Many small molecules require derivatization to increase their volatility and to be amenable to gas chromatographic (GC) separation. Derivatization is usually time-consuming, and typical batch-wise procedures increase sample variability. Sequential automation of derivatization via robotic liquid handling enables the overlapping of sample preparation and analysis, maximizing time efficiency and minimizing variability. Herein, a protocol for the fully automated, two-stage derivatization of human blood-based samples in line with GC-[Orbitrap] mass spectrometry (MS)-based metabolomics is described. The protocol delivers a sample-to-sample runtime of 31 min, being suitable for better throughput routine metabolomic analysis. Key features • Direct and rapid methoximation on vial followed by silylation of metabolites in various blood matrices. • Measure ~40 samples per 24 h, identifying > 70 metabolites. • Quantitative reproducibility of routinely measured metabolites with coefficients of variation (CVs) < 30%. • Requires a Thermo ScientificTM TriPlusTM RSH (or comparable) autosampler equipped with incubator/agitator, cooled drawer, and automatic tool change (ATC) station equipped with liquid handling tools. Graphical overview Workflow for profiling metabolites in human blood using automated derivatization.

• Publikační typ
• časopisecké články MeSH

Newborn screening for Phenylketonuria enables early detection and timely treatment with a phenylalanine-restricted diet to prevent severe neurological impairment. Although effective and in use for 60 years, screening, diagnostic, and treatment practices still vary widely across countries and centers. To evaluate the Phenylketonuria newborn screening practices internationally, we designed a survey with questions focusing on the laboratory aspect of the screening system. We analyzed 24 completed surveys from 23 countries. Most participants used the same sampling age range of 48-72 h; they used tandem mass spectrometry and commercial non-derivatized kits to measure phenylalanine (Phe), and had non-negative cut-off values (COV) set mostly at 120 μmol/L of Phe. Participants mostly used genetic analysis of blood and detailed amino acid analysis from blood plasma as their confirmatory methods and set the COV for the initiation of dietary therapy at 360 μmol/L of Phe. There were striking differences in practice as well. While most participants reported a 48-72 h range for age at sampling, that range was overall quite diverse Screening COV varied as well. Additional screening parameters, e.g., the phenylalanine/tyrosine ratio were used by some participants to determine the screening result. Some participants included testing for tetrahydrobiopterin deficiency, or galactosemia in their diagnostic process. Results together showed that there is room to select a best practice from the many practices applied. Such a best practice of PKU-NBS parameters and post-screening parameters could then serve as a generally applicable guideline.

• Publikační typ
• časopisecké články MeSH

Biotransformation of steroids by fungi has been raised as a successful, eco-friendly, and cost-effective biotechnological alternative for chemical derivatization. Endophytic fungi live inside vegetal tissues without causing damage to the host plant, making available unique enzymes that carry out uncommon reactions. Moreover, using nanofibrous membranes as support for immobilizing fungal cells is a powerful strategy to improve their performance by enabling the combined action of adsorption and transformation processes, along with increasing the stability of the fungal cell. In the present study, we report the use of polyacrylonitrile nanofibrous membrane (PAN NFM) produced by electrospinning as supporting material for immobilizing the endophytic fungus Penicillium citrinum H7 aiming the biotransformation of progesterone. The PAN@H7 NFM displayed a high progesterone transformation efficiency (above 90%). The investigation of the biotransformation pathway of progesterone allowed the putative structural characterization of its main fungal metabolite by GC-MS analysis. The oxidative potential of P. citrinum H7 was selective for the C-17 position of the steroidal nucleus.

• MeSH
• biotransformace MeSH
• nanovlákna * chemie   MeSH
• progesteron MeSH
• Publikační typ
• časopisecké články MeSH

The pathogenic fungus Aspergillus fumigatus utilizes a cyclic ferrioxamine E (FOXE) siderophore to acquire iron from the host. Biomimetic FOXE analogues were labeled with gallium-68 for molecular imaging with PET. [68Ga]Ga(III)-FOXE analogues were internalized in A. fumigatus cells via Sit1. Uptake of [68Ga]Ga(III)-FOX 2-5, the most structurally alike analogue to FOXE, was high by both A. fumigatus and bacterial Staphylococcus aureus. However, altering the ring size provoked species-specific uptake between these two microbes: ring size shortening by one methylene unit (FOX 2-4) increased uptake by A. fumigatus compared to that by S. aureus, whereas lengthening the ring (FOX 2-6 and 3-5) had the opposite effect. These results were consistent both in vitro and in vivo, including PET imaging in infection models. Overall, this study provided valuable structural insights into the specificity of siderophore uptake and, for the first time, opened up ways for selective targeting and imaging of microbial pathogens by siderophore derivatization.

• MeSH
• Aspergillus fumigatus * metabolismus   chemie   MeSH
• aspergilóza * diagnostické zobrazování   mikrobiologie   MeSH
• biomimetické materiály chemie   metabolismus   MeSH
• cyklické peptidy MeSH
• deferoxamin chemie   MeSH
• druhová specificita MeSH
• myši MeSH
• pozitronová emisní tomografie * metody   MeSH
• radioizotopy galia * chemie   MeSH
• siderofory * chemie   metabolismus   MeSH
• Staphylococcus aureus * metabolismus   MeSH
• železité sloučeniny chemie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Lipidy hrají klíčové role v mnoha biologických procesech. Většina lipidů obsahuje dlouhé alifatické řetězce, jejichž variabilní struktura přispívá k rozmanitým fyzikálně-chemickým vlastnostem a biologickým funkcím těchto molekul. Alifatické řetězce lipidů mohou být rovné nebo rozvětvené, s dvojnými či trojnými vazbami, případně s různými funkčními skupinami. V posledních letech došlo k významnému pokroku ve strukturní analýze lipidů pomocí hmotnostní spektrometrie. Nové metody jsou založeny na využití netradičních fragmentačních technik a nových derivatizačních reakcí. S jejich pomocí lze určovat podrobnou strukturu alifatických řetězců, včetně určení polohy násobných vazeb, funkčních skupin a uspořádání alifatických řetězců v komplexních lipidech. Tento přehledový článek navazuje na dva předchozí, které se zabývaly určováním poloh dvojných vazeb (Chem. Listy 117, 684 (2023), Chem. Listy 117, 747 (2023)). Tento text je zaměřen na určení polohy methylového větvení, kyslíkatých funkčních skupin, karbocyklů a stereospecifické polohy acylového řetězce na glycerolu.

Lipids play key roles in many biological processes. Most lipids contain long aliphatic chains whose variable structure contributes to diverse physicochemical properties and biological functions of these molecules. The aliphatic chains of lipids can be straight or branched, with double or triple bonds, or with various functional groups. In recent years, mass spectrometry has made significant progress in the structural analysis of lipids. New methods are based on unconventional fragmentation techniques and new derivatization reactions. They can be used to determine the detailed structure of aliphatic chains, including the position of multiple bonds, functional groups, and the arrangement of aliphatic chains in complex lipids. This review article is a follow-up to two previous articles that dealt with the determination of double bond positions (Chem. Listy 117, 684 (2023), Chem. Listy 117, 747 (2023)). Here, we focus on determining the position of methyl branching, oxygen-containing functional groups, carbocyclic structures, and the stereospecific position of the acyl chain on glycerol.

Pharmacophores such as hydroxyethylamine (HEA) and phthalimide (PHT) have been identified as potential synthons for the development of compounds against various parasitic infections. In order to further advance our progress, we conducted an experiment utilising a collection of PHT and HEA derivatives through phenotypic screening against a diverse set of protist parasites. This approach led to the identification of a number of compounds that exhibited significant effects on the survival of Entamoeba histolytica, Trypanosoma brucei, and multiple life-cycle stages of Leishmania spp. The Leishmania hits were pursued due to the pressing necessity to expand our repertoire of reliable, cost-effective, and efficient medications for the treatment of leishmaniases. Antileishmanials must possess the essential capability to efficiently penetrate the host cells and their compartments in the disease context, to effectively eliminate the intracellular parasite. Hence, we performed a study to assess the effectiveness of eradicating L. infantum intracellular amastigotes in a model of macrophage infection. Among eleven L. infantum growth inhibitors with low-micromolar potency, PHT-39, which carries a trifluoromethyl substitution, demonstrated the highest efficacy in the intramacrophage assay, with an EC50 of 1.2 +/- 3.2 μM. Cytotoxicity testing of PHT-39 in HepG2 cells indicated a promising selectivity of over 90-fold. A chemogenomic profiling approach was conducted using an orthology-based method to elucidate the mode of action of PHT-39. This genome-wide RNA interference library of T. brucei identified sensitivity determinants for PHT-39, which included a P-type ATPase that is crucial for the uptake of miltefosine and amphotericin, strongly indicating a shared route for cellular entry. Notwithstanding the favourable properties and demonstrated efficacy in the Plasmodium berghei infection model, PHT-39 was unable to eradicate L. major infection in a murine infection model of cutaneous leishmaniasis. Currently, PHT-39 is undergoing derivatization to optimize its pharmacological characteristics.

• MeSH
• amfotericin B terapeutické užití   MeSH
• antiprotozoální látky * farmakologie   terapeutické užití   MeSH
• ftalimidy farmakologie   terapeutické užití   MeSH
• Leishmania infantum * MeSH
• Leishmania * MeSH
• leishmanióza kožní * parazitologie   MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The application of polymer-based drug delivery systems is advantageous for improved pharmacokinetics, controlled drug release, and decreased side effects of therapeutics for inflammatory disease. Herein, we describe the synthesis and characterization of linear N-(2-hydroxypropyl)methacrylamide-based polymer conjugates designed for controlled release of the anti-inflammatory drug dexamethasone through pH-sensitive bonds. The tailored release rates were achieved by modifying DEX with four oxo-acids introducing reactive oxo groups to the DEX derivatives. Refinement of reaction conditions yielded four well-defined polymer conjugates with varied release profiles which were more pronounced at the lower pH in cell lysosomes. In vitro evaluations in murine peritoneal macrophages, human synovial fibroblasts, and human peripheral blood mononuclear cells demonstrated that neither drug derivatization nor polymer conjugation affected cytotoxicity or anti-inflammatory properties. Subsequent in vivo tests using a murine arthritis model validated the superior anti-inflammatory efficacy of the prepared DEX-bearing conjugates with lower release rates. These nanomedicines showed much higher therapeutic activity compared to the faster release systems or DEX itself.

• MeSH
• antiflogistika terapeutické užití   MeSH
• dexamethason MeSH
• doxorubicin chemie   MeSH
• leukocyty mononukleární * MeSH
• lidé MeSH
• myši MeSH
• nanomedicína MeSH
• nosiče léků chemie   MeSH
• polymery chemie   MeSH
• revmatické nemoci * MeSH
• uvolňování léčiv MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Multidrug resistance is the major obstacle to cancer chemotherapy. Modulation of P-glycoprotein and drug combination approaches have been considered important strategies to overcome drug resistance. PURPOSE: Aiming at generating a small library of Amaryllidaceae-type alkaloids to overcome drug resistance, two major alkaloids, isolated from Pancratium maritimum, lycorine (1), and 2α-10bα-dihydroxy-9-O-demethylhomolycorine (2), were derivatized, giving rise to nineteen derivatives (3 - 21). METHODS: The main chemical transformation of lycorine resulted from the cleavage of ring E of the diacetylated lycorine derivative (3) to obtain compounds that have carbamate and amine functions (5 - 16), while acylation of compound 2 provided derivatives 17 - 21. Compounds 1 - 21 were evaluated for their effects on cytotoxicity, and drug resistance reversal, using resistant human ovarian carcinoma cells (HOC/ADR), overexpressing P-glycoprotein (P-gp/ABCB1), as model. RESULTS: Excluding lycorine (1) (IC50 values of 1.2- 2.5 μM), the compounds were not cytotoxic or showed moderate/weak cytotoxicity. Chemo-sensitization assays were performed by studying the in vitro interaction between the compounds and the anticancer drug doxorubicin. Most of the compounds have shown synergistic interactions with doxorubicin. Compounds 5, 6, 9 - 14, bearing both carbamate and aromatic amine moieties, were found to have the highest sensitization rate, reducing the dose of doxorubicin 5-35 times, highlighting their potential to reverse drug resistance in combination chemotherapy. Selected compounds (4 - 6, 9 - 14, and 21), able of re-sensitizing resistant cancer cells, were further evaluated as P-gp inhibitors. Compound 11, which has a para‐methoxy-N-methylbenzylamine moiety, was the strongest inhibitor. In the ATPase assay, compounds 9-11 and 13 behaved as verapamil, suggesting competitive inhibition of P-gp. At the same time, none of these compounds affected P-gp expression at the mRNA or protein level. CONCLUSIONS: This study provided evidence of the potential of Amaryllidaceae alkaloids as lead candidates for the development of MDR reversal agents.

• MeSH
• adenokarcinom * MeSH
• alkaloidy amarylkovitých * farmakologie   MeSH
• alkaloidy * farmakologie   MeSH
• chemorezistence MeSH
• doxorubicin farmakologie   MeSH
• fenantridiny * MeSH
• karbamáty farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• P-glykoprotein metabolismus   MeSH
• P-glykoproteiny metabolismus   MeSH
• protinádorové látky * farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Edibles are the only source of nutrients and energy for humans. However, ingredients of edibles have undergone many physicochemical changes during preparation and storage. Aging, hydrolysis, oxidation, and rancidity are some of the major changes that not only change the native flavor, texture, and taste of food but also destroy the nutritive value and jeopardize public health. The major reasons for the production of harmful metabolites, chemicals, and toxins are poor processing, inappropriate storage, and microbial spoilage, which are lethal to consumers. In addition, the emergence of new pollutants has intensified the need for advanced and rapid food analysis techniques to detect such toxins. The issue with the detection of toxins in food samples is the nonvolatile nature and absence of detectable chromophores; hence, normal conventional techniques need additional derivatization. Mass spectrometry (MS) offers high sensitivity, selectivity, and capability to handle complex mixtures, making it an ideal analytical technique for the identification and quantification of food toxins. Recent technological advancements, such as high-resolution MS and tandem mass spectrometry (MS/MS), have significantly improved sensitivity, enabling the detection of food toxins at ultralow levels. Moreover, the emergence of ambient ionization techniques has facilitated rapid in situ analysis of samples with lower time and resources. Despite numerous advantages, the widespread adoption of MS in routine food safety monitoring faces certain challenges such as instrument cost, complexity, data analysis, and standardization of methods. Nevertheless, the continuous advancements in MS-technology and its integration with complementary techniques hold promising prospects for revolutionizing food safety monitoring. This review discusses the application of MS in detecting various food toxins including mycotoxins, marine biotoxins, and plant-derived toxins. It also explores the implementation of untargeted approaches, such as metabolomics and proteomics, for the discovery of novel and emerging food toxins, enhancing our understanding of potential hazards in the food supply chain.

• MeSH
• analýza potravin MeSH
• lidé MeSH
• mořské toxiny MeSH
• mykotoxiny * MeSH
• referenční standardy MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

meso-Methyl BODIPY photocages stand out for their absorption properties and easy chromophore derivatization. However, their low uncaging efficiencies often hinder applications requiring release of protected substrates in high amounts. In this study, we demonstrate that the sulfonothioated BODIPY group photocleaves a sulfonylthio group from the meso-methyl position with a 10-fold higher quantum yield than the most efficient leaving groups studied to date. Photocleavage, observed in solution and in cells, is accompanied by the spatiotemporally controlled photorelease of H2Sn. For this reason, sulfonothioated BODIPY may be applied in cell signaling, redox homeostasis, and metabolic regulation studies.

• MeSH
• signální transdukce * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH