Liquid Chromatography-electrospray Tandem Mass Spectrometry Research Articles

Characterization of the covalent binding of cyanidin-3-glucoside to bovine serum albumin and its inhibition mechanism for advanced nonenzymatic glycosylation reactions.

Nonenzymatic glycosylation of proteins can generate advanced glycosylation end products, which are closely associated with the pathogenesis of certain chronic physiological diseases and aging. In this study, we characterized the covalent binding of cyanidin-3-glucoside (C3G) to bovine serum albumin (BSA) and investigated the mechanism by which this covalent binding inhibits the nonenzymatic glycosylation of BSA. The results indicated that the covalent interaction between C3G and BSA stabilized the protein's secondary structure. Through liquid chromatography-electrospray ionization tandem mass spectrometry analysis, we identified the covalent binding sites of C3G on BSA as lysine, arginine, asparagine, glutamine, and cysteine residues. This covalent interaction significantly suppressed the nonenzymatic glycosylation of BSA, consequently reducing the formation of nonenzymatic glycosylation products. C3G competitively binds to nonenzymatic glycosylation sites (e.g., lysine and arginine) on BSA, thereby impeding the glycosylation process and preventing the misfolding and structural alterations of BSA induced by fructose. Furthermore, the covalent attachment of C3G to BSA preserves the secondary structure of BSA and hinders subsequent nonenzymatic glycosylation events.

Characterization of the covalent binding of cyanidin-3-glucoside to bovine serum albumin and its inhibition mechanism for advanced nonenzymatic glycosylation reactions.

Nonenzymatic glycosylation of proteins can generate advanced glycosylation end products, which are closely associated with the pathogenesis of certain chronic physiological diseases and aging. In this study, we characterized the covalent binding of cyanidin-3-glucoside (C3G) to bovine serum albumin (BSA) and investigated the mechanism by which this covalent binding inhibits the nonenzymatic glycosylation of BSA. The results indicated that the covalent interaction between C3G and BSA stabilized the protein's secondary structure. Through liquid chromatography-electrospray ionization tandem mass spectrometry analysis, we identified the covalent binding sites of C3G on BSA as lysine, arginine, asparagine, glutamine, and cysteine residues. This covalent interaction significantly suppressed the nonenzymatic glycosylation of BSA, consequently reducing the formation of nonenzymatic glycosylation products. C3G competitively binds to nonenzymatic glycosylation sites (e.g., lysine and arginine) on BSA, thereby impeding the glycosylation process and preventing the misfolding and structural alterations of BSA induced by fructose. Furthermore, the covalent attachment of C3G to BSA preserves the secondary structure of BSA and hinders subsequent nonenzymatic glycosylation events.

Quantitative analysis of erythromycin, its major metabolite and clarithromycin in chicken tissues and eggs via QuEChERS extraction coupled with ultrahigh-performance liquid chromatography-tandem mass spectrometry

A simple, rapid and novel method involving ultrahigh-performance liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (UHPLC-ESI–MS/MS) was developed to simultaneously detect erythromycin, its major metabolite and clarithromycin in chicken tissues (muscle, liver and kidney) and eggs (whole egg, albumen and yolk). Samples were extracted using acetonitrile–water (80:20, v/v), and a Cleanert MAS-Q cartridge was used to perform quick, easy, cheap, effective, rugged, and safe (QuEChERS) purification. The average recoveries were 87.78–104.22 %, and the corresponding intraday and interday relative standard deviations were less than 7.10 %. The decision limits and detection capabilities of the chicken tissues and eggs were 2.15–105.21 μg/kg and 2.26–110.42 μg/kg, respectively. For chicken tissues and eggs, the limits of detection and limits of quantification were 0.5 μg/kg and 2.0 μg/kg, respectively. The proposed method was successfully employed to analyse real samples, demonstrating its applicability.

High-Performance Liquid Chromatographic Quantification of the Plant Hormone Abscisic Acid at ppb Levels in Plant Samples after a Single Immunoaffinity Column Cleanup.

High-performance liquid chromatography with ultraviolet detection (HPLC-UV) is a common analysis technique due to its high versatility and simple operation. In the present study, HPLC-UV detection was integrated with immunoaffinity cleanup (IAC) of the sample extracts. The matrix effect was greatly reduced, and the limit of detection was as low as 1 ng/g of free abscisic acid (ABA) in fresh plant tissues. A monoclonal antibody 3F1 (mAb 3F1) was developed to specifically recognize free ABA but not ABA analogues. The mAb 3F1-immobilized immunoaffinity column exhibited a capacity of 850 ng/mL and an elution efficiency of 88.8-105% for standards. The extraction recoveries of the column for ABA ranged from 80.4 to 108.9%. ABA content was detected in various plant samples with IAC-HPLC-UV. The results were verified with ultraperformance liquid chromatography-electrospray tandem mass spectrometry. IAC-HPLC-UV can be a sensitive and cost-efficient method for plant hormone analysis.

Bioguided isolation, identification and bioactivity evaluation of anti-fatigue constituents from Schizophyllum commune

This study aims to clarify the specific anti-fatigue components of Schizophyllum commune (S.commune) and analyze its potential anti-fatigue mechanism. The main anti-fatigue active ingredient of S.commune was locked in n-butanol extract (SPE-n) by activity evaluation. Twelve compounds were identified by high performance liquid chromatography-electrospray tandem mass spectrometry (LC–ESI–MS/MS). The anti-fatigue effect of morusin is the most predominant among these 12 ingredients. The determination of biochemical indices showed that morusin could increase liver glycogen reserves, improve the activity of antioxidant enzymes in liver, and reduce reactive oxygen species (ROS) content in muscle tissue, thereby reducing myocyte damage. Further studies revealed that morusin could reduce the level of oxidative stress by activating Nrf2/HO-1 pathway, thus alleviating the fatigue of mice caused by exhaustive exercise. The current findings provide a theoretical basis for the development of natural anti-fatigue functional food.

Comprehensive profiling of amino acids and derivatives in biological samples: A robust UHPLC-MS/MS method for investigating acute lung injury

The severe respiratory dysfunctions associated with acute lung injury (ALI) and its sequelae have a high morbidity and mortality rate, are multifactorial, and lack a viable treatment. Considering the critical function that amino acids and derivatives play in the genesis of illnesses and the regulation of metabolic processes, monitoring the levels of metabolites associated with amino acids in biological matrices is necessary and interesting to study their pathological mechanisms. Exploring the dynamics of amino acids and derivatives level and searching for biomarkers provides improved clinical ideas for the diagnosis and treatment of ALI. Therefore, we developed an ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS) method that can simultaneously determine the amino acid and derivatives metabolic levels to study amino acid profiles in different biological samples to facilitate clinical research of ALI. In this study, 48 amino acids and derivatives, including neurotransmitters, polyamines, purines, and other types, were quantified simultaneously in a fast, high-throughput, sensitive, and reliable manner within a 15-minute run time without derivatization. No relevant studies have been reported to quantify these 48 amino acid metabolites in three biological samples simultaneously. Satisfactory linearity (R > 0.995), inter-day and intra-day accuracy (85.17–112.67 % and 85.29–111.60 %, respectively), inter-day and intra-day precision (RSD < 13.80 % and RSD < 12.01 %, respectively), matrix effects (81.00 %–118.00 %), recovery (85.09 %–114.65 %) and stability (RSD < 14.72 %) were all demonstrated by the optimized method's successful validation for all analytes. In addition, the suggested method was effectively implemented in plasma, urine, and lung tissue from normal mice and mice with ALI, with the aim of finding potential biomarkers associated with ALI. Potential biomarkers were screened through multivariate statistical analysis and volcanic map analysis, and the changes of markers in ALI were again identified through heat map analysis and correlation analysis with biochemical indicators, which provided ideas and references for subsequent mechanism studies. Here, the technique created in this work offers a quick and dependable way to perform an integrated analysis of amino acids in a variety of biological materials, which can provide research ideas for understanding the physiopathological state of various diseases.

Anti-Cholinergic Effects of the Phenolic Extract from the Astragalus crenatus Plant: A Computational and Network Pharmacology Study.

Investigations into cholinesterase inhibition have received attention from researchers in recent years for the treatment of Alzheimer's disease. Cholinesterase enzymes, namely, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), hold pivotal significance in Alzheimer's disease (AD) treatment. In this study, we utilized the ethanolic extract of Astragalus crenatus followed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to separate and identify at least 21 compounds in the extract. Rosmarinic acid exhibited the highest concentration (96.675 ± 1.3 mg/g extract), succeeded by hesperidin (79.613 ± 1.2 mg/g extract), hesperetin (75.102 ± 1.4 mg/g extract), rutin (68.156 ± 1.6 mg/g extract), chlorogenic acid (67.645 ± 1.5 mg/g extract), fisetin (66.647 ± 2.3 mg/g extract), and hyperoside (63.173 ± 1.5 mg/g extract). A. crenatus extract efficiently inhibited both AChE and BChE activities in a dosage-dependent manner. Molecular docking was employed to scrutinize the anticholinesterase mechanisms of the identified phytocompounds. Notably, a network pharmacology analysis was executed for the most efficacious compound. Based on binding energies, hesperidin emerged as the most potent inhibitor against both AChE and BChE, exhibiting scores of -10.5 Kcal/mol and -9.8 Kcal/mol, respectively. Due to its dual inhibition of AChE and BChE activities, hesperidin from Astragalus crenatus holds promise for the development of novel therapeutics aimed at neurological disorders, particularly AD.

Breast milk concentrations of acetaminophen and diclofenac - unexpectedly high mammary transfer of the general-purpose drug acetaminophen

BackgroundBreastfeeding is considered to be the most effective way of ensuring the health and survival of newborns. However, mammary transfer of drugs administered to mothers to breastfeeding infants remains a pressing concern. Acetaminophen and diclofenac sodium are widely prescribed analgesics for postpartum pain relief, but there have been few recent reports on the mammary transfer of these drugs, despite advances in analytic techniques.MethodsWe conducted a study on 20 postpartum mothers from August 2019–March 2020. Blood and milk samples from participants were analyzed using liquid chromatography-electrospray ionization tandem mass spectrometry within 24 hours after oral administration of acetaminophen and diclofenac sodium. The area under the concentration-time curve (AUC) was calculated from the concentration curve obtained by a naive pooled-data approach.ResultsFor acetaminophen, AUC was 36,053 ng/mL.h and 37,768 ng/mL.h in plasma and breast milk, respectively, with a milk-to-plasma drug concentration ratio of 1.048. For diclofenac, the AUC was 0.227 ng/mL.h and 0.021 ng/mL.h, in plasma and breast milk, respectively, with a milk-to-plasma drug concentration ratio of 0.093.ConclusionsWhile diclofenac sodium showed low mammary transfer, acetaminophen showed a relatively high milk-to-plasma drug concentration ratio. Given recent studies suggesting potential connections between acetaminophen use during pregnancy and risks to developmental prognosis in children, we believe that adequate information regarding the fact that acetaminophen is easily transferred to breast milk should be provided to mothers.

Seizure progression is slowed by enhancing neurosteroid availability in the brain of epileptic rats.

Trilostane is a 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase inhibitor able to produce a manyfold increase in brain levels of various neurosteroids, including allopregnanolone. We previously found that treatment with trilostane can slow down epileptogenesis in the kainic acid (KA) model of temporal lobe epilepsy. It is unknown whether trilostane may have a similar effect on the progression of epilepsy severity, as observed in KA-treated rats. Consequently, we investigated the effects of trilostane (50 mg/kg/day, 1 week) in epileptic rats, given 64 days after KA administration. Seizures were monitored by video-electrocorticographic recordings before and during the treatment with trilostane or vehicle (sesame oil), and neurosteroid levels were measured in serum and cerebral tissue using liquid chromatography-electrospray tandem mass spectrometry after treatment. Pregnenolone sulfate, pregnenolone, progesterone, 5α-dihydroprogesterone, and allopregnanolone peripheral levels were massively increased by trilostane. With the only exception of hippocampal pregnenolone sulfate, the other neurosteroids augmented in both the neocortex and hippocampus. Only pregnanolone levels were not upregulated by trilostane. As expected, a significant increase in the seizure occurrence was observed in rats receiving the vehicle, but not in the trilostane group. This suggests that the increased availability of neurosteroids produced a disease-modifying effect in the brain of epileptic rats.

Abstract A068: Proteomic analysis of keratin 17 positive cells from laser-capture microdissected pancreatic ductal adenocarcinoma

Abstract Introduction. Although patients with pancreatic ductal adenocarcinoma (PDAC) have one of the lowest survival of all carcinomas, there is considerable variation in survival and disease progression, even when accounting for factors such as age and pathologic stage. Our group identified keratin 17 (K17) as a negative prognostic and predictive biomarker in PDAC. Patients with high K17 expression in their tumors have shorter overall survival as well as decreased response to chemotherapeutic agents, including gemcitabine. K17 has also emerged as an established biomarker of the basal molecular subtype of PDAC. In this study, we aim to identify proteins which correlate, directly or indirectly, with K17 expression via a combined approach using immunohistochemistry (IHC) followed by laser capture microdissection (LCM) and mass spectrometry. Methods. We selected 5 PDAC cases of low K17 expression and 5 PDAC cases of high K17 expression from the archival collections from the Stony Brook Cancer Center Biobank. A modified indirect immunoperoxidase approach was performed on sections mounted on LCM-compatible membrane slides. Carcinoma cells that were either K17 positive or negative by IHC were precisely dissected using LMD7 microscope (Leica) from 10-µM thick sections, followed by a modified tissue digestion and protein extraction protocol. Collision-induced fragment mass information and peptide abundance values were obtained by a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS). Spectral cutoffs were analyzed at both &amp;lt;5% and 1% false discovery rates. The human UniProt dataset was used for data alignment. Results. Mass spectrometry yielded over 1600 detected proteins with approximately 50 of which correlated strongly with K17 expression. Our preliminary results show that keratin 5, a known marker of basal-like PDAC, is positively correlated with keratin 17. Genes whose increased expression contribute to the Moffitt classification of basal molecular subtype of PDAC, such as S100A2, keratin 6A and keratin 7, also showed positive correlation with K17 in our study. The remainder of proteins associated with K17 expression include those involved with cell adhesion, cell-extracellular matrix interaction and smooth muscle contraction pathways, with specific types and classes being identified, pending in-depth analysis. Proteins with inverse relationship to K17 expression included those involved in intracellular organelle trafficking and apoptosis pathways. Conclusion. Our findings demonstrate the utility of mass spectrometry-based spatial proteomic analysis of microdissected cell populations from formalin-fixed, paraffin embedded samples. These methods have the potential to uncover complementary biomarkers to K17 that could enhance prognostic accuracy or serve as therapeutic targets and provide mechanistic insight into the mechanisms through which K17 drives tumor aggression. Citation Format: Ji Dong K. Bai, John D. Haley, Natalia Marchenko, Luisa F. Escobar-Hoyos, Kenneth R. Shroyer. Proteomic analysis of keratin 17 positive cells from laser-capture microdissected pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A068.

Association of phthalate exposure with all-cause mortality across renal function status: The U.S. National Health and Nutrition Examination Survey, 2005–2018

BackgroundWide phthalate exposure has been associated with both declines in renal function and an elevated risk of mortality. Whether phthalate-associated risk of premature mortality differs by renal function status remains unclear. MethodsThis study included 9605 adults from the U.S. National Health and Nutrition Examination Survey. Urinary concentrations of 11 phthalate metabolites were assessed using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. According to estimated glomerular filtration rate (eGFR), participants were grouped as having normal or modestly declined renal functions, or chronic kidney disease (CKD). Multivariable Cox regression models estimated all-cause mortality associated with phthalate exposure, overall and by renal function status. ResultsOverall, Mono-n-butyl phthalate (MnBP), Mono-benzyl phthalate (MBzP), Mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and Mono-(2-ethyl-5-carbox-ypentyl) phthalate (MECPP) were associated with an elevated risk of mortality (P-trend across tertile <0.05). Moreover, significant interactions were observed between eGFR and MEHHP, MEOHP, MECPP, DEHP in the whole population (P for interactions <0.05). After stratification by renal function, total Di (2-ethylhexyl) phthalate (DEHP) was additionally found to be associated with mortality risk in the CKD group (HR = 1.12; 95% CI: 1.01, 1.25). Co-exposure to the 11 phthalate metabolites was associated with a higher risk of all-cause mortality in the CKD (HR = 1.47; 95% CI: 1.18, 1.84) and modestly declined renal function group (HR = 1.25; 95% CI: 1.09, 1.44). ConclusionsThe associations between phthalate exposure and risk of all-cause mortality were primarily observed in CKD patients, reinforcing the need for monitoring phthalate exposure in this patient population.

Antioxidant, AChE inhibitory, and anticancer effects of Verbascum thapsus extract.

Verbascum thapsus (VT) is a medicinal plant that is used in folk medicine to treat a variety of ailments. For this study, the biological functions of VT methanol extract were determined in vitro. The plant's methanol extract was created through the maceration process. The phytochemical composition of plant extracts was investigated using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The antioxidant capacity of the extract was determined using the DPPH (2,2-diphenyl-1-picrylhydrazil) and ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) tests and its cytotoxicity was assessed using the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole)) assay on the Caco-2 (human colorectal adenocarcinoma cells), LNCaP (Lymph Node Carcinoma of the Prostate), and HEK293 cell lines (Human embryonic kidney 293 cells) used to model colon, prostate, and non-cancerous cells. VT extract showed low DPPH and ABTS radical scavenging activities compared to standard antioxidants at 30 mg/ml concentration. In addition, it was determined that VT extract inhibited acetylcholinesterase enzyme.

Development and application of a multiple reaction monitoring method for the simultaneous quantification of sodium channels Nav1.1, Nav1.2, and Nav1.6 in solubilized membrane proteins from stable HEK293 cell lines, rodents, and human brain tissues

RationaleNav1.1, 1.2, and 1.6 are transmembrane proteins acting as voltage‐gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development.MethodsUnique peptides for Nav1.1, Nav1.2, and Nav1.6 were selected as quantotropic peptides for each protein and used for their quantification in membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra‐high‐performance liquid chromatography–electrospray ionization tandem mass spectrometry.ResultsNav 1.1, 1.2, and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1 ± 0.2, 6.4 ± 1.2, and 4.0 ± 0.6 fmol/μg membrane protein, respectively. In brains, Nav1.2 showed the highest expression, with approximately three times higher (P &lt; 0.003) in rodents than in humans at 3.05 ± 0.57, with 3.35 ± 0.56 in mouse and rat brains and 1.09 ± 0.27 fmol/μg in human brain. Both Nav1.1 and 1.6 expressions were much lower in the brains, with approximately 40% less expression in human Nav1.1 than rodent Nav1.1 at 0.49 ± 0.1 (mouse), 0.43 ± 0.3 (rat), and 0.28 ± 0.04 (humans); whereas Nav1.6 had approximately 60% less expression in humans than rodents at 0.27 ± 0.09 (mouse), 0.26 ± 0.06 (rat), and 0.11 ± 0.02 (humans) fmol/μg membrane proteins.ConclusionsMultiple reaction monitoring was used to quantify sodium channels Nav1.1, 1.2, and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mice, rats, and humans. We found significant differences in the expression of these channels in mouse, rat, and human brains. Nav expression ranking among the three species was Nav1.2 ≫ Nav1.1 &gt; Nav1.6, with the human brain expressing much lower concentrations overall compared to rodent brain.

Influence of industrial blanching, cutting, and freezing treatments on in vitro gastrointestinal digestion stability of orange (Citrus sinensis L.) and lemon (Citrus limon L.) peel polyphenols.

Citrus peels, which are often discarded as waste in the food-processing industry, are rich sources of polyphenols. The aim of the current study was to investigate the influence of an industrial freezing process along with blanching and cutting pretreatments on the in vitro gastrointestinal digestion stability of orange (Citrus sinensis L.) and lemon (Citrus limon L.) peel polyphenols. The major polyphenols were identified and quantified with ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI-MS/MS) and high performance liquid chromatography-photodiode array detector (HPLC-PDA), respectively. The results revealed that hesperidin and chlorogenic acid were the predominant flavonoid and phenolic acids in orange peels whereas, for lemon peels, eriocitrin and caffeic acid were the dominant flavonoid and phenolic acids, respectively. Blanching pretreatment enhanced the levels of major flavonoids in orange and lemon peels (by 39-82% and 54-146%, respectively) (P < 0.05) after in vitro gastrointestinal digestion. On the other hand, the application of cutting treatment after blanching significantly reduced the levels of flavonoids (23-62%) (P < 0.05); however, there was no statistically significant difference between the phenolic acid levels of cut and uncut citrus peels. Overall, the bioaccessibility of individual flavonoids and phenolic acids from frozen orange peels was generally slightly lower than that of untreated peels (9-34% and 9-49%, respectively). Nevertheless, frozen lemon peels contained higher bioaccessible flavonoids and phenolic acids in comparison with untreated peels (40-172% and 32-98%, respectively). These results suggest that industrial freezing steps could largely preserve the bioaccessibility of polyphenols in orange and lemon peels. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Green Extraction of Hodgsonia heteroclita Oilseed Cake Powder to Obtain Optimal Antioxidants and Health Benefits.

Most biowaste produced by domestic food preparation and food processing has no value, is difficult to manage, and is detrimental to the environment. Oil extraction from Hodgsonia heteroclita seeds produces large amounts of oilseed cake powder (OCP) as biowaste. The extraction of residual phytochemicals using simple and eco-friendly methods can increase the economic utility of OCP. This study optimized the extraction process for Hodgsonia heteroclita OCP using a Box-Behnken design and response surface methodology. The optimized extraction condition was 30 °C for 5 h in 50% (v/v) ethanol, giving a total phenolic content (TPC) of 414.23 mg of gallic acid equivalent/100 g dry weight (DW). Phytochemical profiles of OCP using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ECI-MS/MS) identified 4-hydroxybenzoic acid and ferulic acid as the major compounds. Antioxidant activities and enzyme inhibitory activities toward the major enzymes involved in obesity (lipase), diabetes (α-amylase, α-glucosidase, and dipeptidyl peptidase IV (DPP IV)), Alzheimer's disease (acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-secretase-1 (BACE-1)), hypertension (angiotensin-converting enzyme, ACE), and genotoxicity were also investigated. Results showed that H. heteroclita OCP possessed antioxidant activity and potential inhibitory activities against BACE-1 and ACE, while also being genome-safe. A simple extraction method for H. heteroclita OCP was developed, demonstrating the enhanced value of its phytochemical and health-promoting qualities.

Comparative analysis of Chrysoporthe cubensis exoproteomes and their specificity for saccharification of sugarcane bagasse

The phytopathogenic fungus Chrysoporthe cubensis is a relevant source of lignocellulolytic enzymes. This work aimed to compare the profile of lignocellulose-degrading proteins secreted by C. cubensis grown under semi-solid state fermentation using wheat bran (WB) and sugarcane bagasse (SB). The exoproteomes of the fungus grown in wheat bran (WBE) and sugarcane bagasse (SBE) were qualitative and quantitatively analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Data are available via ProteomeXchange with identifier PXD046075. Label-free proteomic analysis of WBE and SBE showed that the fungus produced a spectrum of carbohydrate-active enzymes (CAZymes) with exclusive characteristics from each extract. While SBE resulted in an enzymatic profile directed towards the depolymerization of cellulose, the enzymes in WBE were more adaptable to the degradation of biomass rich in hemicellulose and other non-lignocellulosic polymers. Saccharification of alkaline pre-treated sugarcane bagasse with SBE promoted glucose release higher than commercial cocktails (8.11 g L−1), while WBE promoted the higher release of xylose (5.71 g L−1). Our results allowed an in-depth knowledge of the complex set of enzymes secreted by C. cubensis responsible for its high lignocellulolytic activity and still provided the identification of promising target proteins for biotechnological applications in the context of biorefinery.

Analyzing antibiotic residues in honey samples using liquid chromatography-tandem mass spectrometry

ABSTRACT This study aimed to present a sensitive, accurate, and precise analytical method for the determination of 32 antibiotics from 5 groups (sulfonamides, macrolides, aminoglycosides, tetracyclines, and quinolones) and some individual antibiotics (lincomycin, griseofulvin, and 5-hydroxy-flunixin) in 63 honey samples collected from Tehran market. In the presented method, the samples were hydrolyzed by 1% HFBA (hepta fluoro butyric acid) in water, purified on Strata XL polymeric reversed-phase cartridges, and finally analyzed by reversed-phase ion-pair liquid chromatography-electrospray ionization tandem mass spectrometry (RP-IP-LC-ESI-MS/MS). Good performance characteristics were gained for recovery, precision, range, and linearity, the limit of detections (LODs), and the limit of quantifications (LOQs). According to the presented results and considering the absence of permissible limits for antibiotics in honey, 74.6% of the tested samples had antibiotic residues more than the LOQ of the method. The results show that the validated method is suitable for simultaneously detecting antibiotic residues in honey.

Investigation of anti-depression effects and potential mechanisms of the ethyl acetate extract of Cynomorium songaricum Rupr. through the integration of in vivo experiments, LC-MS/MS chemical analysis, and a systems biology approach

Background:Cynomorium songaricum Rupr. has long been used as an anti-inflammatory, antidepressant, and anti-aging agent in traditional Chinese medicine in Asia. Its ethyl acetate extract (ECS) has been identified as the main antioxidant component with neuroprotective and estrogen-like effects. However, the potential of ECS in treating depression has not been explored yet.Methods: We identified the primary metabolites in ECS in this study using liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS). Network analysis was used to find the potential targets and pathways associated with the anti-neuroinflammatory depression action of the ECS. In addition, we established a corticosterone (CORT)-induced depression mouse model to assess ECS’s antidepressant effects by monitoring various behavioral changes (e.g., sucrose preference, forced swimming, tail suspension, and open field tests) and biochemical indices of the hippocampus, and validating the network analysis results. Significant pathways underwent verification through western blotting based on network analysis prediction.Results: Our study demonstrates that ECS possesses significant antidepressant activity. The LC-MS/MS analysis of ECS identified 30 main metabolites, including phloridzin, phlorizin, ursolic acid, and naringenin, as well as other flavonoids, terpenoids, and phenolic acids. These metabolites were found to be associated with 64 candidate target proteins related to neuroinflammatory depression from the database, and ten hub proteins were identified through filtration: CXCL8, ICAM1, NOS2, SELP, TNF, IL6, APP, ACHE, MAOA and ADA. Functional enrichment analyses of the candidate targets revealed their primary roles in regulating cytokine production, inflammatory response, cytokine activity, and tumor necrosis factor receptor binding. In vivo, ECS improved hippocampal neuroinflammation in the mouse model. Specifically, ECS reduced the expression of inflammatory factors in the hippocampus, inhibited M1 microglial cell polarization, and alleviated depression through the regulation of the NF-κB-NLRP3 inflammation pathway.Conclusion: Based on experimental and network analysis, this study revealed for the first time that ECS exerted antidepression effect via anti-neuroinflammation. Our research provides valuable information on the use of ECS as an alternative therapeutic approach for depression.

Optimization of pressurized ethanol extraction for efficient recovery of hyperoside and other valuable polar antioxidant-rich extracts from Betula pendula Roth leaves

Silver birch (Betula pendula Roth) leaves, known for their long-standing uses in traditional medicine, are bioactive compound-rich by-products of the birch logging industry, abundantly available throughout the northern hemisphere. Considering the current demands for the sustainable valorization of various plant materials and their processing by-products, this study aimed to optimize pressurized ethanol extraction (PLE-EtOH) parameters in order to produce hyperoside and other valuable polar antioxidant-rich extracts from silver birch leaves. A central composite design along with response surface methodology (CCD-RSM) was used to optimize the temperature and time of PLE-EtOH for yield, total phenolic (TPC) and flavonoid (TFC) content, Trolox equivalent antioxidant capacity (TEAC) measured in oxygen radical absorbance capacity (ORAC), cupric ion reducing antioxidant capacity (CUPRAC), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays, and hyperoside content measured by ultraperformance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI/MS2) analysis. The optimal PLE-EtOH conditions were 86 °C and 39 min, which produced an extract with 32.2% yield, high TPC (97.3 mg gallic acid equivalents/g), TFC (37.3 mg quercetin equivalents/g) and proanthocyanidin (301.2 mg catechin hydrate equivalents/g) content, also strong in vitro antioxidant capacity in ORAC, CUPRAC and ABTS assays (999.3, 297.0 and 392.3 mg Trolox equivalents/g, respectively). Hyperoside (54.1 mg/g), betuloside (18.3 mg/g) and chlorogenic acid (11.5 mg/g) were the prime phenolic compounds in the PLE-EtOH extract. Additionally, a high sun protection factor (37.4 at 1 mg/mL) was determined by in vitro method for this extract. Such products from B. pendula leaves have potential cosmetical, nutraceutical, and pharmaceutical applications.

Functional Characterization of AP2/ERF Transcription Factors during Flower Development and Anthocyanin Biosynthesis Related Candidate Genes in Lycoris.

The APETALA2/ethylene-responsive transcription factor (AP2/ERF) family has been extensively investigated because of its significant involvement in plant development, growth, fruit ripening, metabolism, and plant stress responses. To date, there has been little investigation into how the AP2/ERF genes influence flower formation and anthocyanin biosynthesis in Lycoris. Herein, 80 putative LrAP2/ERF transcription factors (TFs) with complete open reading frames (ORFs) were retrieved from the Lycoris transcriptome sequence data, which could be divided into five subfamilies dependent on their complete protein sequences. Furthermore, our findings demonstrated that genes belonging to the same subfamily had structural similarities and conserved motifs. LrAP2/ERF genes were analyzed for playing an important role in plant growth, water deprivation, and flower formation by means of gene ontology (GO) enrichment analysis. The expression pattern of the LrAP2/ERF genes differed across tissues and might be important for Lycoris growth and flower development. In response to methyl jasmonate (MeJA) exposure and drought stress, the expression of each LrAP2/ERF gene varied across tissues and time. Moreover, a total of 20 anthocyanin components were characterized using ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) analysis, and pelargonidin-3-O-glucoside-5-O-arabinoside was identified as the major anthocyanin aglycone responsible for the coloration of the red petals in Lycoris. In addition, we mapped the relationships between genes and metabolites and found that LrAP2/ERF16 is strongly linked to pelargonidin accumulation in Lycoris petals. These findings provide the basic conceptual groundwork for future research into the molecular underpinnings and regulation mechanisms of AP2/ERF TFs in anthocyanin accumulation and Lycoris floral development.