Fluorescent ligands for adenosine receptors

Interest is increasing in developing fluorescent ligands for characterization of adenosine receptors (ARs), which hold a promise of usefulness in the drug discovery process. The size of a strategically labeled AR ligand can be greatly increased after the attachment of a fluorophore. The choice of dye moiety (e.g. Alexa Fluor 488), attachment point and linker length can alter the selectivity and potency of the parent molecule. Fluorescent derivatives of adenosine agonists and antagonists (e.g. XAC and other heterocyclic antagonist scaffolds) have been synthesized and characterized pharmacologically. Some are useful AR probes for flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer, and scanning confocal microscopy. Thus, the approach of fluorescent labeled GPCR ligands, including those for ARs, is a growing dynamic research field.     

Antioxidant and Anti-Inflammatory Effects in RAW264.7 Macrophages of Malvidin,a Major Red Wine Polyphenol

Background

Red wine polyphenols can prevent cardiovascular and inflammatory diseases. Resveratrol, the most extensively studied constituent, is unlikely to solely account for these beneficial effects because of its rather low abundance and bioavailability. Malvidin is far the most abundant polyphenol in red wine; however, very limited data are available about its effect on inflammatory processes and kinase signaling pathways.

Methods & Findings

The present study was carried out by using RAW 264.7 macrophages stimulated by bacterial lipopolysaccharide in the presence and absence of malvidin. From the cells, activation of nuclear factor-kappaB, mitogen-activated protein kinase, protein kinase B/Akt and poly ADP-ribose polymerase, reactive oxygen species production, mitogen-activated protein kinase phosphatase-1 expression and mitochondrial depolarization were determined. We found that malvidin attenuated lipopolysaccharide-induced nuclear factor-kappaB, poly ADP-ribose polymerase and mitogen-activated protein kinase activation, reactive oxygen species production and mitochondrial depolarization, while upregulated the compensatory processes; mitogen-activated protein kinase phosphatase-1 expression and Akt activation.

Conclusions

These effects of malvidin may explain the previous findings and at least partially account for the positive effects of moderate red wine consumption on inflammation-mediated chronic maladies such as obesity, diabetes, hypertension and cardiovascular disease.     

An Evolution-Guided Analysis Reveals a Multi-Signaling Regulation of Fas by Tyrosine Phosphorylation and its Implication in Human Cancers

Demonstrations of both pro-apoptotic and pro-survival abilities of Fas (TNFRSF6/CD95/APO-1) have led to a shift from the exclusive “Fas apoptosis” to “Fas multisignals” paradigm and the acceptance that Fas-related therapies face a major challenge, as it remains unclear what determines the mode of Fas signaling. Through protein evolution analysis, which reveals unconventional substitutions of Fas tyrosine during divergent evolution, evolution-guided tyrosine-phosphorylated Fas proxy, and site-specific phosphorylation detection, we show that the Fas signaling outcome is determined by the tyrosine phosphorylation status of its death domain. The phosphorylation dominantly turns off the Fas-mediated apoptotic signal, while turning on the pro-survival signal. We show that while phosphorylations at Y232 and Y291 share some common functions, their contributions to Fas signaling differ at several levels. The findings that Fas tyrosine phosphorylation is regulated by Src family kinases (SFKs) and the phosphatase SHP-1 and that Y291 phosphorylation primes clathrin-dependent Fas endocytosis, which contributes to Fas pro-survival signaling, reveals for the first time the mechanistic link between SFK/SHP-1-dependent Fas tyrosine phosphorylation, internalization route, and signaling choice. We also demonstrate that levels of phosphorylated Y232 and Y291 differ among human cancer types and differentially respond to anticancer therapy, suggesting context-dependent involvement of Fas phosphorylation in cancer. This report provides a new insight into the control of TNF receptor multisignaling by receptor phosphorylation and its implication in cancer biology, which brings us a step closer to overcoming the challenge in handling Fas signaling in treatments of cancer as well as other pathologies such as autoimmune and degenerative diseases.     

Does AMH Reflect Follicle Number Similarly in Women with and without PCOS?

Context

Increased Anti-Mullerian Hormone in polycystic ovary syndrome, may be due to overactive follicles rather than reflect antral follicle count.

Objective

Does Anti-Mullerian Hormone reflect antral follicle count similarly in women with or without polycystic ovary syndrome or polycystic ovarian morphology?

Design

Cross-sectional, case-control.

Setting

Women who delivered preterm in 1999–2006. For each index woman, a woman with a term delivery was identified.

Patients

Participation rate was 69%. Between 2006–2008, 262 women were included, and diagnosed to have polycystic ovary syndrome, polycystic ovarian morphology or to be normal controls.

Intervention(s)

Blood tests, a clinical examination and vaginal ultrasound.

Main Outcome Measure(s)

Anti-Mullerian Hormone / antral follicle count -ratio, SHBG, androstenedione and insulin, to test potential influence on the Anti-Mullerian Hormone / antral follicle count -ratio.

Results

Mean Anti-Mullerian Hormone / antral follicle count ratio in women with polycystic ovary syndrome or polycystic ovarian morphology was similar to that of the controls (polycystic ovary syndrome: 1,2 p = 0,10 polycystic ovarian morphology: 1,2, p = 0,27 Controls 1,3). Anti-Mullerian Hormone showed a positive linear correlation to antral follicle count in all groups. Multivariate analysis did not change the results.

Conclusions

We confirmed the positive correlation between AMH and follicle count. Anti-Mullerian Hormone seems to be a reliable predictor of antral follicle count, independent of polycystic ovary syndrome diagnosis or ovarian morphology.     

Inhaled Methane Limits the Mitochondrial Electron Transport Chain Dysfunction during Experimental Liver Ischemia-Reperfusion Injury

Background

Methanogenesis can indicate the fermentation activity of the gastrointestinal anaerobic flora. Methane also has a demonstrated anti-inflammatory potential. We hypothesized that enriched methane inhalation can influence the respiratory activity of the liver mitochondria after an ischemia-reperfusion (IR) challenge.

Methods

The activity of oxidative phosphorylation system complexes was determined after in vitro methane treatment of intact liver mitochondria. Anesthetized Sprague-Dawley rats subjected to standardized 60-min warm hepatic ischemia inhaled normoxic air (n = 6) or normoxic air containing 2.2% methane, from 50 min of ischemia and throughout the 60-min reperfusion period (n = 6). Measurement data were compared with those on sham-operated animals (n = 6 each). Liver biopsy samples were subjected to high-resolution respirometry; whole-blood superoxide and hydrogen peroxide production was measured; hepatocyte apoptosis was detected with TUNEL staining and in vivo fluorescence laser scanning microscopy.

Results

Significantly decreased complex II-linked basal respiration was found in the normoxic IR group at 55 min of ischemia and a lower respiratory capacity (~60%) and after 5 min of reperfusion. Methane inhalation preserved the maximal respiratory capacity at 55 min of ischemia and significantly improved the basal respiration during the first 30 min of reperfusion. The IR-induced cytochrome c activity, reactive oxygen species (ROS) production and hepatocyte apoptosis were also significantly reduced.

Conclusions

The normoxic IR injury was accompanied by significant functional damage of the inner mitochondrial membrane, increased cytochrome c activity, enhanced ROS production and apoptosis. An elevated methane intake confers significant protection against mitochondrial dysfunction and reduces the oxidative damage of the hepatocytes.     

Bioluminescence‐based assays for assessing eco‐ and genotoxicity of airborne emissions

Ecotoxicological tests (or bioassays) are controlled, reproducible tests where ecological responses are determined quantitatively. Due to numerous difficulties arising when airborne emissions are sampled, relatively few ecotoxicological assays have been applied. Aerosol particles are generally collected on a filter, which limits the quantity of the sample, thus also limiting the range of available test organisms. Bacterial bioassays require low sample quantity, and make a good choice when eco‐ or genotoxicity of the sample are to be determined. Of bacterial assays, the bioluminescence inhibition test has been proven applicable for assessing toxicity of airborne contaminants. In this paper diverse test protocols and their modifications are reviewed. Copyright © 2016 John Wiley & Sons, Ltd.     

When to measure plumage reflectance: a lesson from Collared Flycatchers Ficedula albicollis

Sexually selected colour traits of bird plumage are widely studied. Although the plumage is replaced only at one or two yearly moults, plumage colour has long been shown to change between moults. Nevertheless, most studies measure colour weeks to months after the courtship period, typically at nestling rearing, and it is unclear whether these measurements yield relevant data concerning the primary process of sexual selection. Here we analyse repeated spectrometric data taken from male Collared Flycatchers during social courtship and nestling rearing. We show that some spectral traits are not correlated between the two measurements and that within‐individual correlation declines significantly with the likely exposure of the plumage area to damage and soiling. There is an overall decline in spectral trait exaggeration during breeding, but trait decline is not closely related to measurement latency, especially not in the damage‐exposed areas. Finally, sexual selection estimates differ depending on whether they are derived from spectra measured during courtship or during nestling rearing. These results suggest that, contrary to current practice, measurements of plumage reflectance should be made during the primary period of sexual signalling. Spectral trait decline during breeding could also be studied as a possible signal for mates and neighbours.     

What is behind the variation in mate quality dependent sex ratio adjustment? – A meta‐analysis

Theory predicts that parents adjust the sex ratio of their brood to the sexually selected traits of their mate because the reproductive success of sons may be more dependent on inherited paternal attractiveness than that of daughters. Empirical studies vary in terms of whether they support the theory, and this variation has often been regarded as evidence against sex ratio adjustment or has been ascribed to methodological differences. Applying phylogenetic meta‐analyses, we aimed to find biological explanations for the variation observed in songbirds. In particular, we tested the role extra‐pair paternity, because infidelity occurs in the majority of these species and may reduce the adaptive value of adjusting brood sex ratio to the phenotype of the social mate. However, we found that the variation in effect sizes was unrelated to the proportion of extra‐pair paternity. Thus future studies should consider that mate quality dependent sex ratio adjustment may be driven by direct (material) rather than indirect (genetic) benefits. We also tested if the effect sizes are influenced by whether the focal male trait is indeed under sexual selection as it is assumed by the sex allocation theory. We found that for male traits with proven role in sexual selection, effect sizes significantly differed from the null expectation of random production of sons and daughters. For male traits with only presumed sexual role in sexual selection, the deviation from the null expectation was less convincing, and the effect sizes were significantly smaller. This result indicates that studies that neglect the assumptions of the hypotheses concerned, may lead to the underestimation of the mean effect size and, eventually, false conclusions.     

A Pilot Clinical Study of Hyperacute Serum Treatment in Osteoarthritic Knee Joint: Cytokine Changes and Clinical Effects

The serum fraction of platelet-rich fibrin (hyperacute serum) has been shown to improve cartilage cell proliferation in in vitro osteoarthritic knee joint models. We hypothesize that hyperacute serum may be a potential regenerative therapeutic for osteoarthritic knees. In this study, the cytokine milieu at the synovial fluid of osteoarthritic knee joints exposed to hyperacute serum intraarticular injections was investigated. Patients with knee osteoarthritis received three injections of autologous hyperacute serum; synovial fluid was harvested before each injection and clinical monitoring was followed-up for 6 months. Forty osteoarthritic-related cytokines, growth factors and structural proteins from synovial fluid were quantified and analysed by Multivariate Factor Analysis. Hyperacute serum provided symptomatic relief regarding pain and joint stability for OA patients. Both patients “with” and “without effusion knees” had improved VAS, KOOS and Lysholm-Tegner scores 6 months after of hyperacute serum treatment. Synovial fluid analysis revealed two main clusters of proteins reacting together as a group, showing strong and significant correlations with their fluctuation patterns after hyperacute serum treatment. In conclusion, hyperacute serum has a positive effect in alleviating symptoms of osteoarthritic knees. Moreover, identified protein clusters may allow the prediction of protein expression, reducing the number of investigated proteins in future studies.