IgE binding synthetic peptides of Alt a 1, a major allergen of Alternaria alternata

Alternaria alternata protein, Alt a 1 is a major allergen associated with allergy in atopic patients. Although the molecule binds strongly to IgE antibody from patients, the epitopes involved have not been identified or defined. In the present study, we synthesized overlapping peptides spanning the whole sequence and evaluated their IgE binding with sera from patients with Alternaria-induced allergy. The results identified four IgE binding linear regions. Two of these regions K41-P50 and Y54-K63 showed consistent reactivity with all four patients studied. The specific epitopes involved in the immune response may be of value in the immunodiagnosis and probably also in specific immunotherapy.     

C-terminal cysteine residues determine the IgE binding of Aspergillus fumigatus allergen Asp f 2

The knowledge of the structure function relationship of the allergen is essential to design allergenic variants with reduced IgE binding capacity but intact T cell reactivity. Asp f 2 is a major allergen from the fungus Aspergillus fumigatus and >90% of A. fumigatus-sensitized individuals displayed IgE binding to Asp f 2. In the present study, we evaluated the involvement of C-terminal cysteine residues in IgE binding conformation of Asp f 2. The deletion mutants were constructed by adding three C-terminal cysteines of the native Asp f 2 one at a time to the non-IgE binding Asp f 2 (68-203). The point mutants of Asp f 2 (68-268) with C204A and C257A substitutions were constructed to study the role of C-terminal cysteines in IgE binding. Immunological evaluation of reduced and alkylated Asp f 2 and its mutants were conducted to determine the contribution of free sulfhydryl groups as well as the disulfide bonds in allergen Ab interaction. Four-fold increase in IgE Ab binding of Asp f 2 (68-267) compared with Asp f 2 (68-266) and complete loss in IgE binding of C204A mutant of Asp f 2 (68-268) indicate the involvement of C(204) and C(267) in IgE binding conformation of Asp f 2. A significant reduction in IgE binding of wild and mutated Asp f 2 after reduction and alkylation emphasizes the importance of cysteine disulfide bonds in epitope Ab interaction. The hypoallergenic variants may be explored further to develop safe immunotherapeutic strategy for allergic disorders.     

Antioxidant defense of rainbow trout (Oncorhynchus mykiss) in relation to dietary n-3 highly unsaturated fatty acids and vitamin E contents

This study examined the modulation of the antioxidant status and related physiological changes in rainbow trout Oncorhynchus mykiss under different levels of dietary n-3 highly unsaturated fatty acids (n-3 HUFA) and vitamin E. Six diets containing 0, 100 or 1000 mg alpha-tocopheryl acetate kg(-1) diet and 20% or 48% n-3 HUFA provided by normal fish oil or DHA concentrated fish oil, respectively, were fed to 100 g size fish for 15 weeks. Growth of fish fed vitamin E deficient diets under both levels of n-3 HUFA were slightly retarded, accompanied by a reduction of hematocrit values, an enlargement of liver and spleen, an elevation of lipid hydroperoxide in red blood cell and the antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase). Supplementation of vitamin E could protect the fish from these adverse effects; however the higher dose was no better compared to the moderate dose. The modulations were clearly seen in fish fed high n-3 HUFA (48%) since they were under greater oxidative stress as indicated by the markers, lipid hydroperoxide and 8-isoprostane. The increased activity of enzymes corresponds to physiological mechanisms combating the elevation of free radicals under oxidative stress and a dietary fatty acid profile-dependent moderate dose of vitamin E is all that is required to function as an effective antioxidant.     

Characterization of GP21 and GP12: Two Potential Probiotic Bacteria Isolated from the Gastrointestinal Tract of Atlantic Cod

This study evaluated the probiotic potential of GP21 (Pseudomonas sp.) and GP12 (Psychrobacter sp.), two bacteria isolated from the intestinal tract of a cold-water fish, Atlantic cod. The antagonistic activity of the two intestinal bacteria against two fish pathogens (Vibrio anguillarum and Aeromonas salmonicida subsp. salmonicida) was studied under different physical conditions. Further, their resistance to physiological barriers and their ability to form biofilms were examined. In addition, a test was conducted to confirm that the isolates were not pathogenic to the host fish. The two bacteria exhibited differences in their antagonism to the pathogens. Both were active against V. anguillarum at mildly acidic conditions over a 5-day period. The activity of GP21 against A. salmonicida was greater at pH 7–8. The maximum antagonistic activity was observed at a temperature of 15°C and at a salt concentration of 15 ppt for both the isolates. They did not produce acids, could release siderophores and tolerated both the acidic environment and the bile salts. Their ability to form biofilms was high around 15°C and when iron was supplemented in the medium at 5 μmol l^?1. There was no mortality of fish during the pathogenicity experiment, confirming the safety of both isolates for further applications. Considering the favorable characteristics identified here, it could be concluded that GP21 and GP12 isolated from the gastrointestinal tract of Atlantic cod are potential probiotic candidates.     

Tunable Plasmonic Resonances in Hexagonally Ordered Gold Nanostructure Arrays with Increasing Interstice Size

Plasmonics - Gold nanostructure arrays were fabricated by combining colloidal lithography with inclined reactive ion etching and inclinded sputtering. Field emission scanning electron microscopy...     

Genotypic Prediction of Tropism of Highly Diverse HIV-1 Strains from Cameroon

Background

The use of CCR5 antagonists involves determination of HIV-1 tropism prior to initiation of treatment. HIV-1 tropism can be assessed either by phenotypic or genotypic methods. Genotypic methods are extensively used for tropism prediction. However, their validation in predicting tropism of viral isolates belonging to group M non-B subtypes remains challenging. In Cameroon, the genetic diversity of HIV-1 strains is the broadest reported worldwide. To facilitate the integration of CCR5 antagonists into clinical practice in this region, there is a need to evaluate the performance of genotypic methods for predicting tropism of highly diverse group M HIV-1 strains.

Methods

Tropism of diverse HIV-1 strains isolated from PBMCs from Cameroon was determined using the GHOST cell assay. Prediction, based on V3 sequences from matched plasma samples, was determined using bioinformatics algorithms and rules based on position 11/25 and net charge applied independently or combined according to Delobel''s and Garrido''s rules. Performance of genotypic methods was evaluated by comparing prediction generated with tropism assigned by the phenotypic assay.

Results

Specificity for predicting R5-tropic virus was high, ranging from 83.7% to 97.7% depending on the genotypic methods used. Sensitivity for X4-tropic viruses was fairly low, ranging from 33.3% to 50%. In our study, overall, genotypic methods were less able to accurately predict X4-tropic virus belonging to subtype CRF02_AG. In addition, it was found that of the methods we used the Garrido rule has the highest sensitivity rate of over 50% with a specificity of 93%.

Conclusion

Our study demonstrated that overall, genotypic methods were less sensitive for accurate prediction of HIV-1 tropism in settings where diverse HIV-1 strains co-circulate. Our data suggest that further optimization of genotypic methods is needed and that larger studies to determine their utility for tropism prediction of diverse HIV-1 strains may be warranted.     

Nucleotide Enrichment of Live Feed: A Promising Protocol for Rearing of Atlantic Cod Gadus morhua Larvae

We investigated the effect of two commercial nucleotide products (NT1 and NT2), administered through live feed, on growth and stress tolerance of Atlantic cod larvae. Expression of genes related to muscle growth (igf-1, igf1r, igf-2, fst, fgf6, myod, and myhc) and nucleotide metabolism (uox, hprt, ndk, and uck) was evaluated during larval development. In addition, the expression of genes related to stress (hif-1α, hif-2α, hif-3α, and mb) was studied after an air exposure stress test. The enrichment of rotifers with nucleotides did not reveal any difference in nucleotide profiles, the exception being the RNA level of the NT1-enriched group that was significantly higher than the unenriched rotifer. Unenriched Artemia showed poor nucleotide profiles compared to enriched Artemia since 5' UMP, 5' GMP, and 5' AMP were observed only in the nucleotide groups. At 38?days post-hatch (dph), NT1 group had significantly higher dry weight (3.1?±?0.1?mg) than the control (CON; 2.3?±?0.1?mg). The treatments did not produce any significant differences in the expression of the key myogenic genes. Among the genes associated with nucleotide metabolism, ndk was down-regulated in NT1 at 38 dph. In the air exposure test, survival was significantly higher in the CON (77?±?6?%) than in NT1 (48?±?3?%) and NT2 (50?±?3?%). After air exposure, mb was expressed at lower levels in NT2 group, hif-2α was induced in NT1 group, and hif-3α was upregulated in all groups. Our findings indicate that the improvement in the nucleotide profile of Artemia upon nucleotide enrichment could eventuate in the rapid growth of larvae.     

Minimum significant ratio of selectivity ratios (MSRSR) and confidence in ratio of selectivity ratios (CRSR): quantitative measures for selectivity ratios obtained by screening assays

Development of inhibitor compounds selective against undesirable targets is critical in drug discovery. Selectivity ratios for candidate compounds are evaluated by dividing potencies from two assays assessing the off-target and target. Because all potency measurements have underlying uncertainty, understanding error propagation is essential to interpreting selectivity data. Assay noise introduces ambiguity in the statistical significance of selectivity ratios, particularly at low replicate numbers when compounds are often prioritized for subsequent testing. The ability to differentiate potency results for any pair of compounds in one assay is evaluated using a metric called minimum significant ratio (MSR). Potency results of one compound tested in a pair of assays can be differentiated by the minimum significant selectivity ratio (MSSR). To differentiate selectivity ratios for any pair of compounds, we extend this concept by proposing two new parameters called the minimum significant ratio of selectivity ratios (MSRSR) and confidence in ratio of selectivity ratios (CRSR). Importantly, these tools can be used after a single selectivity measurement. We describe these methods and illustrate their usefulness using structure-activity relationship data from a Janus kinase inhibitor project, in which these tools informed a cogent retesting strategy and enabled rapid and objective decision making.     

Binding of peptides corresponding to the carboxy-terminal region of human-β-defensins-1–3 with model membranes investigated by isothermal titration calorimetry

Human-β-defensins HBD-1–3 are important components of the innate immune system. Synthetic peptides Phd-1–3 with a single disulphide bond, spanning the cationic C-terminal region of HBD-1–3, have antimicrobial activity. The interaction of Phd-1–3 with model membranes was investigated using isothermal titration calorimetry (ITC) and steady-state fluorescence polarization to understand the biophysical basis for the mechanism of antimicrobial action. Calorimetric titration of POPE:POPG (7:3) vesicles with peptides at 25 °C and 37 °C showed complex profiles with two distinct regions of heat changes. The data indicate binding of Phd-1–3 at 37 °C to both negative and zwitterionic lipid vesicles is exothermic with low enthalpy values (ΔH ~ ? 1.3 to ? 2.8 kcal/mol) as compared to amphipathic helical antibacterial peptides. The adsorption of peptides to negatively charged lipid membranes is modulated by electrostatic interactions that are described by surface partition equilibrium model using Gouy–Chapman theory. However, this model could not explain the isotherms of peptide binding to zwitterionic lipid vesicles. Fluorescence polarization of TMA-DPH (1-[4-(trimethylammonio) phenyl]-6-phenyl-1,3,5-hexatriene) and DPH (1,6-diphenyl-1,3,5-hexatriene) located in the head group and acyl chain region respectively, indicates that the peptides interact with interfacial region of negatively charged membranes. Based on the results obtained, we conclude that adsorption of cationic peptides Phd-1–3 on lipid surface do not result in conformational change or pore formation. It is proposed that interaction of Phd-1–3 with the negatively charged lipid head group causes membrane destabilization, which in turn affects the efficient functioning of cytoplasmic membrane proteins in bacteria, resulting in cell death.     

Clostridium acetobutylicum 8-oxoguanine DNA glycosylase (Ogg) differs from eukaryotic Oggs with respect to opposite base discrimination

During repair of damaged DNA, the oxidized base 8-oxoguanine (8-oxoG) is removed by 8-oxoguanine-DNA glycosylase (Ogg) in eukaryotes and most archaea, whereas in most bacteria it is removed by formamidopyrimidine-DNA glycosylase (Fpg). We report the first characterization of a bacterial Ogg, Clostridium acetobutylicum Ogg (CacOgg). Like human OGG1 and Escherichia coli Fpg (EcoFpg), CacOgg excised 8-oxoguanine. However, unlike hOGG1 and EcoFpg, CacOgg showed little preference for the base opposite the damage during base excision and removed 8-oxoguanine from single-stranded DNA. Thus, our results showed unambiguous qualitative functional differences in vitro between CacOgg and both hOGG1 and EcoFpg. CacOgg differs in sequence from the eukaryotic enzymes at two sequence positions, M132 and F179, which align with amino acids (R154 and Y203) in human OGG1 (hOGG1) found to be involved in opposite base interaction. To address the sequence basis for functional differences with respect to opposite base interactions, we prepared three CacOgg variants, M132R, F179Y, and M132R/F179Y. All three variants showed a substantial increase in specificity for 8-oxoG.C relative to 8-oxoG.A. While we were unable to definitively associate these qualitative functional differences with differences in selective pressure between eukaryotes, Clostridia, and other bacteria, our results are consistent with the idea that evolution of Ogg function is based on kinetic control of repair.