Development, characterization, and technical applications of an atlantic killifish AhR-2 specific monoclonal antibody (mAb 5B6)

The aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor normally found in the cytoplasmic compartment of cells held by chaperones and immunophilin-like proteins. Ligand binding dissociates the AhR/ligand from chaperone proteins, allowing translocation to the nucleus with subsequent transcription of a suite of responsive genes, most notably Phase I, II, and III drug metabolism genes. Select environmental contaminants such as co-planar PCBs, planar polyaromatic hydrocarbons (PAHs), and halogenated aromatic hydrocarbons (HAHs) are potent AhR agonists, with 2,3,7,8 -tetrachlorodibenzodioxin (TCDD) being one of the most potent. Adverse effects of exposure to these potent environmental AhR ligands include immune suppression, reproductive, and developmental disorders. Mammals express a single AhR protein, while fish express both AhR1 and AhR2. However, to date only the AhR2 protein appears to be involved in mediating the toxic effects of known xenobiotic AhR ligands. Using bacterial expression plasmid systems, a recombinant Atlantic killifish, Fundulus heteroclitus , AhR2 protein was expressed and used to produce a monoclonal antibody (mAb 5B6) that does not cross-react with AhR1. AhR2 expression can be detected with abundance in the cytosol of individual cells and in select organs. By testing the antibody against paraffin-embedded tissues, it was found that mAb 5B6 requires microwaving tissues under high pH conditions to properly recognize its epitope. . High levels of AhR2 protein are detected in the liver, spleen, intestine, and anterior kidney. Experimental exposures to the potent AhR2 ligand PCB-126 induce expression of both AhR2 and CYP1A proteins in most tissues, especially the intestine and liver. Basal expression of AhR2 protein was determined in livers of Atlantic killifish collected at the US-EPA Superfund site in Portsmouth VA, a site heavily contaminated with creosote and containing very high levels of PAHs. Several livers from the Superfund site harbored aggressive tumors and other hepatic lesions. AhR2 protein expression was high in normal tissue, but not cells within lesions. Overall, CYP1A protein expression patterns mirror those of AhR2 protein. This is the first study to examine AhR2 expression in tissues isolated from fish collected in the field, and like CYP1A, this protein may be a sentinel biomarker in future studies

AhR-related Activities in a Creosote-Adapted Population of Adult Atlantic Killifish, Fundulus heteroclitus, Two Decades Post-EPA Superfund Status

Atlantic killifish, Fundulus heteroclitus, have adapted to, or at least developed a level of resistance to creosote-based polycyclic aromatic hydrocarbons (PAHs) found at the Atlantic Wood (AW) superfund site in the southern branch of the Elizabeth River, VA USA. Historically, 90+% of the mummichogs at this site have hepatic lesions of varying severity, and 30+% of these fish have tumors of various origin. Many xenobiotics, including select PAHs found at the Atlantic Wood site, are ligands for the aryl hydrocarbon receptor (AhR), which is a promiscuous ligand-dependent transcription factor involved in developmental regulation, environmental sensing, and induction of a suite of phase I, II, and III drug metabolizing enzymes. Subsequent to the discovery of the AW killifish population, investigators demonstrated that these fish were recalcitrant to AhR activation and CYP1A induction following exposure to model PAHs. Moreover, killifish embryos collected from the AW site are still resistant to the cardiac and craniofacial deformities typically associated with exposure to PAHs and sediments collected from the AW site. In this study, the issue of CYP1A inducibility in AW fish collected in situ was re-examined nearly 2 decades after first discovery, and shown that the gene expression of this sentinel biomarker of AhR activation was significantly higher in intestines in comparison to a reference population. The livers of AW mummichogs were examined by IHC to discover that CYP1A and AhR2 protein patterns reflect tissue damage from lesions, and/or intrinsic cellular physiology of tumors, rather than recalcitrant induction of CYP1A. Gene expression profiles also indicated that liver COX-2 is elevated in livers of these fish. A monoclonal antibody was then generated against COX-2 (mAb CX53-1) and used to observe the possible role of COX-2 and inflammation in Atlantic Wood fish liver lesions. COX-2 protein expression was very high in macrophage aggregates and surrounding tumors, suggesting chronic inflammation. Further characterization of the gut innate immune system focusing on eosinophilic cell infiltration and lysozyme levels demonstrated higher activity. Overall, these findings suggest that AW fish are not necessarily recalcitrant to CYP1A induction, and thus seem to have active functional AhR2 protein(s) in non-damaged tissues. These fish are also under chronic inflammatory stress

Do I feel ill because I crave for work or do I crave for work because I feel ill? A longitudinal analysis of work craving, self-regulation, and health

Background The theory of work craving defines workaholism as a pathological work addiction which comprises: (a) obsessive–compulsive desire to work, (b) anticipation of self-worth compensatory incentives from working, (c) anticipation of reduction of negative emotions or withdrawal symptoms from working, and (d) neurotic perfectionism. Research has shown that workaholism is associated with adverse health outcomes. However, the antecedents of workaholism and the causal direction of the relationship with health have been largely neglected. Aims In the present longitudinal study, we expect that work craving is predicted by deficits in emotional self-regulation (i.e., low action orientation) and mediates the relationship between self-regulation deficits and symptoms of psychological distress. We expected work craving to have an effect on later psychological distress symptoms, but not psychological distress symptoms to have an effect on later work craving. Methods In a sample of 170 German employees, a half-longitudinal design using two times of measurement was implemented to specify the paths of two different structural equation models of mediation: (a) action orientation to later work craving and work craving to later psychological distress, and alternatively, (b) the temporal order of action orientation to later distress and distress to later work craving. Results Our data indicated that work craving partially mediated the relationship between self-regulation deficits and psychological distress, but psychological distress symptoms were not found to increase later work craving. Conclusions The presented longitudinal study indicates important mechanisms of work craving, especially by highlighting the influence of self-regulation deficits on work craving and, in turn, psychological distress

AHR-related activities in a creosote-adapted population of adult atlantic killifish, Fundulus heteroclitus, two decades post-EPA superfund status at the Atlantic Wood Site, Portsmouth, VA USA

Atlantic killifish, Fundulus heteroclitus, are adapted to creosote-based PAHs at the US EPA Superfund site known as Atlantic Wood (AW) on the southern branch of the Elizabeth River, VA USA. Subsequent to the discovery of the AW population in the early 1990s, these fish were shown to be recalcitrant to CYP1A induction by PAHs under experimental conditions, and even to the time of this study, killifish embryos collected from the AW site are resistant to developmental deformities typically associated with exposure to PAHs in reference fish. Historically, however, 90 +% of the adult killifish at this site have proliferative hepatic lesions including cancer of varying severity. Several PAHs at this site are known to be ligands for the aryl hydrocarbon receptor (AHR). In this study, AHR-related activities in AW fish collected between 2011 and 2013 were re-examined nearly 2 decades after first discovery. This study shows that CYP1A mRNA expression is three-fold higher in intestines of AW killifish compared to a reference population. Using immunohistochemistry, CYP1A staining in intestines was uniformly positive compared to negative staining in reference fish. Livers of AW killifish were examined by IHC to show that CYP1A and AHR2 protein expression reflect lesions-specific patterns, probably representing differences in intrinsic cellular physiology of the spectrum of proliferative lesions comprising the hepatocarcinogenic process. We also found that COX2 mRNA expression levels were higher in AW fish livers compared to those in the reference population, suggesting a state of chronic inflammation. Overall, these findings suggest that adult AW fish are responsive to AHR signaling, and do express CYP1A and AHR2 proteins in intestines at a level above what was observed in the reference population. (C) 2016 Elsevier B.V. All rights reserved

Investigation into Polyphenol Profile and Biological Activities of Enriched Persimmon/Apple Smoothies during Storage

Smoothies are becoming an increasingly popular product as a healthy alternative to snacks. The consumer expects from this product that, apart from its nutritional value, it will also be qualitatively stable during storage. Therefore, in this study, original smoothies obtained with persimmon fruit puree and apple juice (Dk/Md) enriched with Arbutus unedo fruits, Myrtus communis purple berry extract, Acca sellowiana, and Crocus sativus petal juice were evaluated for their polyphenol composition, antioxidant activity, and inhibition on targeted digestive enzymes, over six months of storage. The amount of polyphenols evaluated by UPLC-PDA analysis decreased in six months from 23.5% for both Dk/Md and enriched C. sativus smoothies to 42.5% for enriched A. sellowiana, with anthocyanins the most sensitive compounds (71.7–100% loss). Values of antioxidant assays generally strongly decreased during the first three months (up to ca. 60%) and to a lesser extent in the following three months (0.4–27%). In addition, inhibitory activity on α-amylase, α-glucosidase, and pancreatic lipase, especially on the last two enzymes, was negatively affected by time storage. The outcome of this study indicates that persimmon fruit is a good option for producing smoothies, and enrichment with other plant extracts can enhance the bioactive compound content and biological activities. It is recommended that appropriate storage strategies to preserve the properties of those smoothies should be developed

An explicit formula for the coefficients in Laplace's method

Laplace's method is one of the fundamental techniques in the asymptotic approximation of integrals. The coefficients appearing in the resulting asymptotic expansion, arise as the coefficients of a convergent or asymptotic series of a function defined in an implicit form. Due to the tedious computation of these coefficients, most standard textbooks on asymptotic approximations of integrals do not give explicit formulas for them. Nevertheless, we can find some more or less explicit representations for the coefficients in the literature: Perron's formula gives them in terms of derivatives of an explicit function; Campbell, Fr\"oman and Walles simplified Perron's method by computing these derivatives using an explicit recurrence relation. The most recent contribution is due to Wojdylo, who rediscovered the Campbell, Fr\"oman and Walles formula and rewrote it in terms of partial ordinary Bell polynomials. In this paper, we provide an alternative representation for the coefficients, which contains ordinary potential polynomials. The proof is based on Perron's formula and a theorem of Comtet. The asymptotic expansions of the gamma function and the incomplete gamma function are given as illustrations.Comment: 14 pages, to appear in Constructive Approximatio

Activity of Antimicrobial Peptides and Conventional Antibiotics against Superantigen Positive Staphylococcus aureus Isolated from the Patients with Neoplastic and Inflammatory Erythrodermia

Superantigens are proteins comprising a group of molecules produced by various microorganisms. They are involved in pathogenesis of several human diseases. The aim of the study was the comparison of susceptibility to antibiotics and antimicrobial peptides (AMPs) of Staphylococcus aureus (SA) strains producing staphylococcal enterotoxins SEA, SEB, SEC, SED, and TSST-1 and nonproducing ones. In the group of the total 28 of the patients with erythrodermia the presence of SA was confirmed in 24 cases. The total of 14 strains of SA excreted enterotoxins SEA, SEC, SED, and TSST-1. We did not observe that strains producing mentioned superantigens were less susceptible to AMPs (aurein 1.2, citropin 1.1, lipopeptide, protegrin 1, tachyplesin 3, temporin A, and uperin 3.6). The opposite situation was observed in conventional antibiotics. SA strains excreting tested superantigens had higher MICs and MBCs than nonproducing ones. The interesting finding considering the high efficacy of AMPs, against all examined strains of SA, makes them attractive candidates for therapeutic implication

Presence of Chlamydophila pneumoniae DNA in blood cells is a frequent event in patients with the late stage of primary cutaneous lymphomas and with atopic dermatitis

Introduction: Microbial infection and associated super antigens have been implicated in the pathogenesis of cutaneous T-cell lymphoma (CTCL), and many patients die from complicating bacterial infections. It has been postulated that Chlamydophila pneumoniae (C. pneumoniae) infection may be involved in the pathogenesis of Mycosis fungoides (MF) but published data are limited and controversial. Aim: To analyze the frequency of (C. pneumoniae) DNA presence in blood samples of lymphoma cases. Material and methods: Using Q-PCR method we analyzed the presence of DNA in the blood samples obtained from 57 patients with CTCL (55 - mycosis fungoides (MF)/Sezary syndrome (SS), one primary cutaneous anaplastic large cell lymphoma (CD30+) and one NKT cell lymphoma) and 3 patients with cutaneous B-cell lymphomas, and 120 individuals from control groups (40 patients with psoriasis, 40 patients with atopic dermatitis and 40 healthy controls). Results: Chlamydophila pneumoniae DNA was identified in 13 of 55 cases in the MF/SS group (23.6%), in 1 patient with CD30+ large cell lymphoma and in 1 of 3 patients with B-cell lymphoma. The presence of C. pneumoniae was confirmed in 1 of 40 psoriatic patients (2.5%), in 5 of 40 patients with atopic dermatitis (12.5%) and in none of 40 healthy individuals. Presence of C. pneumoniae DNA in MF patients was strongly associated with disease progression; rs = 0.756; p = 0.0123 for groups IA -> IVB, and was noted more frequently in advanced (III + IV) stages than in early (I-II) stages (p = 0.0139). There are no differences in the mean age of MF/SS patients with and without infection. Conclusions: The presence of C. pneumoniae DNA in the blood cells is a frequent event in late stages of MF/SS and may be explained by Th2 shift and suppression of the immune system during the course of the disease.Peer reviewe

Impact of power ultrasound on chemical and physicochemical quality indicators of strawberries dried by convection

[EN] A study on the quality parameters of strawberries dehydrated by convection assisted by power ultrasound (US) at 40 70 C and 30 and 60W has been carried out for the first time. In general, the quality of US-treated samples was higher than that of commercial samples. Even under the most severe conditions used (US at 70 C and 60 W), high values of vitamin C retention (>65%) and scarce advance of Maillard reaction (2-furoylmethyl derivatives of Lys and Arg < 90 mg 100 g 1 protein) were observed. Rehydration ratio was not affected by the power applied and the obtained values were similar to those of convectively-treated samples. According to the results here presented, US is a suitable example of an emerging and environmentally friendly technology that accelerates convective drying, allowing the obtainment of dried strawberries with premium quality.This work has been funded by Ministry of Economy and Competitiveness of Spain and ERDF (FEDER) (Project DPI2012-37466-C03-03), Fun-c-Food (CSD2007-00063 Consolider-INGENIO 2010), CYTED IBEROFUN (P109AC0302) and Comunidad de Valencia, Project PROMETEO/2010/062. J.G.S. also thanks CSIC and the EU for a predoctoral JAE grant. A.C.S. thanks the Spanish Ministry of Economy and Competitiveness for a Ramon y Cajal contract.Gamboa-Santos, J.; Montilla, A.; Cortijo Soria, A.; Cárcel Carrión, JA.; García Pérez, JV.; Villamiel, M. (2014). Impact of power ultrasound on chemical and physicochemical quality indicators of strawberries dried by convection. Food Chemistry. 161:40-46. https://doi.org/10.1016/j.foodchem.2014.03.106S404616

Phytopharmacologic preparations as predictors of plant bioactivity: A particular approach to Echinacea purpurea (L.) Moench antioxidant properties

Objective A large body of evidence has confirmed a multitude of health benefits of plant products and their derived formulations. Echinacea purpurea (L.) Moench is a good example, widely used due to its therapeutic properties. In the present study, the chemical composition of the different samples and antioxidant properties of E. purpurea hydroethanolic and aqueous extracts obtained from dry or fresh raw material were evaluated and compared with dietary supplements based on the same plant (tablets and syrup), to determine the most active phytopharmacologic preparation or formulation. Methods Chemical composition of the different samples was assessed through the determination of free sugars, organic acids and tocopherols. The in vitro antioxidant properties were determined using four assays: 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging activity, reducing power, inhibition of b-carotene bleaching and inhibition of lipid peroxidation by thiobarbituric acid reactive substances (TBARS) assay. Total phenolics and flavonoids were also determined. Results Overall, the hydroethanolic extract of fresh plant revealed the highest activity, directly related with its higher contents in phenolic (229.22 ± 4.38 mg gallic acid equivalent [GAE]/mL), flavonoids (124.83 ± 7.47 mg GAE/mL), organic acids (8.89 ± 0.10 g/100 g), and tocopherols (4.55 ± 0.02 mg/100 g). Tablets followed by syrup revealed the worst effect, positively correlated with the lowest abundance in bioactive molecules. The weak in vitro antioxidant potential of commercial phytopharmacologic formulations could be related to their chemical composition, including the addition of excipients. Conclusion Further studies are necessary to deepen knowledge on this area, namely focusing on in vivo experiments, to establish upcoming guidelines to improve the quality and bioavailability of phytopharmacologic formulations.The authors are grateful to Foundation for Science and Technology (FCT, Portugal) for financial support to the research center CIMO (strategic project PEst-OE/AGR/UI0690/2014) and L. Barros researcher contract under “Programa Compromisso com Ciência – 2008”