A combined observational and Mendelian randomization investigation reveals NMR-measured analytes to be risk factors of major cardiovascular diseases.

Dyslipidaemias is the leading risk factor of several major cardiovascular diseases (CVDs), but there is still a lack of sufficient evidence supporting a causal role of lipoprotein subspecies in CVDs. In this study, we comprehensively investigated several lipoproteins and their subspecies, as well as other metabolites, in relation to coronary heart disease (CHD), heart failure (HF) and ischemic stroke (IS) longitudinally and by Mendelian randomization (MR) leveraging NMR-measured metabolomic data from 118,012 UK Biobank participants. We found that 123, 110 and 36 analytes were longitudinally associated with myocardial infarction, HF and IS (FDR < 0.05), respectively, and 25 of those were associated with all three outcomes. MR analysis suggested that genetically predicted levels of 70, 58 and 7 analytes were associated with CHD, HF and IS (FDR < 0.05), respectively. Two analytes, ApoB/ApoA1 and M-HDL-C were associated with all three CVD outcomes in the MR analyses, and the results for M-HDL-C were concordant in both observational and MR analyses. Our results implied that the apoB/apoA1 ratio and cholesterol in medium size HDL were particularly of importance to understand the shared pathophysiology of CHD, HF and IS and thus should be further investigated for the prevention of all three CVDs.

Differential Solvent DEEP-STD NMR and MD Simulations Enable the Determinants of the Molecular Recognition of Heparin Oligosaccharides by Antithrombin to Be Disentangled.

The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.

Plasma Metabolite Profiling in the Search for Early-Stage Biomarkers for Lung Cancer: Some Important Breakthroughs.

Lung cancer is the leading cause of cancer-related mortality worldwide. In order to improve its overall survival, early diagnosis is required. Since current screening methods still face some pitfalls, such as high false positive rates for low-dose computed tomography, researchers are still looking for early biomarkers to complement existing screening techniques in order to provide a safe, faster, and more accurate diagnosis. Biomarkers are biological molecules found in body fluids, such as plasma, that can be used to diagnose a condition or disease. Metabolomics has already been shown to be a powerful tool in the search for cancer biomarkers since cancer cells are characterized by impaired metabolism, resulting in an adapted plasma metabolite profile. The metabolite profile can be determined using nuclear magnetic resonance, or NMR. Although metabolomics and NMR metabolite profiling of blood plasma are still under investigation, there is already evidence for its potential for early-stage lung cancer diagnosis, therapy response, and follow-up monitoring. This review highlights some key breakthroughs in this research field, where the most significant biomarkers will be discussed in relation to their metabolic pathways and in light of the altered cancer metabolism.

Characterizing human postprandial metabolic response using multiway data analysis.

INTRODUCTION: Analysis of time-resolved postprandial metabolomics data can improve our understanding of the human metabolism by revealing similarities and differences in postprandial responses of individuals. Traditional data analysis methods often rely on data summaries or univariate approaches focusing on one metabolite at a time. OBJECTIVES: Our goal is to provide a comprehensive picture in terms of the changes in the human metabolism in response to a meal challenge test, by revealing static and dynamic markers of phenotypes, i.e., subject stratifications, related clusters of metabolites, and their temporal profiles. METHODS: We analyze Nuclear Magnetic Resonance (NMR) spectroscopy measurements of plasma samples collected during a meal challenge test from 299 individuals from the COPSAC2000 cohort using a Nightingale NMR panel at the fasting and postprandial states (15, 30, 60, 90, 120, 150, 240 min). We investigate the postprandial dynamics of the metabolism as reflected in the dynamic behaviour of the measured metabolites. The data is arranged as a three-way array: subjects by metabolites by time. We analyze the fasting state data to reveal static patterns of subject group differences using principal component analysis (PCA), and fasting state-corrected postprandial data using the CANDECOMP/PARAFAC (CP) tensor factorization to reveal dynamic markers of group differences. RESULTS: Our analysis reveals dynamic markers consisting of certain metabolite groups and their temporal profiles showing differences among males according to their body mass index (BMI) in response to the meal challenge. We also show that certain lipoproteins relate to the group difference differently in the fasting vs. dynamic state. Furthermore, while similar dynamic patterns are observed in males and females, the BMI-related group difference is observed only in males in the dynamic state. CONCLUSION: The CP model is an effective approach to analyze time-resolved postprandial metabolomics data, and provides a compact but a comprehensive summary of the postprandial data revealing replicable and interpretable dynamic markers crucial to advance our understanding of changes in the metabolism in response to a meal challenge.

Studying protein stability in crowded environments by NMR.

Most proteins perform their functions in crowded and complex cellular environments where weak interactions are ubiquitous between biomolecules. These complex environments can modulate the protein folding energy landscape and hence affect protein stability. NMR is a nondestructive and effective method to quantify the kinetics and equilibrium thermodynamic stability of proteins at an atomic level within crowded environments and living cells. Here, we review NMR methods that can be used to measure protein stability, as well as findings of studies on protein stability in crowded environments mimicked by polymer and protein crowders and in living cells. The important effects of chemical interactions on protein stability are highlighted and compared to spatial excluded volume effects.

The critical importance of conditions: Reconciling GPCR functionality and biophysical findings.

G-protein-coupled receptor (GPCR) activation relies on conformational sampling, a nuanced but functionally key behavior well suited to elucidation by nuclear magnetic resonance (NMR) spectroscopy. In this issue of Structure, Thakur et al.1 demonstrate that judicious choice of experimental conditions for 19F NMR studies of a GPCR enables rationalization of functional and pharmacological behavior, leading to testable hypotheses correlating structure, dynamics, and function.

A modular motion compensation pipeline for prospective respiratory motion correction of multi-nuclear MR spectroscopy.

Magnetic resonance (MR) acquisitions of the torso are frequently affected by respiratory motion with detrimental effects on signal quality. The motion of organs inside the body is typically decoupled from surface motion and is best captured using rapid MR imaging (MRI). We propose a pipeline for prospective motion correction of the target organ using MR image navigators providing absolute motion estimates in millimeters. Our method is designed to feature multi-nuclear interleaving for non-proton MR acquisitions and to tolerate local transmit coils with inhomogeneous field and sensitivity distributions. OpenCV object tracking was introduced for rapid estimation of in-plane displacements in 2D MR images. A full three-dimensional translation vector was derived by combining displacements from slices of multiple and arbitrary orientations. The pipeline was implemented on 3 T and 7 T MR scanners and tested in phantoms and volunteers. Fast motion handling was achieved with low-resolution 2D MR image navigators and direct implementation of OpenCV into the MR scanner's reconstruction pipeline. Motion-phantom measurements demonstrate high tracking precision and accuracy with minor processing latency. The feasibility of the pipeline for reliable in-vivo motion extraction was shown on heart and kidney data. Organ motion was manually assessed by independent operators to quantify tracking performance. Object tracking performed convincingly on 7774 navigator images from phantom scans and different organs in volunteers. In particular the kernelized correlation filter (KCF) achieved similar accuracy (74%) as scored from inter-operator comparison (82%) while processing at a rate of over 100 frames per second. We conclude that fast 2D MR navigator images and computer vision object tracking can be used for accurate and rapid prospective motion correction. This and the modular structure of the pipeline allows for the proposed method to be used in imaging of moving organs and in challenging applications like cardiac magnetic resonance spectroscopy (MRS) or magnetic resonance imaging (MRI) guided radiotherapy.

The diagnostic challenge of lack of choline level elevation on 1H-MR spectroscopy in grade II-III gliomas.

The accurate diagnosis of brain tumour is very important in modern neuro-oncology medicine. Magnetic resonance spectroscopy (MRS) is supposed to be a promising tool for detecting cancerous lesions. However, the interpretation of MRS data is complicated by the fact that not all cancerous lesions exhibit elevated choline (Cho) levels. The main goal of our study was to investigate the lack of Cho lesion /Cho ref elevation in the population of grade II-III gliomas. 89 cases of gliomas grade II and III were used for the retrospective analysis - glioma (astrocytoma or oligodendroglioma) grade II (74 out of 89 cases [83%]) and III (15 out of 89 cases [17%]) underwent conventional MRI extended by MRS before treatment. Histopathological diagnosis was obtained either by biopsy or surgical resection. Gliomas were classified to the group of no-choline elevation when the ratio of choline measured within the tumour (Cho lesion ) to choline from NABT (Cho ref ) were equal to or lower than 1. Significant differences were observed between ratios of Cho lesion /Cr lesion calculated for no-choline elevation and glial tumour groups as well as in the NAA lesion /Cr lesion ratio between the no-choline elevation group and glial tumour group. With consistent data concerning choline level elevation and slightly lower NAA value, the Cho lesion /NAA lesion ratio is significantly higher in the WHO II glial tumour group compared to the no-choline elevation cases ( p < 0.000). In the current study the results demonstrated possibility of lack of choline elevation in patients with grade II-III gliomas, so it is important to remember that the lack of elevated choline levels does not exclude neoplastic lesion.

Benchtop NMR Coupled with Chemometrics: A Workflow for Unveiling Hidden Drug Ingredients in Honey-Based Supplements.

Recently, benchtop nuclear magnetic resonance (NMR) spectrometers utilizing permanent magnets have emerged as versatile tools with applications across various fields, including food and pharmaceuticals. Their efficacy is further enhanced when coupled with chemometric methods. This study presents an innovative approach to leveraging a compact benchtop NMR spectrometer coupled with chemometrics for screening honey-based food supplements adulterated with active pharmaceutical ingredients. Initially, fifty samples seized by French customs were analyzed using a 60 MHz benchtop spectrometer. The investigation unveiled the presence of tadalafil in 37 samples, sildenafil in 5 samples, and a combination of flibanserin with tadalafil in 1 sample. After conducting comprehensive qualitative and quantitative characterization of the samples, we propose a chemometric workflow to provide an efficient screening of honey samples using the NMR dataset. This pipeline, utilizing partial least squares discriminant analysis (PLS-DA) models, enables the classification of samples as either adulterated or non-adulterated, as well as the identification of the presence of tadalafil or sildenafil. Additionally, PLS regression models are employed to predict the quantitative content of these adulterants. Through blind analysis, this workflow allows for the detection and quantification of adulterants in these honey supplements.

Associations among NMR-measured inflammatory and metabolic biomarkers and accelerated aging in cardiac catheterization patients.

Research into aging has grown substantially with the creation of molecular biomarkers of biological age that can be used to determine age acceleration. Concurrently, nuclear magnetic resonance (NMR) assessment of biomarkers of inflammation and metabolism provides researchers with new ways to examine intermediate risk factors for chronic disease. We used data from a cardiac catheterization cohort to examine associations between biomarkers of cardiometabolic health and accelerated aging assessed using both gene expression (Transcriptomic Age) and DNA methylation (Hannum Age, GrimAge, Horvath Age, and Phenotypic Age). Linear regression models were used to associate accelerated aging with each outcome (cardiometabolic health biomarkers) while adjusting for chronological age, sex, race, and neighborhood socioeconomic status. Our study shows a robust association between GlycA and GrimAge (5.71, 95% CI = 4.36, 7.05, P = 7.94 × 10-16), Hannum Age (1.81, 95% CI = 0.65, 2.98, P = 2.30 × 10-3), and Phenotypic Age (2.88, 95% CI = 1.91, 3.87, P = 1.21 × 10-8). We also saw inverse associations between apolipoprotein A-1 and aging biomarkers. These associations provide insight into the relationship between aging and cardiometabolic health that may be informative for vulnerable populations.

Resolving the intricate binding of neomycin B to multiple binding motifs of a neomycin-sensing riboswitch aptamer by native top-down mass spectrometry and NMR spectroscopy.

Understanding small molecule binding to RNA can be complicated by an intricate interplay between binding stoichiometry, multiple binding motifs, different occupancies of different binding motifs, and changes in the structure of the RNA under study. Here, we use native top-down mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy to experimentally resolve these factors and gain a better understanding of the interactions between neomycin B and the 40 nt aptamer domain of a neomycin-sensing riboswitch engineered in yeast. Data from collisionally activated dissociation of the 1:1, 1:2 and 1:3 RNA-neomycin B complexes identified a third binding motif C of the riboswitch in addition to the two motifs A and B found in our previous study, and provided occupancies of the different binding motifs for each complex stoichiometry. Binding of a fourth neomycin B molecule was unspecific according to both MS and NMR data. Intriguingly, all major changes in the aptamer structure can be induced by the binding of the first neomycin B molecule regardless of whether it binds to motif A or B as evidenced by stoichiometry-resolved MS data together with titration data from 1H NMR spectroscopy in the imino proton region. Specific binding of the second and third neomycin B molecules further stabilizes the riboswitch aptamer, thereby allowing for a gradual response to increasing concentrations of neomycin B, which likely leads to a fine-tuning of the cellular regulatory mechanism.

4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation.

Despite the importance of proline conformational equilibria (trans versus cis amide and exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0-3 ppm) when a trans X-Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5-12 ppm) difference in chemical shift in a cis X-Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e., the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e., pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n → π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.

NMR Metabolomics of Primary Ovarian Cancer Cells in Comparison to Established Cisplatin-Resistant and -Sensitive Cell Lines.

Cancer cell lines are frequently used in metabolomics, such as in vitro tumor models. In particular, A2780 cells are commonly used as a model for ovarian cancer to evaluate the effects of drug treatment. Here, we compare the NMR metabolomics profiles of A2780 and cisplatin-resistant A2780 cells with those of cells derived from 10 patients with high-grade serous ovarian carcinoma (collected during primary cytoreduction before any chemotherapeutic treatment). Our analysis reveals a substantial similarity among all primary cells but significant differences between them and both A2780 and cisplatin-resistant A2780 cells. Notably, the patient-derived cells are closer to the resistant A2780 cells when considering the exo-metabolome, whereas they are essentially equidistant from A2780 and A2780-resistant cells in terms of the endo-metabolome. This behavior results from dissimilarities in the levels of several metabolites attributable to the differential modulation of underlying biochemical pathways. The patient-derived cells are those with the most pronounced glycolytic phenotype, whereas A2780-resistant cells mainly diverge from the others due to alterations in a few specific metabolites already known as markers of resistance.

Test-Retest Reproducibility of Reduced-Field-of-View Density-Weighted CRT MRSI at 3T.

Quantifying an imaging modality's ability to reproduce results is important for establishing its utility. In magnetic resonance spectroscopic imaging (MRSI), new acquisition protocols are regularly introduced which improve upon their precursors with respect to signal-to-noise ratio (SNR), total acquisition duration, and nominal voxel resolution. This study has quantified the within-subject and between-subject reproducibility of one such new protocol (reduced-field-of-view density-weighted concentric ring trajectory (rFOV-DW-CRT) MRSI) by calculating the coefficient of variance of data acquired from a test-retest experiment. The posterior cingulate cortex (PCC) and the right superior corona radiata (SCR) were selected as the regions of interest (ROIs) for grey matter (GM) and white matter (WM), respectively. CVs for between-subject and within-subject were consistently around or below 15% for Glx, tCho, and Myo-Ins, and below 5% for tNAA and tCr.

Correlation between abnormal cellular immune and changes of magnetic resonance spectroscopy in patients with Alzheimer's disease.

BACKGROUND: Evidence from previous studies indicates that neuroinflammation contributes to the onset of Alzheimer's Disease (AD). Moreover, cellular dysfunction is induced by impaired signaling of neurotransmitters. This study aimed to explore the correlation between cellular immune dysfunction and neurotransmitter changes through cranial Magnetic Resonance Spectroscopy (MRS) in AD patients. METHODS: Here, 32 AD, 40 Vascular Dementia (VD), and 35 Non-Dementia Elderly Control (NDE) cases were enrolled. Flow cytometry was performed to characterize lymphocyte subsets in plasma samples. The IL-1ß and Caspase-1 levels were detected by ELISA. The NLRP3 expression level was measured by Western Blot (WB). The equivalence of N-acetylaspartate (NAA), Creatine (Cr), Choline (Cho), and Inositol (MI) in bilateral hippocampi of patients was examined by MRS. The association of NAA/Cr or MI/Cr ratios with the proportion of T lymphocyte subsets or NK cell subsets was determined through single-factor correlation analysis. RESULTS: The proportion of T lymphocyte subsets was significantly lower in the AD group than in the NDE group (P < 0.01). On the other hand, the Caspase-1, NLRP3, and IL-1ß protein expression levels were significantly higher in the AD group than in the other groups. Further analysis showed that the NAA/Cr ratio was lower in the AD group than in the NDE group. Additionally, a significant positive correlation was found between the NAA/Cr ratio and the MMSE score (r = 0.81, P < 0.01). Moreover, a significant positive correlation was observed between the NAA/Cr and T lymphocyte ratios. The NAA/Cr ratio was significantly negatively correlated with the proportion of NK cells in the blood (r = ï¼0.83, P < 0.01). A significant negative correlation was also recorded between the MI/Cr and T cell ratios in blood samples. CONCLUSIONS: Impaired cellular immune dysfunction in AD patients was significantly correlated with abnormal MRS. Neuroimmune dysfunction may contribute to the pathogenesis of AD and alter the metabolism of neurotransmitters such as aspartic acid and MI in the brains of AD patients. TRIAL REGISTRATION: Not applicable.

The impact of spectral basis set composition on estimated levels of cingulate glutamate and its associations with different personality traits.

BACKGROUND: 1H-MRS is increasingly used in basic and clinical research to explain brain function and alterations respectively. In psychosis research it is now one of the main tools to investigate imbalances in the glutamatergic system. Interestingly, however, the findings are extremely variable even within patients of similar disease states. One reason may be the variability in analysis strategies, despite suggestions for standardization. Therefore, our study aimed to investigate the extent to which the basis set configuration- which metabolites are included in the basis set used for analysis- would affect the spectral fit and estimated glutamate (Glu) concentrations in the anterior cingulate cortex (ACC), and whether any changes in levels of glutamate would be associated with psychotic-like experiences and autistic traits. METHODS: To ensure comparability, we utilized five different exemplar basis sets, used in research, and two different analysis tools, r-based spant applying the ABfit method and Osprey using the LCModel. RESULTS: Our findings revealed that the types of metabolites included in the basis set significantly affected the glutamate concentration. We observed that three basis sets led to more consistent results across different concentration types (i.e., absolute Glu in mol/kg, Glx (glutamate + glutamine), Glu/tCr), spectral fit and quality measurements. Interestingly, all three basis sets included phosphocreatine. Importantly, our findings also revealed that glutamate levels were differently associated with both schizotypal and autistic traits depending on basis set configuration and analysis tool, with the same three basis sets showing more consistent results. CONCLUSIONS: Our study highlights that scientific results may be significantly altered depending on the choices of metabolites included in the basis set, and with that emphasizes the importance of carefully selecting the configuration of the basis set to ensure accurate and consistent results, when using MR spectroscopy. Overall, our study points out the need for standardized analysis pipelines and reporting.

NMR methods to detect fluoride binding and transport by membrane proteins.

Solid-state nuclear magnetic resonance (NMR) methods can probe the motions of membrane proteins in liposomes at the atomic level, and propel the understanding of biomolecular processes for which static structures cannot provide a satisfactory description. High-resolution crystallography snapshots have provided a structural basis for fluoride channels. NMR is a powerful tool to build upon these snapshots and depict a dynamic picture of fluoride channels in native-like lipid bilayers. In this contribution, we discuss solid-state and solution NMR experiments to detect fluoride binding and transport by fluoride channels. Ongoing developments in membrane protein sample preparation and ssNMR methodology, particularly in using 1H, 19F and 13C-detection schemes, offer additional opportunities to study structure and functional aspects of fluoride channels.

Peak Resembling N-acetylaspartate (NAA) on Magnetic Resonance Spectroscopy of Brain Metastases.

Background and Objectives: Differentiating between a high-grade glioma (HGG) and solitary cerebral metastasis presents a challenge when using standard magnetic resonance imaging (MRI) alone. Magnetic resonance spectroscopy (MRS), an advanced MRI technique, may assist in resolving this diagnostic dilemma. N-acetylaspartate (NAA), an amino acid found uniquely in the central nervous system and in high concentrations in neurons, typically suggests HGG over metastatic lesions in spectra from ring-enhancing lesions. This study investigates exceptions to this norm. Materials and Methods: We conducted an MRS study on 49 histologically confirmed and previously untreated patients with brain metastases, employing single-voxel (SVS) techniques with short and long echo times, as well as magnetic resonance spectroscopic imaging (MRSI). Results: In our cohort, 44 out of 49 (90%) patients demonstrated a typical MR spectroscopic profile consistent with secondary deposits: a Cho peak, very low or absent Cr, absence of NAA, and the presence of lipids. A peak at approximately 2 ppm, termed the "NAA-like peak", was present in spectra obtained with both short and long echo times. Among the MRS data from 49 individuals, we observed a peak at 2.0 ppm in five brain metastases from mucinous carcinoma of the breast, mucinous non-small-cell lung adenocarcinoma, two metastatic melanomas, and one metastatic non-small-cell lung cancer. Pathohistological verification of mucin in two of these five cases suggested this peak likely represents N-acetyl glycoproteins, indicative of mucin expression in cancer cells. Conclusions: The identification of a prominent peak at 2.0 ppm could be a valuable diagnostic marker for distinguishing single ring-enhancing lesions, potentially associated with mucin-expressing metastases, offering a new avenue for diagnostic specificity in challenging cases.

Uncovering the Molecular Composition and Architecture of the Bacillus subtilis Biofilm via Solid-State NMR Spectroscopy.

The complex and dynamic compositions of biofilms, along with their sophisticated structural assembly mechanisms, endow them with exceptional capabilities to thrive in diverse conditions that are typically unfavorable for individual cells. Characterizing biofilms in their native state is significantly challenging due to their intrinsic complexities and the limited availability of noninvasive techniques. Here, we utilized solid-state nuclear magnetic resonance (NMR) spectroscopy to analyze Bacillus subtilis biofilms in-depth. Our data uncover a dynamically distinct organization within the biofilm: a dominant, hydrophilic, and mobile framework interspersed with minor, rigid cores of limited water accessibility. In these heterogeneous rigid cores, the major components are largely self-assembled. TasA fibers, the most robust elements, further provide a degree of mechanical support for the cell aggregates and some lipid vesicles. Notably, rigid cell aggregates can persist even without the major extracellular polymeric substance (EPS) polymers, although this leads to slight variations in their rigidity and water accessibility. Exopolysaccharides are exclusively present in the mobile domain, playing a pivotal role in its water retention property. Specifically, all water molecules are tightly bound within the biofilm matrix. These findings reveal a dual-layered defensive strategy within the biofilm: a diffusion barrier through limited water mobility in the mobile phase and a physical barrier posed by limited water accessibility in the rigid phase. Complementing these discoveries, our comprehensive, in situ compositional analysis is not only essential for delineating the sophisticated biofilm architecture but also reveals the presence of alternative genetic mechanisms for synthesizing exopolysaccharides beyond the known pathway.

Quantification of Biopharmaceutically Relevant Nonionic Surfactant Excipients Using Benchtop qNMR.

Nonionic surfactant excipients (NISEs) are commonly added to biologics formulations to mitigate the effects of stress incurred by the active biotherapeutic during manufacturing, transport, and storage. During manufacturing, NISEs are added by dilution of a stock solution directly into a protein formulation, and their accurate addition is critical in maintaining the quality and integrity of the drug product and thus ensuring patient safety. This is especially true for the common NISEs, polysorbates 20 and 80 (PS20 and PS80, respectively) and poloxamer 188 (P188). With the increasing diversity of biologic modalities within modern pharmaceutical pipelines, there is thus a critical need to develop and deploy convenient and user-accessible analytical techniques that can rapidly and reliably quantify these NISEs under biopharmaceutically relevant conditions. We thus pursued 60 MHz benchtop quantitative NMR (qNMR) as a nondestructive and user-friendly analytical technique for the quantification of PS20, PS80, and P188 under such conditions. We demonstrated the ability of benchtop qNMR (1) to quantify simulated PS20, PS80, and P188 stock solutions representative of those used during the drug substance (DS) formulation step in biomanufacturing and (2) to quantify these NISEs at and below their target concentrations (≤0.025% w/v) directly in biologics formulations containing histidine, sucrose, and one of three biotherapeutic modalities (monoclonal antibody, antibody-drug conjugate, and Fc-fusion protein). Our results demonstrate that benchtop qNMR offers a fit-for-purpose, reliable, user-friendly, and green analytical route by which NISE of interest to the biopharmaceutical industry may be readily and reliably quantified. We conclude that benchtop qNMR has the potential to be applied to other excipient formulation components in the presence of various biological modalities as well as the potential for routine integration within analytical and QC laboratories across pharmaceutical development and manufacturing sites.