Ferric Reducing Antioxidant Power and Square Wave Voltammetry for Assay of Low Molecular Weight Antioxidants in Blood Plasma: Performance and Comparison of Methods

The purpose of the present study was to employ two methods—square wave voltammetry (SWV) performed on screen printed sensors and ferric reducing antioxidant power (FRAP)—as suitable tools for the assay of low-molecular-weight antioxidants (LMWAs). LMWAs were assayed by both methods and the resulting data were statistically compared. Plasma samples from five Cinereous vultures accidentally intoxicated with lead were used to represent real biological matrices with different levels of LMWAs. Blood was collected from the birds prior to and one month after treatment with Ca-EDTA. SWV resulted in two peaks. The first peak, with the potential value of 466 ± 15 mV, was recognized as ascorbic and uric acids, while the second one (743 ± 30 mV) represented glutathione, tocopherol, ascorbic acid and in a minor effect by uric acid, too. Contribution of individual antioxidants was recognized by separate assays of LMWA standards. Correlation between peaks 1 and 2 as well as the sum of the two peaks and FRAP was analysed. While peak 1 and the sum of peaks were in close correlation to FRAP results (correlation coefficient of 0.97), the relation between peak 2 and FRAP may be expressed using a correlation coefficient of 0.64. The determination of thiols by the Ellman assay confirmed the accuracy of SWV. Levels of glutathione and other similar structures were stable in the chosen model and it may be concluded that SWV is appropriate for assay of LMWAs in plasma samples. The methods employed in the study were advantageous in minimal sample volume consumption and fast acquisition of results

Biochemical responses and oxidative stress in Francisella tularensis infection: a European brown hare model

<p>Abstract</p> <p>Background</p> <p>The aim of the present study was to investigate biochemical and oxidative stress responses to experimental <it>F. tularensis </it>infection in European brown hares, an important source of human tularemia infections.</p> <p>Methods</p> <p>For these purposes we compared the development of an array of biochemical parameters measured in blood plasma using standard procedures of dry chemistry as well as electrochemical devices following a subcutaneous infection with a wild <it>Francisella tularensis </it>subsp. <it>holarctica </it>strain (a single dose of 2.6 × 10⁹CFU <it>pro toto</it>).</p> <p>Results</p> <p>Subcutaneous inoculation of a single dose with 2.6 × 10⁹colony forming units of a wild <it>F. tularensis </it>strain <it>pro toto </it>resulted in the death of two out of five hares. Plasma chemistry profiles were examined on days 2 to 35 post-infection. When compared to controls, the total protein, urea, lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase were increased, while albumin, glucose and amylase were decreased. Both uric and ascorbic acids and glutathione dropped on day 2 and then increased significantly on days 6 to 12 and 6 to 14 post-inoculation, respectively. There was a two-fold increase in lipid peroxidation on days 4 to 8 post-inoculation.</p> <p>Conclusions</p> <p>Contrary to all expectations, the present study demonstrates that the European brown hare shows relatively low susceptibility to tularemia. Therefore, the circumstances of tularemia in hares under natural conditions should be further studied.</p

Comparison of a high-carbohydrate and high-protein breakfast effect on plasma ghrelin, obestatin, NPY and PYY levels in women with anorexia and bulimia nervosa

Abstract Background The present study investigated plasma levels of gut-brain axis peptides ghrelin, obestatin, NPY and PYY after consumption of a high-carbohydrate (HC) and high-protein (HP) breakfast in patients with anorexia nervosa, bulimia nervosa and in healthy controls. These peptides play an important role in regulation of energy homeostasis and their secretion is disturbed under condition of eating disorders. As various types of consumed macronutrients may induce different plasma hormone responses, so we examined these responses in women patients with eating disorders and compared them with those of healthy controls. Methods We examined plasma hormone responses to HC and HP breakfast in patients with AN (n = 14; age: 24.6 ± 1.8 years, BMI: 15.3 ± 0.7), BN (n = 15; age: 23.2 ± 1.7 years, BMI: 20.5 ± 0.9) and healthy controls (n = 14; age: 24.9 ± 1.4 years, BMI: 21.1 ± 0.8). Blood samples were drawn from the cubital vein, the first blood drawn was collected before meal, and then 30, 60, 90, 120 and 150 min after breakfast consumption. Plasma hormone levels were determined by commercially available RIA kits. Results Fasting and postprandial plasma obestatin levels were significantly increased in both AN and BN patients, while plasma ghrelin levels were significantly increased in AN patients only. After breakfast consumption, plasma levels of ghrelin and obestatin decreased, although they were still above the range of values of healthy controls. Fasting NPY plasma levels were significantly increased in AN and BN patients and did not change postprandially. Fasting PYY levels were comparable in AN, BN and healthy controls, but postprandially significantly increased after HP breakfast in AN and BN patients. Different reactions to breakfast consumption was found for ghrelin and PYY among investigated groups, while for obestatin and NPY these reactions were similar in all groups. Conclusions Significant increase of obestatin and NPY in AN and BN patients may indicate their important role as the markers of eating disorders. Different reactions of ghrelin and PYY to breakfast consumption among groups suggest that role of these hormones in regulation of energy homeostasis can be adjusted in dependence to acute status of eating disorder.</p

Inflammation-Based Scores Increase the Prognostic Value of Circulating Tumor Cells in Primary Breast Cancer

A correlation between circulating tumor cells (CTCs) and monocytes in metastatic breast cancer (BC), where CTCs and monocyte-to-lymphocyte ratio (MLR) were predictors of overall survival (OS), was recently shown. Herein, we aimed to assess the association between CTCs and the complete blood count (CBC)-derived inflammation-based scores in 284 primary BC patients. CTCs were determined in CD45-depleted peripheral blood mononuclear cells by real time-PCR. This method allowed us to detect a subset of CTCs with an epithelial-to-mesenchymal transition phenotype (CTC EMT), previously associated with inferior outcomes in primary BC. In the present study, CTC EMT positivity (hazard ratio (HR) = 2.4; 95% CI 1.20&ndash;4.66, p = 0.013) and elevated neutrophil-to-lymphocyte ratio (NLR) (HR = 2.20; 95% CI 1.07&ndash;4.55; p = 0.033) were associated with shorter progression-free survival (PFS) in primary BC patients. Multivariate analysis showed that CTC EMT-positive patients with NLR &ge; 3 had 8.6 times increased risk of disease recurrence (95% CI 2.35&ndash;31.48, p = 0.001) compared with CTC EMT-negative patients with NLR &lt; 3. Similarly, disease recurrence was 13.14 times more likely in CTC EMT-positive patients with MLR &ge; 0.34 (95% CI 4.35&ndash;39.67, p &lt; 0.001). Given its low methodological and financial demands, the CBC-derived inflammation-based score determination could, after broader validation, significantly improve the prognostication of BC patients

Illuminating the mechanism and allosteric behavior of NanoLuc luciferase

Abstract NanoLuc, a superior β-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational techniques are combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. Structurally, binding to the allosteric site prevents simultaneous binding to the catalytic site, and vice versa, through concerted conformational changes. We demonstrate that restructuration of the allosteric site can boost the luminescent reaction in the remote active site. Mechanistically, an intra-barrel arginine coordinates the imidazopyrazinone component of luciferin, which reacts with O2 via a radical charge-transfer mechanism, and then it also protonates the resulting excited amide product to form a light-emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, fine-tunes the blue color emitter to secure a high emission intensity. This information is critical to engineering the next-generation of ultrasensitive bioluminescent reporters