Determination of intracellular cytokines IFN-γ and IL-4 in canine T lymphocytes by flow cytometry following whole-blood culture

This report describes a whole-blood flow cytometric method for the determination of intracellular cytokines IFN-γ and IL-4 in canine T lymphocyte subpopulations. Conjugated anti-cytokine antibodies and commercially available reagents for cell fixation and permeabilization were used. Canine peripheral blood was cultured with a combination of phorbol-12-myristate-13-acetate (PMA) and ionomycin to promote cytokine synthesis in each cell, along with monensin to increase the sensitivity of the method by retaining IFN-γ and IL-4 within the cell to detectable levels. The optimum concentrations of PMA and ionomycin were determined. Maximum IFN-γ expression from both CD4+ and CD8+ T lymphocytes was detected after 6 h of incubation of cell culture, while maximum IL-4 production took 6 h from CD4+ cells and 4 h from CD8+ cells. This method is a simple immunologic technique for measuring intracellular cytokines which could be of value in the investigation of canine immunological response mainly in various intracellular and extracellular infections, since IFN-γ and IL-4 are considered key cytokines activating the cellular and humoral immunity, respectively.     

Current status of insecticide resistance in Q biotype Bemisia tabaci populations from Crete

BACKGROUND: A major problem of crop protection in Crete, Greece, is the control of Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) with chemical insecticides owing to the rapid development of resistance. The aim of this study was to investigate the establishment of resistance and the underlying mechanisms to major insecticide classes with classical bioassays and known biochemical resistance markers. RESULTS: During a 2005–2007 survey, 53 Q biotype populations were collected. Application history records showed extensive use of neonicotinoids, organophosphates, carbamates and pyrethroids. High resistance levels were identified in the majority of populations (>80%) for imidacloprid (RF: 38–1958×) and α‐cypermethrin (RF: 30–600×). Low resistance levels (RF < 12) were observed for pirimiphos‐methyl. A strong correlation between resistance to imidacloprid and the number of applications with neonicotinoids was observed. Significant correlations were observed between COE and P450‐dependent monoxygenase activity with resistance to α‐cypermethrin and imidacloprid respectively. A propoxur‐based AChE diagnostic test indicated that iAChE was widespread in most populations. Resistance levels for α‐cypermethrin were increased when compared with a previous survey (2002–2003). Differentiation of LC₅₀ values between localities was observed for imidacloprid only. CONCLUSION: Bemisia tabaci resistance evolved differently in each of the three insecticides studied. Imidacloprid resistance seems less established and less persistent than α‐cypermethrin resistance. The low resistance levels for pirimiphos‐methyl suggest absence of cross‐resistance with other organophosphates or carbamates used. Copyright © 2008 Society of Chemical Industry     

Acetylcholinesterase point mutations in European strains of Tetranychus urticae (Acari: Tetranychidae) resistant to organophosphates

BACKROUND: In Tetranychus urticae Koch, acetylcholinesterase insensitivity is often involved in organophosphate (OP) and carbamate (CARB) resistance. By combining toxicological, biochemical and molecular data from three reference laboratory and three OP selected strains (OP strains), the AChE1 mutations associated with resistance in T. urticae were characterised. RESULTS: The resistance ratios of the OP strains varied from 9 to 43 for pirimiphos‐methyl, from 78 to 586 for chlorpyrifos, from 8 to 333 for methomyl and from 137 to 4164 for dimethoate. The insecticide concentration needed to inhibit 50% of the AChE1 activity was, in the OP strains, at least 2.7, 55, 58 and 31 times higher for the OP pirimiphos‐methyl, chlorpyrifos oxon, paraoxon and omethoate respectively, and 87 times higher for the CARB carbaryl. By comparing the AChE1 sequence, four amino acid substitutions were detected in the OP strains: (1) F331W (Torpedo numbering) in all the three OP strains; (2) T280A found in the three OP strains but not in all clones; (3) G328A, found in two OP strains; (4) A201S found in only one OP strain. CONCLUSIONS: Four AChE1 mutations were found in resistant strains of T. urticae, and three of them, F331W, G328A and A201S, are possibly involved in resistance to OP and CARB insecticides. Among them, F331W is probably the most important and the most common in T. urticae. It can be easily detected by the diagnostic PCR‐RLFP assay developed in this study. Copyright © 2009 Society of Chemical Industry     

In Vitro Anti-Orthohantavirus Activity of the High-and Low-Molecular-Weight Fractions of Fucoidan from the Brown Alga Fucus evanescens

The Hantaan orthohantavirus (genovariant Amur–AMRV) is a rodent-borne zoonotic virus; it is the causative agent of haemorrhagic fever with renal syndrome in humans. The currently limited therapeutic options require the development of effective anti-orthohantavirus drugs. The ability of native fucoidan from Fucus evanescens (FeF) and its enzymatically prepared high-molecular-weight (FeHMP) and low-molecular-weight (FeLMP) fractions to inhibit different stages of AMRV infection in Vero cells was studied. The structures of derivatives obtained were determined using nuclear magnetic resonance (NMR) spectroscopy. We found that fucoidan and its derivatives exhibited significant antiviral activity by affecting the early stages of the AMRV lifecycle, notably virus attachment and penetration. The FeHMP and FeLMP fractions showed the highest anti-adsorption activity by inhibiting AMRV focus formation, with a selective index (SI) > 110; FeF had an SI of ~70. The FeLMP fraction showed a greater virucidal effect compared with FeF and the FeHMP fraction. It was shown by molecular docking that 2O-sulphated fucotetrasaccharide, a main component of the FeLMP fraction, is able to bind with the AMRV envelope glycoproteins Gn/Gc and with integrin β3 to prevent virus–cell interactions. The relatively small size of these sites of interactions explains the higher anti-AMRV activity of the FeLMP fraction.     

Polar constituents from the aerial parts of Origanum vulgare L. Ssp. hirtum growing wild in Greece

From the polar extracts of Origanum vulgare L. ssp. hirtum 19 compounds have been isolated. The structures and relative stereochemistry have been elucidated by spectroscopic analysis and determined as apigenin, luteolin, chrysoeriol, diosmetin, quercetin, eriodictyol, cosmoside, vicenin-2, caffeic acid, p-menth-3-ene-1,2-diol 1-O-beta-glucopyranoside, thymoquinol 2-O-beta-glucopyranoside, thymoquinol 5-O-beta-glucopyranoside, thymoquinol 2,5-O-beta-diglucopyranoside, 12-hydroxyjasmonic acid, 12-hydroxyjasmonic acid 12-O-beta-glucopyranoside, lithospermic acid B, rosmarinic acid, 10-epi-lithospermic acid, and epi-lithospermic acid B. The three latter products display unusual stereochemistry of the 3,4-hydroxyphenyllactic acid unit(s), which to the authors' best knowledge has never been reported before in similar compounds. Moreover, lithospermic acid B (and its stereoisomers), p-menth-3-ene-1,2-diol 1-O-beta-glucopyranoside, 12-hydroxyjasmonic acid, and 12-hydroxyjasmonic acid 12-O-beta-glucopyranoside were isolated for the first time from Origanum species.     

Nature-inspired soluble iron-rich humic compounds: new look at the structure and properties

Purpose

Humic substances (HS) being natural polyelectrolyte macromolecules with complex and disordered molecular structures are a key component of the terrestrial ecosystem. They have remarkable influence on environmental behavior of iron, the essential nutrient for plants. They might be considered as environmental friendly iron deficiency correctors free of synthetic iron (III) chelates disadvantages. The main goal of this study was to obtain water-soluble iron-rich humic compounds (IRHCs) and to evaluate their efficiency as chlorosis correctors.

Materials and methods

A facile preparation technique of IRHCs based on low-cost and available parent material was developed. The iron-containing precursor (ferrous sulfate) was added dropwisely into alkaline potassium humate solution under vigorous stirring and pH-control. A detailed characterization both of organic and inorganic parts of the compounds was provided, the iron species identification was carried out jointly by EXAFS and Mössbauer spectroscopy. Bioassay experiments were performed using cucumber Cucumis sativus L. as target plants. Plants were grown in modified Hoagland nutrient solution, prepared on deionized water and containing iron in the form of IRHCs. Total iron content in dry plants measured by spectrophotometry after oxidative digestion and the chlorophyll a content determined after acetone extraction from fresh plant were used as parameters illustrating plants functional status under iron deficiency condition.

Results and discussion

The high solubility (up to130 g/l) and iron content (about 11 wt%) of the IRHCs obtained allow considering them to be perspective for practical applications. A set of analytical methods has shown that the main iron species in IRHCs are finely dispersed iron (III) oxide and hydroxide nanoparticles. An application of the precursor solution acidification allows to obtain compounds containing a significant part of total iron (up to 30 %) in the form of partly disordered iron (II–III) hydroxysulphate green rust GR(SO₄ ^2?). Bioavailability of iron from IRHCs was demonstrated using bioassay in cucumber plants grown up on hydroponics under iron deficiency conditions.

Conclusions

The application of iron oxides chemistry for humic substance containing solution was proved to be an effective approach to synthesis of IRHCs. Using bioassay on cucumber plants C. sativus L. under iron deficiency conditions, the efficiency of compounds obtained as chlorosis correctors was demonstrated. Application of water-soluble IRHCs led to significant increase of chlorophyll a content (up to 415 % of the blank) and iron content in plants (up to 364 % of the blank) grown up on hydroponics.     

A Spatially and Temporally Disaggregated Anthropogenic Emission Inventory in the Southern Balkan Region

ICP Forest Monitoring data collected for more than 10 years made it possible to analyse key factors responsible for changes in forest conditions on a regional European scale. Observation routines may vary between different countries, which makes it difficult to gain more insight into stress-effect relationships. The effects on defoliation of Scots pine (Pinus sylvestris L.) of air concentrations and wet deposition of acidifying compounds, as well as effects of meteorological, site, stand and tree variables were investigated in Lithuania to test the hypothesis that spatial and temporal changes in pine defoliation were closely associated with atmospheric inputs of acidity. Over the period 1994–2004 crown defoliation of more than 8,000 pine trees was monitored in totally 45 stands distributed among three National Parks, where Integrated Monitoring Stations were situated. Air concentrations of SO2, and $ {\text{SO}}^{{{\text{2 - }}}}_{{\text{4}}} $ and $ {\text{NH}}^{{\text{ + }}}_{{\text{4}}} $ deposition, as well as spring and summer precipitation and mean winter temperature were shown to be the key factors affecting defoliation. The acidifying compounds accounted for nearly 58% of the variance in pine defoliation. Meteorological factors increased the degree of explanation to 65%, and stand and site variables to 79%.     

Oviposition responses of culex pipiens to a synthetic racemic Culex quinquefasciatus oviposition aggregation pheromone

The oviposition pheromone of Culex quinquefasciatus was synthesized in a racemic form in a simple (five steps), efficient, high yielding (45% total yield), and low cost way (use of relatively low cost reagents). Our synthetic racemic pheromone (SRP) was tested in the laboratory for its bioactivity on Culex pipiens biotype molestus, which is a member of the species complex that Culex quinquefasciatusbelongs. In the testing conditions, bioactivity at the doses of 0.01, 0.1, 1, and 10 microg per cage was found with the best bioactivity achieved at 1 mug per cage. The effectiveness of our SRP offers a capable tool for improving mosquito oviposition traps for surveillance or even control programs.     

Sensory properties and aromatic composition of baked snacks containing brewer's spent grain

The incorporation of brewer's spent grain (BSG) into baked snacks (crispy-slices) was investigated in terms of nutritional, sensory and aromatic properties. Wheat flour was replaced with BSG at the levels of 10, 15 and 25%. Snacks containing 10% BSG exhibited high Crispiness index (Ci), low Crispiness work (Wc) and a high number of peaks during texture analysis, indicating that the crispiness of the samples was not negatively affected. However higher levels of BSG affected the texture and the crumb structure of snacks and the results were significantly different to the 100% wheat control. Addition of BSG altered the odour profile of the snacks as shown by the volatile profiles, however sensory results indicated that BSG-containing snacks at a level of 10% were highly acceptable and highlighted the possibility of using BSG as a baking ingredient in the formulation of enhanced fibre baked snacks.