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Summary of Achieved Results (TiReD Project)

The project "Time-resolved magnetic resonance to investigate dynamic events in biological systems and biotransformations" (TiReD) successfully fulfilled all three of its main objectives: developing novel data processing strategies, creating experimental and analytical techniques tailored for real-time monitoring of biological events, and applying these approaches to systems of biological relevance.

Key Achievements by Task

  • Task 1: Time-Domain Preprocessing & Data Analysis Tools

    • Demonstrated that standard augmentation strategies in Multivariate Curve Resolution (MCR) perform better than tensorial augmentation by minimizing computational overhead while preserving spectral information and filtering noise.

    • Released open-source software tools, including TrAGICo (extracting trends, shifts, and intensities from NMR series), pyIHM (featuring an optimized alignment algorithm for indirect hard modeling), and t1ttune (automating the setup and optimization of NMR relaxation experiments).

    • Implemented Fast Iterative Filtering (FIF) for baseline correction and developed a ridge-regularized reference deconvolution technique to enhance resolution, signal-to-noise ratio, and linewidths in low-field dDNP experiments.

  • Task 2: Machine Learning Models & Ligand Screening

    • Developed machine learning pipelines (PLSDA+SVM and Boruta+Random Forest Classifier) to classify intact cell phenotypes from low-resolution in-cell 1H NMR spectra.

    • Performed computational design and high-throughput screening of a ~300-compound targeted library against non-canonical DNA structures (i-motifs and G-quadruplexes), utilizing Principal Component Analysis (PCA) on $^1text{H}$ NMR spectra to rapidly identify effective binders.

  • Task 3: Real-Time Monitoring of Biological Systems

    • Bacterial Fermentation: Established an in vitro real-time 1H NMR monitoring and MCR-ALS chemometric deconvolution workflow to track kinetic profiles of 11 major metabolites during Lactic Acid Bacteria fermentation over 24 hours.

    • Hepatic Cell Metabolism: Monitored live human hepatocytes (HepG2) inside an NMR bioreactor over 72 hours, revealing metabolic correlations between linoleic acid supplementation, lipid accumulation, and glutathione depletion.

    • In-Cell 19F NMR Drug Screening: Applied bioreactor-assisted 19F NMR to analyze target engagement, membrane permeability rates, and quantitative intracellular binding affinities of novel fluorinated sulfonamides against Carbonic Anhydrase (CA) isoforms in living human cells.

Dissemination & Research Outputs

  • Publications & Software: Produced 9 peer-reviewed journal publications and published 3 public open-source software repositories on GitHub (T1Ttune, pyihm, tragico).

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