Deciphering the Conformations of Glutathione Oxidized Peptide: A Comparative NMR Study in Solution and Solid-State Environments.

Glutathione (GSH) and its oxidized dimer (GSSG) play an important role in living systems as an antioxidant, balancing the presence of reactive oxygen species (ROS). The central thiol (-S-S-) bond in GSSG can undergo free rotation, providing multiple conformations with respect to the S-S bridge. The six titratable sites of GSSG, which are influenced by pH variations, affect these conformations in solution, whereas in solids, additionally crystal packing effects come into play. In view of differing reports about the structure of GSSG in literature, we have here conducted an extensive reexamination of its conformations using NMR, and contrasting results have been obtained for solution and solid state. In solution, the existence of more than one antiparallel orientation of the monomer unit with different hydrogen bonding schemes has been indicated by NOE and amide temperature coefficient results. On the other hand, in the solid-state, a 1H-1H double-quantum (DQ) to 13C single-quantum (SQ) correlation study has confirmed a parallel orientation, consistent with the reported X-ray crystal structure. Experimentally assigned solid-state NMR resonances have been validated using GIPAW calculations incorporated in the Quantum ESPRESSO package.

Oxidative stress-induced degradation of Brinzolamide: Isolation and in-depth characterization of unique hydroxylamine and oxime degradation products.

Since the safety and efficacy of therapeutic products are strongly related to their stability and purity, impurities including the unavoidable degradation products may affect the pharmacological effect. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines Q3A requires the identification of process impurities and as well as degradation products in any drug substance to assess the inherent stability of the drug. The present work involves an ICH-guided degradation study for the Brinzolamide (BRZ), a topical ophthalmic drug which is generally used to lower the intraocular pressure (IOP) during glaucoma. Under oxidative stress at room temperature for 20 h, four degradation products (namely BRZ-Pk1, BRZ-PK2, BRZ-Pk3, and BRZ-Pk4) are isolated using advanced chromatographic techniques. Upon confirming the masses of the compounds using High-resolution mass spectrometry (HRMS), functional groups are identified with the help of Fourier-transform infrared spectroscopy (FT-IR). Extensive 1-dimensional (1D) and 2-dimensional (2D) Nuclear Magnetic Resonance spectroscopic (NMR) experiments especially 1D nOe, 1H-13C-HSQC and 1H-13C-HMBC unequivocally confirm the structures. Among the four compounds analyzed, three (BRZ-Pk1, BRZ-Pk2, and BRZ-Pk4) are novel, while BRZ-Pk3 was previously reported solely with mass spectrometric data. Nitrogen-based 2D NMR experiments are crucial for determining the oxidation state of hydroxylamine and oxime products within the molecules, and 1D nOe measurements help confirming E/Z isomerism (geometrical isomerism) for BRZ-Pk2 and BRZ-Pk4. All the proposed structures are justified with appropriate analytical data. The proposed mechanisms are expected to help in identifying the possible degradation pathways for similar pharmaceutical candidates.

MRI of GlycoNOE in the human liver using GraspNOE-Dixon.

PURPOSE: The objective of this study was to develop a new MRI technique for non-invasive, free-breathing imaging of glycogen in the human liver using the nuclear Overhauser effect (NOE). METHODS: The proposed method, called GraspNOE-Dixon, uses a novel MRI sequence that combines steady-state saturation-transfer preparation with multi-echo golden-angle radial stack-of-stars sampling. Multi-echo acquisition enables fat/water-separated imaging for quantification of water-specific NOE. Image reconstruction is performed using the improved golden-angle radial sparse parallel imaging (GRASP-Pro) technique to exploit spatiotemporal correlations in dynamic images. To evaluate the proposed technique, imaging experiments were first performed on glycogen phantoms, followed by in vivo studies involving healthy volunteers and patients with fatty liver disease. In addition, a comparative assessment of signal changes before and after a 12-h fasting period was performed. RESULTS: Evaluation experiments on glycogen phantoms showed a robust linear correlation between the NOE signal and glycogen concentration. In vivo experiments demonstrated motion-robust NOE-weighted images, with potential for further acceleration. In subjects with varying liver fat content, the fat/water separation approach resulted in distortion-free Z-spectra, enabling the quantification of glycogen NOE. An approximately one-third reduction in the NOE signal was observed following a 12-h fasting period, consistent with a decrease in glycogen level. CONCLUSION: This study introduces a clinically feasible imaging technique, GraspNOE-Dixon, for free-breathing volumetric multi-echo imaging of hepatic glycogen at 3 T. The motion robust imaging technique developed here may also have applications in other body areas beyond liver imaging.

Deciding treatment end point in necrotizing otitis externa: validation of a standardized clinical response assessment strategy with positron emission tomography findings.

OBJECTIVES: Primary: To explore criteria for treatment endpoint in NOE. Secondary: To study correlation of inflammatory markers, Erythrocyte sedimentation Rate (ESR) and C-reactive protein (CRP) with disease status. METHODS: Prospective cohort study conducted in a tertiary care hospital over two years (2021-2023) consisted 28 patients with NOE. Treatment culmination point was decided based on symptoms control and correlated with PET-scan findings. Clinical response was analysed with respect to the serum inflammatory markers and PET Scan findings. RESULTS: There was fair degree of agreement between clinical resolution and resolution of findings on PET scan (kappa coefficient - 0.76 [95% CI; 0.40,1.00]). Inflammatory markers showed statistically significant decline with clinical resolution but failed to return to normal. CONCLUSION: Decision to terminate treatment of NOE can be reliably made on clinical grounds in patients remaining asymptomatic for three weeks. Resolution of inflammation on PET scan is in congruity with the clinical remission .

Insecticidal activity of Thymus pallescens de Noë and Cymbogon citratus essential oils against Sitophilus zeamais and Tribolium castaneum.

The thrust of the study was to determine the chemical composition of the essential oils extracted from Thymus pallescens de Noé and Cymbogon citratus Stapf. as well as to evaluate their efficacy in controlling Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst) in either single or combined populations. Carvacrol (56.04%) and geraniol (20.86%) were identified as the major constituents of T. pallescens and C. citratus respectively. The tested essential oils showed pronounced insecticidal activity against the pest species in relation with the applied doses. T. pallescens EO had the highest efficacy and S. zeamais was found to be more susceptible to both individual and combined treatments. With reference to the contact and fumigation assessments, T. pallescens EO effectuated corrected mortality rates ranging from 42.5-100% to 25-100% in S. zeamais with corresponding lethal concentration (LC50) values of 17.7 µl/ml and 15µL/L air respectively. Whereas, the T. pallescens EO exhibited corrected mortality rates of 42.5-100% and 20-100% with corresponding LC50 values of 18.1 µl/ml and 15.5 µL/L air against T. castaneum in contact and fumigation assessments, respectively. The corrected mortality rates increased for both insect species when using combination treatments, with significant increases in the LC50 values, ranging from 8.59 to 49.9% for both pest species. Analysis of energy biomarkers in the treated insects indicate significantly increased protein and carbohydrate contents and decreased lipids levels. The study therefore demonstrated the bio-insecticidal toxicity of the EOs from T. pallescens and C. citratus against two important maize post-harvest pests, concurrently revealing significant positive and negative insecticidal activity gradients in relation to single or combined populations.

It is time to reconsider the use of naturally-occurring endotoxins in endotoxin recovery studies: Part 2 of a BioPhorum harmonized endotoxin recovery study.

The evaluation of Naturally Occurring Endotoxins (NOEs) for Low Endotoxin Recovery (LER) studies has been a topic in the industry and regulatory agencies have been hesitant to endorse NOE use in LER studies over purified Lipopolysaccharide (LPS) standards such as Control Standard Endotoxin (CSE) or Reference Standard Endotoxin (RSE). In a recent study involving 11 BioPhorum member companies across 13 sites, NOEs prepared in high and low nutrient conditions were evaluated in two common monoclonal antibody buffer formulations: 10 mM Sodium Citrate, 0.05 % Polysorbate 80, pH 6.0 and 20 mM Histidine, 0.05 % Polysorbate 80, pH 6.0. 12 g-negative bacterial isolates were used to prepare NOE analytes, which were spiked into the formulation buffers. Additionally, the NOEs were spiked into Limulus Amebocyte Lysate (LAL) reagent water as controls and purified LPS into the citrate/polysorbate buffer as the LER control. Results showed the average of three runs per organism was >50 % recovery, at the conclusion of the 7-day period, regardless of nutrient culture preparation conditions. Furthermore, purified LPS controls became undetectable (<50 % recovery) in the citrate/polysorbate buffer, highlighting the presence of LER. These findings highlight the potential value of using NOEs from relevant manufacturing facilities to assess overall risk when purified LPS recovery is insufficient.

Optimization of reverse NOE pumping experiments in the analysis of complex systems with densely crowded NMR spectra.

We present an optimization of Reverse NOE-pumping (RNP) in order to observe the 1H signals of ligands bound to proteins. Although various ligand-based NMR screening methods have been proposed, the most frequently used method has been Saturation-Transfer Difference (STD), owing to the relatively easy setup of experiments. Yet the critical point of STD is the selective irradiation of protein without irradiating ligand, and thus the STD technique is unable to observe 1H ligand signals, which resonate across the entire 1H spectral width. In the present study, the RNP experiment has been improved to develop an effective NMR-based screening technique. The optimized RNP spectra reveal less subtraction artifacts and phase distortion than the original RNP spectra, indicating its applicability to any type of ligand molecules.

Diagnostic accuracy of eosinophil-to-lymphocyte ratio and eosinophil-to-neutrophil ratio as biomarkers for differentiating between fungal and bacterial infection in necrotising otitis externa.

INTRODUCTION: Necrotizing otitis externa (NOE) is a serious, progressive, and potentially life-threatening infection of the external auditory canal, affecting soft tissue and bone. The most common organism causing NOE is Pseudomonas Aeruginosa and less common are Fungal infections. When managing a patient with NOE, a culture is taken from the EAC in order to tailor the appropriate antimicrobial treatment, however commonly, the culture is sterile. Inflammation biomarkers may be used as adjuncts to inform on the differential diagnosis and as prognostic markers. AIM: To characterize and compare values and ratios of components of the complete blood count (CBC) at admission, at patients with positive swab culture. METHODS: A retrospective study of NOE patients was conducted. We included all patients admitted between the years 2001-2023, for whom a culture swab tested positive. We compared CBC findings at hospitalization between bacteria and fungi-positive culture patients. RESULTS: Eosinophils-to-Neutrophils Ratio (ENR) was significantly lower in the fungal group compared to the bacterial group 0.023 ± 0.02 and 0.04 ± 0.03, respectively (p-value = 0.025). Eosinophils-to-Leukocyte Ratio (ELR) was significantly lower in the fungal group compared to the bacterial group 0.058 ± 0.04 and 0.12 ± 0.1 respectively (p-value = 0.009). For definition of ELR ≤ 0.1 we found that, sensitivity was 88% (95%CI = 0.679-0.979) and NPV 90% (95%CI = 0.709-0.982). For definition of ENR ≤ 0.03 sensitivity was 88% (95%CI = 0.679-0.979) and NPV 88% (95%CI = 0.679-0.979). CONCLUSION: Lower values of ELR and ENR in patients with NOE are associated with fungal infection and can serve as a tool in adjusting an appropriate antimicrobial therapy in cases of sterile or when no culture is available.

Spatiotemporal Dynamics of Neuroinflammation Relate to Behavioral Recovery in Rats with Spinal Cord Injury.

PURPOSE: The degree and dynamic progression of neuroinflammation after traumatic spinal cord injuries (SCI) are crucial determinants of the severity of injury and potential for recovery. We used Positron Emission Tomography (PET) to monitor neuroinflammation longitudinally, correlating it with Chemical Exchange Saturation Transfer (CEST) Magnetic Resonance Imaging (MRI) and behavior in contusion-injured rats. These studies help validate CEST metrics and confirm how imaging may be used to evaluate the efficacy of therapies and understand their mechanisms of action. PROCEDURES: 12 SCI and 4 sham surgery rats were subjected to CEST MRI and PET-Translocator Protein (TSPO) scans for 8 weeks following injury. Z-spectra from the SCI were analyzed using a 5-Lorentzian pool model for fitting. Weekly motor and somatosensory behavior were correlated with imaging metrics, which were validated through post-mortem histological and immuo-staining using ionized calcium-binding adaptor protein-1 (iba-1, microglia) and glial fibrillary acidic protein (GFAP, astrocytes). RESULTS: PET-TSPO showed widespread inflammation and post-mortem histology confirmed the presence of activated microglia. Changes in CEST and nuclear Overhauser Effect (NOE) peaks at 3.5 ppm and -1.6 ppm respectively were largest within the first week after injury and more pronounced in rostral versus caudal segments. These temporal indices of neuroinflammation corresponded to the recovery of locomotor behaviors and somatic sensation in rats with moderate contusion injury. The results confirm that CEST MRI metrics are sensitive indices of states of neuroinflammation within injured spinal cords. CONCLUSIONS: The detection of dynamic spatiotemporal features of neuroinflammation progression underscores the importance of considering their timings and locations for neuroprotective and anti-inflammatory therapies. The availability of noninvasive MRI indices of neuroinflammation may facilitate clinical trials aimed at treatments that promote recovery after SCI.

Nuclear Overhauser enhancement imaging at -1.6 ppm in rat brain at 4.7T.

PURPOSE: A new nuclear Overhauser enhancement (NOE)-mediated saturation transfer signal at around -1.6 ppm, termed NOE(-1.6), has been reported at high fields of 7T and 9.4T previously. This study aims to validate the presence of this signal at a relatively low field of 4.7T and evaluate its variations in different brain regions and tumors. METHODS: Rats were injected with monocrystalline iron oxide nanoparticles to reduce the NOE(-1.6) signal. CEST signals were measured using different saturation powers before and after injection to assess the presence of this signal. Multiple-pool Lorentzian fits, with/without inclusion of the NOE(-1.6) pool, were performed on CEST Z-spectra obtained from healthy rat brains and rats with 9L tumors. These fits aimed to further validate the presence of the NOE(-1.6) signal and quantify its amplitude. RESULTS: The NOE(-1.6) signal exhibited a dramatic change following the injection of monocrystalline iron oxide nanoparticles, confirming its presence at 4.7T. The NOE(-1.6) signal reached its peak at a saturation power of ∼0.75 µT, indicating an optimized power level. The multiple-pool Lorentzian fit without the NOE(-1.6) pool showed higher residuals around -1.6 ppm compared to the fit with this pool, further supporting the presence of this signal. The NOE(-1.6) signal did not exhibit significant variation in the corpus callosum and caudate putamen regions, but it showed a significant decrease in tumors, which aligns with previous findings at 9.4T. CONCLUSION: This study successfully demonstrated the presence of the NOE(-1.6) signal at 4.7T, which provides valuable insights into its potential applications at lower field strengths.

Phytochemical analysis, antioxidant and hypoglycemic activities of a methanol extract from Stachys brachyclada de Noé ex Coss. leaves.

Stachys brachyclada de Noé ex Coss. (Lamiaceae) is a quite rare medicinal plant endemic to the Mediterranean basin. In this study, seven secondary metabolites from a methanol extract of its leaves have been isolated and identified by a combination of chromatographic and spectroscopic methods (1D and 2D NMR experiments and ESIMS analysis). They include one ethyl 4-hydroxybenzoate (1), three acylated flavone glycosides (2-4), one diapigenin derivative (5) and two flavone aglycones (6-7). Stachysetin (5) was found the major compound of the extract (74.0 mg/g of dry matter). Moreover, the produced extract showed the ability in inhibiting the α-glucosidase enzyme (IC50 = 13.7 µg/mL), in quenching the radical 1,1-diphenyl-2-picrylhydrazyl (EC50 = 74.6 µg/mL), and in reducing the intracellular oxidative stress level in Human Dermal Fibroblast (64% inhibition at 50 µg/mL).

Investigation of relayed nuclear Overhauser enhancement effect at -1.6 ppm in an ischemic stroke model.

Background: When an ischemic stroke happens, it triggers a complex signalling cascade that may eventually lead to neuronal cell death if no reperfusion. Recently, the relayed nuclear Overhauser enhancement effect at -1.6 ppm [NOE(-1.6 ppm)] has been postulated may allow for a more in-depth analysis of the ischemic injury. This study assessed the potential utility of NOE(-1.6 ppm) in an ischemic stroke model. Methods: Diffusion-weighted imaging, perfusion-weighted imaging, and chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) data were acquired from five rats that underwent scans at 9.4 T after middle cerebral artery occlusion. Results: The apparent diffusion coefficient (ADC), cerebral blood flow (CBF), and apparent exchange-dependent relaxations (AREX) at 3.5 ppm and NOE(-1.6 ppm) were quantified. AREX(3.5 ppm) and NOE(-1.6 ppm) were found to be hypointense and exhibited different signal patterns within the ischemic tissue. The NOE(-1.6 ppm) deficit areas were equal to or larger than the ADC deficit areas, but smaller than the AREX(3.5 ppm) deficit areas. This suggested that NOE(-1.6 ppm) might further delineate the acidotic tissue estimated using AREX(3.5 ppm). Since NOE(-1.6 ppm) is closely related to membrane phospholipids, NOE(-1.6 ppm) potentially highlighted at-risk tissue affected by lipid peroxidation and membrane damage. Altogether, the ADC/NOE(-1.6 ppm)/AREX(3.5 ppm)/CBF mismatches revealed four zones of increasing sizes within the ischemic tissue, potentially reflecting different pathophysiological information. Conclusions: Using CEST coupled with ADC and CBF, the ischemic tissue may thus potentially be separated into four zones to better understand the pathophysiology after stroke and improve ischemic tissue fate definition. Further verification of the potential utility of NOE(-1.6 ppm) may therefore lead to a more precise diagnosis.

Early-stage mapping of macromolecular content in APPNL-F mouse model of Alzheimer's disease using nuclear Overhauser effect MRI.

Non-invasive methods of detecting early-stage Alzheimer's disease (AD) can provide valuable insight into disease pathology, improving the diagnosis and treatment of AD. Nuclear Overhauser enhancement (NOE) MRI is a technique that provides image contrast sensitive to lipid and protein content in the brain. These macromolecules have been shown to be altered in Alzheimer's pathology, with early disruptions in cell membrane integrity and signaling pathways leading to the buildup of amyloid-beta plaques and neurofibrillary tangles. We used template-based analyzes of NOE MRI data and the characteristic Z-spectrum, with parameters optimized for increase specificity to NOE, to detect changes in lipids and proteins in an AD mouse model that recapitulates features of human AD. We find changes in NOE contrast in the hippocampus, hypothalamus, entorhinal cortex, and fimbria, with these changes likely attributed to disruptions in the phospholipid bilayer of cell membranes in both gray and white matter regions. This study suggests that NOE MRI may be a useful tool for monitoring early-stage changes in lipid-mediated metabolism in AD and other disorders with high spatial resolution.

Structural Studies of Monounsaturated and ω-3 Polyunsaturated Free Fatty Acids in Solution with the Combined Use οf NMR and DFT Calculations-Comparison with the Liquid State.

Molecular structures, in chloroform and DMSO solution, of the free fatty acids (FFAs) caproleic acid, oleic acid, α-linolenic acid, eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA) are reported with the combined use of NMR and DFT calculations. Variable temperature and concentration chemical shifts of the COOH protons, transient 1D NOE experiments and DFT calculations demonstrate the major contribution of low molecular weight aggregates of dimerized fatty acids through intermolecular hydrogen bond interactions of the carboxylic groups, with parallel and antiparallel interdigitated structures even at the low concentration of 20 mM in CDCl3. For the dimeric DHA, a structural model of an intermolecular hydrogen bond through carboxylic groups and an intermolecular hydrogen bond between the carboxylic group of one molecule and the ω-3 double bond of a second molecule is shown to play a role. In DMSO-d6 solution, NMR and DFT studies show that the carboxylic groups form strong intermolecular hydrogen bond interactions with a single discrete solvation molecule of DMSO. These solvation species form parallel and antiparallel interdigitated structures of low molecular weight, as in chloroform solution. This structural motif, therefore, is an intrinsic property of the FFAs, which is not strongly affected by the length and degree of unsaturation of the chain and the hydrogen bond ability of the solvent.

Measuring chemical exchange saturation transfer exchange rates in the human brain using a particle swarm optimisation algorithm.

The z-spectrum contains many pools with different exchange rates and T2 values, which can make it difficult to interpret in vivo data and complicates the design of experiments aimed at providing sensitivity to one pool. This work aims to characterise the main pools observable with MRI at 7T in the human brain. To achieve this, we acquired z-spectra at multiple saturation powers in the human brain at 7T. We used simulations to optimise the use of particle swarm optimisation (PSO) to fit these data, validating this approach using further simulations and creatine phantoms. We then used the PSO to fit data from grey and white matter for the pool size, exchange rate, and T2 of five proton pools (magnetisation transfer, amides, amines, nuclear Overhauser enhancement NOE-3.5ppm and NOE-1.7ppm in addition to water). We then devised an approach for using PSO to fit z-spectra while limiting the computational burden, and we investigated the sensitivity of the fit to T2 and k for three overlapping pools. We used this to measure the exchange rate of creatine and to show that it varied with temperature, as expected. In the brain we measured a significantly larger pool size in white matter than in grey matter for the magnetisation transfer pool and the NOE-3.5ppm pool. For all other parameters we found no significant difference between grey and white matter. We showed that PSO can be used to fit z-spectra acquired at a range of B1 to provide information about peak position, amplitude, exchange rate, and T2 in vivo in the human brain. These data could provide more sensitivity to change in some clinical conditions and will also provide key information for further experimental design.

Fast WASABI post-processing: Access to rapid B0 and B1 correction in clinical routine for CEST MRI.

CEST MRI methods, such as APT and NOE imaging reveal biomarkers with significant diagnostic potential due to their ability to access molecular tissue information. Regardless of the technique used, CEST MRI data are affected by static magnetic B0 and radiofrequency B1 field inhomogeneities that degrade their contrast. For this reason, the correction of B0 field-induced artefacts is essential, whereas accounting for B1 field inhomogeneities have shown significant improvements in image readability. In a previous work, an MRI protocol called WASABI was presented, which can map simultaneously B0 and B1 field inhomogeneities, while maintaining the same sequence and readout types as used for CEST MRI. Despite the highly satisfactory quality of B0 and B1 maps computed from the WASABI data, the post-processing method is based on an exhaustive search of a four-parameter space and an additional four-parameter non-linear model fitting step. This leads to long post-processing times that are prohibitive in clinical practice. This work provides a new method for fast post-processing of WASABI data with outstanding acceleration of the parameter estimation procedure and without compromising its stability. The resulting computational acceleration makes the WASABI technique suitable for clinical use. The stability of the method is demonstrated on phantom data and clinical 3 Tesla in vivo data.

Bromination of endo-7-norbornene derivatives revisited: failure of a computational NMR method in elucidating the configuration of an organic structure.

Previously we reported on the bromination of endo-7-bromonorbornene at different temperatures yielding mixtures of addition products. The structural elucidations of the formed compounds were achieved by NMR spectroscopy. Particularly, the γ-gauche effect and long-range couplings were instrumental in assigning the stereochemistry of the adducts. However, in a recent paper, Novitskiy and Kutateladze claimed that based on an applied machine learning-augmented DFT method for computational NMR that the structure of the product, (1R,2R,3S,4S,7s)-2,3,7-tribromobicyclo[2.2.1]heptane was wrong. With the aid of their computational method, they revised a number of published structures, including ours, and assigned our product the structure (1R,2S,3R,4S,7r)-2,3,7-tribromobicyclo[2.2.1]heptane. To fit their revised structure, they proposed an alternative mechanism featuring a skeletal rearrangement without the intermediacy of a carbocation. Herein, we are not only confirming the structure originally assigned by us through crucial NMR experiments, we also present the ultimate structural proof by means of X-ray crystallography. Moreover, we disprove the mechanism proposed by the aforementioned authors based on sound mechanistic reasoning and point to an oversight by the authors that led them to an erroneous mechanistic pathway.

Liver-cell protective pyridones from the fungi Tolypocladium album dws120.

Five previously undescribed pyridone derivatives, tolypyridones I-M, were identified from the solid rice medium fermented by Tolypocladium album dws120, along with two known compounds tolypyridone A (or trichodin A) and pyridoxatin. Their planar structures and partial relative configurations have been determined by careful interpretation of their spectroscopic data. The full assignment of the relative and absolute configurations of tolypyridones I-M was achieved by gauge-independent atomic orbital 13C NMR calculation, quantitative nuclear Overhauser effects based interatomic distance calculation, and electronic circular dichroism calculation. In addition, we have fully determined the configuration of tolypyridone A by X-ray diffraction analysis. In bioassay, tolypyridones I was able to restore cell viability and inhibit the release of alanine aminotransferase and aspartate aminotransferase for ethanol-induced LO2 cells, suggesting its potential as a liver protective agent.

Evaluation of contributors to amide proton transfer-weighted imaging and nuclear Overhauser enhancement-weighted imaging contrast in tumors at a high magnetic field.

PURPOSE: The purpose is to evaluate the relative contribution from confounding factors (T1 weighting and magnetization transfer) to the CEST ratio (CESTR)-quantified amide proton transfer (APT) and nuclear Overhauser enhancement (NOE) (-3.5) in tumors as well as whether the CESTR can reflect the distribution of the solute concentration (fs ). METHODS: We first provided a signal model that shows the separate dependence of CESTR on these confounding factors and the clean CEST/NOE effects quantified by an apparent exchange-dependent relaxation (AREX) method. We then measured the change in these effects in the 9-L tumor model in rats, through which we calculated the relative contribution of each confounding factor. fs was also fitted, and its correlations with the CESTR and AREX were assessed to evaluate their capabilities to reflect fs . RESULTS: The CESTR-quantified APT shows "positive" contrast in tumors, which arises primarily from R1w at low powers and both R1w and magnetization transfer at high powers. CESTR-quantified NOE (-3.5) shows no or weak contrast in tumors, which is due to the cancelation of R1w and NOE (-3.5), which have opposite contributions. CESTR-quantified APT has a stronger correlation with APT fs than AREX-quantified APT. CESTR-quantified NOE (-3.5) has a weaker correlation with NOE (-3.5) fs than AREX-quantified NOE (-3.5). CONCLUSION: CESTR reflects a combined effect of T1 weighting and CEST/NOE. Both factors depend on fs , which contributes positively to the dependence of CESTR on fs in APT imaging and enhances its correlation with fs . In contrast, these factors have opposite contributions to its dependence on fs in NOE (-3.5) imaging, thereby weakening the correlation.

A Comparison of Bonded and Nonbonded Zinc(II) Force Fields with NMR Data.

Classical molecular dynamics (MD) simulations are widely used to inspect the behavior of zinc(II)-proteins at the atomic level, hence the need to properly model the zinc(II) ion and the interaction with its ligands. Different approaches have been developed to represent zinc(II) sites, with the bonded and nonbonded models being the most used. In the present work, we tested the well-known zinc AMBER force field (ZAFF) and a recently developed nonbonded force field (NBFF) to assess how accurately they reproduce the dynamic behavior of zinc(II)-proteins. For this, we selected as benchmark six zinc-fingers. This superfamily is extremely heterogenous in terms of architecture, binding mode, function, and reactivity. From repeated MD simulations, we computed the order parameter (S2) of all backbone N-H bond vectors in each system. These data were superimposed to heteronuclear Overhauser effect measurements taken by NMR spectroscopy. This provides a quantitative estimate of the accuracy of the FFs in reproducing protein dynamics, leveraging the information about the protein backbone mobility contained in the NMR data. The correlation between the MD-computed S2 and the experimental data indicated that both tested FFs reproduce well the dynamic behavior of zinc(II)-proteins, with comparable accuracy. Thus, along with ZAFF, NBFF represents a useful tool to simulate metalloproteins with the advantage of being extensible to diverse systems such as those bearing dinuclear metal sites.