The profile of secondary metabolites occurring in apple cuticular layer is not only characteristic for particular apple cultivar, but also dynamically reflects various external factors in growing environment. In this study, the possibility to authenticate apple samples based on analysis of their cuticular layer extracts was investigated. Ultra-high-performance liquid chromatography coupled to high resolution tandem mass spectrometry (UHPLC-HRMS/MS) was employed for obtaining metabolomic fingerprints. In total, 274 authentic apple samples, four cultivars harvested within the seasons 2020 to 2022 in the Czech Republic and Poland were analysed. The generated complex data processed by univariate and multivariate statistical methods enabled to build classification models for discrimination of apple cultivars as well as their geographical origin. The models showed very good performance in differentiation of Czech and Polish samples for three of four cultivars: ‘Gala’, ‘Golden Delicious’ and ‘Idared’, moreover, the validity of the models was verified over harvest seasons. The diagnostic markers, besides metabolites of triterpene biosynthetic pathway, mainly represented wax esters. ‘Jonagold’, which is known to be susceptible to mutations, was the only cultivar for which the unambiguous classification of geographical origin was not possible.

Abstract: Peptide pools composed of short amino acid sequences have proven to be versatile tools in various research areas and clinical applications. They are powerful tools for epitope mapping, immunotherapy, and vaccine development. Their importance lies in their ability to map complex protein structures, enabling a comprehensive understanding of immune responses and facilitating the identification of potential therapeutic agents. The application of peptide pools also extends to the field of personalized medicine, offering tailored solutions for diseases such as cancer and infectious diseases. Peptide pools are complex mixtures of immunostimulatory antigens primarily intended for T-cell stimulation. They are commercially available in many different compositions and variants. However, unlike other reagents that consist of only one or a few compounds, peptide pools are highly complex products with limited stability. This makes their quality control a major challenge. Quantitative peptide analysis usually requires sophisticated methods, in most cases isotope-labeled standards and reference materials. None of these are routinely available for these products. Synthesis and purification of all labeled peptides might be required. Usually, this approach would be prohibitively laborious and expensive. Therefore, an approach is needed to provide a practical and feasible method for quality control of peptide pools. With insufficient quality control, the use of such products could lead to incorrect experimental results, which would worsen the well-known reproducibility crisis in the biomedical sciences. Here we propose the use of ultra-high performance liquid chromatography (UHPLC) with two detectors, a standard UV detector (at 214 nm) for quantitative analysis and a high-resolution mass spectrometer (HRMS) for identity confirmation. To be cost-efficient and fast, quantification and identification are performed in one chromatographic run. An optimized protocol is shown, and different peak integration methods are compared and discussed. This work was performed using a peptide pool known as CEF, which consists of 32 peptides derived from cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza virus, ranging from 8 to 12 amino acids in length.

Recently, pharmaceutical research has been focused on the design of new antibacterial drugs with higher selectivity towards several strains. Major issues concern the possibility to obtain com-pounds with fewer side effects, at the same time effectively overcoming the problem of antimi-crobial resistance. Several solutions include the synthesis of new pharmacophores starting from piperazine or morpholine core units. Mass spectrometry-based techniques offer important sup-port for the structural characterization of newly synthesized compounds to design safer and more effective drugs for various medical conditions. Here, two new piperazine derivatives and four new morpholine derivatives were synthesized and structurally characterized through a combined approach of FT-ICR and LTQ mass spectrometry. The support of both high-resolution and low-resolution mass spectrometric data namely accurate mass measurements, isotopic distribu-tion and MSn spectra, was crucial to confirm the success of the synthesis. These compounds were further evaluated for inhibitory activity against a total of twenty-nine Gram-positive and Gram-negative bacteria to determine the action spectrum and the antimicrobial effectiveness. Results demonstrated compounds’ antimicrobial activity against many tested bacterial species, providing an inhibitory effect linked to different chemical structure and suggesting that the new-synthesized derivatives could be considered as promising antimicrobial agents.

Plant species are precursors of a wide variety of secondary metabolites that, besides having useful activity for themselves, can also be used by humans for their consumption and economic benefit. Pepper (Capsicum annuum L.) fruit is not only a common food and spice source, but it also stands out for containing high amounts of antioxidants (such as vitamins C and A), polyphenols and capsaicinoids. Particular attention has been paid to capsaicin, whose anti-inflammatory, antiproliferative and analgesic activities, have been proven. Due to the potential interest in pepper metabolites for human use, in this project, we carried out an investigation to identify new bioactive compounds of this crop is carried out. To achieve this, we developed a metabolomic approach, using an HPLC (high-performance liquid chromatography) separative technique coupled to metabolite identification by high resolution mass spectrometry (HRMS). After chromatographic analysis and data processing against metabolic databases, 12 differential bioactive compounds were identified in sweet pepper fruits, including quercetin and its derivatives, L-tryptophan, phytosphingosin, FAD, gingerglycolipid A, tetrahydropentoxylin, blumenol C glucoside, colnelenic acid and capsoside A. The abundance of these metabolites varied depending on the ripening stage of the fruits, either immature green or ripe red. We also studied the variation of these 12 metabolites upon treatment with exogenous nitric oxide (NO), a free radical gas involved in a good number of physiological processes in higher plants such as germination, growth, flowering, senescence, and fruit ripening, among others. Overall, it was found that the content of the analysed metabolites was modulated by the ripening stage and by the presence of NO. The metabolic pattern followed by quercetin and its derivatives, as a consequence of the ripening stage and NO treatment, was also corroborated by transcriptomic analysis of genes involved in the synthesis of these compounds. This opens new research windows on the pepper fruit’s bioactive compounds with nutraceutical potentiality, where biotechnological strategies can be applied for optimizing the level of these beneficial compounds.

Food authorities aim to safeguard our food. This requires sensitive analyses to guarantee detection of both banned and regulated substances at low concentrations. At the same time, broad screening methods are needed to identify new emerging risks. For this purpose effect-based bioassays combined with mass spectrometric analyses offer an advantage. During the regular monitoring of dioxins in agricultural products, a discrepancy was observed between the results of the DR CALUX (Dioxin-Responsive Chemical Activated Luciferase gene Expression) bioassay and the confirmatory gas chromatographic high resolution mass spectrometric (GC-HRMS) analysis in egg and broiler fat samples. The response in the bioassay was high, suggesting a clear exceedance of the maximum limits of dioxins in these samples, yet regulated dioxins or dl-PCBs were not detected by GC/HRMS analysis . Ultimately, a broad screening analysis using GC-HRMS resulted in the identification of 2,3,7,8-tetrabromo-dibenzofuran (2,3,7,8-TBDF) in both egg and broiler fat. To investigate the potential; source of this brominated furan contaminant, different samples were analysed: bedding material, poultry feed, feed additives (choline chloride and L-lysine) and seaweed. The poultry feed and feed additives all contained 2,3,7,8-TBDF. Using a feed to food converter, it became clear that the poultry feed was probably the source of 2,3,7,8-TBDF in broilers and eggs through a feed additive like L-lysine or choline chloride. This study underlines the importance of using a combination of effect based screening assays with sensitive analytical methods to detect potential new and emerging risks.

N-acyl hydrazone (NAH) has been remarked as a promising scaffold in drug design, given its versatility, ease of synthesis, and appealing biological activities (i.e., antimicrobial, antitumoral, analgesic, and anti-inflammatory properties). In the global context of increasing resistance of pathogenic bacteria to antibiotics, NAHs represent potential solutions for developing improved treatment alternatives. Thus, this study presents 6 new derivatives of (EZ)-N'-benzylidene-(2RS)-2-(6-chloro-9H-carbazol-2-yl)propanohydrazide that were obtained through a microwave-assisted synthesis method. In more detail, we have joined two pharmacophore fragments in a single molecule, represented by an NSAID-type carprofen structure and a hydrazone-type structure, obtaining a new series of NSAID-N-acyl hydrazone derivatives that were further characterized spectrally using FT-IR, NMR, and HRMS investigations. Moreover, the compounds have been evaluated for their tuberculostatic activity, testing their effects on four M. tuberculosis strains (two of them susceptible to rifampicin (RIF) and isoniazid (INH), one susceptible to RIF and resistant to INH and one resistant to both RIF and INH). The results of our research highlight the potential of the prepared compounds in fighting against antibiotic-resistant M. tuberculosis strains.

The main aims of this study were (1) to identify and compare the major biologically active compounds in the metabolome of ligulate and tubular flowers of two C. officinalis cultivars, ‘Golden Sea’ (GS) and ‘Paradise Garden’ (PG), and (2) to investigate the effect of different proportions of ligulate and tubular flowers in the inflorescences on their metabolome. Ultra-performance liquid chromatography with photodiode array detection and a Q Exactive Orbitrap high-resolution mass spectrometer were used to analyse the flower metabolome. Analysis of the metabolomics data revealed that polar lipids showed the most significant differences between flowers. Tubular flowers of PG and GS cultivars contained higher levels of 31 and 34 out of 36 lipids, respectively. Furthermore, tubular flowers of both cultivars contained significantly higher levels of phenolamides and caffeoylquinic acids, but lower levels of triterpenoids. To compare the metabolite content of the two C. officinalis cultivars, we analysed the data for their inflorescences. The study showed that GS inflorescences contained significantly higher levels of phenolic compounds and lipids than PG inflorescences. This GS advantage was due to two factors: firstly, the higher content of these metabolites in tubular flowers and, secondly, the higher proportion of tubular flowers in GS inflorescences.

Streptomyces, being one of the most promising genera due to its ability to synthesize a variety of bioactive secondary metabolites of pharmaceutical interest, here studied in relation to its genomic and metabolomic potential. Coinciding with the increase in sequenced data, mining of bacterial genomes for biosynthetic gene clusters (BGCs) has become a routine component of natural product discovery. Herein, we describe the isolation and characterization of a Streptomyces tendae VITAKN with quorum sensing inhibitory activity (QSI) that was isolated from southern coastal parts of India. The nearly complete genome consists of 8,621,231bp with a GC content of 72.2%. Utilizing the BiG-SCAPE-CORASON platform, a sequence similarity network predicted from this strain was evaluated through sequence similarity analysis with the MIBiG database and existing 3,365 BGCs predicted by antiSMASH analysis of publicly available complete Streptomyces genomes. Crude extract analyzed on LC-HRMS/MS and Global Natural Product Social Molecular Networking (GNPS) online workflow using dereplication resulted in the identification of cyclic dipeptides (2,5-diketopiperazines, DKPs) in the extract, which are known to possess QSI activity. Our results highlight the potential use of genomic mining coupled with LC-HRMS/MS and bionformatic tools (GNPS) as a potent approach for metabolome studies in discovering novel QSI lead compounds. This study also provides the biosynthetic diversity of these BGCs and an assessment of the predicted chemical space yet to be discovered.