Liquid Chromatography-tandem Mass Spectrometry Analysis Research Articles

Simultaneous detection of myostatin-targeting monoclonal antibodies in dried blood spots and plasma using liquid chromatography-tandem mass spectrometry with field asymmetric ion mobility spectrometry

Transforming growth factor-β superfamily members, such as myostatin, growth/differentiation factor 11, and activin A, negatively regulate skeletal muscle mass. Inhibitors targeting these cytokines or activin receptor type IIB have the potential to treat muscular diseases and enhance physical performance. However, because of their effects on muscle mass and potential misuse, they are strictly prohibited in sports. Given the high potential for misuse as a doping agent in sports, effective analytical methods for these prohibited antibodies targeting these specific cytokines or their receptor are critically needed. In this study, we aimed to develop and validate a multitarget method to detect the prohibited transforming growth factor-β superfamily-targeting monoclonal antibodies, such as landogrozumab, domagrozumab, and the activin receptor type IIB-targeting antibody, bimagrumab, in human plasma and dried blood spot (DBS) samples using liquid chromatography-tandem mass spectrometry. Antibodies were purified from both the DBS and plasma samples using protein G magnetic beads and field-asymmetric ion mobility spectrometry (FAIMS) to minimize interference, followed by liquid chromatography-tandem mass spectrometry analysis. The validation process included tests for specificity, selectivity, linearity, limit of detection (LOD), limit of identification, precision, recovery, carryover effect, and matrix effect. The LODs for the target antibodies were identical in both DBS and plasma samples at 0.1µg/mL for landogrozumab heavy and light chains, as well as 0.25µg/mL for the domagrozumab light chain and 0.25µg/mL for the bimagrumab heavy chain. However, the heavy chain of domagrozumab exhibited an LOD of 0.5µg/mL in DBS and 1µg/mL in plasma. The analytical method demonstrated strong linearity, with R² values greater than 0.99 for both plasma and DBS, and no carryover effect. Precision (CV%) was below 15% at both middle (1 or 5µg/mL; specific to the heavy chain of domagrozumab in plasma) and high (10µg/mL) concentrations and was less than 20% at the LOD. The selectivity and specificity indicated no interference in the analysis of target mAbs in different blood samples. Recovery was 31.6–49.8% for DBS and 51.4–85.3% for plasma, with no significant matrix effect. This study provides an effective method for doping analysis and novel protein detection.

Towards the non-invasive determination of estradiol levels: development and validation of an LC-MS/MS assay for quantification of salivary estradiol at sub-pg/mL level

BackgroundEstradiol (E2) is a female sex hormone involved in several biological processes. Although E2 levels are commonly measured in blood samples, the use of non-invasive techniques (e.g. determination of salivary E2) would allow for the collection of repeated samples and the inclusion of a greater number of participants. Immunoassay-based techniques to measure salivary E2 failed to accurately mirror the variations observed in the plasmatic concentrations of E2 during the menstrual cycle probably due to the high sensitivity required (in the sub-pg/mL range). Therefore, sensitive and rugged analytical methods for the determination of salivary E2 are required. For this, we developed and validated an analytical methodology for the accurate determination of salivary E2. ResultsThe method is based on chemical derivatization with 1,2-dimethyl-1H-imidazole-5-sulphonyl chloride and liquid chromatography-tandem mass spectrometry analysis by summing highly-specific SRM transitions. This strategy allowed for increasing the sensitivity of the method. The validation of the method showed an accurate and precise quantification of E2 in 1 mL of saliva even at 250 fg/mL (97% accuracy and 15% RSD intra-day, and 104% accuracy and 18% RSD inter-day). In order to evaluate its efficacy, we analysed saliva samples from 5 healthy female volunteers collected during a whole menstrual cycle. Our analyses showed that the variations in the concentration of E2 in the measured samples mirrored those expected during a complete menstrual cycle. Additionally, we validated the suitability of our method for determining salivary E2 levels during pregnancy. SignificanceTo the best of our knowledge, this is the first method that allows to precisely and accurately measuring E2 in saliva samples along the whole menstrual cycle of healthy females. It is also suitable for the determination of estradiol during pregnancy. Its high sensitivity makes this strategy ideal for the evaluation of the role of hormone production in women’s health.

Liquid chromatography-tandem mass spectrometry analysis of nitrogen mustard degradation products after derivatization with benzoylimidazole

Nitrogen mustards are highly toxic compounds and strictly controlled by the Chemical Weapons Convention. When those agents are used, exposure must be confirmed by analyzing biological samples from victims to allow prompt medication and reduction of secondary damage. In this study, a derivatization-liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method to analyze nitrogen mustard degradation products (ethanolamines; EAs) in human urine was developed. Benzoylimidazole (BzI) was used as a derivatization reagent, and two highly hydrophilic EAs (methyldiethanolamine and ethyldiethanolamine) were analyzed by using a reversed-phase column with sufficient retention. For urine samples, after solid-phase extraction with cation exchange cartridge and derivatization, EAs were analyzed at low limits of detection (5 ng/mL). The calibration curves had a wide linear range (50–1000 ng/mL), and the quantitation results were sufficient for screening. Compared with our previous derivatization-LC-MS/MS method using pentafluorobenzoyl chloride as a derivatization reagent and hydrophilic interaction liquid chromatography (HILIC)-MS/MS method, not only the limits of detection but also the operability and throughput were improved. The performance of the newly developed derivatization reagent was also evaluated using density functional theory (DFT) calculations.

Di(2-ethylhexyl) phthalate disrupts circadian rhythm associated with changes in metabolites and cytochrome P450 gene expression in Caenorhabditis elegans

The plasticizer di(2-ethylhexyl) phthalate (DEHP) is a widespread environmental pollutant due to its extensive use. While circadian rhythms are inherent in most living organisms, the detrimental effects of DEHP on circadian rhythm and the underlying mechanisms remain largely unknown. This study investigated the influence of early developmental exposure to DEHP on circadian rhythm and explored the possible relationship between circadian disruption and DEHP metabolism in the model organism Caenorhabditis elegans. We observed that DEHP disrupted circadian rhythm in a dose-dependent fashion. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that DEHP-induced circadian disruption accompanies with altered proportions of DEHP metabolites in C. elegans. RNA sequencing data demonstrated that DEHP-induced circadian rhythm disruption caused differential gene expression. Moreover, DEHP-induced circadian disruption coincided with attenuated inductions of DEHP-induced cytochrome P450 genes, cyp-35A2, cyp-35A3, and cyp-35A4. Notably, cyp-35A2 mRNA exhibited circadian rhythm with entrainment, but DEHP exposure disrupted this rhythm. Our findings suggest that DEHP exposure disrupts circadian rhythm, which is associated with changes in DEHP metabolites and cytochrome P450 gene expression in C. elegans. Given the ubiquitous nature of DEHP pollution and the prevalence of circadian rhythms in living organisms, this study implies a potential negative impact of DEHP on circadian rhythm and DEHP metabolism in organisms.

Caspase-3 interactins with calpain and cathepsin L: implications for protein stability and quality in fish fillets during postmortem storage

The degradation of structural proteins during fish postmortem storage is a critical factor affecting meat quality, leading to economic losses in the seafood industry. The complex interplay between caspase-3 and structural protein stability during fish postmortem storage remains largely unexplored. By treating grass carp fillets with a caspase-3 inhibitor, we establish a model for inhibiting apoptosis, delineating the role of caspase-3 in protein degradation. The effect of caspase-3 on grass carp proteins following postmortem storage and its cascade interaction with calpain and cathepsin L was further investigated. The result demonstrated that the destabilisation of the mitochondrial membrane during apoptosis led to modifications in the B-cell lymphoma (Bcl) family proteins, subsequently triggered the activation of caspase-9/caspase-3. Additionally, caspase-3 reduced the expression of the calpastatin and lysosomal membrane protein 1 (LAMP-1) gene, resulting in increased calpain and cathepsin L activity. SDS-PAGE and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated that caspase-3 inhibitor treatment reduced actin fractures and preserved critical structural proteins. This investigation unveils the molecular mechanisms through which caspase-3 affects protein degradation in fish fillets during storage, providing valuable insights into the postmortem degradation processes.

Quantification of guanidine in environmental samples using benzoin derivatization and LC-MS analysis

The recent discovery of guanidine-dependent riboswitches in many microbes raised interest in the biological function and metabolism of this nitrogen-rich compound. However, very little is known about the concentrations of guanidine in the environment. Several methods have been published for quantifying guanidine and guanidino compounds in human urine and blood, often relying on derivatization followed by fluorescence detection. We adapted this analytical approach using benzoin as the derivatization agent to sensitively and selectively quantify guanidine in environmental samples, thereby facilitating future research on the biological and environmental roles of guanidine. This adapted method was applied to human urine, raw wastewater, and biological growth media as relevant matrices. Our liquid chromatography-tandem mass spectrometry analyses of the derivatized solutions identified a different major derivatization product than previously reported. This product was consistently observed across various substrates (guanidine, methylguanidine, and arginine) and derivatization agents (benzoin and anisoin). We observed a constant background signal, restricting our analyses to a lower limit of quantification of 50 nM. Despite this limitation, our method allowed for the quantification of guanidine concentrations significantly lower than those reported in previous derivatization-based studies.•Selective and sensitive detection of guanidine by LC-MS.•Method development and validation for robust detection of guanidine in environmental samples.•Reduction of sample preparation steps and reduced usage of toxic chemicals compared to previous methods.

Bioaccessibility and antioxidant activity of fermented chrysanthemum rice wine on In vitro simulated digestion

The purpose of this study was to investigate the bioaccessibility of total phenolic (TPC), total flavonoid (TFC), chlorogenic acid and antioxidant activity in fermented chrysanthemum rice wine (FRW) under in vitro gastrointestinal digestion model compared with rice wine (RW). The fermented samples were digested and the bioactive components and antioxidant activity during gastric phase (GP) and intestinal phase (IP) were analyzed. The results showed that TPC, TFC, ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and oxygen radical absorbance capacity (ORAC) decreased during the gastrointestinal digestion stage. TFC was higher in FRW during in vitro gastrointestinal digestion stage and increased to a maximum of 0.483 mg/mL at IP stage. 2.2- azinobis-3- ethyl-benzothiazoline-6 - sulfonic acid (ABTS) reached its maximum value of 91.62% in the late stage of intestinal digestion. The hydroxyl radical scavenging activity (HRSA) increased to 77.94% in the IP-2 phase. In addition, a total of 158 (FRW) and 131 (RW) polyphenols, including flavonoids, isoflavones, phenols and coumarins, were detected and identified by Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) analysis. The biotransformation was carried out between the isomers of phenolic compounds in FRW, where 4,5-dicaffeoylquinic acid might be changed into neochlorogenic acid and chlorogenic acid. Correlation analysis showed that 46.15% of metabolites in FRW were positively correlated with the decrease of DPPH, FRAP and ORAC, while 53.85% were positively correlated with the increase of ABTS and HRSA. The results showed higher bioavailability and antioxidative activity in FRW after in vitro digestion.

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.

Sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC–MS/MS) Analysis of Alkaloid Nitrogen Oxides at Three Different Processing Stages of Binglang

Sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC–MS/MS) Analysis of Alkaloid Nitrogen Oxides at Three Different Processing Stages of Binglang

Prediction of Flavor Potential of Ocimum basilicum L. Side-Stream Phytoconstituents, Using Liquid Chromatography–Tandem Mass Spectrometry Analysis and In Silico Techniques

Although post-distillation side-streams of basil (Ocimum basilicum L.) pose significant economic and environmental challenges, they also bring forth new opportunities in the flavor industry. Thus, the objective of the current study was to assess the phenolic profile of basil side-stream extracts to identify key compounds and to evaluate their taste properties, using liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis, flavor prediction tools and molecular docking. In particular, 52 phytoconstituents, mainly phenolic acids, salvianolic acids, flavonoids and fatty acids derivatives, were elucidated in the side-streams of two different basil varieties (Minimum and Genovese) harvested and distilled in early and late autumn, highlighting the effect of pre-harvest factors on basil’s phenolic fingerprint. Furthermore, the results of tests undertaken using taste prediction tools showed that most of the identified compounds were very likely to taste bitter, while six of them (caffeoylferuloyltartaric acid, isoquercetin, lithospermic acid A, sagerinic acid, salvianolic acids C and F) presented a high bitterant capacity (70–90%). Moreover, according to molecular docking studies, these compounds exhibited a stronger binding affinity to the hTAS2R46 bitter receptor compared to its known agonist, strychnine. This outcome and consequently their bitterness were mainly attributed to interactions with Glu265, Thr180 and/or Trp88 through the formation of direct hydrogen bonds. Therefore, the present results provide insights into the taste profiles of basil side-streams, leading to more sustainable and innovative uses of aromatic herbs residues.

Squaraine-Linked Magnetic Covalent Organic Framework as a Solid-Phase Extraction Absorbent to Determine Trace Phenylpyrazoles.

Phenylpyrazoles are widely used pesticides in the food industry. It is highly desirable to develop efficient pre-treatment and analysis methods to extract and detect phenylpyrazoles in complex food matrices. Herein, the study reports novel squaraine-linked zwitterionic core-shell magnetic covalent organic frameworks (MCOFs), which are found to be excellent pretreatment materials for the detection of trace phenylpyrazoles in samples. By coupling MCOFs to magnetic solid-phase extraction (MSPE) with Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) analysis, the detection of phenylpyrazoles (fipronil, fipronil sulfone, fipronil sulfide, fipronil de-sulfoxide, fipronil desulfinyl, ethiprole, and flufiprole) is achieved and shows good linearity at concentrations of 1-800µg L-1 (R2 ≥ 0.9930). The limit of detection (LOD), limit of quantification (LOQ), and recovery rates are 0.01-0.50µg kg-1, 0.04-1.72µg kg-1, and 70.96-115.32%, respectively. More importantly, this method is successfully applied to determine the phenylpyrazoles in commercial egg, poultry, milk, jujube, cabbage, tea, and rice with a detection rate of ≈0.04%. Therefore, the developed method may contribute to a new strategy for the purification and multi-target extraction of complex food matrices.

Improved skeletal muscle mass and strength through Protamex-mediated hydrolysis of perilla seed cake: Elevated rosmarinic acid levels as a contributing factor

Perilla seed cake (PSC) is a byproduct of oil extraction from perilla seeds. It is rich in proteins and bioactive compounds. PSC was enzymatically hydrolyzed to form PSC hydrolysate (PSCH) to enhance the absorption of PSC, and their effects on muscle health in mice were compared. High performance liquid chromatography-tandem mass spectrometry analysis revealed that PSC contains several polyphenols, including rosmarinic acid (RA), caffeic acid, apigenin, and luteolin. The hydrolysate showed 1.44- and 7.04-fold increases in RA and caffeic acid contents, respectively, compared to those of PSC. The intake of PSC, PSCH, and RA significantly improved muscle mass and exercise performance in mice by upregulating protein synthesis, myogenic differentiation, oxidative muscle fiber formation, fatty acid oxidation, and mitochondrial biogenesis; however, PSCH had better promoting effects than PSC. In conclusion, PSCH improves muscle health through its bioactive compounds (particularly RA), indicating the potential of PSCH and RA in functional foods.

Proteomic analysis revealing mechanisms of κ-carrageenan modulating the gelling properties of heat-induced microbial transglutaminase cross-linked myofibrillar protein gels

The effects of κ-carrageenan (KC, 0.2 %, w/w) on the gelling properties and microstructures of heat-treated myofibrillar protein (MP) gels mediated by various levels (0, 0.1, 0.3, and 0.5 %, w/w) of microbial transglutaminase (TG) were investigated. The results revealed that regardless of TG level, the existence of KC significantly promoted the forming of disulphide bonds but limited the generation of large-sized protein polymers in MP gel treated with TG alone, as well as subsequently enhanced the water retention ability and gel strength of mixed MP gel, especially in the sample adding 0.3% TG and 0.2% KC, which was clearly verified by the forming of more denser and continuous gel network. Moreover, TG-induced changes in protein structure led to the alteration in the protein profiles of different MP gels, and an obvious down-regulation trend of myosin (Q9TV63, Q9TV62, P79293, and Q9TV61) was identified in the TG-0.3 group. Furthermore, liquid chromatography–tandem mass spectrometry analysis demonstrated that TG mainly induces protein crosslinking in the light meromyosin domain of the myosin rod. Meanwhile, in the presence of KC, the crosslinking-site percentage of the TG-0.3 group decreased from 37.7% to 36.2%, indicating that KC potentially reduces TG-induced crosslinking in MP gels. Thus, our findings illustrate the critical mechanisms underlying the modulation of KC on the gelling properties of TG-mediated MP gels at the molecular level, which providing potential guideline for the combined applications of TG and KC in the meat industry.

Determination of ten bisphenols and five parabens in urine by solid supported liquid-liquid extraction and liquid chromatography-tandem mass spectrometry

Bisphenols (BPs) and parabens (PBs) are of great concern for environmental pollution and human health because of their endocrine-disrupting effects and potential health hazards. Urinary biomonitoring of BPs and PBs can provide basic data for human internal exposure evaluation, which is a prerequisite for accurately assessing their health risks. In this study, we developed a new pretreatment procedure based on solid supported liquid-liquid extraction (SLE) for the simultaneous separation of ten BPs and five PBs in human urine, followed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. In the instrumental analysis, the HPLC conditions and MS/MS parameters were comprehensively optimized. Accurate qualitative and quantitative determination of ten BPs and five PBs was achieved by introducing a ternary gradient elution system of water, methanol, and acetonitrile for LC separation. During sample pretreatment, the extraction solvent and elution volume were optimized. Specifically, urine samples were held at room temperature and centrifuged at 3000 r/min for 10 min. The supernatant (2 mL) was then transferred to a glass tube, and the pH was adjusted to 5.0 using HCl (0.5 mL; 0.1 mol/L) and NaAc-HAc buffer (1.5 mL). Thereafter, β-glucuronidase-arylsulfatase (20 μL) and surrogate standard solutions (10 ng;13C12-BPS,13C12-BPAF,13C6-MeP, and 13C6-BuP) were added, and the mixture was incubated in a shaker bath in the dark at 37 ℃ for 16 h. After incubation, the hydrolyzed sample (4 mL) was loaded onto an SLE cartridge and equilibrated for a minimum of 5 min to ensure the solution was completely absorbed by the packing material. Subsequently, the target chemicals were eluted with a mixed ethyl acetate/n-hexane solution (3∶7, v/v; 15 mL). Separation of the targets was performed on a ZORBAX SB-C18 reversed-phase column (250 mm×4.6 mm, 5 μm) using an acetonitrile-methanol-water system as the mobile phase. The method was verified by spiking mixed urine samples at three levels (1, 5, and 50 μg/L), with the recoveries ranging from 84.3% to 119.8%. Except for bisphenols (BPS), whose matrix effect was calculated as -21.8%, the matrix effects of other analytes were lower than 20%, indicating low matrix interference. The linear ranges of the analytes varied from 0.1-500 μg/L to 1-500 μg/L, with correlation coefficients higher than 0.995. The method limits of quantification for target chemicals ranged from 0.03 to 0.30 μg/L, and the relative standard deviations of intra- and inter-day experiments were 1.4%-8.4% and 5.7%-14.6%, respectively, suggesting high stability and reproducibility. The method was successfully applied to the determination of ten BPs and five PBs in 10 urine samples from a general population. The concentrations of target chemicals in the human urine samples varied. Methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), and bisphenol A (BPA) were detected in all samples, with median mass concentrations of 1.10, 0.60, 0.21, and 0.55 μg/L, respectively. The detection rates of the other chemicals were less than 50%, which may be related to the production and use of specific chemicals, their bioavailability, and biological metabolism in humans.

Exploring the anticancer potential of Cytotoxin 10 from Naja kaouthia venom: Mechanistic insights from breast and lung cancer cell lines

Breast and lung cancers are the leading causes of cancer-related deaths in the world. Although considerable progress has been made in the field of cancer therapy, quest to discover potent, safe and cost-effective alternatives especially from natural sources is being pursued. Snake venom, which is a treasure trove of various peptides and proteins including natural toxins that specifically target tissues and receptors in the envenomated victims. Many such proteins are being explored for their therapeutic potential against various diseases including cancers. Here, we report the mechanism of cytotoxic activity of crude venom and a purified protein, Cytotoxin from the monocled cobra (Naja kaouthia), an elapid snake with neurotoxic venom prominently found in the North-East India. The crude venom showed significant cytotoxicity against breast (MCF-7and MDA-MB-231) and lung (A549, NCI-H522) cancer cell lines. Bioassay-guided fractionation using RP-HPLC showed highest cytotoxic activity in peak P9. Liquid chromatography-tandem mass spectrometry (ESI-LC-MS/MS) analysis was employed and the fraction is identified as Cytotoxin 10 which showed comparable cytotoxicity against the experimental cell lines. Cytotoxin 10 also exhibited apoptosis in MCF-7 and A549 cell lines using AO/EtBr and flow cytometry analysis. Expressions of apoptosis related proteins e.g. Bax, Bcl2, Caspase-7 and PARP were also studied following Cytotoxin 10 treatment in both cell lines. Molecular docking experiments performed to investigate the interactions between Cytotoxin 10 and the apoptotic proteins revealed favourable binding scores compared to their corresponding inhibitors. Interestingly, Cytotoxin 10 inhibited migration and adhesion in a time and dose-dependent manner in both MCF-7 and A549 cells. This is the first report elucidating the mechanism of cytotoxic activity of Cytotoxin 10 purified from Naja kaouthia venom of North-East India origin and could pave the way for development of potential therapeutic strategies against breast and lung cancer.

Characterization of Siderophore Produced by Bacillus velezensis YL2021 and Its Application in Controlling Rice Sheath Blight and Rice Blast.

Bacillus velezensis YL2021 has extensive antimicrobial activities against phytopathogens, and its genome harbors a catechol-type siderophore biosynthesis gene cluster. Here, we describe the characterization of siderophore produced by strain YL2021 and its antimicrobial activity in vitro and in vivo. A few types of siderophores were detected by chrome azurol S plates coupled with Arnow's test, purified and identified by Reversed-phase high-performance liquid chromatography (RP-HPLC). We found that strain YL2021 can produce different antimicrobial compounds under low-iron M9 medium or iron-sufficient LB medium although antimicrobial activities can be easily observed on the two media as described above in vitro. Strain YL2021 can produce at least three catechol-type siderophores in low-iron M9 medium while no siderophore was produced in LB medium. Among them, the main antimicrobial siderophore produced by strain YL2021 was bacillibactin, with m/z of 882, based on the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, which has broad-spectrum antimicrobial activities against Gram-positive, Gram-negative bacteria, the oomycete Phytophthora capsici and phytopathogenic fungi. Moreover, the antifungal activity of siderophore including bacillibactin observed in vitro was correlated with control efficacies against rice sheath blight disease caused by Rhizoctonia solani and rice blast disease caused by Magnaporthe oryzae in vivo. Collectively, the results demonstrate that siderophore including bacillibactin produced by Bacillus velezensis YL2021 is a promising biocontrol agent for application in rice disease control.

Plasma proteomic profiles of patients with HIV infection and coinfection with hepatitis B/C virus undergoing anti‑retroviral therapy.

Chronic liver disease is becoming a leading cause of illness and mortality in patients living with human immunodeficiency virus (HIV; PLWH) undergoing suppressive anti-retroviral therapy. Its primary etiology is coinfection with hepatitis B and C virus (HBV and HCV, respectively). Chronic liver inflammation and fibrosis can potentially lead to the development of hepatocellular carcinoma (HCC). Therefore, monitoring of the disease progression in PLWH is required. The present study aimed to explore plasma protein profiles of PLWH and those coinfected with HBV and HCV using shotgun proteomics. HIV-monoinfected, HIV/HBV-coinfected, HIV/HCV-coinfected and uninfected control individuals were recruited. Patients in the three virus-infected groups had significantly higher levels of liver fibrosis indices (fibrosis-4 score and aspartate aminotransferase to platelet ratio index) compared with the control group. Liquid chromatography-tandem mass spectrometry analysis of plasma samples identified 1,074 proteins that were differentially expressed, where subsequent partial least squares-discriminant analysis model demonstrated clear clustering of proteomes from the four sample groups; 18 proteins that were significantly differentially expressed. Heatmap analysis identified two main groups of proteins, six proteins being upregulated only in the HIV/HBV-coinfection group and 10 proteins downregulated in all three virally infected groups. STITCH 5.0 analysis predicted an interaction network containing two identified proteins in the latter group, specifically ubiquitin interaction motif-containing 1 (UIMC1) and haptoglobin (HP), which are part of the profibrogenic TGF-1β/SMAD, inflammatory TNF and tumor suppressor BRCA1 pathways. Expression levels of UIMC1 and HP were significantly lower in HIV-infected groups compared with those in uninfected controls. Altogether, these proteomics data provide protein expression profiles potentially associated with HIV infection and coinfection with HBV/HCV, which may be applied to predict progression to advanced liver disease or HCC in PLWH.

Extraction and Quantification of Phenolic Compounds in Maize.

Plants accumulate hundreds of thousands of specialized metabolites that participate in their interactions with the environment. Among these compounds, phenolics represent a large class, and they play important physiological roles, such as providing a first barrier against pathogens, cues to pollinators, and radiation protection. Maize is one of the most important crops worldwide for food, animal feed, and biofuels, and it has the potential to accumulate different phenolics in vegetative tissues as well as in seeds. Recent studies have identified a large number of phenolic compounds-with a diversity of chemical decorations-in different maize tissues, but these likely represent just a fraction of the metabolic diversity of maize. In this protocol, we describe a specific method for the extraction and quantification of maize phenolic compounds by ultra-high-pressure liquid chromatography-tandem multiple reaction monitoring mass spectrometry (UHPLC-MRM-MS/MS) analysis. We provide detailed instructions for the extraction of phenolics using acidic methanol, and for the quantification of 33 different compounds in maize stems, including flavonoids, phenolic acids, and lignin precursors.

Quantification of florfenicol and its metabolites in fillets of Nile tilapia: Synthesis of metabolites and validation of an on-line solid-phase extraction-ultra high-performance liquid chromatography-tandem mass spectrometry method

This study concerns the synthesis of the florfenicol (FF) metabolites florfenicol amine (FFA), florfenicol alcohol (FFOH), and monochloroflorfenicol (FFCl), for their subsequent use as reference standards in On-line solid-phase extraction-ultra high-performance liquid chromatography-tandem mass spectrometry (SPE-UHPLC-MS/MS) analysis. The metabolites were characterized using 1H and 13C NMR, as well as HRMS, and their purities were confirmed by quantitative NMR to ensure analytical reliability. Validation of the developed analytical method showed that it presented acceptable performance, with linearity >0.99 for all the target analytes, accuracies within ±10 % of nominal concentrations, and intra- and inter-day precisions within 15 %. Application of this method to fillets from fish that had been treated with florfenicol (dose of 10 mg/kg bw daily) demonstrated its effectiveness in consistently detecting FF and its metabolites throughout the treatment. The results emphasized the utility of the method for enhancing pharmacokinetic and residue depletion research. The ability to precisely monitor the drug and its metabolites in treated fish provides important insights into florfenicol metabolism, laying the groundwork for further comprehensive profiling studies of metabolites in fish tissue.

Efficacy of BRAF/MEK-inhibitor therapy for epithelioid glioblastoma with a novel BRAFV600 mutation

Epithelioid glioblastoma (eGB), a very aggressive and rare brain tumour, is associated with a dismal median overall survival. Effective therapies for patients with eGB, particularly with leptomeningeal dissemination, are still lacking. Here, we describe a case of a 25-year-old male diagnosed with an intramedullary cervical tumour with subsequent leptomeningeal disease. Histopathology identified a highly necrotising, epithelioid-type tumour with high cell density, most compatible with the diagnosis of an eGB. DNA analysis revealed an unprecedented B-Raf protooncogene, serine/threonine kinase (BRAF) gene variant in exon 15 (ENST00000288602.6, c.1799_1810delinsATG, p.(V600_W604delinsDG)), triggering activation of the mitogen-activated protein kinase (MAPK) pathway. Consequently, we initiated MAPK inhibitor (MAPKi) therapy, utilizing a combination of BRAF and mitogen-activated protein kinase kinase (MEK) inhibitors. Liquid chromatography–tandem mass spectrometry analysis confirmed the drugs’ presence in the patient’s cerebrospinal fluid, indicating their capacity to cross the blood-brain barrier. Remarkably, the patient responded very well to therapy and transitioned from a near-comatose state to significantly improved health, sustained for over three months. This study highlights that MAPKi, particularly targeted towards novel BRAFV600 mutations, might offer promising advancements in eGB treatment strategies.