Triantafyllos Kaloudis

Microcystins (MC) are a critical class of toxic cyanobacterial secondary metabolites, with specific variants such as MC-LR strictly regulated in drinking water. Despite their extensive structural diversity, comprising over 300 known congeners, comprehensive environmental monitoring is severely hindered by the limited availability of commercial reference standards and public tandem mass spectrometry (MS/MS) spectra. To address this analytical bottleneck, we developed a non-targeted mass spectrometry-based computational workflow leveraging the Mass Spectrometry Query Language (MassQL) to enable comprehensive MC profiling. By systematically analyzing the chemical space and fragmentation patterns of all MC congeners documented in the CyanoMetDB database, we classified the congeners into nine structurally distinct groups. Subsequently, we designed and optimized a suite of MassQL queries targeting the conserved MC backbone, group-specific motifs, and individual constituent amino acids. The performance of these custom queries was rigorously evaluated following Feature-Based Molecular Networking (FBMN). Application across three independent cyanobacterial bloom datasets comprising both in-house and public data acquired via qTOF and Orbitrap high-resolution mass spectrometry platforms demonstrated highly satisfactory analytical performance based on recall, precision, F1-scores, and Matthews Correlation Coefficients (MCC). We further demonstrated that MassQL functions as a powerful MC candidate prioritization tool orthogonal to molecular networking. By interrogating MS/MS data for specific structural motifs, this query-based approach enabled the discovery and annotation of novel MC congeners lacking public library spectra, significantly enhancing non-targeted screening for water quality and risk assessment.

Microbial metabolites play a critical role in regulating ecosystems, including the human body and its microbiota. However, understanding the physiologically relevant role of these molecules, especially through liquid chromatography tandem mass spectrometry (LC-MS/MS)-based untargeted metabolomics, poses significant challenges and often requires manual parsing of a large amount of literature, databases, and webpages. To address this gap, we established the Collaborative Microbial Metabolite Center knowledgebase (CMMC-KB), a platform that fosters collaborative efforts within the scientific community to curate knowledge about microbial metabolites. The CMMC-KB aims to collect comprehensive information about microbial molecules originating from microbial biosynthesis, drug metabolism, exposure-related molecules, food, host-derived molecules, and, whenever available, their known activities. Molecules from other sources, including host-produced, dietary, and pharmaceutical compounds, are also included. By enabling direct integration of this knowledgebase with downstream analytical tools, including molecular networking, we can deepen insights into microbiota and their metabolites, ultimately advancing our understanding of microbial ecosystems.

A plethora of synthetic and natural organic compounds end up in water bodies, threatening public health and ecosystems. When conventional water treatment methods prove insufficient for removing harmful contaminants, Advanced Oxidation and Reduction Processes (AOPs/ARPs) offer a promising alternative by generating reactive species (RS) to degrade them. Liquid Chromatography coupled with High-Resolution tandem MS (LC-HR-MS/MS) has been widely employed to elucidate degradation pathways by detecting transformation products (TPs) produced during AOP/ARP. However, most reported studies are targeted at certain suspect TPs, rather than exploiting the whole information provided by LC-HR-MS/MS. Here, we applied an advanced computational metabolomics pipeline to explore non-targeted HR-MS/MS data, including molecular networking and chemical proportionality, to provide new insights and a more comprehensive view of the degradation pathways in AOP/ARP. We selected the cyclic-peptide cyanobacterial hepatotoxin, microcystin-RR (MC-RR) as a substrate and applied the principles of radiation chemistry of water to selectively produce RS in known yields. Degradation of MC-RR was observed with all RS and degradation rate constants followed the order: H• > eaq- > HO• > HOO•/O2-•, ranging from 0.0021 Gy-1 (HOO•/O2-•) to 0.0729 Gy-1 (H•). By combining LC-HR-MS/MS with computational analysis tools, we were able to annotate both novel and known TPs, indicating degradation pathways that include hydroxylation and isomerization under oxidative conditions, as well as hydrogenation and guanidine/methoxy group reduction under reductive conditions. The proposed approach enhances the elucidation of degradation pathways in AOPs/ARPs and has potential for studying and optimizing water treatment technologies, for decontamination by various organic micropollutants.

Investigating the volatiles isolated from a commercial spirulina (Arthrospira platensis) dietary supplement by gas chromatography–olfactometry (GC–O) in combination with an aroma extract dilution analysis (AEDA) resulted in 29 odor events with flavor dilution (FD) factors between 8 and 2048. Identification experiments, including various offline and online fractionation approaches, led to the structure assignment of 30 odorants, among which the most potent were sweaty 2- and 3-methylbutanoic acid (FD 2048), roasty, earthy, shrimp-like 2-ethyl-3,5-dimethylpyrazine (FD 2048), vinegar-like acetic acid (FD 1024), and floral, violet-like β-ionone (FD 1024). Static headspace dilution analysis revealed sulfuric, cabbage-like methanethiol (FD factor ≥ 32) as an additional potent odorant. In summary, 31 important spirulina odorants were identified in this study, and 14 were reported for the first time as spirulina constituents. Our data will provide a basis for future odor optimization of spirulina-based food products.

Many water utilities are at least occasionally affected by unpleasant taste and odour (T&O) in drinking water. For decades, aesthetic water quality has been of secondary concern to water producers, with water safety being the primary focus. However, there has been a recent shift towards prioritising consumer satisfaction, encompassing not only services, but also water quality, including T&O issues, which can negatively impact the supplier's reputation. Starting to address a T&O problem until consumers' complaints become massive is too late and puts water producers under great stress to take effective action in a timely manner. Rather, a preventive approach is necessary. The most effective approach is to include T&O as a hazard to assess and manage in the context of developing a water safety plan (WSP) for the supply system. The development of a WSP provides an excellent platform for including the stakeholders needed to control the source of T&O events, as this often requires multistakeholder cooperation. Our review provides a comprehensive guide to addressing T&O occurrences and shows how this can be included in the framework of WSP development. It identifies supporting tools and illustrates the information given with a number of examples from water suppliers' practice.

Spirulina, a cyanobacterium widely used as a food supplement due to its high nutrient value, contains volatile organic compounds (VOCs). It is crucial to assess the presence of VOCs in commercial spirulina products, as they could influence sensory quality, various processes, and technological aspects. In this study, the volatile profiles of seventeen commercial spirulina food supplements were determined using headspace solid-phase microextraction (HS-SPME), coupled with gas chromatography-mass spectrometry (GC-MS). The identification of volatile compounds was achieved using a workflow that combined data processing with software tools and reference databases, as well as retention indices (RI) and elution order data. A total of 128 VOCs were identified as belonging to chemical groups of alkanes (47.2%), ketones (25.7%), aldehydes (10.9%), alcohols (8.4%), furans (3.7%), alkenes (1.8%), esters (1.1%), pyrazines (0.8%), and other compounds (0.4%). Major volatiles among all samples were hydrocarbons, especially heptadecane and heptadec-8-ene, followed by ketones (i.e., 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one, β-ionone, 2,2,6-trimethylcyclohexan-1-one), aldehydes (i.e., hexanal), and the alcohol oct-1-en-3-ol. Several volatiles were found in spirulina dietary supplements for the first time, including 6,10-dimethylundeca-5,9-dien-2-one (geranylacetone), 6,10,14-trimethylpentadecan-2-one, hept-2-enal, octanal, nonanal, oct-2-en-1-ol, heptan-1-ol, nonan-1-ol, tetradec-9-en-1-ol, 4,4-dimethylcyclohex-2-en-1-ol, 2,6-diethylpyrazine, and 1-(2,5-dimethylfuran-3-yl) ethanone. The methodology used for VOC analysis ensured high accuracy, reliability, and confidence in compound identification. Results reveal a wide variety of volatiles in commercial spirulina products, with numerous newly discovered compounds, prompting further research on sensory quality and production methods.

Cyanopeptolins (CPs) are one of the most commonly occurring class of cyanobacterial nonribosomal peptides. For the majority of these compounds, protease inhibition has been reported. In the current work, the structural diversity of cyanopeptolins produced by Nostoc edaphicum CCNP1411 was explored. As a result, 93 CPs, including 79 new variants, were detected and structurally characterized based on their mass fragmentation spectra. CPs isolated in higher amounts were additionally characterized by NMR. To the best of our knowledge, this is the highest number of cyanopeptides found in one strain. The biological assays performed with the 34 isolated CPs confirmed the significance of the amino acid located between Thr and the unique 3-amino-6-hydroxy-2-piperidone (Ahp) on the activity of the compounds against serine protease and HeLa cancer cells.