed using a noncompartmental analysis. The results revealed no significant age-related differences in the PK of lidocaine and its metabolites. Among the three age groups, over 90 % of patient achieved a lidocaine concentration within a safe and effective range when the dosage was normalized to 1.5 mg/kg. In conclusion, age-based dosage adjustment was unnecessary for intravenous lidocaine in patients below 86 years undergoing general anesthesia.

Integrative analysis of the ovarian metabolome and transcriptome of the Yaoshan chicken and its improved hybrids.

Introduction: Laying performance is a key factor affecting production efficiency in poultry, but its molecular mechanism is still indistinct. In this study, Yaoshan chickens, a local breed in Guizhou, China, and merchant chickens (GYR) with higher egg yield after the three-line cross improvement hybridization of Yaoshan chickens were used as animal samples. Methods: To explore the regulatory mechanism of the diversities in laying performance, RNA-seq and ultra-performance liquid chromatographytandem mass spectrometry (UPLC-MS/MS) were used to describe the transcriptional and metabolic profiles of the ovaries of Yaoshan and GYR chickens. Results: At the transcriptional level, 288 differentially expressed genes were upregulated in Yaoshan chickens and 353 differentially expressed genes were upregulated in GYR chickens. In addition, GSEA showed that ECM-receptor interactions and the TGF-β signaling pathway were restrained, resulting in increased egg production in GYR chickens. Furthermore, the upregulation of thiamine and carnitine was identified by metabolomic analysis to facilitate the laying performance of hens. Finally, comprehensive analyses of the transcriptome and metabolome found that thiamine and carnitine were negatively correlated with ECM-receptor interactions and the TGF-β signaling pathway, which jointly regulate the laying performance of Yaoshan chickens and GYR chickens. Discussion: Taken together, our research delineates differences in the transcriptional and metabolic profiles of the ovaries of Yaoshan and GYR chickens during the peak egg production period and provides new hypotheses and clues for further research on poultry egg production performance and the improvement of economic benefits.

Discrimination of overheated pasteurized milk using mass spectrometry-based proteomics

Milk is one of the most widely consumed foods globally. To protect consumer interests, it is essential to establish an analytical method to detect the degree of heating in milk. A novel approach using nano liquid chromatography-orbitrap fusion mass spectrometer was developed for screening and identifing thermally sensitive peptides markers in the milk heating process (below 100 °C). This method integrates untargeted proteomics and chemometric tools to analyze protein quantitation data from differently heat-treated milk. Thirteen potential markers were screened out and identified, and further confirmed using by standard substances. Then, the accurate concentrations of 13 potential markers determined by isotope-dilution ultra-performance liquid chromatography-tandem triple quadrupole mass spectrometry were further mining the highly specific and thermally sensitive peptides markers. And Four peptides—INLFDTPLETQYVR, FELLGCELNGCTEPLGLK, QFQFIQVAGR, and GEADALNLDGGYIYTAGK—were selected as marker peptides to differentiate normal pasteurized milk from overheated pasteurized milk. The concentrations of INLFDTPLETQYVR ranges from 150 ± 11 µg/L to 350 ± 23 µg/L, while the concentrations of FELLGCELNGCTEPLGLK ranges from 40 ± 5 µg/L to 92 ± 3 µg/L, can distinguish normal pasteurized milk from overheated pasteurized milk. QFQFIQVAGR indicates overheated pasteurized milk at 230 ± 21 µg/L, and GEADALNLDGGYIYTAGK signifies 750 ± 43 µg/L. This study provides new insights for distinguishing overheated pasteurized milk.

P-474 State-Trait Anxiety Inventory (STAI) punctuations as a non-invasive method for evaluating endometrial prognosis associated with cortisol and estradiol endometrial levels in IVF patients

Abstract Study question Are STAI State punctuations a potential non-invasive marker of endometrial prognosis in IVF patients? Summary answer Being stressed (determined by STAI State punctuations) is positively and negatively correlated with endometrial cortisol and estradiol levels, respectively, and highly associated with pregnancy consecution. What is known already Despite it is well known IVF treatments are a source of stress, this factor is frequently overlooked mainly due to the difficulty of stress evaluation and the lack of accurate biomarkers, not being standardized in the clinical setting. Steroid hormones are essential for reproductive physiology and highly related to well-known stress biomarkers such as cortisol. The lack of correlation between endometrial and serum steroid levels creates the need to look for non-invasive tools reflecting the endometrial microenvironment. In this study, by molecularly and psychologically characterizing patients, we aim to unveil non-invasive potential markers of stress associated to pregnancy consecution. Study design, size, duration This prospective cohort study included a total of 74 IVF patients (<45 years old, no uterine or systemic pathologies and good quality embryos) who underwent endometrial biopsy collection in mid-secretory phase for metabolites measurement between 2019 and 2023. These patients also underwent psychological evaluation by stress related questionnaires. Participants/materials, setting, methods The endometrial concentration of eleven steroid metabolites was measured by ultra-performance liquid chromatography-tandem mass spectrometry. Patients also underwent stress and anxiety evaluation (STAI), and reproductive outcomes in the first embryo transfer after biopsy collection were followed up. Wilcoxon and Barnard’s test were applied to compare the mean and proportion of stressed/unstressed, pregnant/not pregnant patients and metabolites levels. Correlations between metabolites’ levels and the proportion of stressed patients were evaluated by Pearson’s correlation coefficient. Main results and the role of chance Patients with STAI State punctuations > 60 presented higher mean endometrial cortisol levels (8.01 ng/g (n = 23) vs 5.08 ng/g (n = 35), p-value = 0.05) and lower mean estrone levels (54.97 ng/g (n = 23) vs 56.31 ng/g (n = 35), p-value = 0.049) than those patients with STAI State punctuations < 60. Also, in patients with high endometrial estradiol levels (>2 ng/g) a higher significant proportion of unstressed patients (STAI State < 60) was observed (88.9% vs 52% of unstressed patients in > 2 ng/g and < =2 ng/g estradiol concentrations, respectively) (n = 34, p-value = 0.036). As expected, a significant positive correlation between endometrial cortisol levels and stress (measured by STAI State questionnaire) (cor = 0.977, p-value = 0.023) and a negative correlation between endometrial estradiol levels and stress (cor = -0.9945, p-value = 0.0055) was evidenced. Additionally, a higher proportion of pregnant patients was observed when endometrial estradiol concentrations were high (>1 ng/g) (78.9% vs 50% pregnant in > 1 ng/g vs < =1 ng/g estradiol respectively, p-value = 0.035) and a higher proportion of not pregnant patients was observed when endometrial cortisol concentrations were high ( > =13.9 ng/g) (80% vs 34.4% not pregnant in > =13.9 ng/g and <13.9 ng/g cortisol respectively, p-value = 0.029). Limitations, reasons for caution Although we have employed validated psychological questionnaires and stress biomarkers, a causal relationship between stress and reproductive outcomes should be further explored. Wider implications of the findings This study demonstrated a significant correlation between stress and cortisol and estradiol levels. Since both metabolites’ levels were also related to pregnancy chances, STAI State psychological evaluation could be implemented in the clinical practice as a non-invasive method to prevent endometrial failure through psychological counseling. Trial registration number Not applicable

P-241 The in vivo human uterine pH and uterine fluid composition differs from pH and composition of clinically used in vitro preimplantation embryo culture media

Abstract Study question To study the pH and uterine fluid composition in the human uterus three days after a positive LH test or ovum pick-up. Summary answer The uterine pH three days after a positive LH test or ovum pick-up was 6.8. Concentrations of 37 components were determined in uterine fluid. What is known already In assisted reproductive technologies (ART), the in vitro culture of human preimplantation embryos is known to play a crucial role in the success of In Vitro Fertilization (IVF) treatments. It affects live birth rates and child outcomes such as birth weight. Numerous commercial embryo culture media are routinely used in clinical practice worldwide, but their composition is primarily based on laboratory-based clinical insights and experimental studies with animal models. Studies on the natural human preimplantation embryo environment during the physiologically relevant time of the menstrual cycle are limited. Study design, size, duration A cross sectional clinical study was conducted to study the uterine pH and the composition of human uterine fluid. The uterine pH was measured in 53 women on the third day after LH surge (N = 46) or ovum pickup (N = 7), as that time in the menstrual cycle is associated with endometrial receptivity for embryo implantation. Twenty-two samples of aspirated uterine fluid, that were collected at the same time, were analyzed for the concentrations of thirty-seven components. Participants/materials, setting, methods Healthy women of reproductive age with normal functional uterine anatomy were included. Uterine pH was determined using pH probe inserted through outer sheath of an Embryo Transfer (ET) catheter at the lithotomy position. Uterine fluid was then aspirated using a syringe connected to outer sheath of this ET catheter. Collected samples were analyzed using a Cobas 8000 chemistry analyser and ultra-performance liquid chromatography-tandem mass spectrometry. Main results and the role of chance The mean pH in the uterine cavity on the third day after a positive LH test (6.75 ± 0.22) or ovum pick-up (6.82 ± 0.18) was clearly lower than the pH used in routine clinical practice for preimplantation embryo culture (pH 7.3 ± 0.1). Calcium, phosphate, lactate(L), pyruvate, albumin and 21 amino acids were present at different concentrations in human uterine fluid compared to most commonly used preimplantation embryo culture media. Immunoglobulins, citrulline, ornithine and uric acid were present in uterine fluid, but not in clinically used culture media. Limitations, reasons for caution As the uterine cavity is only one part of the reproductive tract, our measurements do not fully represent the complete in vivo environment of preimplantation embryo development. We only studied 37 components, recognizing that uterine fluid likely contains additional components. Wider implications of the findings The observed differences between the uterine pH and the composition of uterine fluid and the pH and composition of preimplantation embryo culture media used in routine clinical practice could be a starting point to further optimize in vitro embryo culture in ART treatments. Trial registration number Not applicable

Quantification of the Plasma Concentration of Vadadustat by High-Performance Liquid Chromatography with Ultraviolet Detection and Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

An inexpensive, simple, and accurate plasma concentration measurement system is needed to actively conduct pharmacokinetic and pharmacodynamic analyses of vadadustat, hypoxia-inducible factor-prolyl hydroxylase inhibitor, in clinical settings. In this study, the authors aimed to develop a method for measuring vadadustat in human plasma that could be applied for therapeutic drug monitoring using high-performance liquid chromatography with ultraviolet detection (HPLC-UV) in a clinical setting. Plasma samples (100 μL) were pretreated with acetonitrile using butyl paraoxybenzoate as an internal standard. Chromatographic separation was performed on a SunShell PFP C18 column (2.6 μm, 4.6 mm × 150 mm). The mobile phase consisted of (A) 20 mM of phosphate buffer (pH 2.4) and (B) acetonitrile (60:40, v/v), delivered isocratically at a flow rate of 1 mL/min. The analytes were detected by UV absorbance at a wavelength of 220 nm, and the column temperature was 40°C. To evaluate the applicability of HPLC-UV in a clinical setting, blood samples were collected at 19 time points from 7 patients who had been taking vadadustat. The calibration curve was linear over the concentration range of 0.2-150 mcg/mL (R2 > 0.99). Intra-assay and interassay accuracy, precision, and stability met the Food and Drug Administration recommendations. The vadadustat plasma concentrations of patients analyzed using the current HPLC-UV method were almost equal to those measured using ultra-performance liquid chromatography-tandem mass spectrometry (mean difference: 0.13 mcg/mL). Large variability in the dose-adjusted plasma concentrations of vadadustat at 12 hours after administration was observed between patients (coefficient of variation = 57.6%). This HPLC-UV method is a simple, accurate quantification method for evaluating plasma concentrations in patients taking vadadustat in a clinical setting.

Innovative approach for mango allergen identification in processed foods using immunomagnetic nanoparticle-enhanced UPLC-MS/MS

Mango (Mangifera indica) is favored for its flavor and nutritional benefits, yet it also poses significant allergenic risks, potentially triggering severe anaphylactic reactions in sensitive individuals. Addressing the urgent need for accurate allergen detection, this study introduces a novel approach utilizing immunomagnetic nanoparticles (IMNPs) combined with ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to identify the 40-kDa major mango allergen in processed foods. By employing 100 nm IMNPs, the method effectively isolates and concentrates the allergens from complex food matrices, facilitating precise UPLC-MS/MS analysis. Demonstrating exceptional specificity, the technique accurately distinguishes mango allergens from those in nine other fruits and vegetables. It achieves low limits of detection (LODs) of 0.032 μg/mL in juice and 0.165 μg/g in cookies. When tested on 34 commercial products and compared with a real-time PCR method approved by the Taiwan FDA, the method validated its high specificity (100%) and sensitivity (92.9%), based on the ingredient labeling of these products. These advancements mark a significant leap in allergen detection technology, offering a sensitive, specific, and practical solution for identifying mango allergen residues in processed foods, thereby enhancing food safety protocols and protecting public health.

Pharmacokinetics of Anti-rheumatic Drugs Methotrexate and Tofacitinib with its Metabolite M9 in Rats by UPLC-MS/MS.

Tofacitinib is an oral JAK inhibitor for the treatment of rheumatoid arthritis (RA). The clinical efficacy and safety of an administered tofacitinib, either monotherapy or in combination with conventional synthetic disease-modifying anti-rheumatic drugs, mainly methotrexate (MTX), have been evaluated. The high plasma concentration with delayed medicine clearance may affect the liver and/or kidney functions. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC- MS/MS) method for the quantitative analysis of methotrexate, tofacitinib, and metabolite M9 in plasma of Sprague Dawley (SD) rats was developed, and its effectiveness was validated as well. Methotrexate, tofacitinib, M9 and fedratinib (internal standard, IS) were separated by gradient elution. The chromatography was performed on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 μm) column with the mobile phases of acetonitrile and 0.1% formic acid aqueous solution with different proportions at the flow rate of 0.30 mL/min. In the positive ionization mode, the analyzes were detected using a Xevo TQ-S triple quadrupole tandem mass spectrometer, with the following mass transition pairs: m/z 313.12 → 148.97 for tofacitinib, m/z 329.10 → 165.00 for M9 and m/z 455.12 → 308.05 for methotrexate. The obtained results manifested good calibration linearity over the ranges of tofacitinib at 0.1-100 ng/mL, M9 at 0.05-100 ng/mL, and methotrexate at 0.05-100 ng/mL. The lower limit of quantifications (LLOQs) of methotrexate, tofacitinib and M9 were 0.05 ng/mL, 0.1 ng/mL and 0.05 ng/mL, respectively. Intra-day and inter-day accuracy values were confirmed with a range of -6.3% to 12.7%, while intra-day and inter-- day precision values were ≤14.4%. Additionally, recoveries were greater than 86.5% for each compound without significant matrix effects. The currently established analytical method exhibited great potential for the evaluation of plasma concentrations of methotrexate, tofacitinib and M9 simultaneously, greatly reducing the detection time, which would serve as a supplementary role in formulating dose decisions to achieve personalized treatment, identify drugs that cause adverse reactions and finally, to assess drug-drug interactions on clinical studies.

Determination of Choline and B Vitamins in a Market Basket of Eight Types of Plant-Based Milk Alts. Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

Determination of Choline and B Vitamins in a Market Basket of Eight Types of Plant-Based Milk Alts. Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

Antioxidant and Alpha-Glucosidase Inhibitory Activity of Durio zibethinus L. Clone 175 (Durian Udang Merah) Shell and UHPLC-ORBITRAP-MS Profiling of the Extract

Durio zibethinus or ‘durian’ is a well-known seasonal fruit of Southeast Asia and has been called the ‘King of Fruit’. The popularity of durian has led to large production of this crop in Malaysia, consequently creating a huge agricultural waste including the shell. Despite illness remedies from various parts of durian, the information on phytochemical constituents and bioactivities of durian shells remained scarce. Therefore, this study aimed to evaluate the total phenolic content (TPC), antioxidant, and α-glucosidase inhibitory activities of D. zibethinus shell extracted with different ethanol percentage (0%, 50%, and 100%). Results showed that the 100% ethanolic extract exhibited the highest 2,2-dipheny-1-picrylhydrazyl (DPPH) free radical scavenging activity with an IC50 value of 96.91 ± 1.09 µg/mL. Furthermore, the 50% ethanol extract exhibited the highest TPC with 130.57 ± 1.92 mg GAE/g crude extract, and nitric oxide (NO) scavenging activity with an IC50 value of 435.30 ± 3.41 µg/mL. Both 50% and 100% ethanolic extracts of D. zibethinusshell exhibited great potential in α-glucosidase inhibitory activity with IC50 values of 1.99 ± 0.90 and 4.53 ± 0.21 µg/mL, respectively. Thus, the bioactive compounds in 100% ethanolic extract of D. zibethinus were profiled by ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. A total of 20 compounds were tentatively identified, including flavonoids, alkaloids, benzofurans, terpenoids, pentose phosphate, organosulfur compounds, organooxygen compounds, polyketides, carotene, carboxylic acid, coumarin, and stigmasterol. In conclusion, durian shell exhibits potential for future applications driven by its inherent pharmacological benefits, consequently contributing to waste reduction.

TLR3-mediated Astrocyte Responses in High and Normal Glucose Adaptation Differently Regulated by Metformin.

Toll-like receptors 3 (TLR3) are innate immune receptors expressed on a wide range of cell types, including glial cells. Inflammatory responses altered by hyperglycemia highlight the need to explore the molecular underpinnings of these changes in cellular models. Therefore, here we estimated TLR3-mediated response of astrocytes cultured at normal (NG, 5 mM) and high (HG, 22.5 mM) glucose concentrations for 48 h before stimulation with polyinosinic:polycytidylic acid Poly(I:C) (PIC) for 6 h. Seahorse Extracellular Flux Analyzer (Seahorse XFp) was used to estimate the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Although adaptation to HG affected ECAR and OCR, the stimulation of cells with PIC had no effect on ECAR. PIC reduced maximal OCR, but this effect disappeared upon adaptation to HG. PIC-stimulated release of cytokines IL-1β, IL-10 was reduced, and that of IL-6 and iNOS was increased in the HG model. Adaptation to HG reduced PIC-stimulated synthesis of COX-derived oxylipins measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Adaptation to HG did not alter PIC-stimulated p38 activity, ERK mitogen-activated protein kinase, STAT3 and ROS production. Metformin exhibited anti-inflammatory activity, reducing PIC-stimulated synthesis of cytokines and oxylipins. Cell adaptation to high glucose concentration altered the sensitivity of astrocytes to TLR3 receptor activation, and the hypoglycemic drug metformin may exert anti-inflammatory effects under these conditions.

The Extracts from Two Antarctic Fish Species, Trematomus newnesi and Trematomus bernacchii, Enhance JEG-3 Cell Migration and Invasion via MMP9 Activation Through Akt/Protein Phosphatase1/β-Catenin Pathway.

This study investigates the impact of extracts derived from Antarctic fish species, Trematomus newnesi and Trematomus bernacchii, on the migration of human placental trophoblast JEG-3 cells, which is a crucial aspect of successful pregnancy. The extracts, obtained from the muscles of these fish, significantly enhance the migration and invasion of JEG-3 cells in in vitro wound healing, Transwell, and collagen invasion assays. These effects are accompanied by an increase in matrix metalloproteinase (MMP) 9 activity, as demonstrated by zymography. Furthermore, the extracts activated Akt and protein phosphatase 1, resulting in the dephosphorylation of β-catenin at Ser33/37/Thr41, as confirmed by western blot analysis. Consequently, MMP9 is upregulated, while metallopeptidase inhibitor 1/3 is downregulated, as verified by western blot and qRT-PCR analyses. Finally, utilizing ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, followed by matching with the Global Natural Product Social Molecular Networking library, the study annotates the compound responsible for the observed migratory activity as taurocholic acid. Importantly, the study confirms that taurocholic acid enhances cell migration in JEG-3 cells. The results of this study emphasize the potential of Antarctic fish extracts in promoting extravillous trophoblast migration and invasion, which are critical for successful pregnancy.

Ecofriendly UPLC-MS/MS method for simultaneous assay of veterinary drugs residues; erythromycin, sulfadiazine and trimethoprim in edible chicken tissues, evaluation of method greenness and whiteness

Antibiotics are the key veterinary drugs used in animal food production. The uncontrolled widespread use of antibiotics may lead to the accumulation of their residues in foodstuff such as milk, eggs, meat and muscles. An Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method was developed for simultaneous determination of triple combination of erythromycin thiocyanate (ERY), sulfadiazine sodium (SFD) and trimethoprim (TMP) in their dosage form and in different chicken tissues including muscles and liver samples. A Simple extraction and cleaning up procedure was applied using 0.1 N EDTA and methanol to remove the interfering substances and hence increase the efficiency of chromatographic separation. Moreover, separation was performed within 1.2 min on Agilent Poroshell 123EC-C18 column using a mobile phase mixture of methanol: water (90:10, v/v), at a flow rate of 0.5 mL/min using quadrupole tandem mass spectrometer detector. The method was validated according to Food and Drug Administration (FDA) guidelines and linearity was achieved in the range of 2.5–1000 ng/mL for ERY and TMP and in the range of for 5–2000 ng/mL for SFD using tamsulosin (TAM) as an internal standard. All the validation results were within the acceptable ranges. Moreover, Three green metric systems including Green Analytical Procedure Index (GAPI), Analytical Eco-Scale and Analytical GREEnness (AGREE) were used to assess the greenness of the developed method. As well as, the novel blue applicability grade index (BAGI) tool was applied to evaluate the practicality of the method. Results of these tools proved the low environmental impact of the method regarding solvent consumption, analysis time, amount of sample needed, sample treatment procedure, and waste product. The developed method has the privilege of high sensitivity and selectivity as well as short analysis time; hence it can be used to test the presence of the residues of studied drugs in various marketed meat samples to assure human safety and maintain public health. Moreover, it is the first developed method for their analysis in liver samples.

Rapid Determination of Tetracyclines in Drinking and Environmental Waters Using Fully Automatic Solid-Phase Extraction with Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry

The abuse and irrational use of tetracyclines (TCs) in human medicine and animal husbandry has become a serious concern, affecting the ecological environment and human health. The aim of this study was to develop a sensitive and selective method using fully automatic solid-phase extraction coupled with ultra-performance liquid chromatography–tandem mass spectrometry for the determination of twelve TCs in water. Four isotope-labeled internal standards for TCs were used to correct matrix effects. Several parameters affecting extraction efficiency were systematically optimized, and the optimum experimental conditions found were 1.0 L water sample with 0.5 g/L Na2EDTA (pH 3.0) extracted and enriched by CNW HLB cartridge and eluted by 4 mL of acetone:methanol (v/v, 1:1). The enrichment factors were up to 798−1059 but only requiring about 60 min per six samples. Under the optimized conditions, the linearity of the method ranged from 0.2 to 100 μg/L for 12 TCs, the detection limits were as low as 0.01−0.15 ng/L, and the recoveries were in the range of 70%–118%, with relative standard deviations less than 15%. The developed method can be successfully utilized for the determination of 12 TCs in pure water, tap water, river water, and mariculture seawater. In summary, three and six TCs were detected in river water and mariculture seawater, respectively, with total concentrations of 0.074–0.520 ng/L (mean 0.248 ng/L) and 0.792–58.369 ng/L (12.629 ng/L), respectively. Tetracycline (TC) and oxytetracycline (OTC) were the dominant TCs in river water, while doxytetracycline (DXC) and OTC were dominant in mariculture seawater.

Development, validation and application of a UPLC-MS/MS method for simultaneous quantification of OPC-61815 and its metabolites tolvaptan, DM-4103 and DM-4107 in human plasma

OPC-61815 is an intravenous formulation vasopressin antagonist designed to treat heart failure patients, especially who have difficulty in oral intake. Tolvaptan together with DM-4103 and DM-4107 are considered as the major metabolites of OPC-61815 biotransformed in the liver via cytochrome P450 (CYP) 3A. An efficient and robust ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of OPC-61815 and its three metabolites in human plasma was developed and fully validated. To our best knowledge, it was the first published method that simultaneously quantified all of these four analytes in only one run. Simple and rapid sample preparation procedure and very short UPLC-MS/MS run time (3.5 min) offered OPC-61815 and its metabolites relatively high throughput detection, which was greatly beneficial to further clinical bio-sample analysis. The method showed good linearity and sufficient sensitivity in the range of 2.00–1000 ng/mL with a low limit of quantitation (2.00 ng/mL) for each analyte. For samples with concentrations above 1000 ng/mL, 100-fold dilution with blank plasma before sample preparation was accepted. High precision and accuracy, high selectivity and satisfactory recovery of this method were demonstrated. For all of the four analytes, no significant matrix effect or carry-over was observed. The stability of analytes and internal standards under different conditions were evaluated to ensure they were stable during the whole period of storage, preparation and detection. Also, re-injection reproducibility was investigated. In addition, the conversion test showed that almost no OPC-61815 converted into DM-4103 and DM-4107 during sample processing, while attention should be paid to the concentration difference between OPC-61815 and tolvaptan in bioanalysis. The developed UPLC-MS/MS method was successfully applied to an open, single and multiple dose administration phase I trial for monitoring the pharmacokinetics of OPC-61815. This work provided a promising way for further pharmacokinetic study of OPC-61815.

Lipidomic signatures in patients with early-onset and late-onset Preeclampsia

BackgroundPreeclampsia is a pregnancy-specific clinical syndrome and can be subdivided into early-onset preeclampsia (EOPE) and late-onset preeclampsia (LOPE) according to the gestational age of delivery. Patients with preeclampsia have aberrant lipid metabolism. This study aims to compare serum lipid profiles of normal pregnant women with EOPE or LOPE and screening potential biomarkers to diagnose EOPE or LOPE.MethodsTwenty normal pregnant controls (NC), 19 EOPE, and 19 LOPE were recruited in this study. Untargeted lipidomics based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to compare their serum lipid profiles.ResultsThe lipid metabolism profiles significantly differ among the NC, EOPE, and LOPE. Compared to the NC, there were 256 and 275 distinct lipids in the EOPE and LOPE, respectively. Furthermore, there were 42 different lipids between the LOPE and EOPE, of which eight were significantly associated with fetal birth weight and maternal urine protein. The five lipids that both differed in the EOPE and LOPE were DGTS (16:3/16:3), LPC (20:3), LPC (22:6), LPE (22:6), PC (18:5e/4:0), and a combination of them were a potential biomarker for predicting EOPE or LOPE. The receiver operating characteristic analysis revealed that the diagnostic power of the combination for distinguishing the EOPE from the NC and for distinguishing the LOPE from the NC can reach 1.000 and 0.992, respectively. The association between the lipid modules and clinical characteristics of EOPE and LOPE was investigated by the weighted gene co-expression network analysis (WGCNA). The results demonstrated that the main different metabolism pathway between the EOPE and LOPE was enriched in glycerophospholipid metabolism.ConclusionsLipid metabolism disorders may be a potential mechanism of the pathogenesis of preeclampsia. Lipid metabolites have the potential to serve as biomarkers in patients with EOPE or LOPE. Furthermore, lipid metabolites correlate with clinical severity indicators for patients with EOPE and LOPE, including fetal birth weight and maternal urine protein levels.

Traditional Korean diet high in one-carbon nutrients increases global DNA methylation: implication for epigenetic diet.

DNA methylation is a major epigenetic phenomenon through which diet affects health and disease. This study aimed to determine the epigenetic influence of the traditional Korean diet (K-diet) on global DNA methylation via one-carbon metabolism. A crossover study was conducted on 52 women. Two diets, a K-diet, high in plant foods and low in calories and animal fat, and a control diet, similar to the diet currently consumed in Korea, were provided to all subjects alternately for 4weeks with a 4-week washout period. Clinical parameters were measured before and after each dietary intervention. Nutrient intake was calculated by using a computer-aided nutritional analysis program. One-carbon metabolites in the serum and global DNA methylation in peripheral mononuclear cells were determined using ultra-performance liquid chromatography-tandem mass spectrometry. The K-diet group consumed more folate (669.9 ± 6.7µg vs. 502.7 ± 3.0, p < 0.001), B6, B12, serine, and choline, and less methionine (992.6 ± 63 vs. 1048.3mg ± 34.1, p < 0.0001) than the control group did. In the K-diet group, the increment of plasma 5-methyltetrahydrofolate (0.08µg/mL ± 0.11 vs 0.02 ± 0.10, p < 0.009) and decrement of L-homocysteine (-70.7 ± 85.0 vs -39.3 ± 69.4, p < 0.0168) were greater than those of the control group. Global DNA methylation was significantly increased in the K-diet group (6.70 ± 3.02% to 9.45 ± 3.69, p < 0.0001) but not in the control group. A K-diet high in one-carbon nutrients can enhance the global DNA methylation status, suggesting an epigenetic mechanism by which the K-diet conveys health effects. Trial registration Korean Clinical Trial Registry (trial number: KCT0005340, 24/08/2020, retrospectively registered).

Beneficial effects of Bacillus mojavensis strain MTC-8 on plant growth, immunity and disease resistance against Magnaporthe oryzae.

Rice blast, a prevalent and highly destructive rice disease that significantly impacts rice yield, is caused by the rice blast fungus. In the present study, a strain named MTC-8, identified as Bacillus mojavensis, was demonstrated has strong antagonistic activity against the rice blast fungus, Rhizoctonia solani, Ustilaginoidea virens, and Bipolaria maydis. The potential biocontrol agents were identified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis and chromatography. Further investigations elucidated the inhibitory mechanism of the isolated compound and demonstrated its ability to suppress spore germination, alter hyphal morphology, disrupt cell membrane integrity, and induce defense-related gene expression in rice. MTC-8 promoted plant growth and may lead to the development of a biocontrol agent that meets agricultural standards. Overall, the Bacillus mojavensis MTC-8 strain exerted beneficial effects on plant growth, immunity and disease resistance against rice blast fungus. In this study, we isolated and purified a bioactive substance from fermentation broth, and the results provide a foundation for the development and application of biopesticides. Elucidation of the inhibitory mechanism against rice blast fungus provides theoretical support for the identification of molecular targets. The successful development of a biocontrol agent lays the groundwork for its practical application in agriculture.