ResearchPad - electron-spin-resonance-spectroscopy https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Effective coupling of rapid freeze-quench to high-frequency electron paramagnetic resonance]]> https://www.researchpad.co/article/elastic_article_7690 We report an easy, efficient and reproducible way to prepare Rapid-Freeze-Quench samples in sub-millimeter capillaries and load these into the probe head of a 275 GHz Electron Paramagnetic Resonance spectrometer. Kinetic data obtained for the binding reaction of azide to myoglobin demonstrate the feasibility of the method for high-frequency EPR. Experiments on the same samples at 9.5 GHz show that only a single series of Rapid-Freeze-Quench samples is required for studies at multiple microwave frequencies.

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<![CDATA[Deoxyarbutin Possesses a Potent Skin-Lightening Capacity with No Discernible Cytotoxicity against Melanosomes]]> https://www.researchpad.co/article/5989dae9ab0ee8fa60bbe85e

Safe and effective ingredients capable of removing undesired hyperpigmentation from facial skin are urgently needed for both pharmaceutical and cosmetic purposes. Deoxyarbutin (4-[(tetrahydro-2H-pyran-2-yl) oxy] phenol, D-Arb) is a glucoside derivative of hydroquinone. Here, we investigated the toxicity and efficacy of D-Arb at the sub-cellular level (directly on melanosomes) and skin pigmentation using in vivo and in vitro models to compare with its parent compound hydroquinone (1,4-benzenediol, HQ). At first, we examined the ultrastructural changes of melanosomes in hyperpigmented guinea pig skin induced by 308-nm monochromatic excimer lightand/or treated with HQ and D-Arb using transmission electron microscopy. The results showed that prominent changes in the melanosomal membrane, such as bulb-like structure and even complete rupture of the outer membranes, were found in the skin after topical application of 5% HQ for 10 days. These changes were barely observed in the skin treated with D-Arb. To further clarify whether membrane toxicity of HQ was a direct result of the compound treatment, we also examinedultrastructural changes of individual melanosomes purified from MNT1 human melanoma cells. Similar observations were obtained from the naked melanosome model in vitro. Finally, we determined the effects of melanosomal fractions exposed to HQ or D-Arb on hydroxyl radical generation in the Fenton reaction utilizing an electron spin resonance assay. D-Arb-treated melanosomesexhibit a moderate hydroxyl radical-scavenging activity, whereas HQ-treated melanosomessignificantly generate more hydroxyl free radicals. This study suggests that D-Arb possesses a potent ability in skin lightening and antioxidation with less melanosome cytotoxicity.

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<![CDATA[Evaluating the Potential of Q-Band ESR Spectroscopy for Dose Reconstruction of Fossil Tooth Enamel]]> https://www.researchpad.co/article/5989da2aab0ee8fa60b82298

The potential of Q-band Electron Spin Resonance (ESR) for quantitative measurements has been scarcely evaluated in the literature and its application for dose reconstruction of fossil tooth enamel with dating purposes remains still quite unknown. Hence, we have performed a comparative study based on several Early to Middle Pleistocene fossil tooth samples using both X- and Q-band spectroscopies. Our results show that Q-band offers a significant improvement in terms of sensitivity and signal resolution: it allows not only to work with reduced amounts of valuable samples (< 4 mg), but also to identify different components of the main composite ESR signal. However, inherent precision of the ESR intensity measurements at Q-band is clearly lower than that achieved at X-band, highlighting the necessity to carry out repeated measurements. All dose values derived from X- and Q-band are nevertheless systematically consistent at either 1 or 2 sigma. In summary, our results indicate that Q-band could now be considered as a reliable tool for ESR dosimetry/dating of fossil teeth although further work is required to improve the repeatability of the measurements.

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<![CDATA[Direct Measurements of Oxygen Gradients in Spheroid Culture System Using Electron Parametric Resonance Oximetry]]> https://www.researchpad.co/article/5989da76ab0ee8fa60b96ae4

Advanced in vitro culture from tissues of different origin includes three-dimensional (3D) organoid micro structures that may mimic conditions in vivo. One example of simple 3D culture is spheroids; ball shaped structures typically used as liver and tumour models. Oxygen is critically important in physiological processes, but is difficult to quantify in 3D culture: and the question arises, how small does a spheroid have to be to have minimal micro-environment formation? This question is of particular importance in the growing field of 3D based models for toxicological assessment. Here, we describe a simple non-invasive approach modified for the quantitative measurement and subsequent evaluation of oxygen gradients in spheroids developed from a non-malignant fish cell line (i.e. RTG-2 cells) using Electron Paramagnetic Resonance (EPR) oximetry. Sonication of the paramagnetic probe Lithium phthalocyanine (LiPc) allows for incorporation of probe particulates into spheroid during its formation. Spectra signal strength after incorporation of probe into spheroid indicated that a volume of 20 μl of probe (stock solution: 0.10 mg/mL) is sufficient to provide a strong spectra across a range of spheroid sizes. The addition of non-toxic probes (that do not produce or consume oxygen) report on oxygen diffusion throughout the spheroid as a function of size. We provide evidence supporting the use of this model over a range of initial cell seeding densities and spheroid sizes with the production of oxygen distribution as a function of these parameters. In our spheroid model, lower cell seeding densities (∼2,500 cells/spheroid) and absolute size (118±32 μm) allow control of factors such as pre-existing stresses (e.g. ∼ 2% normoxic/hypoxic interface) for more accurate measurement of treatment response. The applied methodology provides an elegant, widely applicable approach to directly characterize spheroid (and other organoid) cultures in biomedical and toxicological research.

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<![CDATA[Use of a cocktail of spin traps for fingerprinting large range of free radicals in biological systems]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc603

It is well established that the formation of radical species centered on various atoms is involved in the mechanism leading to the development of several diseases or to the appearance of deleterious effects of toxic molecules. The detection of free radical is possible using Electron Paramagnetic Resonance (EPR) spectroscopy and the spin trapping technique. The classical EPR spin-trapping technique can be considered as a “hypothesis-driven” approach because it requires an a priori assumption regarding the nature of the free radical in order to select the most appropriate spin-trap. We here describe a “data-driven” approach using EPR and a cocktail of spin-traps. The rationale for using this cocktail was that it would cover a wide range of biologically relevant free radicals and have a large range of hydrophilicity and lipophilicity in order to trap free radicals produced in different cellular compartments. As a proof-of-concept, we validated the ability of the system to measure a large variety of free radicals (O-, N-, C-, or S- centered) in well characterized conditions, and we illustrated the ability of the technique to unambiguously detect free radical production in cells exposed to chemicals known to be radical-mediated toxic agents.

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<![CDATA[Effects of BM-573 on Endothelial Dependent Relaxation and Increased Blood Pressure at Early Stages of Atherosclerosis]]> https://www.researchpad.co/article/5989da0dab0ee8fa60b7875d

Endothelial dysfunction is considered to be an early event in atherosclerosis and plays a pivotal role in the development, progression and clinical complications of atherosclerosis. Previous studies have shown the beneficial effects of combined inhibition of thromboxane synthase and antagonism of thromboxane receptors by BM-573 on atherosclerosis; however our knowledge about the beneficial effects of BM-573 on endothelial function and increased blood pressure related to early stage of atherosclerosis is limited. In the present study, we investigated the effects of short-term (3μM, 1 hour) and chronic (10mg/L, 8 weeks) treatments with BM-573 on vasodilatory function, nitric oxide (NO) bioavailability, oxidative stress and systolic blood pressure in 15 weeks old apolipoprotein E-deficient (ApoE-KO) mice. ApoE-KO mice showed a reduced endothelium-derived relaxation. In addition, NO bioavailability was reduced and oxidative stress and blood pressure were increased in ApoE-KO mice versus wild-type mice. BM-573 treatments were able to improve the relaxation profile in ApoE-KO mice. Short-term effects of BM-573 were mainly mediated by an increased phosphorylation of both eNOS and Akt, whereas BM-573 in vivo treatment also reduced oxidative stress and restored NO bioavailability. In addition, chronic administration of BM-573 reduced systolic blood pressure in ApoE-KO mice. In conclusion, pharmacological modulation of TxA2 biosynthesis and biological activities by dual TP antagonism/TxAS inhibition with BM-573, already known to prevent plaque formation, has the potential to correct vasodilatory dysfunction at the early stages of atherosclerosis.

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