Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 μm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.

• Keywords
• High-throughput lipidomics, Mass spectrometry, Plasma, Quantitation, Reversed-phase, Serum, Ultrahigh-performance liquid chromatography,
• MeSH
• Chromatography, Reverse-Phase * methods   MeSH
• Mass Spectrometry methods   MeSH
• Liquid Chromatography-Mass Spectrometry MeSH
• Humans MeSH
• Lipidomics * methods   MeSH
• Lipids * analysis   MeSH
• High-Throughput Screening Assays methods   MeSH
• Software MeSH
• Tandem Mass Spectrometry methods   MeSH
• Chromatography, High Pressure Liquid methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Lipids * MeSH

Tick-borne encephalitis virus (TBEV) is a neurotropic orthoflavivirus that invades the central nervous system, leading to severe neurological manifestations. In this study, we developed a reporter virus comprising TurboGFP-expressing TBEV (tGFP-TBEV) as a versatile tool for advancing TBEV research. The tGFP-TBEV facilitates quantitative measurement of viral replication, enables precise tracking of individual infected cells, and supports high-throughput screening of potential antiviral compounds and virus-neutralization assays. Furthermore, tGFP-TBEV proved effective as a model for studying TBEV infection in rat organotypic cerebellar slices cultured ex vivo and for visualizing TBEV infection in the mouse brain. Using tissue-clearing protocols and light-sheet fluorescence microscopy, we achieved high-resolution, three-dimensional mapping of the TBEV distribution in the mouse brain. This analysis uncovered distinct patterns of TBEV tropism, with infections concentrated in regions associated with neurogenesis, olfactory processing, and specific neuroanatomical pathways. The ability to visualize infection at both the cellular and whole-organ level provides a new tool for detailed investigations into viral tropism, replication, and interactions with host tissues, paving the way for deeper insights into TBEV biology and the pathogenesis of tick-borne encephalitis.

• Keywords
• TBEV, light-sheet microscopy, neurotropism, organotypic cerebellar slices, reporter viruses, tissue clearing,
• MeSH
• Encephalitis, Tick-Borne * virology   MeSH
• Rats MeSH
• Humans MeSH
• Luminescent Proteins genetics   metabolism   MeSH
• Brain * virology   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Virus Replication MeSH
• Genes, Reporter MeSH
• Viral Tropism MeSH
• Encephalitis Viruses, Tick-Borne * genetics   physiology   MeSH
• Imaging, Three-Dimensional MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Luminescent Proteins MeSH

Forward-directed genetic screens are extremely powerful in identifying novel genes involved in a specific biological process, including various chromatin regulatory pathways. However, the traditional ways of genetic mapping are time- and cost-demanding. Recently, the whole process was revolutionized by the development of mapping-by-sequencing (MBS) protocols. In MBS, the causal mutations and their positions within genes are identified directly by whole-genome sequencing and bioinformatics analysis of the bulk of mutant plants selected based on the mutant phenotype from a segregating population. MBS increases precision and economizes the mapping. Here, we describe a general protocol and provide practical tips on how to proceed with the mapping-by-sequencing on the example of Arabidopsis forward-directed genetic screen designed to identify mutants sensitive to a specific type of DNA damage. The described protocol is generally applicable to a wide range of genetic screens in various inbreeding species with a reference genome sequence.

• Keywords
• DNA damage repair, DNA-protein crosslinks, Forward genetics, Genetic mapping, High-throughput sequencing, Mapping-by-sequencing, SNP calling, Zebularine,
• MeSH
• Arabidopsis * genetics   MeSH
• Phenotype MeSH
• Genome, Plant MeSH
• Chromosome Mapping * methods   MeSH
• Mutation MeSH
• Whole Genome Sequencing methods   MeSH
• Computational Biology methods   MeSH
• High-Throughput Nucleotide Sequencing * methods   MeSH
• Publication type
• Journal Article MeSH

A series of triterpenoids of the lupane, taraxastane, friedelane and baccharane type were oxidized using selenium dioxide (SeO2) and benzeneseleninic anhydride (BSA) under various conditions. Depending on the reaction conditions, different reaction pathways were observed, including dehydrogenation, allylic oxidation, and 1,2-diketone formation. In this way, derivatives functionalized in the triterpene core (especially in rings A, D, and E), difficult to obtain by other methods, can be easily prepared. In some cases, rarely observed α-phenylseleno-ketones were isolated. An unexpected reaction involving the cleavage of the carbon-carbon double bond was observed in the presence of stoichiometric amounts of osmium tetroxide. Further transformations of selected intermediates facilitated the synthesis of new, functionally enriched derivatives. The key reaction pathways were investigated using density functional theory (DFT), focusing on bond length variations and transition states, revealing energetically favored pathways and critical transition structures, including covalent and noncovalent interactions. Solvent and isomerization equilibrium effects were proposed to explain the experimentally observed discrepancies. Cytotoxic activity of selected derivatives was investigated. Derivatives 4 and 38 showed strongest cytotoxicity in cancer cells and fibroblasts (IC50 2.6-26.4 μM); some compounds were selective for G-361 or HeLa cells. These results suggest that they may find application in pharmaceuticals.

• Keywords
• BSA oxidation, Cytotoxic activity, Cytotoxicity of O-Mesylates, DFT calculations, Oxidation of triterpenoids, SeO(2) oxidation, α-phenylseleno-ketone,
• MeSH
• Humans MeSH
• Molecular Structure MeSH
• Cell Line, Tumor MeSH
• Oxidation-Reduction MeSH
• Pentacyclic Triterpenes MeSH
• Cell Proliferation drug effects   MeSH
• Antineoplastic Agents * pharmacology   chemistry   chemical synthesis   MeSH
• Drug Screening Assays, Antitumor MeSH
• Selenium * chemistry   MeSH
• Density Functional Theory MeSH
• Triterpenes * chemistry   pharmacology   chemical synthesis   MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• lupane MeSH Browser
• Pentacyclic Triterpenes MeSH
• Antineoplastic Agents * MeSH
• Selenium * MeSH
• Triterpenes * MeSH

OBJECTIVE: This study presents the design and synthesis of a new series of human carbonic anhydrase (hCA) inhibitors based on a 5-methyl/phenyl-7-(7'-oxycoumarin)-[1,2,4]triazolo[1,5-a]pyrimidine scaffold. METHODS: The chemical structures of novel coumarin-based triazolopyrimidines 3a-u were confirmed after using NMR and MS analyses. Their inhibitory profiles were evaluated against a panel of five hCA isoforms. Molecular docking simulations were conducted to elucidate the binding modes of compounds 3d and 3s with hCA IX and XII isoforms. Selected derivatives 3d and 3g were tested for their antiproliferative effects on the medulloblastoma HD-MB03 and the glioblastoma U87MG cell lines. Additionally, compounds 3d and 3g were evaluated alone or in combination with cisplatin (cis-Pt) for their ability to induce apoptosis in HD-MB03 cells. RESULTS: In vitro kinetic studies demonstrated that all 5-methyl triazolopyrimidine derivatives (3a-r) selectively inhibited the tumor-associated hCA isoforms (hCA IX and XII), with KI values ranging from 0.75 to 10.5 μM, while hCA I, II, IV isoforms were not significantly inhibited (KIs > 100 μM). Compound 3d emerged as the most potent and selective inhibitor, with KIs of 0.92 and 0.75 μM for hCA IX and XII, respectively. This derivative significantly suppressed cell proliferation in human brain tumor cell lines, particularly HD-MB03, when it was studied for its adjuvant effects in combination with cisplatin. CONCLUSION: In this study, we have identified compound 3d as a selective inhibitor of the isoforms hCA IX and XII, showing minimal inhibition over hCA I, II, and IV isoenzymes (selectivity indices > 100). Its moderate inhibitory effects on hCA IX and XII at submicromolar levels were paralleled by significant antiproliferative activity against HD-MB03 cells. These findings underscore the potential of compound 3d as a promising candidate for further therapeutic development, especially in combination with clinically used chemotherapeutic agents.

• Keywords
• 2, 4]triazolo[1, 5-a]pyrimidine, Carbonic anhydrase inhibitors, [1, antiproliferative activity, coumarin, isoform selectivity, structure– activity relationship.,
• MeSH
• Antigens, Neoplasm metabolism   MeSH
• Apoptosis drug effects   MeSH
• Carbonic Anhydrase Inhibitors * pharmacology   chemical synthesis   chemistry   MeSH
• Carbonic Anhydrase IX * antagonists & inhibitors   metabolism   MeSH
• Carbonic Anhydrases * metabolism   MeSH
• Humans MeSH
• Molecular Structure MeSH
• Cell Line, Tumor MeSH
• Cell Proliferation drug effects   MeSH
• Antineoplastic Agents * pharmacology   chemical synthesis   chemistry   MeSH
• Pyrimidines * pharmacology   chemistry   chemical synthesis   MeSH
• Drug Design * MeSH
• Drug Screening Assays, Antitumor MeSH
• Molecular Docking Simulation MeSH
• Triazoles * chemistry   pharmacology   chemical synthesis   MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Neoplasm MeSH
• CA9 protein, human MeSH Browser
• carbonic anhydrase XII MeSH Browser
• Carbonic Anhydrase Inhibitors * MeSH
• Carbonic Anhydrase IX * MeSH
• Carbonic Anhydrases * MeSH
• Antineoplastic Agents * MeSH
• Pyrimidines * MeSH
• Triazoles * MeSH

Histone deacetylases (HDACs) are essential epigenetic regulators, with HDAC6 overexpression linked to estrogen receptor (ER) activity and breast cancer progression. While several HDAC6 inhibitors have been investigated, their clinical success remains limited due to toxicity and off-target effects, necessitating the discovery of novel, selective inhibitors. This study employs a multi-stage computational approach to identify potent HDAC6 inhibitors for breast cancer therapy. A large-scale virtual screening of 264 834 compounds was conducted, followed by molecular docking, molecular dynamics (MD) simulations (100 ns), molecular mechanics/generalized born surface area (MM/GBSA) binding free energy calculations, and absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions. The HDI-3 emerged as the most promising candidate among replicate simulations, exhibiting a substantially favorable MM/GBSA binding free energy of -130.67 kcal/mol-indicative of strong thermodynamic stability and stronger binding affinity compared to reference inhibitors Trichostatin A and Ricolinostat. Molecular dynamics simulations revealed that HDI-3 maintained structural stability, persistent key interactions with active site residues (ASP649, HIS651, ASP742), and low conformational fluctuations. The ADMET evaluation confirmed HDI-3's favorable pharmacokinetic properties, including optimal bioavailability, non-mutagenicity, and low hepatotoxicity. Essential dynamics and principal component analysis further validated its stable binding profile. While these findings highlight HDI-3 as a selective and pharmacologically viable HDAC6 inhibitor, it is important to acknowledge that the results are entirely computational. Therefore, experimental validation is essential to confirm the compound's efficacy and safety. This integrated computational pipeline provides an efficient strategy to accelerate targeted drug discovery, laying the groundwork for future experimental investigations.

• Keywords
• ADMET, HDAC6 inhibitors, MD simulation, MM/GBSA, breast cancer, molecular docking, virtual screening,
• Publication type
• Journal Article MeSH

SARS-CoV-2 exoribonuclease (ExoN) is essential for viral replication, contributing to proofreading, RNA synthesis, and genomic RNA recombination. As such, it represents a promising target for antiviral drugs. Several low-molecular-weight inhibitors, including disulfiram and aurintricarboxylic acid (ATA), have been reported to inhibit ExoN activity. Computational studies have also suggested that various natural phenolic compounds may inhibit ExoN; however, their inhibitory potency remains largely unknown. In this study, we systematically evaluated the inhibitory potency of 60 phenolic phytochemicals, including flavonoids, phenolic acids, coumarins, and other related compounds, using a dual-assay approach, with ATA as the reference inhibitor. Initially, we used nano-differential scanning fluorimetry to assess the thermal stabilization or destabilization of the enzyme induced by compound binding. Subsequently, we performed a TBE-PAGE-based enzymatic activity assay to examine ExoN activity inhibition. Selected compounds were then validated using a FRET-based enzymatic assay. While none of the compounds achieved the ATA's inhibitory efficacy, three compounds demonstrated measurable inhibitory activity: myricetin (IC50 = 142 µM), ellagic acid (IC50 = 44.4 µM), and shikonin (IC50 = 7.92 µM). Our dual assay approach, complemented by crosslinking experiments, revealed that shikonin exhibits a distinct inhibitory mechanism, possibly involving the disruption of ExoN subunit interactions. These findings emphasize the necessity of experimental validation following in silico screening, particularly for promiscuous chemicals such as phenolic natural products. This approach may help to narrow down rationally designed compounds for further optimization.

• Keywords
• Exoribonuclease, Inhibitors, Nsp14, Phenolic natural products, SARS-CoV-2, Shikonin,
• Publication type
• Journal Article MeSH

Carbon dots (CDs) are complex carbon-based nanomaterials with exceptional photoluminescence characteristics and great promise for sustainable metal-free photocatalysis. However, their structural heterogeneity poses a major challenge for the rational design and prediction of photocatalytic performance. To overcome this limitation, we propose a bottom-up strategy centered on CD-inspired systems with well-defined molecular architecture. Specifically, we computationally screened 5700 stacked polycyclic aromatic hydrocarbon aggregates, as representatives of CD aromatic domains, to identify donor-acceptor pairs capable of efficient charge separation under photoexcitation. Using a few carefully chosen molecular descriptors and a computationally efficient protocol, we identified best candidate systems for oxidative and reductive quenching pathways. Subsequent time-dependent density functional theory analysis confirmed that these systems exhibit key photocatalytic features: a charge-transfer character in the lowest excited state, well-separated bright local excitations, favorable redox potentials, and propensity for extended aggregation with core-surface charge separation. Our approach not only offers a practical design route for CD-like photocatalysts but also provides the fundamental understanding needed to engineer tunable, efficient, and sustainable donor-acceptor photocatalytic systems.

• Publication type
• Journal Article MeSH

The therapeutic potential of HDAC inhibitors containing a hydroxamic acid moiety as a zinc-binding group (ZBG) is limited in clinical use due to their potential mutagenicity. In addition, hydroxamic acids often exhibit off-target effects that can lead to undesirable toxicity. Therefore, the development of HDAC inhibitors with alternative ZBGs has proven to be a promising approach to overcome these drawbacks. HDAC inhibitors carrying alkyl hydrazide as ZBG have recently been published as selective inhibitors for different HDAC subtypes. In the present study, a ligand-based virtual screening workflow, employing a classification categorical model, was developed and applied for a designed targeted chemical space. The two most promising hits from the virtual screening were synthesized and evaluated by in vitro enzyme inhibition assays. Both hits showed strong inhibition of HDAC11 with IC50 values in the nanomolar range. In addition, the compounds showed good selectivity towards HDAC11 at a concentration of 1 μM, only HDAC8 was also significantly inhibited among all tested subtypes. Finally, the binding mode of the selected candidates was investigated by docking against different HDACs, followed by molecular dynamics simulations and metadynamics studies to provide insights for further chemical optimization.

• Keywords
• Alkyl hydrazides, AlphaFold, Classification model, Docking, HDAC11, Molecular dynamics simulation, Virtual screening,
• MeSH
• Histone Deacetylases * chemistry   metabolism   MeSH
• Hydrazines * chemistry   pharmacology   MeSH
• Histone Deacetylase Inhibitors * chemistry   pharmacology   MeSH
• Humans MeSH
• Drug Design MeSH
• Molecular Dynamics Simulation * MeSH
• Molecular Docking Simulation * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• HDAC11 protein, human MeSH Browser
• Histone Deacetylases * MeSH
• Hydrazines * MeSH
• Histone Deacetylase Inhibitors * MeSH

BACKGROUND AND OBJECTIVE: Patient-ventilator asynchronies (PVA) are associated with ventilator-induced lung injury and increased mortality. Current detection methods rely on static thresholds, extensive preprocessing, or proprietary ventilator data. This study aimed to develop and validate a fully online, real-time system that detects and classifies PVAs directly from ventilator screen data while alerting clinicians based on severity. METHODS: The SmartAlert system was developed using ventilator screen recordings from ICU patients. It extracts pressure and flow waveforms from video recordings, converts them into time-series data, and employs deep neural networks to classify asynchronies and assign alarm levels from no urgency to most urgent. A dataset of 381,280 double-breath units was independently annotated by two expert intensivists. Two deep learning models were trained: one for alarm prediction and another for asynchrony classification (ineffective triggering, double cycling, high inspiratory effort, no asynchrony). Performance was evaluated using accuracy, sensitivity, specificity, and AUC-ROC, compared to expert consensus. RESULTS: SmartAlert demonstrated strong performance for alarm level prediction (overall accuracy: 83.8 %, weighted AUC-ROC: 0.943 [95 % CI: 0.941-0.945]) and PVA classification (weighted accuracy: 89.3 %, weighted AUC-ROC: 0.951 [95 % CI: 0.950-0.953]). It showed high specificity for urgent alarms (99.9 % for level 3) and PVA types (98.5 % for ineffective triggering, 96.9 % for double cycling, 94.8 % for high inspiratory effort). CONCLUSIONS: We developed and internally validated SmartAlert, an automated system that detects PVAs, classifies severity, and alerts clinicians in real time. Its potential to reduce alarm fatigue, optimize ventilator settings, and improve patient outcomes remains to be tested in clinical trials.

• Keywords
• Deep neural networks, Mechanical ventilation, Patient-ventilator asynchrony, Real-time monitoring,
• MeSH
• Patient-Ventilator Asynchrony MeSH
• Deep Learning MeSH
• Intensive Care Units * MeSH
• Clinical Alarms MeSH
• Humans MeSH
• Ventilators, Mechanical * MeSH
• Neural Networks, Computer MeSH
• Reproducibility of Results MeSH
• ROC Curve MeSH
• Machine Learning * MeSH
• Respiration, Artificial * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH