Integrating genomics and multiplatform metabolomics enables metabolite quantitative trait loci detection in breeding-relevant apple germplasm.

Apple (Malus × domestica) has commercial and nutritional value, but breeding constraints of tree crops limit varietal improvement. Marker-assisted selection minimises these drawbacks, but breeders lack applications for targeting fruit phytochemicals. To understand genotype-phytochemical associations in apples, we have developed a high-throughput integration strategy for genomic and multiplatform metabolomics data. Here, 124 apple genotypes, including members of three pedigree-connected breeding families alongside diverse cultivars and wild selections, were genotyped and phenotyped. Metabolite genome-wide association studies (mGWAS) were conducted with c. 10 000 single nucleotide polymorphisms and phenotypic data acquired via LC-MS and 1 H NMR untargeted metabolomics. Putative metabolite quantitative trait loci (mQTL) were then validated via pedigree-based analyses (PBA). Using our developed method, 519, 726 and 177 putative mQTL were detected in LC-MS positive and negative ionisation modes, and NMR, respectively. mQTL were indicated on each chromosome, with hotspots on linkage groups 16 and 17. A chlorogenic acid mQTL was discovered on chromosome 17 via mGWAS and validated with a two-step PBA, enabling discovery of novel candidate gene-metabolite relationships. Complementary data from three metabolomics approaches and dual genomics analyses increased confidence in validity of compound annotation and mQTL detection. Our platform demonstrates the utility of multiomic integration to advance data-driven, phytochemical-based plant breeding.

Polyphenols Weaken Pea Protein Gel by Formation of Large Aggregates with Diminished Noncovalent Interactions.

Polyphenols are well-known native cross-linkers and gel strengthening agents for many animal proteins. However, their role in modifying plant protein gels remains unclear. In this study, multiple techniques were applied to unravel the influence of green tea polyphenols (GTP) on pea protein gels and the underlying mechanisms. We found that the elasticity and viscosity of pea protein gels decreased with increased GTP. The protein backbone became less rigid when GTP was present based on shortened T1ρH in relaxation solid-state NMR measurements. Electron microscopy and small-angle X-ray scattering showed that gels weakened by GTP possessed disrupted networks with the presence of large protein aggregates. Solvent extraction and molecular dynamic simulation revealed a reduction in hydrophobic interactions and hydrogen bonds among proteins in gels containing GTP. The current findings may be applicable to other plant proteins for greater control of gel structures in the presence of polyphenols, expanding their utilization in food and biomedical applications.

Analysis and Authentication of Avocado Oil Using High Resolution NMR Spectroscopy.

Avocado oil is a food product of high commercial and nutritional value. As a result, it can be a subject of adulteration similar to other high-value edible oils, such as olive oil. For olive oil and many other foods products, NMR spectroscopy has been successfully used for authentication and quality assessment. In this study, we apply NMR analysis to avocado oil to differentiate it from other oils including olive, canola, high-oleic (HO) safflower, HO sunflower and soybean oil using commercial and lab-made samples of avocado oils. NMR allowed the rapid analysis of the fatty acid profile and detection of minor compounds, such as sterols, oxidation products, and hydrolysis products, which can be used to assess oil quality and authenticity. The NMR assignment was conducted using traditional 2D NMR and the novel NOAH super-sequences. Combining chemometrics with NMR enabled us to differentiate between avocado oil and other oils. Avocado oil has compositional similarities with other vegetable oils, such as HO sunflower and HO safflower oil, which can be used as potential adulterants. Despite these similarities, NMR-based metabolomics captured differences in the levels of certain compounds including fatty acids, terpenes, sterols, and oxidation products to detect adulteration and for quality control purposes.

Enzymatic Preparation of Bioactive Peptides Exhibiting ACE Inhibitory Activity from Soybean and Velvet Bean: A Systematic Review.

The Angiotensin-I-converting enzyme (ACE) is a peptidase with a significant role in the regulation of blood pressure. Within this work, a systematic review on the enzymatic preparation of Angiotensin-I-Converting Enzyme inhibitory (ACEi) peptides is presented. The systematic review is conducted by following PRISMA guidelines. Soybeans and velvet beans are known to have high protein contents that make them suitable as sources of parent proteins for the production of ACEi peptides. Endopeptidase is commonly used in the preparation of soybean-based ACEi peptides, whereas for velvet bean, a combination of both endo- and exopeptidase is frequently used. Soybean glycinin is the preferred substrate for the preparation of ACEi peptides. It contains proline as one of its major amino acids, which exhibits a potent significance in inhibiting ACE. The best enzymatic treatments for producing ACEi peptides from soybean are as follows: proteolytic activity by Protease P (Amano-P from Aspergillus sp.), a temperature of 37 °C, a reaction time of 18 h, pH 8.2, and an E/S ratio of 2%. On the other hand, the best enzymatic conditions for producing peptide hydrolysates with high ACEi activity are through sequential hydrolytic activity by the combination of pepsin-pancreatic, an E/S ratio for each enzyme is 10%, the temperature and reaction time for each proteolysis are 37 °C and 0.74 h, respectively, pH for pepsin is 2.0, whereas for pancreatin it is 7.0. As an underutilized pulse, the studies on the enzymatic hydrolysis of velvet bean proteins in producing ACEi peptides are limited. Conclusively, the activity of soybean-based ACEi peptides is found to depend on their molecular sizes, the amino acid residues, and positions. Hydrophobic amino acids with nonpolar side chains, positively charged, branched, and cyclic or aromatic residues are generally preferred for ACEi peptides.

NMR-Based Analysis of Pomegranate Juice Using Untargeted Metabolomics Coupled with Nested and Quantitative Approaches.

Pomegranate juice is a complex mixture of structurally diverse compounds appearing in various concentrations. The composition of the final product depends on several factors, such as the fruit variety and the addition of adulterants. Its diverse composition makes pomegranate juice an excellent system for assessing the potential of an analytical method for targeted and untargeted analysis. Here, we tested the ability of 1D and 2D NMR spectroscopy techniques for the targeted and untargeted metabolite analysis of pomegranate juice. The NMR spectra assignment was performed using the novel NOAH sequences and spiking with model compounds. Several metabolites, including sugars, organic acids, and amino acids, were identified and quantified in a rapid and simultaneous manner. Five internal standards were tested, with potassium hydrogen phthalate and dimethylmalonic acid found to be the most appropriate, based on their shorter T1 relaxation times and spectral simplicity, while MnCl2 was successfully applied as a relaxation agent for the reduction of the experimental time. Among the pulse sequences that were tested for their quantitative potential, the Carr-Purcell-Meiboom-Gill gave the best results. The quantitative, QEC-HSQC experiment was also found to be very promising for mixture analysis. Additionally, the potential of 1D/2D NMR-based untargeted analysis was successfully tested on two cases, namely, differentiation between cultivars and detection of adulteration with apple juice. This study demonstrates the proof of concept for 1D and 2D NMR methods in the targeted and untargeted analysis of pomegranate juice and can be extended to other complex matrices.

Quantitative Measurement of a Chiral Drug in a Complex Matrix: A J-Compensated Quantitative HSQC NMR Method.

We developed a J-compensated QHSQC NMR method for the quantitative measurement of enantiopurity and concentration of levofloxacin in a complex drug matrix. The pulse sequence can achieve uniform signal responses by the suppression of the heteronuclear coupling modulation and alleviation of the homonuclear coupling evolution. Furthermore, we discuss the influence of relaxation on peak intensities and propose the selection conditions of reference signals to achieve accurate quantifications. The evaluation of the methodology shows that the results obtained by selected peaks are in accordance with theoretical analysis with good reliability and linearity. The enantiomeric separation and quantification of levofloxacin in creams are achieved by using a chiral solvating agent, a reference compound, and the J-compensated QHSQC pulse sequence. To our knowledge, this is the first example of QHSQC methodology to quantify enantiomers in the complex matrix.

Dose-Dependent Effects of Triclocarban Exposure on Lipid Homeostasis in Rats.

Environmental exposure to triclocarban (TCC), a common antibacterial agent widely used in thousands of personal care products, poses a potential risk for human health. Previous in vitro studies about biological effects of TCC have yielded a variety of inconsistent results and apparently not been verified in vivo. In the current study, dose-dependent effects of TCC exposure on lipid homeostasis in rats were investigated using a combination of untargeted 1H NMR metabolomics, targeted metabolite profiling (LC/GC-MS), histopathological assessments, and biological assays. Our results revealed that TCC dose-dependently activated aryl hydrocarbon receptor (AHR) and its transcriptional targets such as Cyp1a1 and Cyp1b1 in the liver of rats, suggesting that TCC may be a potent AHR agonist. Although TCC exhibited dose-dependent toxicity, oral exposure with relatively low dose TCC caused more significant hepatic lipogenesis of rats than relatively high and moderate doses of TCC. It was mainly manifested by histopathological observations and promotion of de novo fatty acid, phospholipid, and ceramide biosynthesis and gut microbiota fermentation. Our findings provide new insights into health effects of TCC exposure with different dosages in vivo, especially on the induction and progression of nonalcoholic fatty liver disease, and further our understanding in the pathogenesis of metabolic diseases induced by environmental pollutants.

Nuclear Magnetic Resonance (NMR) Spectroscopy in Food Science: A Comprehensive Review.

Nuclear magnetic resonance (NMR) spectroscopy is a robust method, which can rapidly analyze mixtures at the molecular level without requiring separation and/or purification steps, making it ideal for applications in food science. Despite its increasing popularity among food scientists, NMR is still an underutilized methodology in this area, mainly due to its high cost, relatively low sensitivity, and the lack of NMR expertise by many food scientists. The aim of this review is to help bridge the knowledge gap that may exist when attempting to apply NMR methodologies to the field of food science. We begin by covering the basic principles required to apply NMR to the study of foods and nutrients. A description of the discipline of chemometrics is provided, as the combination of NMR with multivariate statistical analysis is a powerful approach for addressing modern challenges in food science. Furthermore, a comprehensive overview of recent and key applications in the areas of compositional analysis, food authentication, quality control, and human nutrition is provided. In addition to standard NMR techniques, more sophisticated NMR applications are also presented, although limitations, gaps, and potentials are discussed. We hope this review will help scientists gain some of the knowledge required to apply the powerful methodology of NMR to the rich and diverse field of food science.

P-N Cooperative Borane Activation and Catalytic Hydroboration by a Distorted Phosphorous Triamide Platform.

Studies of the stoichiometric and catalytic reactivity of a geometrically constrained phosphorous triamide 1 with pinacolborane (HBpin) are reported. The addition of HBpin to phosphorous triamide 1 results in cleavage of the B-H bond of pinacolborane through addition across the electrophilic phosphorus and nucleophilic N-methylanilide sites in a cooperative fashion. The kinetics of this process of were investigated by NMR spectroscopy, with the determined overall second-order empirical rate law given by ν = -k[1][HBpin], where k = 4.76 × 10-5 M-1 s-1 at 25 °C. The B-H bond activation process produces P-hydrido-1,3,2-diazaphospholene intermediate 2, which exhibits hydridic reactivity capable of reacting with imines to give phosphorous triamide intermediates, as confirmed by independent synthesis. These phosphorous triamide intermediates are typically short lived, evolving with elimination of the N-borylamine product of imine hydroboration with regeneration of the deformed phosphorous triamide 1. The kinetics of this latter process are shown to be first-order, indicative of a unimolecular mechanism. Consequently, catalytic hydroboration of a variety of imine substrates can be realized with 1 as the catalyst and HBpin as the terminal reagent. A mechanistic proposal implicating a P-N cooperative mechanism for catalysis that incorporates the various independently verified stoichiometric steps is presented, and a comparison to related phosphorus-based systems is offered.

Orthogonal Comparison of GC-MS and 1H NMR Spectroscopy for Short Chain Fatty Acid Quantitation.

Short chain fatty acids (SCFAs) are important regulators of host physiology and metabolism and may contribute to obesity and associated metabolic diseases. Interest in SCFAs has increased in part due to the recognized importance of how production of SCFAs by the microbiota may signal to the host. Therefore, reliable, reproducible, and affordable methods for SCFA profiling are required for accurate identification and quantitation. In the current study, four different methods for SCFA (acetic acid, propionic acid, and butyric acid) extraction and quantitation were compared using two independent platforms including gas chromatography coupled with mass spectrometry (GC-MS) and 1H nuclear magnetic resonance (NMR) spectroscopy. Sensitivity, recovery, repeatability, matrix effect, and validation using mouse fecal samples were determined across all methods. The GC-MS propyl esterification method exhibited superior sensitivity for acetic acid and butyric acid measurement (LOD < 0.01 µg mL-1, LOQ < 0.1 µg mL-1) and recovery accuracy (99.4%-108.3% recovery rate for 100 µg mL-1 SCFA mixed standard spike in and 97.8%-101.8% recovery rate for 250 µg mL-1 SCFAs mixed standard spike in). NMR methods by either quantitation relative to an internal standard or quantitation using a calibration curve yielded better repeatability and minimal matrix effects compared to GC-MS methods. All methods generated good calibration curve linearity (R2 > 0.99) and comparable measurement of fecal SCFA concentration. Lastly, these methods were used to quantitate fecal SCFAs obtained from conventionally raised (CONV-R) and germ free (GF) mice. Results from global metabolomic analysis of feces generated by 1H NMR and bomb calorimetry were used to further validate these approaches.

Antioxidant Drug Tempol Promotes Functional Metabolic Changes in the Gut Microbiota.

Recent studies have identified the important role of the gut microbiota in the pathogenesis and progression of obesity and related metabolic disorders. The antioxidant tempol was shown to prevent or reduce weight gain and modulate the gut microbiota community in mice; however, the mechanism by which tempol modulates weight gain/loss with respect to the host and gut microbiota has not been clearly established. Here we show that tempol (0, 1, 10, and 50 mg/kg p.o. for 5 days) decreased cecal bacterial fermentation and increased fecal energy excretion in a dose-dependent manner. Liver (1)H NMR-based metabolomics identified a dose-dependent decrease in glycogen and glucose, enhanced glucogenic and ketogenic activity (tyrosine and phenylalanine), and increased activation of the glycolysis pathway. Serum (1)H NMR-based metabolomics indicated that tempol promotes enhanced glucose catabolism. Hepatic gene expression was significantly altered as demonstrated by an increase in Pepck and G6pase and a decrease in Hnf4a, ChREBP, Fabp1, and Cd36 mRNAs. No significant change in the liver and serum metabolomic profiles was observed in germ-free mice, thus establishing a significant role for the gut microbiota in mediating the beneficial metabolic effects of tempol. These results demonstrate that tempol modulates the gut microbial community and its function, resulting in reduced host energy availability and a significant shift in liver metabolism toward a more catabolic state.

Reversing methanogenesis to capture methane for liquid biofuel precursors.

BACKGROUND: Energy from remote methane reserves is transformative; however, unintended release of this potent greenhouse gas makes it imperative to convert methane efficiently into more readily transported biofuels. No pure microbial culture that grows on methane anaerobically has been isolated, despite that methane capture through anaerobic processes is more efficient than aerobic ones. RESULTS: Here we engineered the archaeal methanogen Methanosarcina acetivorans to grow anaerobically on methane as a pure culture and to convert methane into the biofuel precursor acetate. To capture methane, we cloned the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable organism, anaerobic methanotrophic archaeal population 1 (ANME-1) from a Black Sea mat, into M. acetivorans to effectively run methanogenesis in reverse. Starting with low-density inocula, M. acetivorans cells producing ANME-1 Mcr consumed up to 9 ± 1 % of methane (corresponding to 109 ± 12 µmol of methane) after 6 weeks of anaerobic growth on methane and utilized 10 mM FeCl3 as an electron acceptor. Accordingly, increases in cell density and total protein were observed as cells grew on methane in a biofilm on solid FeCl3. When incubated on methane for 5 days, high-densities of ANME-1 Mcr-producing M. acetivorans cells consumed 15 ± 2 % methane (corresponding to 143 ± 16 µmol of methane), and produced 10.3 ± 0.8 mM acetate (corresponding to 52 ± 4 µmol of acetate). We further confirmed the growth on methane and acetate production using (13)C isotopic labeling of methane and bicarbonate coupled with nuclear magnetic resonance and gas chromatography/mass spectroscopy, as well as RNA sequencing. CONCLUSIONS: We anticipate that our metabolically-engineered strain will provide insights into how methane is cycled in the environment by Archaea as well as will possibly be utilized to convert remote sources of methane into more easily transported biofuels via acetate.

Dynamic Coupling at the Ångström Scale.

While momentum transfer from active particles to their immediate surroundings has been studied for both synthetic and biological micron-scale systems, a similar phenomenon was presumed unlikely to exist at smaller length scales due to the dominance of viscosity in the ultralow Reynolds number regime. Using diffusion NMR spectroscopy, we studied the motion of two passive tracers--tetramethylsilane and benzene--dissolved in an organic solution of active Grubbs catalyst. Significant enhancements in diffusion were observed for both the tracers and the catalyst as a function of reaction rate. A similar behavior was also observed for the enzyme urease in aqueous solution. Surprisingly, momentum transfer at the molecular scale closely resembles that reported for microscale systems and appears to be independent of swimming mechanism. Our work provides new insight into the role of active particles on advection and mixing at the Ångström scale.

Metabolomics Reveals that Aryl Hydrocarbon Receptor Activation by Environmental Chemicals Induces Systemic Metabolic Dysfunction in Mice.

Environmental exposure to dioxins and dioxin-like compounds poses a significant health risk for human health. Developing a better understanding of the mechanisms of toxicity through activation of the aryl hydrocarbon receptor (AHR) is likely to improve the reliability of risk assessment. In this study, the AHR-dependent metabolic response of mice exposed to 2,3,7,8-tetrachlorodibenzofuran (TCDF) was assessed using global (1)H nuclear magnetic resonance (NMR)-based metabolomics and targeted metabolite profiling of extracts obtained from serum and liver. (1)H NMR analyses revealed that TCDF exposure suppressed gluconeogenesis and glycogenolysis, stimulated lipogenesis, and triggered inflammatory gene expression in an Ahr-dependent manner. Targeted analyses using gas chromatography coupled with mass spectrometry showed TCDF treatment altered the ratio of unsaturated/saturated fatty acids. Consistent with this observation, an increase in hepatic expression of stearoyl coenzyme A desaturase 1 was observed. In addition, TCDF exposure resulted in inhibition of de novo fatty acid biosynthesis manifested by down-regulation of acetyl-CoA, malonyl-CoA, and palmitoyl-CoA metabolites and related mRNA levels. In contrast, no significant changes in the levels of glucose and lipid were observed in serum and liver obtained from Ahr-null mice following TCDF treatment, thus strongly supporting the important role of the AHR in mediating the metabolic effects seen following TCDF exposure.

Solution structure of the major G-quadruplex formed in the human VEGF promoter in K+: insights into loop interactions of the parallel G-quadruplexes.

Vascular endothelial growth factor (VEGF) proximal promoter region contains a poly G/C-rich element that is essential for basal and inducible VEGF expression. The guanine-rich strand on this tract has been shown to form the DNA G-quadruplex structure, whose stabilization by small molecules can suppress VEGF expression. We report here the nuclear magnetic resonance structure of the major intramolecular G-quadruplex formed in this region in K(+) solution using the 22mer VEGF promoter sequence with G-to-T mutations of two loop residues. Our results have unambiguously demonstrated that the major G-quadruplex formed in the VEGF promoter in K(+) solution is a parallel-stranded structure with a 1:4:1 loop-size arrangement. A unique capping structure was shown to form in this 1:4:1 G-quadruplex. Parallel-stranded G-quadruplexes are commonly found in the human promoter sequences. The nuclear magnetic resonance structure of the major VEGF G-quadruplex shows that the 4-nt middle loop plays a central role for the specific capping structures and in stabilizing the most favored folding pattern. It is thus suggested that each parallel G-quadruplex likely adopts unique capping and loop structures by the specific middle loops and flanking segments, which together determine the overall structure and specific recognition sites of small molecules or proteins. LAY SUMMARY: The human VEGF is a key regulator of angiogenesis and plays an important role in tumor survival, growth and metastasis. VEGF overexpression is frequently found in a wide range of human tumors; the VEGF pathway has become an attractive target for cancer therapeutics. DNA G-quadruplexes have been shown to form in the proximal promoter region of VEGF and are amenable to small molecule drug targeting for VEGF suppression. The detailed molecular structure of the major VEGF promoter G-quadruplex reported here will provide an important basis for structure-based rational development of small molecule drugs targeting the VEGF G-quadruplex for gene suppression.

Identification of Compounds That Impact Consumer Flavor Liking of American-European Hazelnut Hybrids Using Nontargeted LC/MS Analysis.

American-European (Corylus americana × Corylus avellana) hazelnut hybrids are being developed for the Midwest-growing region of the United States. However, an inadequate understanding of the compounds that impact the consumer acceptance of hazelnuts limits breeding programs. Nontargeted liquid chromatography/mass spectrometry (LC/MS) chemical profiles of 12 roasted hybrid hazelnut samples and the corresponding consumer flavor liking scores were modeled by orthogonal partial least squares with good fit and predictive ability (R2Y > 0.9, Q2 > 0.9) to identify compounds that impact nut liking. The five most predictive compounds (1-5) were negatively correlated to flavor liking, selected as putative markers, purified by multidimensional preparative LC/MS, structurally elucidated (nuclear magnetic resonance, MS), quantified, and validated for sensory relevance. Compound 1 was identified as 1″-O-3'-b-glucofuranosyl-1'-O-1-b-glucofuranosyl-(2,6-dihydroxyphenyl)-ethan-4-one. Compounds 2 and 4 were identified as rotamers of 2-(3-hydroxy-2-oxoindolin-3-yl) acetic acid 3-O-6'-galactopyranosyl-2″-(2″oxoindolin-3″yl) acetate, whereas compounds 3 and 5 were identified as rotamers of 1″-O-1'-b-glucofuranosyl-9-O-6'-b-glucopyranosyl-2″-(2″-oxoindolin-3″yl) acetate. Sensory evaluation determined that all compounds were characterized by bitterness and/or astringency. The sensory threshold values of compounds 1-5 were determined to be below the concentrations reported in 91, 83, 41, 25, and 41% of all 12 hybrid hazelnut samples, respectively, indicating they contributed to aversive flavor attributes.

Analysis and authentication of avocado oil by low-field benchtop NMR spectroscopy and chemometrics.

Avocado oil is a nutritious, edible oil produced from avocado fruit. It has high commercial value and is increasing in popularity, thus powerful analytical methods are needed to ensure its quality and authenticity. Recent advancements in low-field (LF) NMR spectroscopy allow for collection of high-quality data despite the use of low magnetic fields produced by non-superconductive magnets. Combined with chemometrics, LF NMR opens new opportunities in food analysis using targeted and untargeted approaches. Here, it was used to determine poly-, mono-, and saturated fatty acids in avocado oil. Although direct signal integration of LF NMR spectra was able to determine certain classes of fatty acids, it had several challenges arising from signal overlapping. Thus, we used partial least square regression and developed models with good prediction performance for fatty acid composition, with residual prediction deviation ranging 3.46-5.53 and root mean squared error of prediction CV ranging 0.46-2.48. In addition, LF NMR, combined with unsupervised and supervised methods, enabled the differentiation of avocado oil from other oils, namely, olive oil, soybean oil, canola oil, high oleic (OL) safflower oil, and high OL sunflower oil. This study showed that LF NMR can be used as an efficient alternative for the compositional analysis and authentication of avocado oil. PRACTICAL APPLICATION: Here, we describe the application of LF-NMR for fatty acid analysis and avocado oil authentication. LF-NMR can be an efficient tool for targeted and untargeted analysis, thus becoming an attractive option for companies, regulatory agencies, and quality control laboratories. This tool is especially important for organizations and entities seeking economic, user-friendly, and sustainable analysis solutions.

c-MYC promoter G-quadruplex formed at the 5'-end of NHE III1 element: insights into biological relevance and parallel-stranded G-quadruplex stability.

We studied the structures and stabilities of G-quadruplexes formed in Myc1234, the region containing the four consecutive 5' runs of guanines of c-MYC promoter NHE III(1,) which have recently been shown to form in a supercoiled plasmid system in aqueous solution. We determined the NMR solution structure of the 1:2:1 parallel-stranded loop isomer, one of the two major loop isomers formed in Myc1234 in K(+) solution. This major loop isomer, although sharing the same folding structure, appears to be markedly less stable than the major loop isomer formed in the single-stranded c-MYC NHE III(1) oligonucleotide, the Myc2345 G-quadruplex. Our NMR structures indicated that the different thermostabilities of the two 1:2:1 parallel c-MYC G-quadruplexes are likely caused by the different base conformations of the single nucleotide loops. The observation of the formation of the Myc1234 G-quadruplex in the supercoiled plasmid thus points to the potential role of supercoiling in the G-quadruplex formation in promoter sequences. We also performed a systematic thermodynamic analysis of modified c-MYC NHE III(1) sequences, which provided quantitative measure of the contributions of various loop sequences to the thermostabilities of parallel-stranded G-quadruplexes. This information is important for understanding the equilibrium of promoter G-quadruplex loop isomers and for their drug targeting.

A comparative evaluation of low-field and high-field NMR untargeted analysis: Authentication of virgin coconut oil adulterated with refined coconut oil as a case study.

Despite the advances in low-field nuclear magnetic resonance (NMR), there are limited spectroscopic applications for untargeted analysis and metabolomics. To evaluate its potential, we combined high-field and low-field NMR with chemometrics for the differentiation between virgin and refined coconut oil and for the detection of adulteration in blended samples. Although low-field NMR has less spectral resolution and sensitivity compared to high-field NMR, it was still able to achieve a differentiation between virgin and refined coconut oils, as well as between virgin coconut oil and blends, using principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and random forest techniques. These techniques were not able to distinguish between blends with different levels of adulteration; however, partial least squares regression (PLSR) enabled the quantification of adulteration levels for both NMR approaches. Given the significant benefits of low-field NMR, including economic and user-friendly analysis and fitting in an industrial environment, this study establishes the proof of concept for its utilization in the challenging scenario of coconut oil authentication. Also, this method has the potential to be used for other similar applications that involve untargeted analysis.

The major G-quadruplex formed in the human platelet-derived growth factor receptor ß promoter adopts a novel broken-strand structure in K+ solution.

Overexpression of platelet-derived growth factor receptor ß (PDGFR-ß) has been associated with cancers and vascular and fibrotic disorders. PDGFR-ß has become an attractive target for the treatment of cancers and fibrotic disorders. DNA G-quadruplexes formed in the GC-rich nuclease hypersensitivity element of the human PDGFR-ß gene promoter have been found to inhibit PDGFR-ß transcriptional activity. Here we determined the major G-quadruplex formed in the PDGFR-ß promoter. Instead of using four continuous runs with three or more guanines, this G-quadruplex adopts a novel folding with a broken G-strand to form a primarily parallel-stranded intramolecular structure with three 1 nucleotide (nt) double-chain-reversal loops and one additional lateral loop. The novel folding of the PDGFR-ß promoter G-quadruplex emphasizes the robustness of parallel-stranded structural motifs with a 1 nt loop. Considering recent progress on G-quadruplexes formed in gene-promoter sequences, we suggest the 1 nt looped G(i)NG(j) motif may have been evolutionarily selected to serve as a stable foundation upon which the promoter G-quadruplexes can build. The novel folding of the PDGFR-ß promoter G-quadruplex may be attractive for small-molecule drugs that specifically target this secondary structure and modulate PDGFR-ß gene expression.