The aim of this article is to summarize recent bioinformatic and statistical developments applicable to NMR-based metabolomics. Extracting relevant information from large multivariate datasets by statistical data analysis strategies may be of considerable complexity. Typical tasks comprise for example classification of specimens, identification of differentially produced metabolites, and estimation of fold changes. In this context it is of prime importance to minimize contributions from unwanted biases and experimental variance prior to these analyses. This is the goal of data normalization. Therefore, special emphasize is given to different data normalization strategies. In the first part, we will discuss the requirements and the pros and cons for a variety of commonly applied strategies. In the second part, we will concentrate on possible solutions in case that the requirements for the standard strategies are not fulfilled. In the last part, very recent developments will be discussed that allow reliable estimation of metabolic signatures for sample classification without prior data normalization. In this contribution special emphasis will be given to techniques that have worked well in our hands.

Model animals are employed in experiments as substitutes for human tissues and fluids, particularly when accessing particular human samples (such as cerebrospinal fluid, brain, ocular tissues, etc.) poses significant challenges or is ethically constrained. Nonhuman primates are frequently regarded as superior animal models for investigating human ophthalmological diseases. However, despite this recognition, the metabolomic composition of ocular tissues in non-human primates remains predominantly unexplored. In this work, we present a dataset on metabolite concentrations in serum and ocular tissues, including aqueous humor (AH), vitreous humor (VH), and lens, in two Macaque species: crab-eating macaque (Macaca fascicularis) and rhesus macaque (Macaca mulatta). A total of 99 compounds were quantified in 45 samples, shedding light on the previously unknown metabolomic profiles of primate eye tissues.

Graviera is a very popular yellow hard cheese produced in mainland Greece and the Aegean islands, and in three PDO (Protected Denomination of Origin) locations. Apart from geographic location, milk type and production practices are also factors that affect cheese composition, and make this dairy product unique in taste and aroma. In this work, 1H NMR spectroscopy in combination with chemometrics has been used to determine the metabolite profile (40 compounds) of graviera cheese produced in different geographic locations, with emphasis on cheeses produced on the island of Crete. Organic acids and amino acids were the main components quantified in the polar cheese fraction, while the fatty acid composition of the lipid fraction was also obtained. Analysis of Variance (Anova) of the dataset showed that γ-aminobutyric acid (GABA), conjugated linoleic acids (CLA) and linoleic acid differentiate gravieras produced in different areas of Crete, and that total amino acid content was higher in cheeses produced in eastern Crete. Targeted Discriminant Analysis models classified gravieras produced in mainland Greece, Cyclades and Crete based on differences in 1,2-diglycerides, sterols, GABA and fatty acid composition. Targeted and untargeted OPLS-DA models were capable of differentiating between gravieras produced in the island of Crete and hold promise as the basis for the authentication of PDO graviera products.

Background: The official standard for quality control of honey is currently based on physicochemical properties. However, this method is time-consuming, cost intensive, and do not lead to information on the originality of honey. This study aims to classify raw stingless bee honeys by bee species origins as a potential classifier using NMR-LCMS-based metabolomics approach. Methods: Raw stingless bee honeys were analysed and classified by bee species origins using proton nuclear magnetic resonance (1H NMR) spectroscopy and an ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-QTOF MS) in combination with chemometrics tools. Results: The honey samples were able to be classified into three different groups based on the bee species origins of Heterotrigona itama, Geniotrigona thoracica, and Tetrigona apicalis. D-Fructofuranose (H. itama honey), β-D-Glucose, D-Xylose, α-D-Glucose (G. thoracica honey), and L-Lactic acid, Acetic acid, L-Alanine (T. apicalis honey) identified via 1H NMR data and the diagnostic ions of UHPLC-QTOF MS were characterized as the discriminant metabolites or putative chemical markers. Conclusion: It could be suggested that the quality of honey in terms of originality and purity can be rapidly determined using classification technique by bee species origins via 1H NMR- and UHPLC-QTOF MS-based metabolomics approach.

The breeding of stress-tolerant cultivated plants that would allow for a reduction in harvest losses and undesirable decrease in quality attributes requires a new quality of knowledge on molecular markers associated with relevant agronomic traits, on quantitative metabolic responses of plants on stress challenges, and on the mechanisms controlling the biosynthesis of these molecules. By combining metabolomics with genomics, transcriptomics and proteomics datasets a more comprehensive knowledge of the composition of crop plants used for food or animal feed is possible. In order to optimize crop trait developments, to enhance crop yields and quality, as well as to guarantee nutritional and health factors, that provides the possibility to create functional food or feedstuffs, the knowledge about the plants’ metabolome is crucial. Next to classical metabolomics studies, this review focusses on several metabolomics based working techniques, such as sensomics, lipidomics, hormonomics and phytometabolomics, which were used to characterize metabolome alterations during abiotic and biotic stress, to find resistant food crops with a preferred quality or at least to produce functional food crops are highlighted.

NMR spin relaxation times have been an instrumental tool in deciphering the local en-vironment of ionic species, the various interactions they engender and the effect of these interactions on their dynamics in conducting media. Of particular importance has been their application in studying the wide range of electrolytes for energy storage, on which this chapter is based. Here we highlight some of the research carried out on electrolytes in recent years using NMR relaxometry techniques. Specifically, studies on liquid electro-lytes, such as ionic liquids and organic solvent; on semi-solid-state electrolytes, such as ionogels and polymer gels, and solid electrolytes such as glasses, glass ceramics and polymers. Although this chapter focuses on a small selection of materials, we believe they demonstrate the breadth of application and the invaluable nature of NMR relaxometry.

Synthesis of biocompatible and bioresorbable composite materials such as “polymer matrix-mineral constituent,” which stimulate the natural growth of living tissues and the restoration of damaged parts of the body, is one of the challenging problems in regenerative medicine and material science. Composite films of bioresorbable polymer of polyvinylpyrrolidone (PVP) and hydroxyapatite (HA) were obtained. HA was synthesized in situ in a solution of polymer mixture. We applied electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) approaches to study their properties. EPR in two frequency ranges allowed to derive spectroscopic parameters of the nitrogen-based light and radiation-induced paramagnetic centers in HA, PVP and PVP-HA with high accuracy. It was shown that PVP did not significantly affect the EPR spectral and relaxation parameters of the radiation-induced centers in HA. Magic angle spinning (MAS) 1H NMR shows the presence of two signals at 4.7 ppm and -2.15 ppm attributed to “free” water and hydroxyl groups, while the single line - to 31P. NMR relaxation measurements for 1H and 31P show that the relaxation decays are a multicomponent process that can be described by three components of the transverse relaxation times. The obtained results demonstrated that the applied magnetic resonance methods can be used for the control of quality of the synthesis of the PVP-HA composites and, potentially, for the development of analytical tools to follow the processes of the sample treatment, resorption, and degradation.

Glycine in its neutral form can exist in the gas phase while its zwitterion form is more stable in water solution.But how many waters are actually necessary to stabilize the zwitterionic structure in the gas phase? Are the intramolecular isotropic spin spin coupling constants sensitive enough to accuse the change in the environment? or the conformer observed? These and related questions have been investigated by a computational study at the level of density functional theory employing the B3LYP functional and the 6-31++G**-J basis set. We found that at least two water molecules explicitly accounted in the super-molecule structure are necessary to stabilize both conformers of glycine within a water polarizable continuum model. At least half of the SSCC’s of both conformers are very stable to changes in the environment and at least four of them differ significantly between Neutral and Zwitterion conformation.

The dysregulation of cellular metabolism is a hallmark of ageing. To understand the metabolic changes that occur as a consequence of the ageing process and to find biomarkers for age-related diseases, we conducted a metabolomic analysis of brain, heart, kidney, liver, lung and spleen in young (9-10 weeks) and old (96-104 weeks) wild type (mixed genetic background of 129/J and C57BL/6) mice using NMR spectroscopy. We found differences in metabolic fingerprints of all tissues and identified several metabolites to be altered in most tissues, suggesting that they may be universal biomarkers of ageing. In addition, we found distinct tissue-clustered sets of metabolites throughout the organism. The associated metabolic changes may reveal novel therapeutic targets for the treatment of ageing and age-related diseases. Moreover, the identified metabolite biomarkers could provide a sensitive molecular read-out to age determine the age of biologic tissues and to validate the effectiveness and potential off-target effects of senolytic drug candidates on both a systemic and tissue-specific level.

: Extra- and intra-cellular activity occurs under the direction of numerous inter-molecular interactions, and in any tissue or cell, molecules are densely packed, thus promoting those molecular interactions. Galectins and chemokines, the focus of this review, are small, protein effector molecules that mediate various cellular functions, in particular cell adhesion and migration, as well as cell signaling/activation. In the past, researchers have reported that combinations of these (and other) effector molecules act separately, yet sometimes in concert, but nevertheless physically apart and via their individual cell receptors. This view that each effector molecule functions independently of the other limits our thinking about functional versatility and cooperation, and, in turn, ignores the prospect of physiologically important inter-molecular interactions, especially when both molecules are present or co-expressed in the same cellular environment. This review is focused on such protein-protein interactions with chemokines and galectins, their homo- and hetero-oligomeric structures that they can form, and the functional consequences of those paired interactions.

The formation of inclusion complexes between alkylsulfonate guests and a cationic pillar[5]arene receptor in water was investigated by NMR and ITC techniques. The results show the formation of host-guest complexes stabilized by electrostatic interactions and hydrophobics effects with binding constants of up to 107 M-1 for the guest with higher hydrophobic character. Structurally, the alkyl chain of the guest is included in the hydrophobic aromatic cavity of the macrocycle while the sulfonate groups are hold in the multicationic portal by ionic interactions.

The recent revival of the study of organic natural products as renewable sources of medicinal drugs, cosmetics, dyes, and materials motivated the creation of general-purpose structural databases. Dereplication, the efficient identification of already reported compounds, relies on the grouping of structural, taxonomic and spectroscopic databases that focus on a particular taxon (species, genus, family, order…). A set of freely available python scripts, CNMRPredict, is proposed for the quick supplementation of taxon-oriented search results from the LOTUS database (lotus.naturalproducts.net) with predicted carbon-13 NMR data from the ACD/Labs (acdlabs.com) CNMR predictor and DB software to provide easily searchable databases. The database construction process is illustrated using Brassica rapa as taxon example.

In the last 20 years, a huge amount of experimental results about halogen bonding (XB) has been produced. Most of the systems have been characterized by solid state X-ray crystallography, whereas in solution the only routine technique is the titration (by using ¹H and ¹⁹F NMR, IR, UV-Vis or Raman spectroscopies, depending on the nature of the system), with the aim of characterizing the strength of the XB interaction. Unfortunately, the titration techniques have many intrinsic limitations and they should be coupled with other, more sophisticated techniques to have an accurate and detailed description of the geometry and stoichiometry of the XB adduct in solution. In this review, it will be shown how crucial information about XB adducts can be obtained by advanced NMR techniques, as Nuclear Overhauser Effect-based Spectroscopies (NOESY, ROESY, HOESY…) and diffusion NMR techniques (PGSE or DOSY).

Alzheimer’s Disease (AD) affects the quality of life of millions of people worldwide and represents one of the biggest challenges for the whole society. The SH-SY5Y neuroblastoma cell line is often used as an in vitro model of neuronal function and is widely applied to study the molecular events leading to AD. In the last few years, basic research on SH-SY5Y cells has provided interesting insights for the discovery of new drugs and biomarkers for improved AD treatment and diagnosis. At the same time, untargeted NMR metabolomics is widely applied on biological fluids for (i) metabolic profile analysis, (ii) screening for differential metabolites, (iii) analysis of metabolic pathways, and (iv) the discovery of new biomarkers. Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) have proved to be powerful methods for processing NMR data, being useful in signal quantization, even if more sophisticated --- typically non--linear --- techniques are needed to obtain compact yet information--rich embeddings for complex spectra. In this paper, a compression technique based on convolutional autoencoders is proposed, which can perform a high dimensionality reduction of the spectral signal (up to more than 300 times), maintaining informative features (guaranteed by a reconstruction error always smaller than 5%). Moreover, before compression, an ad hoc preprocessing method was devised to remedy the scarcity of available data. The compressed spectral data were then used to train some SVM classifiers to distinguish diseased from healthy cells, achieving an accuracy close to 78%, a significantly better performance with respect to using PCA--compressed data.

Functional osseoconductive coatings based on hydroxylapatite (HAp) and applied preferentially by atmospheric plasma spraying to medical implant surfaces are a mainstay of modern implantology. During contact with the hot plasma jet, HAp particles melt incongruently and undergo complex dehydration and decomposition reactions that alter their phase composition and crystallographic symmetry, and thus, the physical and biological properties of the coatings. Surface analytical methods such as laser-Raman and nuclear magnetic resonance (NMR) spectroscopies are useful tools to assess the structural changes of HAp imposed by heat treatment during their flight along the hot plasma jet. In this contribution, the controversial information on the existence or non-existence of oxyapatite, i.e. fully dehydrated HAp as a thermodynamically stable compound is highlighted.

Metabolomics is a powerful set of methods that uses analytical techniques to identify and quantify metabolites in biological samples, providing a snapshot of the metabolic state of a biological system. In medicine, metabolomics can help diagnose diseases, reveal molecular basis of a disease, and monitor treatment responses, while in agriculture, it can improve crop yields and plant breeding. However, animal metabolomics faces several challenges due to the complexity and diversity of animal metabolomes, the lack of standardized protocols, and the difficulty in interpreting metabolomic data. The current dataset includes quantitative metabolomic profiles of eye lens tissues from 26 bird species (111 specimens) that can aid researchers in developing new experiments, mathematical models, and integrations with other omics data. The dataset includes raw 1H NMR spectra, protocols for sample preparation, and data preprocessing with the final table containing information on 89 reliably identified and quantified metabolites. The dataset is quantitative, making it relevant for supplementing with new specimens or comparison groups, followed by data mining and expected new interpretations. It was obtained from bird specimens collected in compliance with ethical standards and reveals potential differences in metabolic pathways due to phylogenetic difference or environmental exposure.

Objectives: One of the home-made herbal remedy being traditionally used against gout is evaluated to identify any new and characteristic marker compound which may be used as characteristic analytical marker to standardize this polyherbal remedy. Materials and method: A distinct and remarkable peak in the HPLC chromatogram of remedy was identified which was further isolated by fraction collector. This fraction was then evaporated and characterized for structure elucidation using set of various techniques such as RP-HPLC,UV, FTIR, TLC, EIMS and NMR. Results: The compound characterized was non polar compound, ester of betulin and palmitoleic acid. Although the remedy has been standardized using set of four markers as discussed in literature but this newly developed marker compound can be used to further standardize this remedy and other herbal preparations comprising of this marker compound.Conclusion: Hence the isolated compound can be used as novel analytical marker in the standardization of this herbal remedy and the other herbal preparations containing such marker compound

Artic root is a well-known plant adaptogen with multipotential pharmacological properties. Thin-layer chromatography (TLC) – screening followed by diode-array high-performance liquid chromatography and nuclear magnetic resonance spectroscopy proved to be a reliable and convenient method for simultaneous determination of quality of various herbal raw materials and supplements. This combination allowed for comparing and differentiating arctic root samples as well as defining their authenticity. The study provided information on the chemical and biological properties of the seven chosen samples as well as qualitative and quantitative evaluation of the quality markers: rosavin, salidroside, and p-tyrosol. The absence of rosavin, salidroside, and p-tyrosol in three samples was detected using TLC-screening and confirmed by HPLC-DAD and NMR. The paper highlighted the importance of quality control and strict regulation for herbal medicine supplements and preparations.

Novel sensing technologies for liquid biopsies offer a promising prospect for the early detection of metabolic conditions through -omics techniques. Indeed, high-field NMR facilities are routinely used for metabolomics investigations on a range of biofluids in order to rapidly recognize unusual metabolic patterns in patients suffering from a range of diseases. However, these techniques are restricted by the prohibitively large size and cost of such facilities, suggesting a possible role for smaller, low-field NMR instruments in biofluid analysis. Herein we describe selected biomolecule validation on a low-field benchtop NMR spectrometer (60 MHz), and present an associated protocol for the analysis of biofluids on compact NMR instruments. We successfully detect common markers of diabetic control at low-to-medium concentrations through optimized experiments, including glucose (≤ 2.6 mmol./L) and acetone (25 μmol./L), and additionally in readily-accessible biofluids. We present a combined protocol for the analysis of these biofluids with low-field NMR spectrometers for metabolomics, and offer a perspective on the future of this technique appealing to point-of-care applications.

1-[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]methanamine compound has been successfully synthesized by reacting p-Toluic hydrazide and glycine via polyphosphoric acid condensation route. The course of the reaction was found to be high yielding and the title compound was spectroscopically characterized by FT-IR, DSC, 13C/1H-NMR and Mass spectrometric techniques.

Pulsed-field gradient (PFG) diffusion experiments can be used to measure anomalous diffusion in many polymer or biological systems. However, it is still complicated to analyze PFG anomalous diffusion, particularly the finite gradient pulse width (FGPW) effect. In practical applications, the FGPW effect may not be neglected such as in clinical diffusion magnetic resonance imaging (MRI). Here, two significantly different methods are proposed to analyze PFG anomalous diffusion: the effective phase shift diffusion equation (EPSDE) method and an observing the signal intensity at the origin method. The EPSDE method describes the phase evolution in virtual phase space, while the method to observe the signal intensity at the origin describes the magnetization evolution in real space. However, these two approaches give the same general PFG signal attenuation including FGPW effect, which can be numerically evaluated by a direct integration method. The direct integration method is fast and without overflow. It is a convenient numerical evaluation method for Mittag-Leffler function type PFG signal attenuation. The methods here provide a clear view of spin evolution under field gradient, and their results will help the analysis of PFG anomalous diffusion.

Four new unusual pentacyclic triterpenoids (1-4) were isolated from the roots of Jasminum sambac (L.) Ait. Their structures were elucidated by 1D and 2D NMR analysis, single crystal X-ray diffraction and HRESIMS.

For more than twenty years chemists use peptidic organocatalysts to transfer acyl groups onto alcohols. The goal was and still is the increase of selectivity towards the substrate of choice. One carefully designed example is a highly enantioselective tetrapeptide organocatalyst for the kinetic resolution of trans-cycloalkane-1,2-diols. Until now, the intermediate responsible for the acyl transfer of this tetrapeptide has never been directly observed. It was proposed computationally that a π-methylhistidine moiety is acylated as an intermediate step in the catalytic cycle. In this study we set out to investigate whether we can detect and characterize this key intermediate using NMR-spectroscopy and mass spectrometry. Different mass spectrometric experiments using a nano-ElectroSpray Ionization (ESI) source and tandem MS-techniques allowed the identification of tetrapeptide acylium ions using different acylation reagents. The complexes of trans-cyclohexane-1,2-diols with the tetrapeptide were also detected. Additionally, we were able to detect acylated tetrapeptides in solution using NMR-spectroscopy and monitor the acetylation reaction of a trans-cyclohexane-1,2-diol. These findings are important steps towards the understanding of this organocatalyst and its high enantioselectivity.

A 9.4 T/400 MHz superconducting magnet with a warm bore of 60 mm is being designed. The 400 MHz NMR magnet is composed of 9 coaxial coils to generate a central field of 9.4 T. The magnetic field homogeneity is better than 4 ppm for 50 mm diameter spherical volume (DSV) and better than 1 ppb for 5 mm DSV. Three types of NbTi strands were used to reduce the cost of the superconducting materials. A preload with 80 MPa was exerted on the superconducting coils and over-banding with stainless steel was adopted to release the stress from the electromagnetic forces. The magnet will be operated in persistent mode with superconducting joints to reduce the field decay. The pulse tube cryocooler with a cooling capacity of 1 W at 4.2 K can be used to reduce the liquid helium evaporation and vibration during operation. The magnet will be equipped with Bi2223/AgAu HTS current leads to reduce the heat losses. In this paper, the design of the 9.4 T NMR superconducting coils, electromagnetic field calculations, and stress analysis of the superconducting coils were presented.

Bulgaria and North Macedonia have a long history of production and use of honey, however, there is an obvious lack of systematic and in-depth research on honey from both countries. Of particular interest is the oak honeydew honey, highly valued by consumers because of its health benefits. Aim of this study was to characterize honeydew and floral honeys from Bulgaria and North Macedonia based on their NMR profiles. 1D and 2D 1H and 13C NMR spectra were measured of 16 North Macedonian and 22 Bulgarian honey samples. 25 individual substances were identified, including quinovose, which was found for the first time in honey. Chemometric methods (PCA - principal component analysis, PLS-DA - partial least squares discriminant analysis, ANOVA) were used to detect similarities and differences between samples, as well as to determine their botanical and geographical origin. Semiquantitative data on individual sugars and some other constituents were obtained; which allowed reliable classification of honey samples by botanical and geographical origin, based on chemometric approaches. The results enabled to distinguish oak honeydew honey from other honey types, and to determine the country of origin. NMR was a rapid and convenient method, avoiding the need for other more time-consuming analytical techniques.

The potential to affect gene expression via G-quadruplex stabilization has been extended to all domains of life, including viruses. Here, we investigate the polymorphism and structures of G-quadruplexes of the human papillomavirus type 52 with UV, CD and NMR spectroscopy and gel electrophoresis. We show that oligonucleotide with five G-tracts folds into several structures and that naturally occurring single nucleotide polymorphisms (SNPs) have profound effects on the structural polymorphism in the context of G-quadruplex forming propensity, conformational heterogeneity and folding stability. With help of SNP analysis, we were able to select one of the predominant forms, formed by G-rich sequence d(G3TAG3CAG4ACACAG3T). This oligonucleotide termed HPV52(1-4) adopts a three G-quartet snap back (3+1) type scaffold with four syn guanine residues, two edgewise loops spanning the same groove, a no-residue V loop and a propeller type loop. The first guanine residue is incorporated in the central G-quartet and all four-guanine residues from G4 stretch are included in the three quartet G-quadruplex core. Modification studies identified several structural elements that are important for stabilization of the described G-quadruplex fold. Our results expand set of G-rich targets in viral genomes and address the fundamental questions regarding folding of G-rich sequences.

Liquid-liquid phase separation (LLPS) of proteins has been found ubiquitously in eukaryotic cells, critical in the controlling of many biological processes through forming a temporary condensed phase with different bimolecular components. TDP-43 is recruited to stress granules in cells and is the main component of TDP-43 granules and proteinaceous amyloid inclusions in patients with amyotrophic lateral sclerosis (ALS). TDP-43 low complexity domain (LCD) is able to demix in solution forming the protein condensed droplets. The molecular interactions regulating its LLPS were investigated at the protein fusion equilibrium stage, where the droplets stopped growing. We found the molecules in the droplet were still liquid-like but with enhanced intermolecular helix-helix interaction in the LCD. The protein would start to aggregate after about 200 minutes of lag time and aggregate slower than at the condition when the protein does not phase separate or the molecules have a reduced intermolecular helical interaction. A structural transition intermediate towards protein aggregation was also discovered involving a decrease of the intermolecular helix-helix interaction and a reduction in the helicity. Therefore, LLPS and the intermolecular helical interaction could help maintain the stability of TDP-43 LCD.

Energy metabolism, including alterations in energy intake and expenditure, is closely related to aging and longevity. Metabolomics studies have recently unraveled changes in metabolite composition in plasma and tissues during aging and have provided critical information to elucidate the molecular basis of aging process. However, the metabolic changes in tissues responsible for food intake and lipid storage have remained unexplored. In this study, we aimed to investigate aging-related metabolic alterations in these tissues. To fill this gap, we employed NMR-based metabolomics in several tissues, including different parts of the intestine (duodenum, jejunum, ileum) and brown/white adipose tissues (BAT, WAT) of young (9-10 weeks) and old (96-104 weeks) wild-type (mixed genetic background of 129/J and C57BL/6) mice. We further included plasma and skeletal muscle of the same mice to verify previous results. Strikingly, we found that duodenum, jejunum, ileum, and WAT do not metabolically age. In contrast, plasma, skeletal muscle, and BAT show a strong metabolic aging phenotype. Overall, we provide first insights into the metabolic changes of tissues essential for nutrient uptake and lipid storage and have identified biomarkers for metabolites that could be further explored to study the molecular mechanisms of aging.

Two secondary waste materials, municipal incinerator bottom ash (MIBA) and sewage sludge ash (SSA), were mixed with clay for ceramic manufacturing in this study. Specimens with 5 different MIBA replacement amount of 0%, 5%,10%, 15%, and 20%(wt) and 3 different SSA replacement amount of 0%, 10%, and 20%(wt) were prepared and then a series of tests and analysis were conducted to investigate how the two materials affect the quality of the final product and to what extent. It concludes that a mix with up to 20% of SSA and 5% of MIBA could result in quality tiles complying with specifications for interior or exterior flooring applications at certain kiln temperatures.

The food additive sorbic acid is considered as an effective preservative for certain cereal products, and propionic acid is commonly added in bakery wares, e.g. bread and fine bakery wares. The aim of this study is to develop and validate a new nuclear magnetic resonance spectroscopy (¹H NMR) method for routine screening and quantification of sorbic and propionic acids in bread and several bakery products for quality control purposes. Results showed that none of the screened samples contained higher concentrations than regulatory maximum limits. However, for some samples, labelling of preservatives was lacking or they were used in food categories, for which the use is not approved. It can be concluded that the developed NMR method can be used for routine screening of bakery products.

Samples of sunflower Waste Cooking Oils (WCOs) subjected to several cycles of frying were treated with water under four different combinations of temperature and pH. The variation of the chemical composition of the oil samples was monitored through headspace Solid-Phase Microextraction (HS-SPME) coupled with gas-chromatographic technique (GC), ¹H NMR spectroscopy and ESI⁺ mass spectrometry measurements. The possibility to modify the chemical composition of the recycled vegetable oil by tuning the water treatment parameters was exploited for the designing of a mini-plant for the production of bio-lubricants. A full description of the proposed prototype is also reported.

Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44 (Leu-8, Ile-44, Val-70). Nevertheless, a variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD recognizes ubiquitin. Here, we describe the structure of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis and NMR spectroscopy of the ¹⁵N isotopically labelled protein, we demonstrate that a Phe-Pro motif in N4BP1 recognizes the canonical hydrophobic patch of ubiquitin. This recognition mode resembles the molecular mechanism evolved in the CUE (Coupling of ubiquitin conjugation to ER degradation) domain family, where an invariant proline, usually following a phenylalanine, is required for binding to ubiquitin. Interestingly, the UBD of N4BP1 is evolutionary related to CUBAN (Cullin binding domain associating with NEDD8) (40% identity and 47% similarity), a protein module that also recognizes the ubiquitin-like NEDD8, which is the closest relative of ubiquitin (58% identity and 80% similarity). By performing circular dichroism and ¹⁵N NMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the UBD of N4BP1 lacks the NEDD8 binding properties observed in CUBAN and it recognizes the Ile44-centered patch of ubiquitin through a dedicated binding site, which share some of the features observed in the CUE domain family. Moreover, we show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both the CUBAN and the UBD of N4BP1 are poly-ubiquitinated in cells. This modification is dependent on the presence of a functional domain. We believe that the structural and functional characterization of this novel UBD will allow a deeper understanding of the molecular mechanisms governing N4BP1 function, while at the same time providing a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved.

The synthesis of four samples of new polyurethanes was evaluated by changing the ratio of the diol monomers used, poly(propylene glycol) (PPG) and D-isosorbide, in the presence of aliphatic isocyanates such as the isophorone diisocyanate (IPDI) and 4,4′-methylenebis(cyclohexyl isocyanate) (HMDI). The thermal properties of the four polymers obtained were determined by DSC, exhibiting Tg values in the range 55-70 ºC, and their molecular structure characterized by FTIR, 1H and 13C NMR spectroscopies. The diffusion coefficients of these polymers in solution were measured by the Pulse Gradient Spin Echo (PGSE) NMR method, enabling the calculation of the corresponding hydrodynamic radii in diluted solution (1.62–2.65 nm). The molecular weights were determined by GPC/SEC and compared with the values determined by quantitative 13C NMR analysis. Finally, the biocompatibility of the polyurethanes was assessed using the HaCaT keratinocyte cell line by the MTT reduction assay method showing values superior to 70% cell viability.

Soups show diverse health functions, which should be related with their nutrient profiles. NMR spectroscopy is a robust, sensitive and rapid method to unveil or identify the chemical composition of food or food-derived metabolites. In the current study, 1H-NMR spectroscopy approach was applied to identify the metabolic profiling of two kinds of home-cooked freshwater fish soups (crucian carp and snakehead fish) before and after in vitro gastrointestinal digestion. The nutritional profiles of these two kinds of soups were identified at first. With the help of OPLS-DA method (Orthogonal Partial Least Squares Discriminant Analysis), these two different kinds freshwater fish under different states were completely discriminated. The metabolites changes in digested fish soups could reveal the information of chemical compounds which play important roles in the body. Furthermore, the metabolic patterns of different kinds of fish soups could reflect the various nutrition profiling characteristics for dietary therapy.

Nanostructured materials have already become an integral part of our daily life. In many applications ion mobility decisively affects the performance of, e.g., batteries and sensors. Nanocrystalline ceramics often exhibit enhanced transport properties due to their heterogeneous structure showing crystalline (defect-rich) grains and disordered interfacial regions. In particular, anion conductivity in nonstructural binary fluorides easily exceeds that of their coarse-grained counterparts. To further increase ion dynamics aliovalent substitution is a practical method to influence the number of (i) defect sites and (ii) the charge carrier density. Here, we used high energy-ball milling to incorporate Y³⁺ ions into the cubic structure of SrF₂. As compared to pure nanocrystalline SrF2 the ionic conductivity of Sr_1-xYxF_2+x with x = 0.3 increased by 4 orders of magnitude reaching 0.8 x 10 ^-5 S/cm^-1 at 450 K. We discuss the effect of YF₃ incorporation on conductivities isotherms determined by both activation energies and Arrhenius pre-factors. The enhancement seen is explained by size mismatch of the cations involved, which are forced to form a cubic crystal structure with extra F anions if x is kept smaller than 0.5

The blood-brain barrier (BBB) is a selectively permeable boundary that separates the circulating blood from the extracellular fluid of the brain and is an essential component for brain homeostasis. In glioblastoma (GBM), the BBB of peritumoral vessels is often disrupted. Pericytes, being important to maintain the BBB integrity, can be functionally modified by GBM cells by inducing proliferation and cell motility via the TGF-β-mediated induction of central epithelial to mesenchymal transition (EMT) factors., We demonstrate that pericytes strengthen the integrity of the BBB in primary endothelial cell/pericyte co-cultures as in vitro BBB model, using TEER measurement of the barrier integrity. In contrast, this effect was abrogated by TGF-β or conditioned medium from TGF-β secreting GBM cells, finally leading to the disruption of a so far intact and tight BBB. TGF-β dramatically changed the metabolic behavior of pericytes, such as shutting down the TCA cycle, driving energy generation from oxidative phosphorylation towards glycolysis, and by shifting the cells towards the activation of pathways that are necessary to produce molecules used for proliferation and cell division. Furthermore, combined metabolomics and RNASeq analyses indicated that the observed functional changes of TGF-β-treated pericytes are closely connected with their behavior.

Multinuclear NMR studies of the gaseous mixtures that involve volatile compounds and ³He atoms are featured in this review. Precise analyses of the ³He and other nuclei resonance frequencies show linear dependences on the gas density. Extrapolation of the gas phase results to the zero-pressure limit gives ν₀(³He) and ν₀(ⁿX) resonance frequencies of nuclei in a single 3-helium atom and nuclei in molecules at a given temperature. The NMR frequency comparison method provides an approach for determining different nuclear magnetic moments. Application of quantum chemical shielding calculations which include a more complete and careful theoretical treatment allows the shielding of isolated molecules to be achieved with great accuracy and precision. They are used for evaluation of nuclear moments without shielding impact on bare nuclei: ^10/11B, ¹³C, ¹⁴N, ¹⁷O, ¹⁹F, ²¹Ne, ²⁹Si, ³¹P, ³³S, ^35/37Cl, ³³S , ⁸³Kr, ^129/131Xe, and ¹⁸³W. On the other hand, new results of nuclear moments were used for reevaluation of absolute nuclear magnetic shielding in molecules under study. Additionally, ³He gas in water solutions of lithium and sodium salts was used for measurements of ^6/7Li and ²³Na magnetic moments and reevaluation of shielding parameters of Li⁺ and Na⁺ water solvated cations. In this paper, guest ³He atoms that play a role in probing the electron density in many host macromolecules are presented as well.

Acrylamide is probably carcinogenic to humans (International Agency for Research on Cancer, group 2A) with major occurrence in heated, mainly carbohydrate-rich foods. For roasted coffee, a European Union benchmark level of 400 µg/kg acrylamide is of importance. Regularly, the acrylamide contents are controlled using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). This reference method is reliable and precise but laborious because of the necessary sample clean-up procedure and instrument requirements. This research investigates the possibility of predicting the acrylamide content from proton nuclear magnetic resonance (NMR) spectra that are already recorded for other purposes of coffee control. In the NMR spectrum acrylamide is not directly quantifiable, so that the aim was to establish a correlation between the reference value and the corresponding NMR spectrum by means of a partial least squares (PLS) regression. Therefore, 40 commercially available coffee samples with already available LC-MS/MS data and NMR spectra were used as calibration data. To test the accuracy and robustness of the model and its limitations, 50 coffee samples with extreme roasting degrees and blends were additionally prepared as test set. The PLS model shows an applicability for the varieties C. arabica and C. canephora, which were medium to very dark roasted using drum or infrared roasters. The root mean square error of prediction (RMSEP) is 79 µg/kg acrylamide (n=32). The PLS model is judged as suitable to predict the acrylamide values of commercially available coffee samples. On the other hand, very light roasts containing more than 1000 µg/kg acrylamide are currently not suitable for PLS prediction.

LC–NMR combines the advantage of the outstanding separation power of liquid chromatography (LC) and the superior structural elucidating capability of nuclear magnetic resonance (NMR). NMR has proved that it is a standout detector for LC by providing maximum structural information about plant originated extracts particularly in its isolating ability of isomeric (same molecular formula) and/or isobaric (same molecular weight) compounds as compared to other detectors. The present review provides an overview of the LC–NMR developmental trends and its application in natural products analysis. The different LC–NMR operational modes are described, as well as how technical improvements assist in establishing this powerful technique as an important analytical tool in the analysis of complex plant-derived compounds. On-flow, stop-flow and loop-storage modes, as well as the new offline mode LC–SPE–NMR and capLC-NMR configurations that avoid the ingestion of expensive deuterated solvents throughout the experiment are mentioned. Utilization of cryogenic probe and microprobe technologies which are the other important promising approaches for guaranteeing the sensitivity issues are also described. Concluding remarks and future outlooks are also discussed.

The article describes facile one-pot, hi-yielding reactions to synthesize substituted 3,4-dimethyl-1H-pyrrole-2-carboxamide (3a–m) and carbohydrazide analogues (5a–l) as potential antifungal and antimicrobial agents. The structural integrity and purity of the synthesized compounds were assigned based on appropriate spectroscopic techniques. Synthesized compounds were assessed in vitro for antifungal and antibacterial activity. The compound 5h, 5i and 5j were found to be the most potent against A. fumigatus, with MIC value of 0.031 mg/mL. The compound 5f bearing a 2,6-dichloro group on the phenyl ring was found to be the most active broad spectrum antibacterial agent with MIC value of 0.039 mg/mL. The mode of action of the most promising antifungal compounds (one representative from each series; 3j and 5h) was established by their molecular docking to the active site of sterol 14α-demethylase. Molecular docking studies revealed a highly spontaneous binding ability of the tested compounds in the access channel away from catalytic heme iron of the enzyme, which suggested that the tested compounds inhibit this enzyme and would avoid heme iron related deleterious side effects observed with existing antifungal compounds.

Toxicologically relevant levels of the psychoactive ∆9-tetrahydocannabinol (∆9-THC) as well as high levels of non-psychoactive cannabinoids potentially occur in CBD (cannabidiol) oils. For consumer protection in the fast-growing CBD oil market, facile and rapid quantitative methods to determine the cannabinoid content are crucial. However, the current standard method, i.e., liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS), requires a time-consuming multistep sample preparation. In this study, a quantitative nuclear magnetic resonance spectroscopy (qNMR) method for screening cannabinoids in CBD oils was developed. Contrary to the HPLC-MS/MS method, this qNMR features a facile sample preparation, i.e., only diluting the CBD oil in deuterochloroform. Pulse length-based concentration determination (PULCON) enables a direct quantification using an external standard. The signal intensities of the cannabinoids were enhanced during the NMR spectra acquisition by means of multiple suppression of the triglycerides which are a major component of the CBD oil matrix. The validation confirmed linearity for CBD, cannabinol (CBN), ∆9-THC and ∆8-THC in hemp seed oil with sufficient recoveries and precision for screening. Comparing the qNMR results to HPLC-MS/MS data for 46 commercial CBD oils verified the qNMR accuracy for ∆9-THC and CBD but with higher limits of detection. The developed qNMR method paves the way for increasing the sample throughput as a complementary screening before HPLC-MS/MS.

The spike protein (S) of SARS-CoV-2 has been shown to bind to the human angiotensin-converting enzyme 2 (ACE2) receptor with much higher affinity compared to other coronaviruses. The binding interface between the ACE2 receptor plays a critical role in the entry mechanism of SARS-CoV-2 virus. There are specific amino acids involved in the interaction between S protein and ACE2 receptor. This specificity is critical for the virus to establish a systemic infection and cause COVID-19 disease. In the ACE2 receptor, the largest number of amino acids that play a crucial role in the mechanism of interaction and recognition with the S protein are located in the C-terminal part which represents the main binding region between ACE2 and S. This fragment is abundant in coordination residues such as aspartates, glutamates and histidine that could be targeted by metal ions. Zn²⁺ ion binds to the ACE2 receptor in its catalytic site and modulates its activity, but it could also contribute to the structural stability of the entire protein. The ability of the human ACE2 receptor to coordinate metal ions, such Zn²⁺, in the same region where it binds to the S protein could have a crucial impact in the mechanism of recognition and interaction of ACE2-S with consequences on their binding affinity that deserve to be investigated. To test this possibility, this study aims to characterize the coordination ability of Zn²⁺, and also Cu²⁺ for comparison, with selected peptide models of the ACE2 binding interface using spectroscopic and potentiometric techniques.

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are central eukaryotic messengers. These very highly phosphorylated molecules can exist in two distinct conformations, a canonical one with five phosphoryl groups in equatorial positions, and a “flipped” axial conformation. Using 13C-labeled InsPs / PP-InsPs, the behavior of these molecules was investigated by 2D-NMR under solution conditions reminiscent of a cytosolic environment. Remarkably, the most highly phosphorylated messenger InsP8 readily adopts both conformations at physiological conditions. Environmental factors - such as pH, metal cation composition, and temperature - strongly influence the conformational equilibrium. Thermodynamic data revealed that the transition of InsP8 from the equatorial to the axial conformation is, in fact, an exothermic process. The speciation of InsPs and PP-InsPs also affects their interaction with protein binding partners; addition of Mg2+ decreased the binding constant Kd of InsP8 to an SPX protein domain. The results illustrate that PP-InsP speciation reacts very sensitively to solution conditions, suggesting it might act as an environment-dependent molecular switch.

Resolving small molecule mixtures by nuclear magnetic resonance (NMR) spectroscopy has been of great interest for a long time for its precision, reproducibility and efficiency. However, spectral analyses for such mixtures are often highly challenging due to overlapping resonance lines and limited chemical shift windows. The existing experimental and theoretical methods to produce shift NMR spectra in dealing with the problem have limited applicability owing to sensitivity issues, inconsistency and / or requirement of prior knowledge. Recently, we have resolved the problem by decoupling multiplet structures in NMR spectra by the wavelet packet transform (WPT) technique. In this work, we developed a scheme for deploying the method in generating highly resolved WPT NMR spectra and predicting the composition of the corresponding molecular mixtures from their 1H NMR spectra in an automated fashion. The four-step spectral analysis scheme consists of calculating WPT spectrum, peak matching with a WPT shift NMR library, followed by two optimization steps in producing the predicted molecular composition of a mixture. The robustness of the method was tested on an augmented dataset of 1000 molecular mixtures, each containing 3 to 7 molecules. The method successfully predicted the constituent molecules with a median true positive rate of 1.0 against the varying compositions, while a median false positive rate of 0.04 was obtained. The approach can be scaled easily for much larger datasets.

Highly reactive decomposition products of (deuterated) chloroform can deteriorate samples dissolved in this commonly used NMR solvent. For sensitive samples (such as peptides, unsaturated fatty acids, vitamins), this can lead to abnormal NMR spectra (e.g. signal shifts depending on pH, attenuation of signals over time due to chemical changes of analytes, new signals from reaction products). Such irreproducibly influenced spectra are especially problematic for non-targeted analysis methods. To prevent these artefacts, chlorine, phosgene and hydrochloric acid need to be eliminated from deuterated chloroform prior to its use. Since the common stabilization methods have proven insufficient for sensitive NMR samples, another purging method has been tested: Mitigation is easily and reliably achieved by washing the deuterated chloroform with concentrated Na2CO3-solution and subsequent desiccation with oven-dried Na2CO3.

Lipoproteins are important cardiovascular (CV) risk biomarkers. This study aimed to investigate the associations of lipoprotein subclasses with micro- and macrovascular biomarkers to better understand how these subclasses relate to atherosclerotic CV diseases. One hundred fifty-eight serum samples from the EXAMIN AGE study, consisting of healthy individuals and CV risk patients, were analyzed by nuclear magnetic resonance (NMR) spectroscopy to quantify lipoprotein subclasses. Microvascular health was quantified by measuring retinal arteriolar and venular diameters. Macrovascular health was quantified by measuring carotid-to-femoral pulse wave velocity (PWV). Nineteen lipoprotein subclasses showed statistically significant associations with retinal vessel diameters and nine with PWV. These lipoprotein subclasses together explained up to 26% of variation (R²=0.26, F(29,121)=2.80, p<0.001) in micro- and 12% (R²=0.12, F(29,124)=1.70, p=0.025) of variation in macrovascular health. High-density (HDL-C) and low-density lipoprotein cholesterol (LDL-C) as well as triglycerides together explained up to 13% (R²=0.13, F(3,143)=8.42, p<0.001) of micro and 8% (R²=0.08, F(3,145)=5.46, p=0.001) of macrovascular variation. Lipoprotein subclasses seem to reflect micro- and macrovascular end organ damage more precisely as compared to only measuring HDL-C, LDL-C and triglycerides. Further studies are needed to analyse how the additional quantification of lipoprotein subclasses can improve CV risk stratification and CV disease prediction.

: Dacarbazine, DAC, 5-(3,3-dimethyltriazeno)imidazol-4-carboxamideis is an imidazole-carboxamide derivative, that is structurally related to purines. DAC belongs to the triazene compounds, which are a group of alkylating agents with antitumour and mutagenic properties. DAC is a non-cell cycle specific drug, active on all phases of cellular cycle. In the frame of this work the 3d-metal complexes (cobalt and copper) with dacarbazine were synthesized. Their spectroscopic properties by the use of FT-IR, FT-Raman and 1HNMR were studied. The structures of dacarbazine and its complexes with copper(II) and cobalt(II) were calculated using DFT methods. The effect of metals on the electronic charge distribution of dacarbazine was discussed on the basis of calculated NBO atomic charges. The reactivity of metal complexes in relation to ligand alone was estimated on the basis of calculated energy of HOMO and LUMO orbitals. The aromaticity of imidazole ring in dacarbazine and the complexes was compared (on the basis of calculated geometric indices of aromaticity). Thermal stability of the investigated 3d-metal complexes with dacarbazine and the products of their thermal decomposition were analyzed.

The innovation of the new psychoactive substances (NPS) market requires the rapid identification of new substances that can be a risk to public health, in order to reduce the damage due to their use. Twelve seized products suspected to contain illicit substances were analyzed by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), gas chromatography coupled to mass spectrometry (GC-MS), and nuclear magnetic resonance spectroscopy (NMR). Synthetic cathinones (SCat) were found in all products, either as a single component or in mixtures. Infrared spectra of all products were consistent with the molecular structure of SCat, showing an intense absorption band at 1700–1674 cm‐1, corresponding to the carbonyl stretching, a medium/strong peak at 1605-1580 cm-1, indicating stretching vibrations in the aromatic ring (C=C) and bands with relative low intensity at frequencies near 2700–2400 cm-1, corresponding to an amine salt. It was possible to identify a total of eight cathinone derivatives by GC-MS and NMR analysis: 4’-methyl-α-pyrrolidinohexanophenone (MPHP), α-pyrrolidinohexanophenone (α-PHP), 3-fluoromethcathinone (3-FMC), methedrone, methylone, buphedrone, N-ethylcathinone, and pentedrone. Among the adulterants found in these samples, caffeine was the most frequently detected substance, followed by ethylphenidate. These results highlight the prevalence of SCat in seized materials of the Portuguese market. Reference standards are usually required for confirmation, but when reference materials are not available, the combination of complementary techniques is fundamental for a rapid and an unequivocal identification of such substances.

Protein oligomerzation is key to countless physiological processes, but also to abnormal amyloid conformations implicated in over 25 mortal human diseases. Angiogenin (h-ANG), a ribonuclease A family member, produces RNA fragments that regulate ribosome formation, the creation of new blood vessels and stress granule function. Too little h-ANG activity leads abnormal protein oligomerization resulting in Amyotrophic Lateral Sclerosis (ALS) or Parkinson’s disease. While a score of disease linked h-ANG mutants has been studied by X-ray diffraction, some elude crystallization. There is also debate regarding the structure that RNA fragments adopt after cleavage by h-ANG. Here, to better understand the beginning of the process that leads to aberrant protein oligomerization, the solution secondary structure and residue-level dynamics of WT h-ANG and two mutants: H13A and R121C, are characterized by multidimensional heteronuclear NMR spectroscopy under near physiological conditions. All three variants are found to adopt well folded and highly rigid structures in solution, although the elements of secondary structure are somewhat shorter than those observed in cystallography studies. R121C alters the environment of nearby residues only. By constrast, the mutation H13A affects local residues as well as nearby active site residues residues K41 and H119. The conformation characterization by CD and 1D 1H NMR spectroscopies of tRNAAla before and after h-ANG cleavage reveals a retention of most duplex structure and little or no G-quadruplex formation.

Due to legal regulations, the rise of globalised (online) commerce and the need for public health protection, the analysis of spirits (alcoholic beverages &gt; 15 % vol) is a task with growing importance for governmental and commercial laboratories. In this article a newly developed method using nuclear magnetic resonance (NMR) spectroscopy for the simultaneous determination of 15 substances relevant for the quality and authenticity assessment of spirits is described. The new method starts with a simple and rapid sample preparation and does not need an internal standard. For each sample a group of 1H-NMR spectra is recorded, among them a 2D spectrum for analyte identification and 1D spectra with suppression of solvent signals for quantification. Using the Pulse Length Based Concentration Determination (PULCON) method, concentrations are calculated from curve fits of the characteristic signals for each analyte. The optimisation of the spectra, their evaluation and the transfer of the results are done fully automatically. Glucose, fructose, sucrose, acetic acid, citric acid, formic acid, ethyl acetate, ethyl lactate, acetaldehyde, ethanol, methanol, n-propanol, isobutanol, isopentanol, 2-phenylethanol and 5-(hydroxymethyl)furfural (HMF) can be quantified with an overall accuracy better than 8 %. This new NMR-based targeted quantification method enables the simultaneous and efficient quantification of relevant spirits ingredients in their typical concentration ranges in one process with good accuracy. It has proven to be a reliable method for all kinds of spirits in routine food control.

Petrosamine (1) – a colored pyridoacridine alkaloid from the Belizean sponge, Petrosia sp. that is also a potent inhibitor of acetylcholine esterase (AChE) – was investigated by spectroscopic and computational methods. Analysis of the petrosamine free energy landscapes, pKa and tautomerism revealed an accurate electronic depiction of the molecular structure of 1 as the di-keto form, with net charge of q = +1, rather than a dication (q = +2) under ambient conditions of isolation-purification. The pronounced solvatochromism (UV-vis) reported for 1, and related analogs, was investigated in detail and is best explained by charge delocalization and stabilization of the ground state (HOMO) of 1 rather than an equilibrium of competing tautomers. Refinement of the molecular structure of 1 by QM methods complements published computational docking studies to define the contact points in the enzyme active site that may improve design of new AChE inhibitors based on the pyridoacridine alkaloid molecular skeleton.

The genetic code conceals a ‘code within the codons’, which hints at biophysical interactions between amino acids and their cognate nucleotides. But research over decades has failed to corroborate systematic biophysical interactions across the code. Using molecular dynamics simulations and NMR, we have analysed interactions between the 20 standard proteinogenic amino acids and four RNA mononucleotides in three charge states. Our simulations show that 50% of amino acids bind best with their anticodonic middle base in the -1 charge state common to the backbone of RNA, while 95% of amino acids interact most strongly with at least one of their codonic or anticodonic bases. Preference for the cognate anticodonic middle base was greater than 99% of randomized assignments. We verify a selection of our results using NMR, and highlight challenges with both techniques for interrogating large numbers of weak interactions. Finally, we extend our simulations to a range of amino acids and dinucleotides, and corroborate similar preferences for cognate nucleotides. Despite some discrepancies between the predicted patterns and those observed in biology, the existence of weak stereochemical interactions means that random RNA sequences could template non-random peptides. This offers a compelling explanation for the emergence of genetic information in biology.

Phospholipid metabolism, including phosphatidylcholine (PC) biosynthesis, is crucial for various biological functions and is associated to longevity. Phosphatidylethanolamine N-methyltransferase (PEMT) is a protein that catalyzes the biosynthesis of PC, the levels of which change in various organs such as brain and kidney during aging. However, the role of PEMT for systemic PC supply is not fully understood. To address how PEMT affects aging-associated energy metabolism in tissues responsible for nutrient absorption, lipid storage and energy consumption, we employed NMR-based metabolomics to study liver, plasma, intestine (duodenum, jejunum, ileum), brown/white adipose tissues (BAT, WAT), and skeletal muscle of young (9–10 weeks) and old (96–104 weeks) wild-type (WT) and PEMT knockout (KO) mice. We found that the effect of PEMT-knockout was tissue-specific and age-dependent. Deficiency of PEMT affected the metabolome of all tissues examined, among which the metabolome of BAT from both young and aged KO mice was dramatically changed in comparison to WT mice, whereas the metabolome of jejunum was only slightly affected. As for aging, the absence of PEMT increased the divergence of metabolome during aging of liver, WAT, duodenum and ileum and decreased the impact on skeletal muscle. Overall, our results suggest that PEMT plays a previously unexplored critical role in both aging and energy metabolism.

Polyphosphate (polyP) accumulating organisms (PAOs) are the key agent to perform enhanced biological phosphorus removal (EBPR) activity, and intracellular polyP plays a key role in this process. Potential associations between EBPR performance and the polyP structure have been suggested, but are yet to be extensively investigated, mainly due to the lack of established methods for polyP characterization in the EBPR system. In this study, we explored and demonstrated that single-cell Raman spectroscopy (SCRS) can be employed for characterizing intracellular polyPs of PAOs in complex environmental samples such as EBPR systems. The results, for the first time, revealed distinct distribution patterns of polyP length (as Raman peak position) in PAOs in lab-scale EBPR reactors that were dominated with different PAO types, as well as among different full-scale EBPR systems with varying configurations. Furthermore, SCRS revealed distinctive polyP composition/features among PAO phenotypic sub-groups, which are likely associated with phylogenetic and/or phenotypic diversity in EBPR communities, highlighting the possible resolving power of SCRS at the microdiversity level. To validate the observed polyP length variations via SCRS, we also performed and compared bulk polyP length characteristics in EBPR biomass using conventional polyacrylamide gel electrophoresis (PAGE) and solution ³¹P nuclear magnetic resonance (³¹P-NMR) methods. The results are consistent with the SCRS findings and confirmed the variations in the polyP lengths among different EBPR systems. Compared to conventional methods, SCRS exhibited advantages as compared to conventional methods, including the ability to characterize in situ the intracellular polyPs at subcellular resolution in a label-free and non-destructive way, and the capability to capture subtle and detailed biochemical fingerprints of cells for phenotypic classification. SCRS also has recognized limitations in comparison with ³¹P-NMR and PAGE, such as the inability to quantitatively detect the average polyP chain length and its distribution. The results provided initial evidence for the potential of SCRS-enabled polyP characterization as an alternative and complementary microbial community phenotyping method to facilitate the phenotype-function (performance) relationship deduction in EBPR systems.

The fine structure of Apricot pectin polysaccharides is discussed from both literature and recent work we have performed. Structural characterization utilized 1D and 2D homo- and hetero-nuclear NMR spectroscopy including results from High Performance Size Exclusion Chromatography (HP-SEC), HP Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) and FTIR spectroscopy. The purified pectin fraction (F1AP) was found to be composed of D-galacturonic acid, L-rhamnose, D-arabinose and D-galactose in a 1.0 : 0.1 : 0.36 : 0.12 molar ratio, respectively, which is consistent with a pectin RG-1 branched with arabinogalactan side chain polysaccharides, and has a weight-average molecular weight of approximately 1588.2 kDa. The slope of the hydrodynamic radius versus the molar mass was 0.57, indicating the presence of coil conformations. The results from HPAEC-PAD, HPSEC and NMR spectroscopy (1H, 13C, zTOCSY, HSQC, HSQCTOXY and HMBC) demonstrated that F1AP fine fraction F1AP1, derived from original F1AP, has a backbone of (1→4)-linked -D-galacturonic acid and -L-rhamnopyranosyl residues branched with arabinogalactan of multiply glycosidic linkages of T- α-Araf, 3-α-Araf, 5-α-Araf, T- α -Arap, 2- α-Arap side chains, having methyl and acetylated groups, and has high molecular weights (1945 kDa). While the other F1A6 fraction consists predominately of an HG region of pectin polysaccharides with low molecular weights (117.5 kDa), less aggregated. The F1AP1 polysaccharide was highly viscous and its hydrodynamic radius was 64,3 nm. The exponent b of Rh-Mw (conformation plot) was determined to be 0.48, indicating a compact flexible coil conformation of the macromolecule in solution. However, the F1AP6 fraction of apricot fruits belong to backbone of repeats units of [-(1→4)-α-D-GalpA-1]n – both HG and partly contents RG-II polysaccharides, bearing heterosugar sidechains, including α-Ara, α -Rha and β-Gal residues and adopt more stiffness rod like conformation.

Nuclear Magnetic Resonance (NMR) and its various forms are extensively utilized in both research and clinical settings to analyse molecules. NMR data pre-processing, while essential to NMR data analysis, can be uniquely complex. Despite the availability of software tools, understanding these processes can be challenging, complicating the selection of appropriate pre-processing steps. In this review, we elucidate pre-processing steps in the time domain from a mathematical and statistical perspective, explaining direct current offset removal, eddy current correction, shift and linear prediction, weighting, zero filling, and domain transformation using plain language and fundamental mathematical formulas. Our objective is to clarify these processes in simple terms and provide general guidance.

Abstract: Agarose is known to form a homogeneous thermoreversible gel in aqueous medium over a critical polymer concentration. The solid-liquid phase transitions are thermoreversible but depend on the molecular structure of the agarose sample tested. Then, in a first step, the structure was characterised by 1H and 13C NMR in D2O and in DMSO which is a solvent of agarose whatever the temperature. A low yield in methyl substituent on the D-galactose unit was determined. Then, evolution of the 1H NMR spectrum was followed as a function of temperature in increasing and decreasing temperature process from 25 to 80°C. A large thermal hysteresis is obtained and discussed. It helps to interpret rheological behaviour. In fact, NMR signals are related to proton relaxation and especially to proton involved in H-bonds between water and -OH agarose for tightly bound water and agarose/agarose in chain packing. In a second step, water was exchanged against ethanol which is a non solvent of agarose. A stable gel was demonstrated and characterised by rheology to be compared with aqueous behaviour. Bound water playing the role of plasticizer is probably removed and the gel is much stronger (and brittle) in ethanol with a larger thermal stability. It is the first time that such gel is characterised without phase transition when passing from a good-solvent to a non-solvent. This extends the domains of application of agarose.

A series of cysteine-based perfluoroaromatic (hexafluorobenzene (I) and decafluorobiphenyl (II) were synthesized and established as a chemoselective and available core to simple or more complex systems since small molecules to biomolecules with interesting properties. As proof of concept of the potential application of perfluorinated derivatives as non-cleavable linkers, some antibody-perfluorinated conjugates were prepared via thiol, demonstrating that the bioconjugation process doesn’t affect to the macromolecular entity. Besides, some molecular properties of synthesized compounds are evaluated using a combination of spectroscopic characterization (FT-IR and 19F-NMR chemical shifts) and theoretical calculations. Moreover, molecular Docking was also developed to predict cysteine-based hexafluorobenzene and decafluorobiphenyl derivatives’ affinity against topoisomerase Il and cyclooxygenase 2 (COX-2). The results suggested that mainly cysteine-based decafluorobiphenyl derivatives could be potential topoisomerase II α and COX-2 inhibitors, becoming potential anticancer agents and candidates for anti-inflammatory treatment.

Resveratrol is a well-known natural polyphenol with a plethora of pharmacological activities. As a potent antioxidant, resveratrol is highly oxidizable, and readily reacts with reactive oxygen species (ROS). Such a reaction not only leads to a decrease in ROS levels in a biological environ-ment but may also generate a wide range of metabolites with altered bioactivities. Inspired by this notion, in the current study, our aim was to take a diversity-oriented chemical approach to study the chemical space of oxidized resveratrol metabolites. Chemical oxidation of resveratrol and a bioactivity-guided isolation strategy using xanthine oxidase (XO) and radical scavenging activities led to the isolation of a diverse group of compounds, including a chlorine-substituted compound (2), two iodine-substituted compounds (3 and 4), two viniferins (5 and 6), an eth-oxy-substituted compound (7) two ethoxy-substituted dimers (8 and 9). Compounds 4, 7, 8 and 9 are reported here for the first time. All compounds without ethoxy-substitution exerted stronger XO inhibition than their parent compound, resveratrol. By enzyme kinetic and in silico docking studies compounds 2, 3 and 4 were identified as potent competitive inhibitors of the enzyme while the viniferins acted as mixed-type inhibitors. Further, compounds 2 and 9 had better DPPH scavenging activity and oxygen radical absorbing capacity than resveratrol. Our results suggest that the antioxidant activity of resveratrol is modulated by the effect of a cascade of chemically stable oxidized metabolites, several of which have significantly altered target specificity as compared to their parent compound.

Crustacean hyaluronidases are 1 poorly understood both in terms of their enzymatic properties and in terms of their structural features. In this work, we have shown that the hepatopancreas homogenate of the red king crab has a hyaluronidase activity that is an order of magnitude higher than its commercial counterpart. Zymography revealed the hyaluronidase activity of the protein roughly from40 to 50 kDa relative to the molecular marker used in electrophoresis. Analysis of the hepatopancreas transcriptome revealed a hyaluronidase sequence with an expected molecular weight of 42.5 kDa. It turned out that the reaction of cleavage of hyaluronate in the presence of a homogenate proceeds by the mechanism of b-elimination, which is well known for bacterial hyaluronidases. Thus, a new hyaluronidase of higher eukaryotes was found and described, which is not integrated into the modern classification of hyaluronidases.

New pharmaceutically acceptable salts of trazodone for the treatment of central nervous system disorders are synthesized and described. Each salt (trazodone hydrogen bromide and trazodone 1-hydroxy-2-naphthoate) was obtained by two or three different methods leading to the same crystalline form. Although trazodone salts are poorly crystalline, single-crystal X-ray diffraction data for trazodone 1-hydroxy-2-naphthoate were collected and analyzed as well as compared to the previously described crystal structure of commercially available trazodone hydrochloride. The powder samples of all new salts were characterized by Fourier transform infrared spectroscopy and 13C solid-state nuclear magnetic resonance spectroscopy. Spectroscopic studies were supported by gauge including projector augmented wave (GIPAW) calculations of carbon chemical shielding constants. The main goal of our research was to find salts with better physicochemical properties and to make an attempt to associate them with both the anion structure and the most prominent interactions exhibited by the protonated trazodone cation. The dissolution profiles of trazodone from tablets prepared from various salts with lactose monohydrate were investigated. The studies revealed that salts with simple anions show a fast release of the drug while the presence of more complex anion, more strongly interacting with the cation, effects a slow-release profile of the active substance and can be used for the preparation of the tables with a delay or prolonged mode of action.

The activation of halogen bonding by the substitution of the pentafluorosulfanyl (SF5) group was studied using a series of SF5-substituted iodobenzenes. The simulated electrostatic potential values of SF5-substituted iodobenzenes, ab initio molecular orbital calculations of intermolecular interactions of SF5-substituted iodobenzenes with pyridine, and the 13C NMR titration experiments of SF5-substituted iodobenzenes in the presence of pyridine or tetra (n-butyl) ammonium chloride (TBAC) indicated the obvious activation of halogen bonding, although this was highly dependent on the position of SF5-substitution on the benzene ring. 3,5-Bis-SF5-iodobenzene was the most effective halogen bond donor followed by o-SF5-substituted iodobenzene, while the m- and p-SF5 substitutions did not activate the halogen bonding of iodobenzenes. The 2:1 halogen bonding complex of 3,5-bis-SF5-iodobenzene and 1,4-diazabicyclo[2.2.2]octane (DABCO) was also confirmed. Since SF5-containing compounds have emerged as promising novel pharmaceutical and agrochemical candidates, the 3,5-bis-SF5-iodobenzene unit should be an attractive fragment of rational drug design capable of halogen bonding with biomolecules.

The peptidyl-prolyl isomerases of the cyclophilin type are distributed throughout human cells, including eight found solely in the nucleus. Nuclear cyclophilins are involved in complexes that regulate chromatin modification, transcription, and pre-mRNA splicing. This review collects what is known about the eight human nuclear cyclophilins: PPIH, PPIE, PPIL1, PPIL2, PPIL3, PPIG, CWC27, and PPWD1. Each “spliceophilin” is evaluated in relation to the spliceosomal complex in which it has been studied, and current work studying the biological roles of these cyclophilins in the nucleus are discussed. All eight of the human splicing complexes available in the PDB are analyzed from the viewpoint of the human spliceophilins. Future directions in structural and cellular biology, and the importance of developing spliceophilin-specific inhibitors, are considered.

Reactions of picolinamides with 1,3-propanesultone in methanol followed by the treatment with ketones led to a series of previously unknown chemical transformations, yielding first pyridinium salts (2a-f) with a protonated endocyclic nitrogen atom and then heterocyclic salts (3a-j) containing a 4-imidazoline ring. The structures of intermediate and final products were determined by IR and 1H, 13C NMR spectroscopy and X-ray study. The effects of the ketone and alcohol structures on the product yield were studied by quantum-chemical calculations. The stability of salts 3a-j towards hydrolysis and alcoholysis makes them excellent candidates for the search of new types of biologically active compounds.

By employing a variation of the polyamidation method using in situ silylated diamines and acid chlorides, it has been possible to obtain a rod-type polyamide: poly(p-phenylene terephthalamide) (PPTA, a polymer used in the high value-added material Kevlar), with a molecular weight much higher than that obtained with the classical, and industrial, polyamidation method. The optimization of the method has consisted in using, together with the silylating agent, a mixture of pyridine and a high pKa tertiary amine. The research has been complemented by a combination of nuclear magnetic resonance and molecular simulation studies, which have determined that the improvements in molecular weight derive mainly from the formation of silylamide groups in the growing polymer.

Through its suggestive name, non-targeted methods (NTMs) do not aim at a predefined "needle in the haystack". Instead, they exploit all the constituents of the haystack. This new form of analytical methods is increasingly finding applications in food and feed testing. However, the concepts, terms, and considerations related to this burgeoning field of analytical testing needs to be propagated for the benefit of ones associated in academic research, commercial development, and official control. This paper addresses the frequently asked questions around notations and terminologies surrounding NTMs. The widespread development and adoption of these methods also necessitates the need to develop approaches to NTM validation, i.e., evaluating the performance characteristics of a method to determine if it is fit-for-purpose. This work aims to provide a roadmap to approaching NTM validation. In doing so, the paper deliberates on the different considerations that influence the approach to validation and provides suggestions thereof.

Fluorine represents a privileged building block in pharmaceutical chemistry. Diethylaminosulfur-trifluoride (DAST) is a reagent commonly used for replacement of alcoholic hydroxyl groups with fluorine and is also known to catalyze water elimination and cyclic Beckmann-rearrangement type reactions. In this work we aimed to use DAST for diversity-oriented semisynthetic transformation of natural products bearing multiple hydroxyl groups to prepare new bioactive compounds. Four ecdysteroids, including a new constituent of Cyanotis arachnoidea, were selected as starting materials for DAST-catalyzed transformations. The newly prepared compounds represented combinations of various structural changes DAST was known to catalyze, and a unique cyclopropane ring closure that was found for the first time. Several compounds demonstrated in vitro antitumor properties. A new 17-N-acetylecdysteroid (13) exerted potent antiproliferative activity and no cytotoxicity on drug susceptible and multi-drug resistant mouse T-cell lymphoma cells. Further, compound 13 acted in significant synergism with doxorubicin without detectable direct ABCB1 inhibition. Our results demonstrate that DAST is a versatile tool for diversity-oriented synthesis to expand chemical space towards new bioactive compounds.

Cold brew coffee is a new trend in coffee industry. This paper presents pilot studies into several aspect of this beverage. Using an online survey, the current practices of cold brew coffee preparation were investigated identifying a rather large variability with a preference for extraction of medium roasted Arabica coffee using 50-100 g/l at 8°C for about 1 day. Sensory testing using ranking and triangle tests showed that cold brew may be preferred over iced coffee (cooled down hot extracted coffee). Extraction experiments at different conditions combined with nuclear magnetic resonance (NMR) analysis showed that the usual extraction time may be longer than necessary as most compounds are extracted within only a few hours, while increasing turbulence (e.g. using ultrasonication) and temperature may additional increase the speed of extraction. NMR analysis also revealed a possible chemical differentiation between cold brew and hot brew using multivariate data analysis. Decreased extraction time and reduced storage times could be beneficial for cold brew product quality as microbiological analysis of commercial samples detected samples with spoilage organisms and contamination with Bacillus cereus.

NMR spectral datasets, especially in systems with limited samples, can be difficult to interpret if they contain multiple chemical components (phases, polymorphs, molecules, crystals, glasses, etc…) and the possibility of overlapping resonances. In this paper, we benchmark several blind source separation techniques for analysis of NMR spectral datasets containing negative intensity. For benchmarking purposes, we generated a large synthetic datasbase of quadrupolar solid-state NMR-like spectra that model spin-lattice T1 relaxation or nutation tip/flip angle experiments. Our benchmarking approach focused exclusively on the ability of blind source separation techniques to reproduce the spectra of the underlying pure components. In general, we find that FastICA (Fast Independent Component Analysis), SIMPLISMA (SIMPLe-to-use-Interactive Self-modeling Mixture Analysis), and NNMF (Non-Negative Matrix Factorization) are top-performing techniques. We demonstrate that dataset normalization approaches prior to blind source separation do not considerably improve outcomes. Within the range of noise levels studied, we did not find drastic changes to the ranking of techniques. The accuracy of FastICA and SIMPLISMA degrades quickly if excess (unreal) pure components are predicted. Our results indicate poor performance of SVD (Singular Value Decomposition) methods, and we propose alternative techniques for matrix initialization. The benchmarked techniques are also applied to real solid state NMR datasets. In general, the recommendations from the synthetic datasets agree with the recommendations and results from the real data analysis. The discussion provides some additional recommendations for spectroscopists applying blind source separation to NMR datasets, and for future benchmark studies. Applications of blind source separation to NMR datasets containing negative intensity may be especially useful for understanding complex and disordered systems with limited samples and mixtures of chemical components.

Bottom-up systems biology entails the construction of kinetic models of cellular pathways by collecting kinetic information on the pathway components (e.g. enzymes) and collating this into a kinetic model, based for example on ordinary differential equations. This requires integration and data transfer between a variety of tools, ranging from data acquisition in kinetics experiments, to fitting and parameter estimation, to model construction, evaluation and validation. Here, we present a workflow that uses the Python programming language, specifically the modules from the SciPy stack, to facilitate this task. Starting from raw kinetics data, acquired either from spectrophotometric assays with microtitre plates or from NMR spectroscopy time courses, we demonstrate the fitting and construction of a kinetic model using scientific Python tools. The analysis takes place in a Jupyter notebook, which keeps all information related to a particular experiment together in one place and thus serves as an e-labbook, enhancing reproducibility and traceability. The Python programming language serves as an ideal foundation for this framework because it is powerful yet relatively easy to learn for the non-programmer, has a large library of scientific routines and active user community, is open-source and extensible, and many computational systems biology software tools are written in Python or have a Python API. Our workflow thus enables investigators to focus on the scientific problem at hand rather than worrying about data integration between disparate platforms.

As a renewable industrial crop, Miscanthus offers numerous advantages, namely high photosynthesis activity (as a C4 plant) and exceptional CO2 fixation rate. These properties make Miscanthus very attractive for industrial exploitation, such as lignin generation. Here, we present a systematic study analyzing the correlation of the lignin structure with Miscanthus genotype and plant portion (stem versus leaf). Specifically, the ratio of the three monolignols and corresponding building blocks as well as the linkages formed between the units have been studied. Depending on the Miscanthus genotype and plant component (leaf versus stem), correlations between chemical structure and properties of the lignins have been determined, i.e. correlations in molecular weight, polydispersity and decomposition temperature. Lignin isolation was performed using non-catalyzed organosolv pulping and the structure analysis includes NREL, FTIR, UV-Vis, HSQC-NMR, TGA, pyrolysis GC/MS. Structural differences were found for stem and leaf-derived lignins. Compared to beech wood lignins, Miscanthus lignins possess lower molecular weight and narrow polydispersities (< 1.5 Miscanthus vs. > 2.5 beech) corresponding to improved homogeneity. In addition to conventional univariate analysis of FTIR spectra, multivariate chemometrics revealed distinct differences for aromatic in-plane deformations of stem versus leaf-derived lignins. These results emphasize the potential of Miscanthus as low-input resource and Miscanthus-derived lignin as promising agricultural feedstock.

In this article/review, the selective interactions of several berberine and palmatine derivatives with various DNA G-quadruplex structures are reported. These derivatives were constructed starting from two natural compounds, berberine and palamatine, through specific synthetic passages following two different schemes for each of them and using several substituents. The details of these synthesis are also described. Indeed, the study of the interactions of these derivative compounds with various G-quadruplex forming sequences was carried out by means of various structural and biochemical techniques. The results show that the presence of suitable side chains are very useful to improve the interaction of the ligands with G-quadruplex structures. Thus, since G-quadruplex formation is promoted by these compounds, which have never been reported before, these may be tested as potential anticancer drugs.

The productivity of forests is often considered to be limited by the availability of phosphorus (P). Knowledge of the role of organic and inorganic P in humid subtropical forest soils is lacking. In this study, we used chemical fractionation and 31P nuclear magnetic resonance (NMR) spectroscopy to characterize the form of P and its distribution in undisturbed perhumid Chamaecyparis forest soils. The toposequence of transects was investigated for the humic layer from summit to footslope and lakeshore. The clay layer combined with a placic-like horizon in the subsoil may affect the distribution of soil P because both total P and organic P (Po) contents in all studied soils decreased with soil depth. In addition, Po content was negatively correlated with soil crystalline Fe oxide content, whereas inorganic P (Pi) content was positively correlated with soil crystalline Fe oxide content and slightly increased with soil depth. Thus, Pi may be mostly adsorbed by soil crystalline Fe oxides in the soils. Among all extractable P fractions, the NaOH-Po fraction appeared to be the major component, followed by NaHCO3-Po; the resin-P and HCl-Pi fractions were lowest. In addition, we found no typical trend for Pi and Po contents in soils with topographical change among the three sites. From the 31P-NMR spectra, the dominant Po form in soils from all study sites was monoesters with similar spectra. The 31P-NMR findings were basically consistent with those from chemical extraction. Soil formation processes may be the critical factor affecting the distribution of soil P. High precipitation and year-round high humidity may be important in the differentiation of the P species in this landscape.

The LiCl and LiNO₃ water solutions in the presence of small amounts of 3-helium have been investigated by means of multinuclear resonance spectroscopy. The resulting concentration dependences of the ³He, ^6,7Li⁺, ¹⁴NO₃^¯ and ³⁵Cl^¯ resonance radiofrequencies are reported in the infinite limit. This data along with new theoretical corrections of shielding lithium ions was analyzed by a known NMR relationship method. Consequently, the nuclear magnetic moments of ⁶Li and ⁷Li were established against that of the helium-3 dipole moment: μ(⁶Li)=+0.822046(5)μ_N and μ(⁷Li)=+3.256418(20)μ_N. The new results were shown to be very close to the previously obtained values of the (ABMR) atomic beam magnetic resonance method. This experiment proves that our helium method is well suited for establishing dipole moments from NMR measurements performed in water solutions. This technique is especially valuable when gaseous substances of the needed element are not available. All shielding constants of species present in water solutions are consistent with new nuclear magnetic moments and these taken as a reference. Both techniques – NMR and ABMR – give practically the same results providing that all shielding corrections are properly made.

Abstract: Filipendula ulmaria, commonly known as meadowsweet, is a wild herbaceous flowering plant widely distributed in Europe. A range of salicylic acid derivatives and flavonol glycosides have been previously associated with the antirheumatic and diuretic properties of F. ulmaria. In the present work, an hydroalcoholic extract of F. ulmaria aerial parts was extensively profiled using an efficient NMR-based dereplication strategy. The approach involves fractionation of the crude extract by centrifugal partition chromatography (CPC), ¹³C NMR analysis of the fractions, 2D-cluster mapping of the whole NMR dataset and finally, structure elucidation using a natural metabolite database, validated by 2D NMR data interpretation and liquid chromatography coupled to mass spectrometry. The chemodiversity of the aerial parts was quite broad, with 28 compounds unambiguously identified, belonging to various biosynthetic classes. The F.ulmaria extract and CPC fractions were screened for their potential to improve skin epidermal barrier function and also skin renewal properties using in vitro assays performed on Normal Human Epidermal Keratinocytes. Fractions containing quercetin, kaempferol glycosides, ursolic acid, pomolic acid, naringenin, β-sitosterol, and tellimagrandins I and II were found to be responsible for the up-regulation of genes related to skin barrier function, epidermal renewal and stress responses. This research is significant as it could provide a natural solution for improving hydration and skin renewal properties.

Reservoir in the Central Structural Zone of the Xihu Sag is the Huagang Formation, dominated by natural gas reservoirs, and the reservoir in the Western Slope Zone is the Pinghu Formation, dominated by light oil and wet gas. In this paper, the Gas Drive Water Displacement- Magnetic Resonance Imaging (GWD-MRI) experiments are used to simulate the charging characteristics of the sandstone migration layer. The centrifugal- Magnetic Resonance (Cen-NMR) experiments simulate the short-term fast trap charging process, and the Semi-Permeable Baffle (SPB) charging experiment simulates the slow trap accumulation process. Studies have shown that there is a start-up pressure for the migration layer charging, and the start-up pressure of the core with a permeability of 0.3mD is about 0.6MPa. Experimental simulations have confirmed that there is a front zone with changed water saturation, with a width of about 1-1.5cm. The migration layer charging mainly has two actions, drive effect and carry effect. The drive effect reduces the water saturation to 70%-80%, and the carry effect can still reduce the water saturation by 5%-10%. The water saturation of rapid charging is mainly affected by the petro-physical. The porosity is high, the water saturation is low. The water saturation decreased significantly with the increase of centrifugal force when the centrifugal force is smaller. If the centrifugal force is greater than 0.8 MPa, the water saturation decreases slowly. The water saturation of the trap slowly charging is basically maintained at 40%-50%, which matches the water saturation of the airtight coring from 40%-55%, the petro-physical do not affect the final water saturation.

Nuclear magnetic resonance (NMR) techniques are widely used to identify pure substances and probe protein dynamics. Edible oil is a complex mixture composed of hydrocarbons, which have a wide range of molecular size distribution. In this research, low field NMR (LF-NMR) relaxation characteristic data of various sample oils were analyzed. We also suggest a new method for prediction size of edible oil molecules using LF-NMR relaxation time distributions. According to the relative molecular mass, carbon chain length, transverse relaxation time of different sample oils, combined with oil viscosity and other factors, the relationship between carbon chain length and transverse relaxation time rate was analyzed. Various oils and fats in the mixed fluid are displayed, reflecting the composition information of different oils. To study the correlation between the rotation correlation time and molecular information of oil molecules. The molecular composition of the resulting fluid determines its properties, such as viscosity and phase behavior. The results show that low-field NMR can obtain information on the composition, macromolecular aggregation, and molecular dynamics of complex fluids. Measurements of grease in the free fluid state show that the relaxation time can reflect the intrinsic properties of the fluid. It is shown that the composition characteristics and states of complex fluids can be measured by low-field nuclear magnetic resonance.

The deep Cretaceous tight sandstone in Kuqa Depression of Tarim foreland basin is an ultra-low porosity and ultra-deep gas-bearing reservoir, which is characterized by small pores, fine throats, and poor connectivity. The wireline logging responses are so complex, and especially, it is difficult to identify fluid types from resistivity logs. Based on acoustic, density, and neutron logs response differences in gas and water layers, effective fluid sensitivity factors are constructed for gas layer identification. From conventional logs, acoustic-neutron porosity difference, density-neutron porosity difference, and triple-porosity ratio are all sensitive parameters to the gas layer. From the NMR logging response mechanism, the density and NMR porosity difference, and T2 geometric mean of the movable fluid are also two sensitive parameters to the gas layer. Based on these parameters, a series of fluid typing charts are constructed and their adaptabilities are analyzed and compared. By contrast, NMR log interpretation is better, and triple-porosity comprehensive method from conventional logs is also effective when NMR logging is not available. Finally, the comprehensive fluid typing strategy by combining some methods for ultra-low porosity tight sandstone is summarized and optimized. This study is another alternative for fluid identification using non-electrical logs.

Recently the number of studies investigating triterpenoid saponins has drastically increased due to their diverse and potentially attractive biological activities. Currently the literature contains chemical structures of few hundreds of triterpenoid saponins of plant and animal origin. Triterpenoid saponins consist of a triterpene aglycone with one or more sugar moieties attached to it. However, due to similar physico-chemical properties, isolation and identification of a large diversity of triterpenoid saponins remain challenging. This study demonstrates a methodology to screen saponins using hyphenated analytical platforms, GC-MS, LC-MS/MS, and LC-SPE-NMR/MS, in the example of two different phenotypes of the model plant Barbarea vulgaris (winter cress), glabrous (G) and pubescent (P) type that are known to differ by their insect resistance. The proposed methodology allows for detailed comparison of saponin profiles from intact plant extracts as well as saponin aglycone profiles from hydrolysed samples. Continuously measured 1D proton NMR data during LC separation along with mass spectrometry data revealed significant differences, including contents of saponins, types of aglycones and numbers of sugar moieties attached to the aglycone. A total of 49 peaks were tentatively identified as saponins from both plants; they are derived from eight types of aglycones and with 2–5 sugar moieties. Identification of two previously known insect-deterrent saponins, hederagenin cellobioside and oleanolic acid cellobioside, demonstrated the applicability of the methodology for relatively rapid screening of bioactive compounds.

Magnetic Resonance Imaging (MRI) is widely used in clinics and research due to its accurate disease identification and non-invasive nature. MRI is based on Nuclear Magnetic Resonance (NMR), which is also extensively employed in various fields. NMR and its modern versions involve intricate pre-processing steps before data analysis. These steps are initially processed in the time domain and subsequently in the frequency domain. While our previous review focused on time domain pre-processing (https://www.preprints.org/manuscript/202310.2032/v1), this review delves into the mathematical and statistical aspects of frequency domain pre-processing. We discuss essential pre-processing steps like phase error correction, baseline correction, solvent filtering, calibration and alignment, reference deconvolution, binning/bucketing and peak picking, peak fitting/deconvolution and compound identification, integration and quantification, normalization and transformation. Furthermore, we offer practical recommendations for each step.

Porphyrinic photosensitizers (PSs) and their nano-sized polymer-based carrier systems are required to exhibit low dark toxicity, avoid side effects, and ensure high in vivo tolerability. Yet, little is known about the intracellular fate of PSs during the dark incubation period and how it is af-fected by nanoparticles. In a systematic study, high resolution magic angle spinning NMR spectroscopy combined with statistical analyses was used to study the metabolic profile of cultured HeLa cells treated with different concentrations of the PS chlorin e4 (Ce4) alone or encapsulated in carrier systems. For the latter, either polyvinylpyrrolidone (PVP) or the micelle forming polyethylene glycol (PEG)-polypropylene glycol triblock copolymer Kolliphor P188 (KP) were used. Diffusion edited spectra indicated Ce4 membrane localization evidenced by Ce4 concentration dependent chemical shift perturbation of the cellular phospholipid choline resonance. The effect was also visible in the presence of KP and PVP, but less pronounced. The appearance of the PEG resonance in the cell spectra pointed towards cell internalization of KP whereas no conclusion could be drawn for PVP that remained NMR-invisible. Multivariate statistical analyses of the cell spectra (PCA, PLS-DA and oPLS) revealed a concentration-dependent metabolic response upon exposure to Ce4 that was attenuated by KP and even more by PVP. Significant Ce4-concentration dependent alterations were mainly found for metabolites involved in the tricarboxylic acid cycle and the phosphatidylcholine metabolism. The data underline an important protective role of the polymeric carriers following cell internalization. Moreover, the current study allowed - to our knowledge for the first time - to trace the intracellular PS localization on an atomic level by NMR methods.

The available magnetic field strength for high resolution NMR in persistent superconducting magnets has recently improved from 23.5 to 28 Tesla, increasing the proton resonance frequency from 1 to 1.2 GHz. For magic-angle spinning (MAS) NMR, this is expected to improve resolution, provided the sample preparation results in homogeneous broadening. We compare two-dimensional (2D) proton detected MAS NMR spectra of four membrane proteins at 950 and 1.2 GHz. We find a consistent improvement in resolution that scales superlinearly with the increase in magnetic field for three of the four examples. In 3D and 4D spectra, which are now routinely acquired, this improvement indicates the ability to resolve at least 2 and 2.5 times as many signals, respectively.

Continuous research into the availability of phosphorus (P) in forest soil is critical for sustainable management of forest ecosystems. In this study, we used sequential chemical extraction and 31P-nuclear magnetic resonance spectroscopy (31P-NMR) to evaluate the form and distribution of inorganic P (Pi) and organic P (Po) in Casuarina forest soils of a subtropical coastal sand dune at Houlong in Taiwan. The soil samples were collected from humic (+2-0 cm) and mineral layers (mineral-I: 0-10, mineral-II: 10-20 cm) at two topographic locations (upland and lowland) by elevation. Sequential chemical extraction revealed that the NaOH-Po fraction, as moderately recalcitrant P, was the dominant form in humic and mineral-I layers in both upland and lowland soils, whereas the cHCl-Pi fraction was the dominant form in the mineral-II layer. Resistant P content, including NaOH-Pi, HCl-Pi, cHCl-Pi, and cHCl-Po fractions, was higher in the upland than lowland in the corresponding layers; however, labile P content, NaHCO3-Po, showed the opposite pattern. Content of resistant Pi (NaOH-Pi, HCl-Pi, and cHCl-Pi) increased significantly with depth, but that of labile Pi (resin-Pi and NaHCO3-Pi) and recalcitrant Po (NaHCO3-Po, NaOH-Po, and cHCl-Po) decreased significantly with depth at both locations. 31P-NMR spectroscopy revealed inorganic orthophosphate and monoesters-P as the major forms in this area. The proportions of Pi and Po evaluated by sequential chemical extraction and 31P-NMR spectroscopy were basically consistent. The results indicated that the soils were in weathered conditions. Furthermore, the P distribution and forms significantly differed between the upland and lowland by variation in elevation and eolian aggradation effects in this coastal sand dune landscape.

JBIR-99 is a secondary metabolite of marine fungi that has been shown to possess strong antibiotic activity. An efficient approach using a combination of size exclusion chromatography with a Sephadex LH-20 and high-speed counter-current chromatography (HSCCC) has been successfully developed for the isolation and purification of a polyketide from the solid-state fermentation of Meyerozyma guilliermondii. The active compound was isolated with purity >95% by HSCCC using an optimized solvent system composed of petroleum ether–ethyl acetate– 95% ethanol–water (5:3:5:3, v/v/v/v) after size exclusion chromatography. This compound was successfully purified in the quantity of 68 mg from 120 mg of the crude extract. The structure of JBIR-99 was elucidated and assigned by 1D, 2D NMR spectroscopic, and positive HRESITOFMS. Moreover, the relative configuration of compound JBIR-99, displaying a quite complex multi-ring structure, is determined by X-ray crystallography for the first time. The purification method developed for JBIR-99 will facilitate the further investigation and development of this antibiotic agent as a lead compound. Furthermore, it is suggested that the combination of size exclusion chromatography and HSCCC could be more widely applied for the isolation and purification of polyketides from marine fungi.

This article has been studied the synthesis of a new derivative of the known alkaloid cytisine contained in the seeds of plants of Cytisus laburnum L. and Thermopsis lanceolata R.Br., both of the Lugiminosae family. The new compound has been obtained from two biologically active compounds such as isoxazole and cytisine. It has been demonstrated that the reaction led to the single-stage method under very mild conditions to obtain some 4-[(3,5-dimethyl-1,2-oxazole-4-yl)sulfonyl]amides. This class of compounds is promising to obtain the new biologically active compounds. This article has examined in detail a structure with using the 1H and 13C NMR and two-dimensional NMR spectroscopy of COSY (1H-1H), HMQC (1H-13C) and NMVS (1H-13C). As a result, the homo- and heteronuclear spin-spin couplings should be established. The X-ray diffraction analysis has determined the spatial structure of a new derivative based on cytisine alkaloid. Thus, its hemorheological activity has been studied.

Lithium titanates are used in various applications, such as anode materials for lithium intercalation (Li4Ti5O12) or breeding materials in fusion reactors (Li2TiO3). Here, we report the formation of nano-crystalline lithium titanates by a mechanochemical approach and present a deeper insight into their structural characteristics by PXRD and solid-state NMR spectroscopy. The compounds were synthesized in a high energy planetary ball mill with varying milling parameters and different grinding tools. NaCl type Li2TiO3 (α-Li2TiO3) was formed by dry milling of lithium hydroxide with titania (rutile or anatase) and by a milling induced structure transformation of monoclinic β Li2TiO3 or spinel type Li4Ti5O12. Heating of mechanochemical prepared α Li2TiO3 induces a phase transformation to the monoclinic phase similar to hydrothermal reaction products, but a higher thermal stability was observed for the mechanochemical formed product. Microstructure and crystallographic structure were characterized by PXRD via Rietveld analysis. Detailed phase analysis shows the formation of the cubic phase from the various educts. A set of two lattice parameters for α Li2TiO3 was refined, depending on the presence of OH- during the milling process. An average crystallite size of less than 15 nm was observed for the mechanochemical generated products. The local Li environment detected by 6Li SPE MAS NMR revealed Li defects in the form of tetrahedral instead of octahedral site occupation. Subsequent adjustment of the structural model for Rietveld refinement leads to better fits, supporting this interpretation.

Hepcidin-25 was identified as the main iron regulator in the human body by binding to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II)-binding site known as ATCUN (amino terminal Cu(II)- and Ni(II)- binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the formed complex of hepcidin-25 with copper could reveal insights into its biological role. The present work is mainly focused on the study of the metal-bound form of hepcidin-25, which could be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25, which was achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandem mass spectrometry MS/MS, high resolution mass spectrometry HRMS) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a 3D model of hepcidin-25 with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or reference material comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.

Peptides of hydrolysates of food proteins are an easily digestible source of amino acids necessary for the body to adapt to physical stress. Commercially significant hydrolysates include whey protein, casein, and other animal proteins. Hydrolysates of plant proteins are gaining popularity, but they are less common, then animal ones. Soy protein isolate is promising for obtaining the hydrolysates due to its affordable price and balanced amino acid profile. However, there are no direct studies showing an improvement in the result of physical activity when eating soy protein isolate hydrolysate (SPIH). In this work, for the first time, the study was conducted on the safety and efficacy of SPIH during physical load on model animals (rats). It was shown that the hydrolysate did not lead to pathological changes in the viscera, food intake, and weight of animals did not differ from the control group (animals consumed whey protein). Under physical load rats enteral fed SPIH showed a tendency to adapt more quickly to physical stress than the control group and the group of animals that was fed by free amino acids. The metabolites of animal blood serum were studied by NMR spectroscopy. It was shown that by the 95th minute after feeding in the group of rats receiving SPIH, the difference of proteinogenic amino acids concentrations in blood between individuals was significantly less than in the groups receiving whey protein or a mixture of amino acids. In other words, individual biochemical and physiological characteristics of individuals did not affect the assimilation of amino acids of hydrolysate.

The fresh bulbs of Stenomesson miniatum, a plant belonging to Amaryllidaceae family with a poorly investigated phytochemical profile, were traditionally employed by Andean healers to treat tumors and abscesses. The aims of this study were to characterize the alkaloid extract from the bulbs of S. miniatum and test its cytotoxic and antibacterial potential. The alkaloid extract was characterized by dereplication using various techniques (CPC, NMR, UPLC-HRMS) and referring to in-home or online databases for spectroscopic data matching. Cytotoxic activities were evaluated on A431 human epidermoid carcinoma cells through a metabolic assay, and on Jurkat human acute T-leukemia cells through a cell-impermeant fluorescent nuclear probe. Antibacterial assays were carried out against Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes by using a standardized microdilution broth method 96-well plate. Eleven known Amaryllidaceae alkaloids were identified together with another compound determined as being an extraction artefact. The alkaloid extract showed good cytotoxic activity against both the tumor cell lines, reaching an IC50 of 3.3 µg/mL against A431 cells and of 10.9 µg/mL against Jurkat cells. The analysis of the fractions allowed the activity to be attributed to the presence of pretazettine and haemathamine. Conversely, no antibacterial activities were recorded for any of the samples.

This study focused on hydrolysis of cosmetic fillers hyaluronic acid (HA) and kinetics of the HA hydrolysis using the homogenate of the red king crab hepatopancreas. Turbidimetric analysis of the reaction mixture revealed a bell-shaped time dependence of aggregation formation. It was shown that the obtained homogenate has the similar activity to the commercially available hyaluronidase. The atomic force microscopy (AFM) examination found that the HA fillers were represented by spherical-like structures. These structures were destroyed under the action of the homogenate of the red king crab hepatopancreas. NMR of the reaction mixture showed that HA degradation lasts for some days, but a maximum rate of the reaction is detected in the first hours of incubation. The preparation with hyaluronidase activity obtained from the red king crab hepatopancreas could be used as potentially safe product for treating filler complications.