LC/MS
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This study deals with the comprehensive phytochemical composition and antiviral activity against SARS-CoV-2 of acidic (non-decarboxylated) and neutral (decarboxylated) ethanolic extracts from seven high-cannabidiol (CBD) and two high-Δ9-tetrahydrocannabinol (Δ9-THC) Cannabis sativa L. genotypes. Their secondary metabolite profiles, phytocannabinoid, terpenoid, and phenolic, were determined by LC-UV, GC-MS, and LC-MS/MS analyses, respectively. All three secondary metabolite profiles, cannabinoid, terpenoid, and phenolic, varied significantly among cannabinoid extracts of different genotypes. The dose-response analyses of their antiviral activity against SARS-CoV-2 showed that only the single predominant phytocannabinoids (CBD or THC) of the neutral extracts exhibited antiviral activity (all IC50 < 10.0 μM). The correlation matrix between phytoconstituent levels and antiviral activity revealed that the phenolic acids, salicylic acid and its glucoside, chlorogenic acid, and ferulic acid, and two flavonoids, abietin, and luteolin, in different cannabinoid extracts from high-CBD genotypes are implicated in the genotype-distinct antagonistic effects on the predominant phytocannabinoid. On the other hand, these analyses also suggested that the other phytocannabinoids and the flavonoid orientin can enrich the extract's pharmacological profiles. Thus, further preclinical studies on cannabinoid extract formulations with adjusted non-phytocannabinoid compositions are warranted to develop supplementary antiviral treatments.
The Global Alliance for Genomics and Health (GA4GH) Phenopacket Schema was released in 2022 and approved by ISO as a standard for sharing clinical and genomic information about an individual, including phenotypic descriptions, numerical measurements, genetic information, diagnoses, and treatments. A phenopacket can be used as an input file for software that supports phenotype-driven genomic diagnostics and for algorithms that facilitate patient classification and stratification for identifying new diseases and treatments. There has been a great need for a collection of phenopackets to test software pipelines and algorithms. Here, we present Phenopacket Store. Phenopacket Store v.0.1.19 includes 6,668 phenopackets representing 475 Mendelian and chromosomal diseases associated with 423 genes and 3,834 unique pathogenic alleles curated from 959 different publications. This represents the first large-scale collection of case-level, standardized phenotypic information derived from case reports in the literature with detailed descriptions of the clinical data and will be useful for many purposes, including the development and testing of software for prioritizing genes and diseases in diagnostic genomics, machine learning analysis of clinical phenotype data, patient stratification, and genotype-phenotype correlations. This corpus also provides best-practice examples for curating literature-derived data using the GA4GH Phenopacket Schema.
- MeSH
- algoritmy MeSH
- databáze genetické MeSH
- fenotyp * MeSH
- genomika * metody MeSH
- lidé MeSH
- software * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
UNLABELLED: Methotrexate is used to manage moderate to severe psoriasis and psoriatic arthritis. Methotrexate acts by inhibiting the enzymes involved in nucleotide synthesis. Methotrexate polyglutamates (MTXPGs) have a higher potency to inhibit Dihydrofolate reductase (DHFR), 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (ATIC), and thymidylate synthase (TS), compared to naïve methotrexate. Among all the MTXPGs, methotrexate polyglutamate three (MTXPG-3) is a more potent inhibitor of DHFR, ATIC, and TS enzymes. MTXPG-3 is anticipated to allow therapeutic drug monitoring in immune-mediated inflammatory diseases. We aim to study MTXPG-3 levels as a biomarker for both efficacy and adverse events among psoriatic patients treated with methotrexate monotherapy. We used the LC-MS/MS (Liquid Chromatography Mass Spectrophotometry) system for measuring erythrocyte MTXPG-3. We recruited 106 patients with psoriasis who were treated with methotrexate. Sixty-one of them had psoriatic arthritis (concomitant or in the past). The mean age was 45.08 ± 13.04 years. After twenty-four weeks of methotrexate treatment, 73(69%) were responders, and 33(31%) were non-responders. Thirty-nine (36%) experienced adverse effects, and 67(64%) did not experience any adverse effects. We observed a significant positive correlation between erythrocyte MTXPG-3 and methotrexate dose per week at weeks 12 and 16 but not at week 24. Erythrocyte MTXPG-3 did not correlate with response or adverse effects. It can be used as a marker of compliance. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12291-024-01269-x.
- Publikační typ
- časopisecké články MeSH
Multidimensional chromatography coupled to tandem mass spectrometry (MS/MS), including simple sample preparation with protein precipitation, anion conversion with ammonium hydroxide, and solid-phase extraction using mixed-mode anion exchange in a 96-well plate format, has been validated for rapid simultaneous analysis of human insulin and its six analogs (lispro, glulisine, glargine, degludec, detemir, and aspart) in human plasma. This method is critical for clinical diagnostics, forensic investigations, and anti-doping efforts due to the widespread use of these substances. In the present study, improved chromatographic resolution was achieved using a first-dimension trap-and-elute configuration with an XBridge C18 (2.1 × 20 mm, 3.5 μm) trap column combined with second dimension separation on a Cortecs Ultra-High-Performance Liquid Chromatography (UHPLC) C18+ (2.1 × 100 mm, 1.6 μm) analytical column implemented within a two-dimensional-LC-MS/MS system. The total chromatographic run time was 11 min. This setup increases both the resolution and sensitivity of the method. A mobile phase consisting of 0.8% formic acid (FA) in water and 0.7% FA in acetonitrile was used for gradient elution. Bovine insulin was used as the internal standard. MS detection was performed in positive electrospray ionization mode, and the ion suppression due to matrix effects was evaluated. Validation criteria included linearity, precision, accuracy, recovery, lower limit of quantitation, matrix effect, and stability tests with and without protease inhibitor cocktail under different conditions (short-term stability, long-term stability, and freeze-thaw stability). The concentration range for all insulins was 50-15 000 pg/mL, with limits of quantification below the therapeutic reference range for all analytes. Intra-run precision ranged from 1.1% to 5.7%, inter-run precision from 0.7% to 5.9%, and overall recovery from 96.9% to 114.3%. The validated method has been implemented successfully by the Department of Forensic Medicine at our hospital for the investigation of unexplained deaths.
Epilepsy, affecting over 50 million people globally, presents a significant neurological challenge. Effective prevention of epileptic seizures relies on proper administration and monitoring of Anti-Seizure Medication (ASMs). Therapeutic Drug Monitoring (TDM) ensures optimal dosage adjustment, minimizing adverse effects and potential drug interactions. While traditional venous blood collection for TDM may be stressful, emerging alternative sampling methods, particularly Dried Blood Spot (DBS) or oral fluid offer less invasive way of sampling. This study aimed to develop and validate an analytical method for the determination of lamotrigine in such alternative samples. The sample, either DBS or oral fluid, was subjected to extraction, evaporation, and reconstitution in 15 % acetonitrile containing 0.1 % formic acid. A Kinetex C18 Polar column was used for liquid chromatographic separation and MS in ESI+ mode was used for detection and quantitation of lamotrigine using an isotopically labelled internal standard according to EMA guidelines. The calibration range of the developed method enables the determination of lamotrigine in the concentration range of 1-30 μg/mL in DBS and 0.5-20 μg/mL in oral fluid. Oral fluid and DBS samples from patients treated with lamotrigine analysed by the developed method were compared to plasma concentrations measured by the hospital's accredited laboratory. Preliminary results indicate a promising potential for these alternative matrices in clinical TDM applications. By offering a less invasive sampling approach, this method improves the accessibility and safety of pharmacotherapy for epilepsy patients. The results of this study lay the foundation for further clinical applications by implementing alternative matrix TDM, which may significantly advance personalized care in epilepsy management.
- MeSH
- antikonvulziva * analýza krev MeSH
- chromatografie kapalinová metody MeSH
- epilepsie farmakoterapie MeSH
- kalibrace MeSH
- kapalinová chromatografie-hmotnostní spektrometrie MeSH
- lamotrigin * analýza krev MeSH
- lidé MeSH
- limita detekce MeSH
- monitorování léčiv * metody MeSH
- reprodukovatelnost výsledků MeSH
- sliny * chemie MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- test suché kapky krve * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
Cholestatic liver diseases are characterized by intrahepatic accumulation of bile acids (BAs), exacerbating liver inflammation, and fibrosis. Dimethyl fumarate (DMF) is a clinically approved anti-inflammatory drug that demonstrated protective effects in several experimental models of liver injury. Still, its effect on BA homeostasis and liver fibrosis has not been thoroughly studied. Herein, we hypothesized that DMF could improve BA homeostasis and mitigate the progression of cholestasis-induced liver fibrosis. The DMF was administered to mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced cholestasis for 4 wk. The content of individual BAs in the plasma, liver, bile, intestine, and feces was measured using the LC-MS method alongside the analysis of liver phenotype and related executive and regulatory pathways. The DMF slowed down the progression of DDC-induced liver fibrosis by suppressing hepatic stellate cell and macrophage activation and by reducing c-Jun N-terminal kinase phosphorylation. Notably, DMF reduced BA cumulation in the plasma and liver of cholestatic mice by increasing BA fecal excretion via their reduced Bacteroidetes phyla-mediated deconjugation in the intestine. In addition, DMF was identified as the antagonist of the mouse farnesoid X receptor in enterocytes. In conclusion, DMF alleviates DDC-induced cholestatic liver injury through pleiotropic action leading to significant anti-inflammatory and antifibrotic activity of the agent. In addition, DMF mitigates BA retention in the liver and plasma by increasing their fecal excretion in cholestatic mice. These findings suggest that DMF warrants further investigation as a potential therapeutic agent for human chronic fibrosing cholestatic liver disorders.NEW & NOTEWORTHY Chronic cholestatic cholangiopathies present a therapeutic challenge due to their complex pathophysiology, where the accumulation of bile acids plays a crucial role. In this study, we found that dimethyl fumarate attenuated cholestatic liver damage in a murine model through its significant anti-inflammatory and antifibrotic activity supported by reduced bile acid accumulation in the plasma and liver via their increased fecal excretion.
- MeSH
- cholestáza * farmakoterapie metabolismus chemicky indukované MeSH
- dimethyl fumarát * farmakologie terapeutické užití MeSH
- jaterní cirhóza * metabolismus farmakoterapie patologie etiologie MeSH
- jaterní hvězdicovité buňky účinky léků metabolismus MeSH
- játra * metabolismus účinky léků patologie MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- žlučové kyseliny a soli * metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The semi-synthetic cannabinoid hexahydrocannabinol (HHC) has become a highly discussed topic in forensic toxicology since 2022 due to its legal availability at this time and its psychoactive effects. This study aimed to investigate the pharmacokinetics, effects, and immunological detectability of HHC after oral (25 mg HHC fruit gum) and inhalative (three puffs from HHC vape) consumption with three participants per group. Serum (up to 48 h), urine (up to five days), and saliva (up to 48 h) samples were collected at different relevant time points and analyzed by HPLC-MS/MS for (9R)/(9S)-HHC, 11-hydroxy-HHC, and (9R)/(9S)-HHC carboxylic acid with a fully validated method. Additionally, immunological detectability was investigated with three different commercially available tests. To address the psychoactive effects, the subjective "high" feeling (scale 0-10) was monitored and different psychophysical tests (e.g. modified Romberg test, walk and turn) were conducted. Overall, the pharmacokinetics and effects of HHC were comparable to tetrahydrocannabinol (THC). However, the route of administration as well as inter-individual factors played a crucial role regarding maximum concentrations, pharmacokinetic profiles, and psychoactive effects.
- MeSH
- agonisté kanabinoidních receptorů farmakokinetika farmakologie MeSH
- aplikace inhalační * MeSH
- aplikace orální * MeSH
- dospělí MeSH
- emoce účinky léků MeSH
- farmakokinetika * MeSH
- imunologické testy MeSH
- kanabinoidy * analýza krev farmakokinetika farmakologie moč MeSH
- kapalinová chromatografie-hmotnostní spektrometrie MeSH
- lidé MeSH
- psychofyziologie * MeSH
- psychotropní léky * analýza krev farmakokinetika farmakologie moč MeSH
- sliny chemie MeSH
- tetrahydrokanabinol farmakokinetika farmakologie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The precise and unambiguous detection and quantification of internal RNA modifications represents a critical step for understanding their physiological functions. The methods of direct RNA sequencing are quickly developing allowing for the precise location of internal RNA marks. This detection is, however, not quantitative and still presents detection limits. One of the biggest remaining challenges in the field is still the detection and quantification of m6A, m6Am, inosine, and m1A modifications of adenosine. The second intriguing and timely question remaining to be addressed is the extent to which individual marks are coregulated or potentially can affect each other. Here, we present a methodological approach to detect and quantify several key mRNA modifications in human total RNA and in mRNA, which is difficult to purify away from contaminating tRNA. We show that the adenosine demethylase FTO primarily targets m6Am marks in noncoding RNAs in HEK293T cells. Surprisingly, we observe little effect of FTO or ALKBH5 depletion on the m6A mRNA levels. Interestingly, the upregulation of ALKBH5 is accompanied by an increase in inosine level in overall mRNA.
- MeSH
- adenosin * analogy a deriváty metabolismus genetika analýza MeSH
- alfa-ketoglutarát-dependentní dioxygenasa, AlkB homolog 5 * metabolismus genetika MeSH
- chromatografie kapalinová metody MeSH
- gen pro FTO * metabolismus genetika MeSH
- HEK293 buňky MeSH
- inosin * metabolismus genetika MeSH
- kapalinová chromatografie-hmotnostní spektrometrie MeSH
- lidé MeSH
- messenger RNA * genetika metabolismus MeSH
- posttranskripční úpravy RNA MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
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
Východiska: Hledání účinných biomarkerů pro včasnou diagnostiku ovariálního karcinomu (ovarian cancer – OC) patří k naléhavým úkolům moderní onkogynekologie. Metabolické profilování pomocí ultra vysokoúčinné kapalinové chromatografie a hmotnostní spektrometrie (ultraigh performance liquid chromatography and mass spectrometry – UHPLC-MS) poskytuje informace o souhrnu všech nízkomolekulárních metabolitů vzorku biologických tekutin pacienta, které odrážejí procesy probíhající v těle. Cílem studie bylo prozkoumat metabolomický profil krevní plazmy a moči pacientek se serózním ovariálním adenokarcinomem pomocí UHPLC-MS. Materiál a metody: K provedení metabolomické analýzy bylo odebráno 60 vzorků krevní plazmy a 60 vzorků moči pacientek s diagnózou serózního karcinomu vaječníků a 20 vzorků zdravých dobrovolníků. Chromatografická separace byla provedena na chromatografu Vanquish Flex UHPLC System (Thermo Scientific, Německo). Analýza hmotnostní spektrometrií byla provedena na Orbitrap Exploris 480 (Thermo Scientific, Německo) vybaveném elektrosprejovým ionizačním zdrojem. Bioinformatická analýza byla provedena pomocí Compound Discoverer Software (Thermo Fisher Scientific, USA), statistická analýza dat byla provedena v programovacím jazyce Python pomocí knihovny SciPy. Výsledky: Pomocí UHPLC-MS bylo v krevní plazmě identifikováno 1 049 metabolitů různých tříd. U pacientek s OC mělo 8 metabolitů významně nižší koncentraci (p < 0,01) ve srovnání se zdravými dárci, zatímco u 19 látek byly zjištěny vyšší hladiny (p < 0,01). Během metabolomického profilování vzorků moči bylo identifikováno 417 metabolitů: 12 látek mělo významně nižší koncentraci ve srovnání se zjevně zdravými jedinci a u 14 látek byly hladiny vyšší (p < 0,01). U pacientek se serózním adenokarcinomem vaječníků byla zjištěna významná změna v metabolomu krevní plazmy a moči, vyjádřená abnormálními koncentracemi lipidů a jejich derivátů, mastných kyselin a jejich derivátů, acylkarnitinů, fosfolipidů, aminokyselin a jejich derivátů, derivátů dusíkatých bází a steroidů. Mezi nejslibnější markery tohoto onemocnění přitom patří kynurenin, kyselina myristová, lysofosfatidylcholin a L-oktanoylkarnitin. Závěr: Odhalené změny v metabolomu se mohou stát základem pro zlepšení přístupů k diagnostice serózního ovariálního adenokarcinomu.
Background: The search for effective biomarkers for ovarian cancer (OC) early diagnosis is an urgent task of modern oncogynecology. Metabolic profiling by ultra-high performance liquid chromatography and mass spectrometry (UHPLC-MS) provides information on the totality of all low molecular weight metabolites of patient’s biological fluids sample, reflecting the processes occurring in the body. The aim of the study was to research blood plasma and urine metabolomic profile of patients with serous ovarian adenocarcinoma by UHPLC-MS. Material and methods: To perform metabolomic analysis, 60 blood plasma samples and 60 urine samples of patients diagnosed with serous ovarian carcinoma and 20 samples of apparently healthy volunteers were taken. Chromatographic separation was performed on a Vanquish Flex UHPLC System chromatograph (Thermo Scientific, Germany). Mass spectrometric analysis was performed on an Orbitrap Exploris 480 (Thermo Scientific, Germany) equipped with an electrospray ionization source. Bioinformatic analysis was performed using Compound Discoverer Software (Thermo Fisher Scientific, USA), statistical data analysis was performed in the Python programming language using the SciPy library. Results: Using UHPLC-MS, 1,049 metabolites of various classes were identified in blood plasma. In patients with OC, 8 metabolites had a significantly lower concentration (P < 0.01) compared with conditionally healthy donors, while the content of 19 compounds, on the contrary, increased (P < 0.01). During the metabolomic profiling of urine samples, 417 metabolites were identified: 12 compounds had a significantly lower concentration compared to apparently healthy individuals, the content of 14 compounds increased (P < 0.01). In patients with ovary serous adenocarcinoma, a significant change in the metabolome of blood plasma and urine was found, expressed in abnormal concentrations of lipids and their derivatives, fatty acids and their derivatives, acylcarnitines, phospholipids, amino acids and their derivatives, derivatives of nitrogenous bases and steroids. At the same time, kynurenine, myristic acid, lysophosphatidylcholine and L-octanoylcarnitine are the most promising markers of this disease. Conclusion: The revealed changes in the metabolome can become the basis for improving approaches to the diagnosis of serous ovarian adenocarcinoma.
