Orsay virus (OrV), infecting Caenorhabditis elegans, is a valuable model for studying host-virus interactions, yet its bipartite RNA genome packaging and assembly mechanisms remain unclear. To investigate these mechanisms, we combine infection assays, single-molecule imaging, and cryo-electron microscopy. We find that the two RNA segments accumulate asymmetrically, and that productive infection depends on coinfection by multiple particles. We therefore asked whether unequal segment availability generates heterogeneous virions. Cryo-EM analysis of OrV, including symmetric and asymmetric reconstructions to 2.5 Ang resolution, unveils uniform internal density, excluding large populations of empty or semi-empty particles. We show the genome forms a semi-ordered network contacting the electropositive capsid interior through sequence-independent interactions with the N-terminus and penton region. We also resolve the capsid-delta linker, identifying the penton as a likely site coordinating capsid assembly. Together, we propose a model in which stochastic genome assortment emerges during assembly modulated by segment-specific replication dynamics.

Glycan-lectin interactions at cell surfaces regulate numerous biological processes but remain challenging to characterize at the molecular level. Glycosylation heterogeneity results in lectin-binding targets-from purified glycoproteins to the cell-surface glycocalyx-presenting multiple glycan epitopes simultaneously. Concurrently, distinct lectins often exhibit overlapping glycan binding selectivity, with similar affinities for widely distributed epitopes. Consequently, how lectins compete and achieve selective recognition at glycoprotein and cell-surface levels remains poorly understood. Here, we introduce 19F lectin tagging, as an NMR-based approach to probe these complex glycan-mediated binding processes. Incorporation of 19F probes into lectins yields simple, background-free spectra, enabling binding studies in complex biological environments, including the cell surface. Importantly, this approach also allows the analysis of lectin mixtures with overlapping glycan specificities while individually resolving their binding behavior. We focus on galectins, a family of multifunctional and N-acetyllactosamine (LacNAc)-binding lectins that regulate diverse processes at the cell surface, to dissect their competitive binding behavior across targets of increasing complexity, from small carbohydrates to glycoproteins and the cell-surface glycocalyx. Our results provide insight into the mechanisms underlying the recognition of the immune checkpoint glycoprotein TIM-3 by galectins and reveal competitive binding between some galectin family members at the cell surface. Collectively, these findings reveal that galectin specificity is not dictated solely by LacNAc recognition but instead arises from the molecular context in which glycans are presented, including multivalency and competition for shared glycan ligands. More broadly, they highlight the potential of 19F lectin tagging to investigate binding events in biologically relevant systems.

Glycan-lectin interactions at cell surfaces regulate numerous biological processes but remain challenging to characterize at the molecular level. Glycosylation heterogeneity results in lectin-binding targets─from purified glycoproteins to the cell-surface glycocalyx─presenting multiple glycan epitopes simultaneously. Concurrently, distinct lectins often exhibit overlapping glycan binding selectivity, with similar affinities for widely distributed epitopes. Consequently, how lectins compete and achieve selective recognition at glycoprotein and cell-surface levels remains poorly understood. Here, we introduce 19F lectin tagging, as an NMR-based approach to probe these complex glycan-mediated binding processes. Incorporation of 19F probes into lectins yields simple, background-free spectra, enabling binding studies in complex biological environments, including the cell surface. Importantly, this approach also allows the analysis of lectin mixtures with overlapping glycan specificities while individually resolving their binding behavior. We focus on galectins, a family of multifunctional and N-acetyllactosamine (LacNAc)-binding lectins that regulate diverse processes at the cell surface, to dissect their competitive binding behavior across targets of increasing complexity, from small carbohydrates to glycoproteins and the cell-surface glycocalyx. Our results provide insight into the mechanisms underlying the recognition of the immune checkpoint glycoprotein TIM-3 by galectins and reveal competitive binding between some galectin family members at the cell surface. Collectively, these findings reveal that galectin specificity is not dictated solely by LacNAc recognition but instead arises from the molecular context in which glycans are presented, including multivalency and competition for shared glycan ligands. More broadly, they highlight the potential of 19F lectin tagging to investigate binding events in biologically relevant systems.

Phosphorylated metabolites, here referred to as phosphometabolites, are sufficiently abundant and widely distributed to provide a condensed representation of metabolism that can be readily accessed through NMR spectroscopy. This study addresses the challenge of precisely quantifying phosphometabolites via quantitative 31P NMR from tissue extracts. We optimized standard operating procedures for enhanced spectral resolution, signal intensity, and accuracy. By amply evaluating solvent and buffer conditions, reference compounds, and paramagnetic relaxation enhancers, we identified optimal conditions for metabolite analysis, including the use of trimethylphosphine oxide for accurate signal referencing due to its short T1 relaxation time and minimal offset and glycine buffer (in D2O) at pD 9.5, where virtually invariant 31P signal frequencies and sample osmolarities are observed, along with maximal NMR detection sensitivity and temperature stability of the pH. These methodological advancements significantly improve the reliability and reproducibility of phosphometabolite characterization, allowing the assignment of up to 60 independent signals in the one-dimensional (1D) 31P spectrum (of a total of 94 peaks), that resulted in the proper quantification of 44 phosphometabolites from different tissular samples.

We report the successful development and translation of high-field nuclear magnetic resonance (NMR) based comprehensive lipoprotein analysis to routine benchtop systems. This demonstrates the potential to reimagine population level cardiovascular disease risk analysis and individual level screening based on blood sampling. Using a quantitative calibration approach, we obtained stable and reproducible results from multiple sites, despite reduced spectral dispersion and sensitivity at lower field strengths. Our study shows that 25 out of 28 major lipoprotein parameters, including key cardiometabolic risk markers, were faithfully measured using benchtop NMR systems within 15 min. This development has significant implications for making a powerful diagnostic tool widely available, enhancing the potential for longitudinal personalized medicine through molecular phenotyping in the clinic.

BACKGROUND:Nuclear magnetic resonance (NMR) spectroscopy enables the characterisation of lipoprotein sub-particles, providing a more detailed lipid profile than the conventional lipid measurements, with potential clinical relevance, particularly in cardiovascular disease (CVD), which remains the leading cause of mortality worldwide. Nonetheless, for clinical implementation, it is essential to first determine the normal variation of lipoprotein parameters by age and sex. METHODS:This cross-sectional study analysed a large dataset of 31,275 serum or plasma samples from five different countries using the B.I.LISA™ NMR-based platform, quantifying 112 lipoprotein parameters, including subclass size and concentration. Lipoprotein parameters from specific cohorts were fitted to a Quantile Generalised Additive Model (QGAM) to calculate the different percentiles as a function of age and sex. FINDINGS:A sub-cohort of individuals belonging to non-oriented cohorts (27,470 individuals) showed that lipoprotein parameters exhibit distinct sex- and age-dependent patterns, with inflection points observed around 44 and 60 years in women and around 60 years in men, aligning with known ageing acceleration models. The sub-cohort of 3021 individuals showing cardiometabolic risk factors was used to evaluate the effect of obesity, hypertension and diabetes in the lipoprotein distribution. Finally, we analysed the lipoprotein parameters that align with SCORE2 (a well-known CVD risk predictor) in an age- and sex-dependent manner. Many NMR-derived parameters effectively distinguish between low and high/very high CVD risk profiles, with very low-density (VLDL)-associated parameters demonstrating the highest sensitivity across a broad age range. INTERPRETATION:Our findings provide reference values for NMR-derived lipoprotein parameters by age and sex, enabling their accurate interpretation in the context of cardiovascular disease risk stratification. FUNDING:The specific funding of this article is provided in the acknowledgements section.

Abstract Background Metabolic syndrome (MetS) is a cluster of medical conditions and risk factors correlating with insulin resistance that increase the risk of developing cardiometabolic health problems. The specific criteria for diagnosing MetS vary among different medical organizations but are typically based on the evaluation of abdominal obesity, high blood pressure, hyperglycemia, and dyslipidemia. A unique, quantitative and independent estimation of the risk of MetS based only on quantitative biomarkers is highly desirable for the comparison between patients and to study the individual progression of the disease in a quantitative manner. Methods We used NMR-based metabolomics on a large cohort of donors (n = 21,323; 37.5% female) to investigate the diagnostic value of serum or serum combined with urine to estimate the MetS risk. Specifically, we have determined 41 circulating metabolites and 112 lipoprotein classes and subclasses in serum samples and this information has been integrated with metabolic profiles extracted from urine samples. Results We have developed MetSCORE, a metabolic model of MetS that combines serum lipoprotein and metabolite information. MetSCORE discriminate patients with MetS (independently identified using the WHO criterium) from general population, with an AUROC of 0.94 (95% CI 0.920–0.952, p < 0.001). MetSCORE is also able to discriminate the intermediate phenotypes, identifying the early risk of MetS in a quantitative way and ranking individuals according to their risk of undergoing MetS (for general population) or according to the severity of the syndrome (for MetS patients). Conclusions We believe that MetSCORE may be an insightful tool for early intervention and lifestyle modifications, potentially preventing the aggravation of metabolic syndrome.

Prototypic receptors for human influenza viruses are N-glycans carrying α2,6-linked sialosides. Due to immune pressure, A/H3N2 influenza viruses have emerged with altered receptor specificities that bind α2,6-linked sialosides presented on extended N-acetyl-lactosamine (LacNAc) chains. Here, binding modes of such drifted hemagglutinin's (HAs) are examined by chemoenzymatic synthesis of N-glycans having 13C-labeled monosaccharides at strategic positions. The labeled glycans are employed in 2D STD-1H by 13C-HSQC NMR experiments to pinpoint which monosaccharides of the extended LacNAc chain engage with evolutionarily distinct HAs. The NMR data in combination with computation and mutagenesis demonstrate that mutations distal to the receptor binding domain of recent HAs create an extended binding site that accommodates with the extended LacNAc chain. A fluorine containing sialoside is used as NMR probe to derive relative binding affinities and confirms the contribution of the extended LacNAc chain for binding.

Glycan-mediated molecular recognition events are essential for life. NMR is widely used to monitor glycan binding to lectins in solution using isolated glycans and lectins. In this context, we herein explore diverse NMR methodologies, from both the receptor and ligand perspectives, to monitor glycan-lectin interactions under experimental conditions mimicking the native milieu inside cells and on cell surface. For the NMR experiments inside cells, galectin-7 is employed as model, since most galectins are soluble and carry out their functions in the cellular micro-environment. Using Danio Rerio oocytes, the 1H-15N HMQC NMR spectrum of a folded galectin has been observed inside cell for the first time, using a glycomimetic ligand (TDG) to overcoming the natural tendency of galectins to bind to numerous galactose-containing receptors within cells. Alternatively, most lectins, other than galectins, are displayed on the cell surface, providing a multivalent presentation to bind their glycan partners in cis (at the same cell) or in trans (on other cells). In this case, ligand-based STD-NMR experiments have been successfully applied to account for the interactions of natural glycans and glycomimetics with Siglec-10. These methodologies provide the proof-of-concept to open the door to the NMR analysis of the recognition of glycans in native-like settings.

Proton nuclear magnetic resonance (NMR) N-acetyl signals (Glyc) from glycoproteins and supramolecular phospholipids composite peak (SPC) from phospholipid quaternary nitrogen methyls in subcompartments of lipoprotein particles) can give important systemic metabolic information, but their absolute quantification is compromised by overlap with interfering resonances from lipoprotein lipids themselves. We present a J-Edited DIffusional (JEDI) proton NMR spectroscopic approach to selectively augment signals from the inflammatory marker peaks Glyc and SPCs in blood serum NMR spectra, which enables direct integration of peaks associated with molecules found in specific compartments. We explore a range of pulse sequences that allow editing based on peak J-modulation, translational diffusion, and T2 relaxation time and validate them for untreated blood serum samples from SARS-CoV-2 infected patients (n = 116) as well as samples from healthy controls and pregnant women with physiological inflammation and hyperlipidemia (n = 631). The data show that JEDI is an improved approach to selectively investigate inflammatory signals in serum and may have widespread diagnostic applicability to disease states associated with systemic inflammation.

The binding of intrinsically disordered proteins to globular ones can require the folding of motifs into α-helices. These interactions offer opportunities for therapeutic intervention but their modulation with small molecules is challenging because they bury large surfaces. Linear peptides that display the residues that are key for binding can be targeted to globular proteins when they form stable helices, which in most cases requires their chemical modification. Here we present rules to design peptides that fold into single α-helices by instead concatenating glutamine side chain to main chain hydrogen bonds recently discovered in polyglutamine helices. The resulting peptides are uncharged, contain only natural amino acids, and their sequences can be optimized to interact with specific targets. Our results provide design rules to obtain single α-helices for a wide range of applications in protein engineering and drug design.

SARS-CoV-2 infection causes a significant reduction in lipoprotein-bound serum phospholipids give rise to supramolecular phospholipid composite (SPC) signals observed in diffusion and relaxation edited 1H NMR spectra. To characterize the chemical structural components and compartmental location of SPC and to understand further its possible diagnostic properties, we applied a Statistical HeterospectroscopY in n-dimensions (SHY-n) approach. This involved statistically linking a series of orthogonal measurements made on the same samples, using independent analytical techniques and instruments, to identify the major individual phospholipid components giving rise to the SPC signals. Thus, an integrated model for SARS-CoV-2 positive and control adults is presented that relates three identified diagnostic subregions of the SPC signal envelope (SPC1, SPC2, and SPC3) generated using diffusion and relaxation edited (DIRE) NMR spectroscopy to lipoprotein and lipid measurements obtained by in vitro diagnostic NMR spectroscopy and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The SPC signals were then correlated sequentially with (a) total phospholipids in lipoprotein subfractions; (b) apolipoproteins B100, A1, and A2 in different lipoproteins and subcompartments; and (c) MS-measured total serum phosphatidylcholines present in the NMR detection range (i.e., PCs: 16.0,18.2; 18.0,18.1; 18.2,18.2; 16.0,18.1; 16.0,20.4; 18.0,18.2; 18.1,18.2), lysophosphatidylcholines (LPCs: 16.0 and 18.2), and sphingomyelin (SM 22.1). The SPC3/SPC2 ratio correlated strongly (r = 0.86) with the apolipoprotein B100/A1 ratio, a well-established marker of cardiovascular disease risk that is markedly elevated during acute SARS-CoV-2 infection. These data indicate the considerable potential of using a serum SPC measurement as a metric of cardiovascular risk based on a single NMR experiment. This is of specific interest in relation to understanding the potential for increased cardiovascular risk in COVID-19 patients and risk persistence in post-acute COVID-19 syndrome (PACS).

A code is defined by the nature of the symbols, which are used to generate information‐storing combinations (e. g. oligo‐ and polymers). Like nucleic acids and proteins, oligo‐ and polysaccharides are ubiquitous, and they are a biochemical platform for establishing molecular messages. Of note, the letters of the sugar code system (third alphabet of life) excel in coding capacity by making an unsurpassed versatility for isomer (code word) formation possible by variability in anomery and linkage position of the glycosidic bond, ring size and branching. The enzymatic machinery for glycan biosynthesis (writers) realizes this enormous potential for building a large vocabulary. It includes possibilities for dynamic editing/erasing as known from nucleic acids and proteins. Matching the glycome diversity, a large panel of sugar receptors (lectins) has developed based on more than a dozen folds. Lectins ‘read’ the glycan‐encoded information. Hydrogen/coordination bonding and ionic pairing together with stacking and C−H/π‐interactions as well as modes of spatial glycan presentation underlie the selectivity and specificity of glycan‐lectin recognition. Modular design of lectins together with glycan display and the nature of the cognate glycoconjugate account for the large number of post‐binding events. They give an entry to the glycan vocabulary its functional, often context‐dependent meaning(s), hereby building the dictionary of the sugar code.

Neue 2D-FOSY-Experimente fokussieren auf ein gekoppeltes Kernspinsystem, d. h. eine posttranslationale Modifikation oder ein struktureller Hotspot im Allgemeinen, mit 3 bis 5 bekannten Frequenzen. Mit höherer Empfindlichkeit und Vielseitigkeit als bei herkömmlichen Experimenten löst FOSY das Problem der spektralen Dispersion und liefert innerhalb weniger Stunden die relevante lokale NMR-Signalzuordnung, wie Vladislav Y. Orekhov et al. in ihrer Zuschrift auf S. 23732 für zwei Phosphorylierungsstellen in IDP Tau mit 441 Residuen zeigen.

While a structural description of the molecular mechanisms guiding ribosome assembly in eukaryotic systems is emerging, bacteria use an unrelated core set of assembly factors for which high-resolution structural information is still missing. To address this, we used single-particle cryo-electron microscopy to visualize the effects of bacterial ribosome assembly factors RimP, RbfA, RsmA, and RsgA on the conformational landscape of the 30S ribosomal subunit and obtained eight snapshots representing late steps in the folding of the decoding center. Analysis of these structures identifies a conserved secondary structure switch in the 16S ribosomal RNA central to decoding site maturation and suggests both a sequential order of action and molecular mechanisms for the assembly factors in coordinating and controlling this switch. Structural and mechanistic parallels between bacterial and eukaryotic systems indicate common folding features inherent to all ribosomes.

A new suite of 2D FOSY experiments focuses onto one coupled nuclear spin system at a time, that is, a post-translational modification or a structural hotspot in general, with three to five known frequencies. With higher sensitivity and versatility than achievable by traditional experiments, FOSY solves the spectral dispersion problem and obtains only the relevant local NMR signal assignment in a few hours as demonstrated by Vladislav Y. Orekhov and co-workers in their Communication on page 23540 for two phosphorylation sites in the 441-residue IDP Tau.

Glycans of cellular glycoconjugates serve as biochemical signals for a multitude of (patho)physiological processes via binding to their receptors (e.g. lectins). In the case of human adhesion/growth-regulatory galectin-1 (Gal-1), small angle neutron scattering and fluorescence correlation spectroscopy have revealed a significant decrease of its gyration radius and increase of its diffusion coefficient upon binding lactose, posing the pertinent question on the nature and region(s) involved in the underlying structural alterations. Requiring neither a neutron source nor labeling, diffusion measurements by 1H NMR spectroscopy are shown here to be sufficiently sensitive to detect this ligand-induced change. In order to figure out which region(s) of Gal-1 is (are) affected at the level of peptides, we first explored the use of H/D exchange mass spectrometry (HDX MS). Hereby, we found a reduction in proton exchange kinetics beyond the lactose-binding site. The measurement of fast HN/H2O exchange by phase-modulated NMR clean chemical exchange (CLEANEX) NMR on 15N-labeled Gal-1 then increased the spatial resolution to the level of individual amino acids. The mapped regions with increased protection from HN/H2O (D2O) exchange that include the reduction of solvent exposure around the interface can underlie the protein's compaction. These structural changes have potential to modulate this galectin's role in lattice formation on the cell surface and its interaction(s) with protein(s) at the F-face.