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.

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.

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.

In vivo hypotensive effect of a chemically characterised extract from the leaves of Lippia alba (Mill.) N.E.Br.

Lippia alba (erva-cidreira) is often mentioned in Brazilian ethnopharmacological studies. Although its leaves have been used to treat hypertension, few studies have evaluated its hypotensive effects. This work aimed to evaluate the haemodynamic effects of Lippia alba methanolic extract and to characterise its chemical composition. Normotensive rats received an intravenous injection of L. alba extract. Systolic, diastolic, mean arterial pressures, and electrocardiographic data were analysed.1H-qNMR and LC-MS were used to assess the chemical composition. L. alba extract had significant hypotensive effects on systolic, diastolic, and mean arterial pressure. Acteoside was identified as major compound (292.6 ± 2.7 mg/g). Sixty-one other compounds were tentatively identified, mainly phenylethanoids, flavonoids, and iridoids. L. alba extract reduces systolic, diastolic, mean arterial pressure, and appears to be associated with a reduction in heart rate. Acteoside, a known hypotensive compound, may be responsible for these effects, but other structurally similar minority compounds may also contribute.

Exploration of plasma biomarkers for Alzheimer's disease by targeted lipid metabolomics based on nuclear magnetic resonance (NMR) spectroscopy.

Alzheimer's disease (AD) is the most common cause of dementia, but the disease lacks convenient and cost-effective alternative biomarkers currently. We utilized targeted lipid metabolomics based on nuclear magnetic resonance (NMR) spectroscopy to identify plasma biomarkers in AD patients. Our study was a cross-sectional study that enrolled 58 AD patients and 40 matched health controls (HCs). Firstly, we identified plasma lipid metabolites that were significantly different between the two groups based on P < 0.05 and variable importance in the projection (VIP) > 1. Then we examined the correlation between the lipid metabolites and cognitive function using partial correlation analysis and assessed the diagnostic ability of the lipid metabolites using receiver operating characteristic (ROC) curves. Seventeen lipoproteins showed significant differences between AD patients and HCs among 114 lipid metabolites. All 17 lipoproteins were subtypes of low-density lipoprotein (LDL). Among them, LDL-3 particle number, LDL-3 apolipoprotein-B, LDL-3 phospholipids, LDL free cholesterol and LDL phospholipids were significantly correlated with cognitive function. The ROC curves showed that LDL-2 triglycerides (TG) and LDL-3 TG could significantly distinguish AD patients from HCs, with the area under the curve (AUC) above 0.7. In addition, we explored a strategy of combined diagnosis that significantly improved the diagnostic efficacy for AD (AUC = 0.879). Our study provides insight into the lipoprotein alterations associated with AD and potential biomarkers for its diagnosis and cognitive function assessment.

Rotameric heterogeneity of conserved tryptophan is responsible for reduced photochemical quantum yield in cyanobacteriochrome slr1393g3.

The red/green cyanobacteriochrome (CBCR) slr1393g3 exhibits a quantum yield of only 8% for its forward photoconversion significantly lower than other species from the same CBCR subfamily. The cause for this reduced photoconversion is not yet clear, although in the related NpR6012g4 dark-state structural heterogeneity of a paramount Trp residue has been proposed to cause the formation of nonproductive subpopulation. However, there is no such information on the equivalent residue in slr1393g3, W496. Here we use solid-state NMR to explore all possible sidechain rotamers of this Trp residue and their local interactions at the atomic level. The indole nitrogen (Nε1) is used as an NMR probe, achieved by site-specific 15N-indole labeling of a quadruply Trp-deleted variant and trehalose vitrification technique. The data reveal a set of seven indole rotamers of W496 with four distinct environments for the Nε1-H group. Only a minority population of 20% is found to retain the π-stacking and hydrogen-bonding interactions with the chromophore in the dark state that has been assigned to account for complete forward photoconversion. Our results demonstrate the direct role of W496 in modulating the forward quantum yield of slr1393g3 via rearrangement of its sidechain rotameric conformations.

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.

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.

Determining Absolute Configuration of Small Molecules by Diffusion NMR Experiments.

Enantiomers are ubiquitous in many areas of science, such as pharmaceuticals, agriculture, and food. Nuclear magnetic resonance (NMR) alone is not able to differentiate enantiomers as their spectra are identical. However, these can be distinguished using chiral auxiliaries (such as chiral complexing agents) that form diastereomeric complexes, but absolute identification is still troublesome, usually requiring a chemical reaction with a chiral derivatizing agent. Here, we propose a new method that uses a hybrid mixture of solvating agents in a simple comparison of diffusion NMR experiments, which can discriminate enantiomers in both frequency and diffusion domains, dubbed CHIMERA (CHIral Micelle Enantiomer Resolving Agent). The new method was assessed for twenty-three small chiral molecules using a combination of BINOL and (-)-DMEB, a chiral surfactant, and initial results indicate that absolute configuration can be obtained from a simple experiment.

Simple and Effective Identification of Local 6π- and Global [4n + 2] Aromaticity of Macrocyclic Conjugated Hydrocarbons by 1H/13C Chemical Shifts and the Corresponding Ring Current Effect.

Structures, 1H/13C chemical shifts, and the ring current effects (spatial magnetic properties: through-space NMR shieldings [TSNMRSs]) of various π-conjugated macrocyclic hydrocarbons and the corresponding charged analogues have been calculated at the B3LYP/6-311G(d,p) theory level using the GIAO perturbation method and employing the nucleus-independent chemical shift (NICS) characterization. The spatial magnetic properties (TSNMRS) are visualized as iso-chemical shielding surfaces (ICSSs) of various size and direction and together with especially the δ(1H)/ppm chemical shifts employed to unequivocally qualify and quantify local 6π-aromaticity of individual benzenoid building blocks and the global ([4n + 2], n > 1) aromaticity of the macrocyclic ring.

UV-vis absorbance spectra, molar extinction coefficients and circular dichroism spectra for the two cyanobacterial metabolites anabaenopeptin A and anabaenopeptin B.

The UV-vis absorbance spectra, molar extinction coefficients and circular dichroism spectra, as well as NMR and high resolution tandem mass spectrometry spectra were determined for two prominent secondary metabolites from cyanobacteria, namely anabaenopeptin A and anabaenopeptin B. The compounds were extracted from the cyanobacterium Planktothrix rubescens CBT929 and purified by flash chromatography and HPLC. Exact amounts of isolated compounds were assessed by quantitative 1H-NMR with internal calibrant ethyl 4-(dimethylamino)benzoate in DMSO­d6 at 298 K with a recycle delay (d1) of 120 s. UV-vis absorbance spectra were recorded in methanol at room temperature. Molar extinction coefficients were determined at 278 nm as 4190 M-1 cm-1 and 2300 M-1 cm-1 in methanol for anabaenopeptin A and anabaenopeptin B, respectively. Circular dichroism spectra and secondary fragmentation mass spectra are also reported.

Ligand-Induced Folding in a Dopamine-Binding DNA Aptamer.

Aptamers are often employed as molecular recognition elements in the development of different types of biosensors. Many of these biosensors take advantage of the aptamer having a ligand-induced structure-formation binding mechanism. However, this binding mechanism is poorly understood. Here we use isothermal titration calorimetry, circular dichroism spectroscopy and NMR spectroscopy to study the binding and ligand-induced structural change exhibited by a dopamine-binding DNA aptamer. We analysed a series of aptamers where we shorten the terminal stem that contains the 5´ and 3´termini of the aptamer sequence. All aptamers bind dopamine in an enthalpically driven process coupled with an unfavorable entropy. A general trend of the aptamer having a weaker binding affinity is observed as the terminal stem is shortened. For all aptamers studied, numerous signals appear in the imino region of the 1H NMR spectrum indicating that new structure forms with ligand binding. However, it is only when this region of structure formation in the aptamer is brought close to the sensor surface that we obtain a functional electrochemical aptamer-based biosensor.

Exploring the dynamics and interactions of the N-myc transactivation domain through solution NMR.

Myc proteins are transcription factors crucial for cell proliferation. They have a C-terminal domain that mediates Max and DNA binding, and an N-terminal disordered region culminating in the transactivation domain (TAD). The TAD participates in many protein-protein interactions, notably with kinases that promote stability (Aurora-A) or degradation (ERK1, GSK3) via the ubiquitin-proteasome system. We probed the structure, dynamics and interactions of N-myc TAD using nuclear magnetic resonance (NMR) spectroscopy following its complete backbone assignment. Chemical shift analysis revealed that N-myc has two regions with clear helical propensity: Trp77-Glu86 and Ala122-Glu132. These regions also have more restricted ps-ns motions than the rest of the TAD, and, along with the phosphodegron, have comparatively high transverse (R2) 15N relaxation rates, indicative of slower timescale dynamics and/or chemical exchange. Collectively these features suggest differential propensities for structure and interaction, either internal or with binding partners, across the TAD. Solution studies on the interaction between N-myc and Aurora-A revealed a previously uncharacterised binding site. The specificity and kinetics of sequential phosphorylation of N-myc by ERK1 and GSK3 were characterised using NMR and resulted in no significant structural changes outside the phosphodegron. When the phosphodegron was doubly phosphorylated, N-myc formed a robust interaction with the Fbxw7-Skp1 complex, but mapping the interaction by NMR suggests a more extensive interface. Our study provides foundational insights into N-myc TAD dynamics and a backbone assignment that will underpin future work on the structure, dynamics, interactions and regulatory post-translational modifications of this key oncoprotein.

In-situ Monitoring of Photocatalysis on Polymeric Carbon Nitride Thin Films.

Polymeric carbon nitride has attracted significant interest in heterogeneous photocatalysis due to its activity under visible-light irradiation. Herein, we report on using carbon nitride-coated NMR tubes for in-situ studies of photocatalytic reaction mechanisms. In a first step, we exploited carbon nitride-coated crimp vials as batch photoreactors for visible photocatalytic fluorinations of unactivated C(sp3)-H bonds, with moderate to excellent yields and reusability over multiple cycles. Eventually, carbon nitride-coated NMR tubes were used as a photoreactor by coupling them with optical fiber irradiation directly inside the spectrometer. This enabled us to follow the reaction with in-situ NMR spectroscopy identifying reactive intermediates otherwise elusive in conventional analyses. The method provides advantages for the study of photocatalytic mechanisms of complex reactions and substantially reduces the need of comparative tests for depicting reaction intermediates and conversion pathways.

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.

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.