Lipoprotein (Sub)Fraction Analysis on the Bruker B.I. LISA Platform.

The Bruker B.I. LISA platform provides a method for human plasma/serum lipoprotein analysis and yields data on the particle numbers and lipids of the main lipoprotein classes (VLDL, IDL, LDL, HDL), and the subfractions within those classes. In order to obtain quantitative and reproducible results, the prescribed protocol, the B.I. Methods, needs to be followed. In this chapter, the B.I. Methods protocol steps relevant for B.I. LISA analyses are described.

NMR-Based Analysis of Cellular Central Energy Metabolism and Exchange Rates.

Cell cultures are widely used in studies to gain mechanistic insights of metabolic processes. The foundation of these studies lies on the quantification of intracellular and extracellular metabolites, and nuclear magnetic resonance (NMR) is one of the key analytical platforms used to this aim. Among the factors influencing the quality of the produced data are the sampling procedures as well as the acquisition and processing of spectroscopic data. Here we provide our workflow for obtaining quantitative metabolic data from adherent mammalian cells using NMR spectroscopy. The described protocol is compatible with other analytical methods like LC- or GC-MS-based lipidomics and untargeted metabolomics from the same sample. We also show how the collected extracellular data can be used to extract exchange flux rates, particularly useful for flux analysis studies and metabolic engineering of human-induced pluripotent stem cells.

NMR-Based Stable Isotope Tracing of Cancer Metabolism.

NMR is widely used for metabolite profiling (metabolomics, metabonomics) particularly of various readily obtainable biofluids such as plasma and urine. It is especially valuable for stable isotope tracer studies to track metabolic pathways under control or perturbed conditions in a wide range of cell models as well as animal models and human subjects. NMR has unique properties for utilizing stable isotopes to edit or simplify otherwise complex spectra acquired in vitro and in vivo, while quantifying the level of enrichment at specific atomic positions in various metabolites (i.e., isotopomer distribution analysis).In this protocol, we give an overview with specific protocols for NMR-based stable isotope-resolved metabolomics, or SIRM, with a workflow from administration of isotope-enriched precursors, via sample preparation through to NMR data collection and reduction. We focus on indirect detection of common NMR-active stable isotopes including 13C, 15N, 31P, and 2H, using a variety of 1H-based two-dimensional experiments. We also include the application and analyses of multiplex tracer experiments.

Metabolomic investigation of fresh beef, lamb and venison using nuclear magnetic resonance spectroscopy in relation to colour stability.

This study investigated changes in the metabolome of fresh beef, lamb, and venison in relation to colour stability during display storage. Changes in meat colour and metabolites in loin muscles (Longissimus lumborum) of beef, lamb and venison were determined under a simulated retail display at 4 °C. Metabolite analysis was performed using nuclear magnetic resonance (NMR) spectroscopy, and 27 metabolites were identified. The stability of fresh meat colour was found to be in the following order: beef > lamb > venison. Several trends were observed, and amino acids and metabolites involved in ATP generation were found to be the most important. Leucine, isoleucine and valine were increased, whereas succinate, inosine monophosphate and choline were decreased over the storage time of all three meat types (p < 0.05). As a reduction in succinate, inosine monophosphate and choline during storage were found for all three meat types, these metabolites could potentially be associated with colour stability.

High-resolution indirect detection of spin-3/2 quadrupolar nuclei in solids using multiple-quantum-filtered through-space D-HMQC experiments.

Through-space heteronuclear correlation experiments under magic-angle spinning (MAS) conditions can provide unique insights into inter-atomic proximities. In particular, it has been shown that experiments based on two consecutive coherence transfers, 1H → I → 1H, like D-HMQC (dipolar-mediated heteronuclear multiple-quantum correlation), are usually more sensitive for the indirect detection via protons of spin-3/2 quadrupolar nuclei with low gyromagnetic ratio. Nevertheless, the resolution is often decreased by the second-order quadrupolar broadening along the indirect dimension. To circumvent this issue, we incorporate an MQMAS (multiple-quantum MAS) quadrupolar filter into the t1 evolution period of the D-HMQC sequence, which results in a novel pulse sequence called D-HMQC-MQ. The triple-quantum coherences evolving during this filter are excited and reconverted using cosine-modulated long-pulses synchronized with the sample rotation to avoid spinning sidebands in the indirect dimension. The desired coherence transfer pathways during this sequence are selected using two nested cogwheel phase cycles with 56 steps. This high-resolution heteronuclear correlation technique is demonstrated experimentally for the indirect detection via 1H of spin-3/2 isotopes, such as 11B, 23Na and 35Cl, in zinc borate hydrate, NaH2PO4 and l-histidine hydrochloride, respectively. We show that this experiment can be applied at high magnetic fields up to 28.2 T for protons subject to chemical shift anisotropies larger than 20 ppm, provided the MAS frequency is sufficiently stable since the D-HMQC-MQ experiment, like the parent D-HMQC, is sensitive to MAS fluctuations, which can produce t1-noise.

TDP-43 Amyloid Fibril Formation via Phase Separation-Related and -Unrelated Pathways.

Intrinsically disordered regions (IDRs) in proteins can undergo liquid-liquid phase separation (LLPS) for functional assembly, but this increases the chance of forming disease-associated amyloid fibrils. Not all amyloid fibrils form through LLPS however, and the importance of LLPS relative to other pathways in fibril formation remains unclear. We investigated this question in TDP-43, a motor neuron disease and dementia-causing protein that undergoes LLPS, using thioflavin T (ThT) fluorescence, NMR, transmission electron microscopy (TEM), and wide-angle X-ray scattering (WAXS) experiments. Using a fluorescence probe modified from ThT strategically designed for targeting protein assembly rather than ß-sheets and supported by TEM images, we propose that the biphasic ThT signals observed under LLPS-favoring conditions are due to the presence of amorphous aggregates. These aggregates represent an intermediate state that diverges from the direct pathway to ß-sheet-dominant fibrils. Under non-LLPS conditions in contrast (at low pH or at physiological conditions in a construct with key LLPS residues removed), the protein forms a hydrogel. Real-time WAXS data, ThT signals, and TEM images collectively demonstrate that the gelation process circumvents LLPS and yet still results in the formation of fibril-like structural networks. We suggest that the IDR of TDP-43 forms disease-causing amyloid fibrils regardless of the formation pathway. Our findings shed light on why both LLPS-promoting and LLPS-inhibiting mutants are found in TDP-43-related diseases.

Nonflammable Ether and Phosphate-Based Liquid Electrolytes for Sodium-Ion Batteries.

This study investigates a group of electrolytes containing NaPF6 or NaBF4 salts in phosphate- and ether-based solvents for high-mass loading sodium-ion batteries. It explores physicochemical properties such as ionic conductivity, dynamic viscosities, and nonflammability. The combination of experimental with computational studies reveals detailed insights into the physicochemical properties of the nonflammable liquid electrolytes. Diglyme-based electrolytes become nonflammable with 50 vol % phosphate solvents, while tetraglyme-based electrolytes require 70 vol %. The solvation structure has been investigated using NMR and is combined with computational studies to provide information about properties such as solvation structure, ionic conductivity, and viscosity. The molecular dynamic simulations confirm the enhanced solvation in diglyme-based liquid electrolytes observed experimentally by 23Na-NMR. Despite lacking sufficient electrochemical stability, this work provides a fundamental understanding of the solvation structure and physicochemical properties of a novel electrolyte system. This is an important contribution to be applied in future electrolyte design rationale.

M, NM-polynomials Based on Reverse, Reduced Reverse Degree and Neighborhood Degree Based Topological Indices with Applications to Bond Energy of Y-Junction Nanotubes.

BACKGROUND: In graph theory, M polynomials like the matching polynomial are very crucial in examining the matching structures within graphs, while NM polynomials extends this to analyze non-matching edges. These polynomials are important in many fields, including chemistry and network architecture. They support the derivation of topological indices for protein structure analysis, network communication optimization, and drug design in QSAR/QSPR investigations. OBJECTIVE: The aim of this paper is to define novel M and NM polynomials for different topological indices and to derive their closed-form expressions, specifically for Y-junction nanotubes. These new polynomials and indices are employed to create a robust QSPR model to predict bond energy in Y-junction nanotubes, that provide high accuracy and reliability in the model's statistical performance. METHOD: This paper introduces new forms of M and NM polynomials tailored to specific topological indices related to reverse and neighborhood reverse properties. We derive closed-form expressions for these indices in Y-junction nanotubes. Furthermore, we develop a QSPR model to predict bond energy in Y-junction nanotubes using the newly defined indices. RESULT: We define novel M and NM polynomials for various topological indices and derive precise expressions for Y-junction nanotubes. Utilizing these indices, we construct a highly accurate QSPR model (R² = 0.999) for predicting bond energy in Y-junction nanotubes, confirming the validity of our polynomial definitions and indices. CONCLUSION: We have presented new M and NM polynomials for different topological indices and derive their expressions specifically for Y-junction nanotubes. With these newly defined indices, we have developed a highly precise QSPR model to predict bond energy, achieving an R² value of 0.999. This work underscores the effectiveness of our polynomial definitions and indices in predicting material properties.

Lipid profiling of RON and DEK-dependent signaling in breast cancer guides discovery of gene networks predictive of poor outcomes.

Recurrent and metastatic breast cancer is frequently treatment resistant. A wealth of evidence suggests that reprogrammed lipid metabolism supports cancer recurrence. Overexpression of the RON and DEK oncoproteins in breast cancer is associated with poor outcome. Both proteins promote cancer metastasis in laboratory models, but their influence on lipid metabolite levels remain unknown. To measure RON- and DEK-dependent steady-state lipid metabolite levels, a nuclear magnetic resonance (NMR)-based approach was utilized. The observed differences identified a lipid metabolism-related gene expression signature that is prognostic of overall survival (OS), distant metastasis-free survival (DMFS), post-progression survival (PPS), and recurrence-free survival (RFS) in patients with breast cancer. RON loss led to decreased cholesterol and sphingomyelin levels, whereas DEK loss increased total fatty acid levels and decreased free glycerol levels. Lipid-related genes were then queried to define a signature that predicts poor outcomes for patients with breast cancer patients. Taken together, RON and DEK differentially regulate lipid metabolism in a manner that predicts and may promote breast cancer metastasis and recurrence.

Germacrane-type sesquiterpenes from Artemisia atrovirens and their anti-inflammatory activity.

Artemisia plants are well-known for their abundant sesquiterpene compounds, which encompass various structural types and exhibit a range of biological activities. In this study, a systematic investigation of Artemisia atrovirens revealed the presence of germacrane-type sesquiterpenes for the first time. This included the discovery of 10 new compounds and three known analogues, among which were two rare dimeric germacrane-type compounds. Their structures were fully characterized through a comprehensive analysis involving MS, IR, 1D- and 2D-NMR spectroscopic data, single crystal X-ray diffraction, density functional theory (DFT) NMR calculations, and time-dependent DFT electronic circular dichroism (TDDFT ECD) calculations. Furthermore, all isolated compounds were evaluated for their anti-inflammatory activity in LPS-stimulated RAW 264.7 murine macrophages. Compound 10 demonstrated a potent inhibitory effect on NO production, with an IC50 value of 4.01 ±â€¯0.09 µM. This study highlights the diverse chemical repertoire of Artemisia species and underscores their potential in drug discovery and development.

Carbon-13 Hyperpolarization of α-Ketocarboxylates with Parahydrogen in Reversible Exchange.

Signal Amplification by Reversible Exchange (SABRE) is a relatively simple and fast hyperpolarization technique that has been used to hyperpolarize the α-ketocarboxylate pyruvate, a central metabolite and the leading hyperpolarized MRI contrast agent. In this work, we show that SABRE can readily be extended to hyperpolarize 13C nuclei at natural abundance on many other α-ketocarboxylates. Hyperpolarization is observed and optimized on pyruvate (P13C=17%) and 2-oxobutyrate (P13C=25%) with alkyl chains in the R-group, oxaloacetate (P13C=11%) and alpha-ketoglutarate (P13C=13%) with carboxylate moieties in the R group, and phenylpyruvate (P13C=2%) and phenylglyoxylate (P13C=2%) with phenyl rings in the R-group. New catalytically active SABRE binding motifs of the substrates to the hyperpolarization transfer catalyst-particularly for oxaloacetate-are observed. We experimentally explore the connection between temperature and exchange rates for all of these SABRE systems and develop a theoretical kinetic model, which is used to fit the hyperpolarization build-up and decay during SABRE activity.

A Miscibility Study of p(MMA-co-HEMA)-Based Polymer Blends by Thermal Analysis and Solid-State NMR Relaxometry.

Ternary amorphous solid dispersions (ASDs) consist of a multicomponent carrier with the aim of improving physical stability or dissolution performance. A polymer blend as a carrier that combines a water-insoluble and a water-soluble polymer may delay the drug release rate, minimizing the risk of precipitation from the supersaturated state. Different microstructures of the ternary ASD may result in different drug release performances; hence, understanding the phase morphology of the polymer blend is crucial prior to drug incorporation. The objective of this study is to investigate the miscibility of the water-insoluble p(MMA-co-HEMA) and water-soluble polymers such as HPC, HPMC, HPMC-AS, and Soluplus. To prepare the polymer blends, p(MMA-co-HEMA) was spray dried in 80/20 and 90/10 (w/w) ratios with one of the water-soluble polymers. Thermal analysis (mDSC and DMA) and solid-state (ss)NMR relaxometry were applied to study the miscibility of these blends. No conclusions regarding miscibility could be drawn from the Tg measurements by thermal analysis. However, phase-separation could be demonstrated in all blends by ssNMR relaxometry. Moreover, by measuring both the T1ρH and T1H relaxation times, domain sizes between 5 and 50 nm could be estimated. This work shows the importance of using complementary analytical techniques to investigate polymer miscibility.

Imino chemical shift assignments of tRNAAsp, tRNAVal and tRNAPhe from Escherichia coli.

Transfer RNAs (tRNAs) are an essential component of the protein synthesis machinery. In order to accomplish their cellular functions, tRNAs go through a highly controlled biogenesis process leading to the production of correctly folded tRNAs. tRNAs in solution adopt the characteristic L-shape form, a stable tertiary conformation imperative for the cellular stability of tRNAs, their thermotolerance, their interaction with protein and RNA complexes and their activity in the translation process. The introduction of post-transcriptional modifications by modification enzymes, the global conformation of tRNAs, and their cellular stability are highly interconnected. We aim to further investigate this existing link by monitoring the maturation of bacterial tRNAs in E. coli extracts using NMR. Here, we report on the 1H, 15N chemical shift assignment of the imino groups and some amino groups of unmodified and modified E. coli tRNAAsp, tRNAVal and tRNAPhe, which are essential for characterizing their maturation process using NMR spectroscopy.

Structure and functional studies of Avt1, a novel peptide from the sea anemone Aulactinia veratra.

Sea anemones are a rich source of peptide toxins spanning a diverse range of biological activities, typically targeting proteins such as ion channels, receptors and transporters. These peptide toxins and their analogues are usually highly stable and selective for their molecular targets, rendering them of interest as molecular tools, insecticides and therapeutics. Recent transcriptomic and proteomic analyses of the sea anemone Aulactinia veratra identified a novel 28-residue peptide, designated Avt1. Avt1 was produced using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The liquid chromatography-mass spectrometry profile of synthetic Avt1 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds. The solution structure determined by NMR revealed that Avt1 adopts an inhibitor cystine knot (ICK) fold, in which a ring is formed by two disulfide bonds with a third disulfide penetrating the ring to create the pseudo-knot. This structure provides ICK peptides with high structural, thermal and proteolytic stability. Consistent with its ICK structure, Avt1 was resistant to proteolysis by trypsin, chymotrypsin and pepsin, although it was not a trypsin inhibitor. Avt1 at 100 nM showed no activity in patch-clamp electrophysiological assays against several mammalian voltage-gated ion channels, but has structural features similar to toxins targeting insect sodium ion channels. Although sequence homologues of Avt1 are found in a number of sea anemones, this is the first representative of this family to be characterised structurally and functionally.

Ammonothermal Synthesis of Luminescent Imidonitridophosphate Ba4P4N8(NH)2:Eu2.

The structural variability of a compound class is an important criterion for the research into phosphor host lattices for phosphor-converted light-emitting diodes (pc-LEDs). Especially, nitridophosphates and the related class of imidonitridophosphates are promising candidates. Recently, the ammonothermal approach has opened a systematic access to this substance class with larger sample quantities. We present the successful ammonothermal synthesis of the imidonitridophosphate Ba4P4N8(NH)2:Eu2+. Its crystal structure is solved by X-ray diffraction and it crystallizes in space group Cc (no. 9) with lattice parameters a = 12.5250(3), b = 12.5566(4), c = 7.3882(2) Å and ß = 102.9793(10)°. For the first time, adamantane-type (imido)nitridophosphate anions [P4N8(NH)2]8- are observed next to metal ions other than alkali metals in a compound. The presence of imide groups in the structure and the identification of preferred positions for the hydrogen atoms are performed using a combination of quantum chemical calculations, Fourier-transform infrared, and solid-state NMR spectroscopy. Eu2+ doped samples exhibit cyan emission (λmax = 498 nm, fwhm = 50 nm/1981 cm-1) when excited with ultraviolet light with an impressive internal quantum efficiency (IQE) of 41 %, which represents the first benchmark for imidonitridophosphates and is promising for potential industrial application of this compound class.

Mechanistic Insights into the Removal of Surfactant-Like Contaminants on Mesoporous Polydopamine Nanospheres from Complex Wastewater Matrices.

The detrimental environmental effects of surfactant-like contaminants (SLCs) with distinctive amphiphilic structures have garnered significant attention, particularly since perfluorooctanesulfonate was classified as a persistent organic pollutant. Despite the numerous absorbents developed for SLCs removal, the underlying interaction mechanisms remain speculative and lack experimental validation. To address this research gap, we elucidate the mechanistic insights into the selective removal of SLCs using mesoporous polydopamine nanospheres (MPDA) fabricated via a novel soft-template method. We employed low-field nuclear magnetic resonance to quantitatively characterize the hydrophilicity of the absorbents using water molecules as probes. The results demonstrated that MPDA with uniform mesopores exhibited a remarkable threefold enhancement in SLCs' adsorption capacity compared to conventional polydopamine particles via intraparticle diffusion. We further demonstrated the dominant effects of electrostatic and hydrophobic interactions on the selective removal of SLCs with MPDA by regulating the isoelectric pH value and performing a comparative analysis. The mechanism-inspired SLC-removal strategy achieved an average removal rate of 76.3% from highly contaminated wastewater. Our findings offer new avenues for applying MPDA as an efficient adsorbent and provide innovative and mechanistic insights for targeted SLC removal in complex wastewater matrices.

Carob seeds as a source of bioactive flavonoid derivatives: isolation, network pharmacology-guided anti-cancer activity, and HPLC standardization.

INTRODUCTION:  Carob, Ceratonia siliqua L. (CS), is a legume well-known for its edible pod pulp. Its seeds are used almost exclusively as a source of the food additive E410. Although a variety of metabolites have been identified by HPLC and LC-MS analysis in CS, reports concerned with their isolation are scarce.   Methodology: In this study, two flavonoid derivatives were isolated from the methanolic extract of CS seeds, namely, quercetin-3-O-rhamnoside and 4'-p-hydroxybenzoylisorhamnetin-3,7-di-O-rhamnoside. Network pharmacology was unusually used as a guide for estimation of the biological potential of the isolated compounds. Finally, the methanolic extract of CS seeds and its ethyl acetate fraction were standardized for their 4'-p-hydroxybenzoylisorhamnetin-3,7-di-O-rhamnoside content by HPLC. RESULTS:  The identified isolates displayed the ability to interfere with the activity of several target proteins associated with renal and colon cancers. Their cytotoxic effect on renal and colorectal cancer cell lines was investigated in comparison to Doxorubicin. The selectivity of the isolated compounds was evaluated on normal human fetal fibroblast cell lines. The isolated 4'-p-hydroxybenzoylisorhamnetin-3,7-di-O-rhamnoside showed very potent cytotoxic activity against the tested cell lines with the highest selectivity. CONCLUSION:  CS seeds can be used as a source of bioactive flavonoid derivatives that can be incorporated in pharmaceutical industries.

Molecular Interactions between Tau Protein and TIA1: Distinguishing Physiological Condensates from Pathological Fibrils.

The interaction of tau protein with other key proteins essential for stress granule formation determines their functional and pathological impact. In a biological framework, the synergy between Alzheimer's associated tau protein and the stress granule core protein TIA1 is widely recognized. However, the molecular details of this association remain unclear. In this study, we throw light on the importance of the state in which the TIA1 exists in mediating its association with the tau protein. Investigations were carried out on the three repeat constructs of tau (K19) and different structures formed by TIA1. Specifically, the condensate formed by TIA1 full-length (TIA1-FL) protein as well as fibril formed by low complexity domain of TIA1 (TIA1-LCD). The dynamics of K19 inside TIA1-FL condensates and the aggregation kinetics of K19 in the presence of TIA1-LCD fibrils were examined using various biophysical techniques. Relaxation-based solution NMR spectroscopic investigations suggest a weak interaction with TIA1 condensates and indicated a reduction in the dynamics of K19 within these TIA1 condensates. In contrast, a significant interaction was observed between K19, and TIA1-LCD fibrils primarily mediated through 321KCGS324 and 306VQIVYKPVDLSKV317. Our findings emphasize that the interaction between Tau and TIA1 varies depending on whether TIA1 is in its physiological condensate form or its pathological fibril state.

Synthesis, characterization, and crystal structure of hexa-kis-(1-methyl-1H-imidazole-κN 3)zinc(II) dinitrate.

The synthesis of the title compound, [Zn(C4H6N2)6](NO3)2, is described. This complex consists of a central zinc metal ion surrounded by six 1-methyl-imidazole ligands, charge balanced by two nitrate anions. The complex crystallizes in the space group P. In the crystal, the nitrate ions are situated within the cavities created by the [Zn(N-Melm)6]2+ cations, serving as counter-ions. The three oxygen atoms of the nitrate ion engage in weak C-H⋯O inter-actions. In addition to single-crystal X-ray diffraction analysis, the complex was characterized using elemental analysis, 1H NMR, 13C NMR, and FTIR spectroscopy.

Characterization of the intracellular polyphosphate granules of the phototrophic green sulfur bacterium Chlorobaculum tepidum.

The ability to generate polyphosphate (polyP) granules is important for survival for bacteria during resistance to diverse environmental stresses, however the genesis of polyP granules is poorly understood. Chlorobaculum tepidum (Cba tepidum) is a thermophilic green sulfur anoxygenic phototrophic bacterium which uses reduced sulfur compounds as electron donors. The presence of electron rich granules inside the Cba tepidum was reported, but no further information was provided. In this work we used cell thin sections at three different time points of cultivation to observe the biogenesis of the inclusions over time, and the in cell total phosphate concentration was monitored over time as well. Furthermore, the elemental analysis (EDS) of the electron rich inclusions showed the presence of phosphorus and oxygen. The existence of polyphosphate was demonstrated by 31P NMR spectroscopy of cell lysates. Finally, we show that the biogenesis of the phosphorus granules correlates with an abundance of proteins that are closely related to polyphosphate metabolism.