LC-MS/MS assay
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Antibiotic resistance is one of the biggest threats to global health. Fungal endophytes are important sources of active natural products with antimicrobial potential. The purpose of this study was to characterize the endophytes coexisting with Helichrysum oocephalum, evaluate their antimicrobial activities, and annotate the endophytes metabolites. Six fungal species, including Fusarium avenaceum and Fusarium tricinctum, were identified. Endophytes were cultured, and their metabolites were extracted. The antimicrobial effects of the extracts were tested against Staphylococcus aureus, Bacillus cereus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans. In addition, anti-biofilm effects of the extracts were examined against P. aeruginosa and S. epidermidis. The metabolites in the most active extract were annotated on the basis of the LC-ESI-QToF-MS/MS data. In anti-biofilm studies, F. avenaceum extract was effective in destroying and inhibiting the biofilm formation of S. epidermidis. LC-MS analysis showed that most of the identified compounds in the active extracts were enniatins (cyclic hexadepsipeptides). However, apicidin derivatives were also annotated. Our results revealed that these endophytes, especially Fusarium species, have antimicrobial activity against S. aureus, B. cereus, and C. albicans and anti-biofilm activity against S. epidermidis. According to the literature, the observed antimicrobial activity can be attributed to the enniatins. However, further phytochemical and pharmacological studies are necessary in this regard.
- MeSH
- antibakteriální látky * farmakologie izolace a purifikace chemie MeSH
- antifungální látky * farmakologie izolace a purifikace chemie MeSH
- antiinfekční látky * farmakologie izolace a purifikace chemie MeSH
- Bacillus cereus účinky léků MeSH
- biofilmy účinky léků MeSH
- Candida albicans účinky léků MeSH
- endofyty * chemie metabolismus izolace a purifikace MeSH
- Escherichia coli účinky léků MeSH
- Fusarium * chemie metabolismus MeSH
- mikrobiální testy citlivosti MeSH
- Pseudomonas aeruginosa účinky léků MeSH
- Staphylococcus aureus účinky léků MeSH
- Staphylococcus epidermidis účinky léků MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
Background: A hallmark of cancer is the presence of an immunosuppressive tumor microenvironment (TME). Immunosuppressive M2 macrophages (MΦs) in the TME facilitate escape from immune surveillance and promote tumor growth; therefore, TME-induced immunosuppression is a potent immunotherapeutic approach to treating cancer. Methods: Cancer cell-secreted proteins were detected by using liquid chromatography-mass spectrometry (LC-MS). Neutralizing antibodies (nAbs) were used to assess which proteins were involved in MΦs polarization and differentiation. The protein-protein interaction was characterized using co-immunoprecipitation and immunofluorescence assays. Cancer-secreted heat shock protein 70 (Hsp70) protein was quantified using an enzyme-linked immunosorbent assay (ELISA). MΦ polarization and tumor growth were assessed in vivo with subcutaneous LLC-GFP tumor models and toll-like receptor 2 (TLR2) knockout mice; in vitro assessments were conducted using TLR2 knockout and both LLC-GFP and LN227 lentiviral-mediated knockdown (KD) cells. Results: Cancer cells released a secreted form of Hsp70 that acted on MΦ TLR2 to upregulate Mer receptor tyrosine kinase (MerTK) and induce MΦ M2 polarization. Hsp70 nAbs led to a reduction in CD14 expression by 75% in THP-1 cells in response to Gli36 EMD-CM. In addition, neutralizing TLR2 nAbs resulted in a 30% and 50% reduction in CD14 expression on THP-1 cells in response to MiaPaCa-2 and Gli36 exosome/microparticle-depleted conditioned media (EMD-CMs), respectively. Hsp70, TLR2, and MerTK formed a protein complex. Tumor growth and intra-tumor M2 MΦs were significantly reduced upon cancer cell Hsp70 knockdown and in TLR2 knockout mice. Conclusions: Cancer-secreted Hsp70 interacts with TLR2, upregulates MerTK on MΦs, and induces immunosuppressive MΦ M2 polarization. This previously unreported action of secreted Hsp70 suggests that disrupting the Hsp70-TLR2-MerTK interaction could serve as a promising immunotherapeutic approach to mitigate TME immunosuppression in solid cancers.
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Heart failure (HF) is becoming an increasingly prevalent issue, particularly among the elderly population. Lipids are closely associated with cardiovascular disease (CVD) pathology. Lipidomics as a comprehensive profiling tool is showing to be promising in the prediction of events and mortality due to CVD as well as identifying novel biomarkers. MATERIALS AND METHODS: In this study, eicosanoids and lipid profiles were measured in order to predict survival in patients with de novo or acute decompensated HF. Our study included 50 patients (16 females, mean age 73 years and 34 males, mean age 71 years) with de novo or acute decompensated chronic HF with a median follow-up of 7 months. Lipids were semiquantified using targeted lipidomic liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Eicosanoid concentrations were determined using a commercially available sandwich ELISA assay. RESULTS: From 736 lipids and 3 eicosanoids, 39 significant lipids were selected (by using the Mann-Whitney U test after Benjamini-Hochberg correction) with the highest number of representatives belonging to the polyunsaturated (PUFA) phosphatidylcholines (PC). PC 42:10 (p = 1.44 × 10-4) was found to be the most statistically significantly elevated in the surviving group with receiver operating characteristics of AUC = 0.84 (p = 3.24 × 10-7). A multivariate supervised discriminant analysis based on the aforementioned lipid panel enabled the classification of the groups of surviving and non-surviving patients with 90 % accuracy. CONCLUSIONS: In the present study we describe a trend in PUFA esterified in PC that were systematically increased in surviving patients with HF. This trend in low-abundant and rarely identified PUFA PC (mainly very long chain PUFA containing PC such as PC 42:10 or PC 40:9 containing FA 22:6, FA 20:5 and FA 20:4) suggests candidate biomarkers.
- Publikační typ
- časopisecké články MeSH
Cylindrospermopsin, a potent hepatotoxin produced by harmful cyanobacterial blooms, poses environmental and human health concerns. We used a 3D human liver in vitro model based on spheroids of HepG2 cells, in combination with molecular and biochemical assays, automated imaging, targeted LC-MS-based proteomics, and lipidomics, to explore cylindrospermopsin effects on lipid metabolism and the processes implicated in hepatic steatosis. Cylindrospermopsin (1 μM, 48 h) did not significantly affect cell viability but partially reduced albumin secretion. However, it increased neutral lipid accumulation in HepG2 spheroids while decreasing phospholipid levels. Simultaneously, cylindrospermopsin upregulated genes for lipogenesis regulation (SREBF1) and triacylglycerol synthesis (DGAT1/2) and downregulated genes for fatty acid synthesis (ACLY, ACCA, FASN, SCD1). Fatty acid uptake, oxidation, and lipid efflux genes were not significantly affected. Targeted proteomics revealed increased levels of perilipin 2 (adipophilin), a major hepatocyte lipid droplet-associated protein. Lipid profiling quantified 246 lipid species in the spheroids, with 28 significantly enriched and 15 downregulated by cylindrospermopsin. Upregulated species included neutral lipids, sphingolipids (e.g., ceramides and dihexosylceramides), and some glycerophospholipids (phosphatidylethanolamines, phosphatidylserines), while phosphatidylcholines and phosphatidylinositols were mostly reduced. It suggests that cylindrospermopsin exposures might contribute to developing and progressing towards hepatic steatosis or metabolic dysfunction-associated steatotic liver disease (MASLD).
- MeSH
- alkaloidy * farmakologie MeSH
- bakteriální toxiny * metabolismus MeSH
- buněčné sféroidy účinky léků metabolismus MeSH
- buňky Hep G2 MeSH
- homeostáza účinky léků MeSH
- játra * metabolismus účinky léků MeSH
- lidé MeSH
- lipidomika MeSH
- lipogeneze účinky léků MeSH
- metabolismus lipidů * účinky léků MeSH
- proteomika MeSH
- toxiny kmene Cyanobacteria * MeSH
- uracil * analogy a deriváty metabolismus MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Pathogenic heterozygous mutations in the progranulin gene (GRN) are a key cause of frontotemporal dementia (FTD), leading to significantly reduced biofluid concentrations of the progranulin protein (PGRN). This has led to a number of ongoing therapeutic trials aiming to treat this form of FTD by increasing PGRN levels in mutation carriers. However, we currently lack a complete understanding of factors that affect PGRN levels and potential variation in measurement methods. Here, we aimed to address this gap in knowledge by systematically reviewing published literature on biofluid PGRN concentrations. METHODS: Published data including biofluid PGRN concentration, age, sex, diagnosis and GRN mutation were collected for 7071 individuals from 75 publications. The majority of analyses (72%) had focused on plasma PGRN concentrations, with many of these (56%) measured with a single assay type (Adipogen) and so the influence of mutation type, age at onset, sex, and diagnosis were investigated in this subset of the data. RESULTS: We established a plasma PGRN concentration cut-off between pathogenic mutation carriers and non-carriers of 74.8 ng/mL using the Adipogen assay based on 3301 individuals, with a CSF concentration cut-off of 3.43 ng/mL. Plasma PGRN concentration varied by GRN mutation type as well as by clinical diagnosis in those without a GRN mutation. Plasma PGRN concentration was significantly higher in women than men in GRN mutation carriers (p = 0.007) with a trend in non-carriers (p = 0.062), and there was a significant but weak positive correlation with age in both GRN mutation carriers and non-carriers. No significant association was seen with weight or with TMEM106B rs1990622 genotype. However, higher plasma PGRN levels were seen in those with the GRN rs5848 CC genotype in both GRN mutation carriers and non-carriers. CONCLUSIONS: These results further support the usefulness of PGRN concentration for the identification of the large majority of pathogenic mutations in the GRN gene. Furthermore, these results highlight the importance of considering additional factors, such as mutation type, sex and age when interpreting PGRN concentrations. This will be particularly important as we enter the era of trials for progranulin-associated FTD.
- MeSH
- frontotemporální demence * genetika patologie MeSH
- lidé MeSH
- membránové proteiny genetika MeSH
- mezibuněčné signální peptidy a proteiny genetika MeSH
- mutace genetika MeSH
- progranuliny genetika MeSH
- proteiny nervové tkáně genetika MeSH
- virulence MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- systematický přehled MeSH
Saxitoxins (STXs) are potent neurotoxins produced by marine dinoflagellates or freshwater cyanobacteria known to cause acute and eventually fatal human intoxications, which are classified as paralytic shellfish poisonings (PSPs). Rapid analysis of STXs in blood plasma can be used for a timely diagnosis and confirmation of PSPs. We developed a fast and simple method of STX extraction based on plasma sample acidification and precipitation by acetonitrile, followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Our approach provides the results ≤30 min, with a limit of detection of 2.8 ng/mL and a lower limit of quantification of 5.0 ng/mL. Within-run and between-run precision experiments showed good reproducibility with ≤15% values. Standard curves for calibration were linear with correlation coefficients ≥0.98 across the assay calibration range (5-200 ng/mL). In an interlaboratory analytical exercise, the method was found to be 100% accurate in determining the presence or absence of STX in human plasma specimens, with recovery values of 86-99%. This simple method for STX determination in animal or human plasma can quickly and reliably diagnose STX exposures and confirm suspected PSP cases to facilitate patient treatment or expedite necessary public health or security actions.
INTRODUCTION: Helichrysum leucocephalum Boiss. (Asteraceae) is an endemic plant to Iran. No reports have studied the cytotoxicity of the plant. The current study aimed to evaluate the cytotoxicity of H. leucocephalum collected from Fars province (Iran) against MCF-7 and HDF cell lines using HPLC-based activity profiling and to annotate the active constituents by LC-ESIQTOF-MS/MS. METHODS: H. leucocephalum was collected from three locations in Fars province. The dried flowers and leaves were separately extracted by percolation using methanol. The crude extracts were fractionated by liquid-liquid partitioning with dichloromethane (DCM) and aqueous methanol. The cytotoxicity of the fractions was evaluated against MCF-7 and HDF cells by Alamarblue assay. HPLC-based activity profiling was used to track the active constituents. LC-MS dereplication strategy was used for the annotation of the compounds in the active time window. LC-MS data were preprocessed by MZmine 3.3.0 and submitted to multivariate analysis to compare the differences and similarities in the metabolites of the samples. RESULTS: The DCM fractions showed a dose-dependent cytotoxicity against the cancerous cells (IC50s, 9.8-105.1 μg/ml). In general, the metabolites of the flowers and their cytotoxicity were higher than the leaves. LCESIMS/MS analyses revealed that prenylated and geranylated α,β-unsaturated spiroketal phloroglucinols were among the active constituents. CONCLUSION: It can be concluded that H. leucocephalum is a rich source of phloroglucinol derivatives with cytotoxic activities. Further phytochemical analysis is needed to characterize the bioactive components.
- Publikační typ
- časopisecké články MeSH
OBJECTIVE: The laboratory diagnosis of inherited metabolic disorders (IMD) has undergone significant development in recent decades, mainly due to the use of mass spectrometry, which allows rapid multicomponent analysis of a wide range of metabolites. Combined with advanced software tools, the diagnosis becomes more efficient as a benefit for both physicians and patients. METHODS: A hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry assay for determination of urinary purines, pyrimidines, N-acylglycines, N-acetylated amino acids, sugars, sugar alcohols and other diagnostically important biomarkers was developed and validated. Evaluation of the results consisting of utilisation of robust scaling and advanced visualization tools is simple and even suitable for urgent requirements. RESULTS: The developed method, covering 65 biomarkers, provides a comprehensive diagnostic platform for 51 IMD. For most analytes, linearity with R2 > 0.99, intra and inter-day accuracy between 80 and 120 % and precision lower than 20 % were achieved. Diagnostic workflow was evaluated on 47 patients and External Quality Assurance samples involving a total of 24 different IMD. Over seven years, more than 2300 urine samples from patients suspected for IMD have been routinely analysed. CONCLUSIONS: This method offers the advantage of a broad coverage of intermediate metabolites of interest and therefore may be a potential alternative and simplification for clinical laboratories that use multiple methods for screening these markers.
AIM: The objective of this study was to evaluate off-label high-dose ceftazidime population pharmacokinetics in cancer patients with suspected or proven extensively drug-resistant (XDR) Pseudomonas aeruginosa infections and then to compare the achievement of the pharmacokinetic/pharmacodynamic (PK/PD) target after standard and off-label high-dose regimens using population model-based simulations. A further aim was to clinically observe the occurrence of adverse effects during the off-label high-dose ceftazidime treatment. METHODS: In patients treated with off-label high-dose ceftazidime (3 g every 6 h), blood samples were collected and ceftazidime serum levels measured using LC-MS/MS. A pharmacokinetic population model was developed using a nonlinear mixed-effects modelling approach and Monte Carlo simulations were then used to compare standard and high-dose regimens for PK/PD target attainment. RESULTS: A total of 14 cancer patients with serious infection suspected of XDR P. aeruginosa aetiology were eligible for PK analysis. XDR P. aeruginosa was confirmed in 10 patients as the causative pathogen. Population ceftazidime volume of distribution was 13.23 L, while clearance started at the baseline of 1.48 L/h and increased by 0.0076 L/h with each 1 mL/min/1.73 m2 of eGFR. High-dose regimen showed significantly higher probability of target attainment (i.e., 86% vs. 56% at MIC of 32 mg/L). This was translated into a very low mortality rate of 20%. Only one case of reversible neurological impairment was observed. CONCLUSION: We proved the superiority of the ceftazidime off-label high-dose regimen in PK/PD target attainment with very low occurrence of adverse effects. The off-label high-dose regimen should be used to optimize treatment of XDR P. aeruginosa infections.
- MeSH
- antibakteriální látky škodlivé účinky farmakokinetika MeSH
- ceftazidim škodlivé účinky farmakokinetika MeSH
- chromatografie kapalinová MeSH
- lidé MeSH
- metoda Monte Carlo MeSH
- mikrobiální testy citlivosti MeSH
- nádory * komplikace farmakoterapie MeSH
- off-label použití léčivého přípravku MeSH
- pseudomonádové infekce * farmakoterapie MeSH
- Pseudomonas aeruginosa MeSH
- tandemová hmotnostní spektrometrie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Byla zavedena a validována jednoduchá metoda s použitím kapalinové chromatografie a tandemové hmotnostní spektrometrie (LC-MS/MS) pro stanovení kolistinu A a kolistinu B v plazmě. Extrakce proteinů z plazmy byla provedena pomocí 1 ml kazet Oasis HLB a chromatografická separace byla provedena na koloně Arion® Polar C18 (250 × 4,6 mm; 5 mm) při 35 °C. Mobilní fáze se skládala z vody obsahující 0,1 % kyseliny mravenčí a methanolu obsahujícího 0,1 % kyseliny mravenčí v poměru 40 : 60 (v/v), při průtoku 0,8 ml/minutu. Eluent byl detekován v režimu pozitivních iontů pomocí ionizace elektrosprejem s následujícími iontovými přechody m/z: kolistin A 585,55 → 101,05; kolistin B 578,5 → 101,15; a IS 602,4 → 101,1; 120,15; 86,15. Byly provedeny testy krátkodobé stability kolistinu a CMS, a to při pokojové teplotě a 37 °C, kdy se stabilita obou složek se zvyšující se teplotou snižuje. Předložený příspěvek je součástí studie Pharmacokinetics of Colistin in Critically Ill Patients With Extracorporeal Membrane Oxygenation (COL-ECMO2022), v níž budou prezentovány další výsledky.
A simple liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the determination of colistin A and colistin B in human plasma was developed and validated. Plasma extraction was performed using Oasis HLB 1 ml cartridges, analysis was performed using Arion® Polar C18 (250×4,6mm; 5mm) column at 35°C. Mobile phases consisted of water containing 0,1% formic acid and methanol containing 0,1% formic acid (40:60, v/v) delivered at a flow rate of 0,8 ml/minute. Eluent was detected in the positive ion mode using electrospray ionization at the following transitions of mass to charge (m/z): colistin A 585,55 → 101,05; colistin B 578,5 → 101,15; and IS 602,4 → 101,1; 120,15; 86,15. Short-term stability tests of colistin and CMS were performed, at room temperature and 37°C, where the stability of both components decreases with increasing temperature. The presented paper is part of the Pharmacokinetics of Colistin in Critically Ill Patients With Extracorporeal Membrane Oxygenation (COL-ECMO2022) study in which further results will be presented.
- MeSH
- chemické techniky analytické * metody přístrojové vybavení MeSH
- chromatografie kapalinová metody přístrojové vybavení MeSH
- hydrolýza MeSH
- kolistin * krev MeSH
- lidé MeSH
- methansulfonáty krev MeSH
- stabilita proteinů MeSH
- statistika jako téma MeSH
- tandemová hmotnostní spektrometrie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- klinická studie MeSH
- práce podpořená grantem MeSH
