Quantitative Serum NMR Spectroscopy Stratifies COVID-19 Patients and Sheds Light on Interfaces of Host Metabolism and the Immune Response with Cytokines and Clinical Parameters.

The complex manifestations of COVID-19 are still not fully decoded on the molecular level. We combined quantitative the nuclear magnetic resonance (NMR) spectroscopy serum analysis of metabolites, lipoproteins and inflammation markers with clinical parameters and a targeted cytokine panel to characterize COVID-19 in a large (534 patient samples, 305 controls) outpatient cohort of recently tested PCR-positive patients. The COVID-19 cohort consisted of patients who were predominantly in the initial phase of the disease and mostly exhibited a milder disease course. Concerning the metabolic profiles of SARS-CoV-2-infected patients, we identified markers of oxidative stress and a severe dysregulation of energy metabolism. NMR markers, such as phenylalanine, inflammatory glycoproteins (Glyc) and their ratio with the previously reported supramolecular phospholipid composite (Glyc/SPC), showed a predictive power comparable to laboratory parameters such as C-reactive protein (CRP) or ferritin. We demonstrated interfaces between the metabolism and the immune system, e.g., we could trace an interleukin (IL-6)-induced transformation of a high-density lipoprotein (HDL) to a pro-inflammatory actor. Finally, we showed that metadata such as age, sex and constitution (e.g., body mass index, BMI) need to be considered when exploring new biomarkers and that adding NMR parameters to existing diagnoses expands the diagnostic toolbox for patient stratification and personalized medicine.

Concurrent J-evolving refocusing pulses.

Conventional refocusing pulses are optimised for a single spin without considering any type of coupling. However, despite the fact that most couplings will result in undesired distortions, refocusing in delay-pulse-delay-type sequences with desired heteronuclear coherence transfer might be enhanced considerably by including coupling evolution into pulse design. We provide a proof of principle study for a Hydrogen-Carbon refocusing pulse sandwich with inherent J-evolution following the previously reported ICEBERG-principle with improved performance in terms of refocusing performance and/or overall effective coherence transfer time. Pulses are optimised using optimal control theory with a newly derived quality factor and z-controls as an efficient tool to speed up calculations. Pulses are characterised in theory and experiment and compared to conventional concurrent refocusing pulses, clearly showing an improvement for the J-evolving pulse sandwich. As a side-product, also efficient J-compensated resfocusing pulse sandwiches - termed BUBU pulses following the nomenclature of previous J-compensated BUBI and BEBEtr pulse sandwiches - have been optimised.

Diffusion and Relaxation Edited Proton NMR Spectroscopy of Plasma Reveals a High-Fidelity Supramolecular Biomarker Signature of SARS-CoV-2 Infection.

We have applied nuclear magnetic resonance spectroscopy based plasma phenotyping to reveal diagnostic molecular signatures of SARS-CoV-2 infection via combined diffusional and relaxation editing (DIRE). We compared plasma from healthy age-matched controls (n = 26) with SARS-CoV-2 negative non-hospitalized respiratory patients and hospitalized respiratory patients (n = 23 and 11 respectively) with SARS-CoV-2 rRT-PCR positive respiratory patients (n = 17, with longitudinal sampling time-points). DIRE data were modelled using principal component analysis and orthogonal projections to latent structures discriminant analysis (O-PLS-DA), with statistical cross-validation indices indicating excellent model generalization for the classification of SARS-CoV-2 positivity for all comparator groups (area under the receiver operator characteristic curve = 1). DIRE spectra show biomarker signal combinations conferred by differential concentrations of metabolites with selected molecular mobility properties. These comprise the following: (a) composite N-acetyl signals from α-1-acid glycoprotein and other glycoproteins (designated GlycA and GlycB) that were elevated in SARS-CoV-2 positive patients [p = 2.52 × 10-10 (GlycA) and 1.25 × 10-9 (GlycB) vs controls], (b) two diagnostic supramolecular phospholipid composite signals that were identified (SPC-A and SPC-B) from the -+N-(CH3)3 choline headgroups of lysophosphatidylcholines carried on plasma glycoproteins and from phospholipids in high-density lipoprotein subfractions (SPC-A) together with a phospholipid component of low-density lipoprotein (SPC-B). The integrals of the summed SPC signals (SPCtotal) were reduced in SARS-CoV-2 positive patients relative to both controls (p = 1.40 × 10-7) and SARS-CoV-2 negative patients (p = 4.52 × 10-8) but were not significantly different between controls and SARS-CoV-2 negative patients. The identity of the SPC signal components was determined using one and two dimensional diffusional, relaxation, and statistical spectroscopic experiments. The SPCtotal/GlycA ratios were also significantly different for control versus SARS-CoV-2 positive patients (p = 1.23 × 10-10) and for SARS-CoV-2 negatives versus positives (p = 1.60 × 10-9). Thus, plasma SPCtotal and SPCtotal/GlycA are proposed as sensitive molecular markers for SARS-CoV-2 positivity that could effectively augment current COVID-19 diagnostics and may have value in functional assessment of the disease recovery process in patients with long-term symptoms.

Comprehensive and High-Throughput Exploration of Chemical Space Using Broadband 19 F†NMR-Based Screening.

Fragment-based lead discovery has become a fundamental approach to identify ligands that efficiently interact with disease-relevant targets. Among the numerous screening techniques, fluorine-detected NMR has gained popularity owing to its high sensitivity, robustness, and ease of use. To effectively explore chemical space, a universal NMR experiment, a rationally designed fragment library, and a sample composition optimized for a maximal number of compounds and minimal measurement time are required. Here, we introduce a comprehensive method that enabled the efficient assembly of a high-quality and diverse library containing nearly 4000 fragments and screening for target-specific binders within days. At the core of the approach is a novel broadband relaxation-edited NMR experiment that covers the entire chemical shift range of drug-like 19 F motifs in a single measurement. Our approach facilitates the identification of diverse binders and the fast ligandability assessment of new targets.

Homonuclear BIRD-decoupled spectra for measuring one-bond couplings with highest resolution: CLIP/CLAP-RESET and constant-time-CLIP/CLAP-RESET.

Heteronuclear one-bond couplings are of interest for various aspects of structural analysis of small organic molecules, including for example the distinction of axial and equatorial protons or the use of RDCs as angular constraints. Such couplings are most easily measured from pure doublets in HSQC-type spectra. Recently, the fully decoupled RESET HSQC experiment was reported and several other so-called pure-shift methods followed that allow for the removal of splittings due to homonuclear scalar interactions in one and two-dimensional NMR. In this work we present broadband homonuclear decoupled CLIP/CLAP-RESET experiments based on an isotope-selective BIRD filter element using a recently reported improved version of Zangger-Sterk data chunking. The concatenated FIDs result in multiplets in which most homonuclear splittings are removed while the heteronuclear one-bond couplings are retained. Couplings can be extracted in an IPAP fashion without scaling of subspectra by the use of optimized coherence transfer elements like the COB-INEPT. The method leads to complete homonuclear decoupling for CH groups and CH3 groups in isotropic samples, but leaves residual splittings with antiphase contributions for e.g. CH2 groups due to (2)JHH coupling evolution that is not affected by the BIRD element. For this case we present a constant-time version of the proposed BIRD decoupling scheme with full homonuclear decoupling. In addition, the effects of strong coupling are discussed. Strong coupling artifacts cannot be circumvented, but the proposed experiments allow their distinct recognition.