The influence of medium on the chemical composition ofBlastomyces dermatitidis

Whole cells and cell walls of the mycelial and yeast forms ofBlastomyces dermatitidis grown in four different media were analyzed for differences in lipid, fatty acid, carbohydrate, and protein contents. The bound (saponifiable) fatty acids of yeast and mycelial whole cells (but not the cell walls) vary considerably in response to growth medium. The percentage of readily extractable lipid varied somewhat in whole cells. The percentage of carbohydrate and protein of whole cells and cell walls are little affected by the medium in which the cells are grown.     

The effect of polymers on the stability of microencapsulated formulations of Bacillus thuringiensis subsp. kurstaki (Bt-KD2) after exposure to Ultra Violet Radiation

We compared the protective effect of three polymers; starch, gelatin and sodium alginate (2, 3, 5%) as coating materials, on the stability of microencapsulated formulation of Bacillus thuringiensis after exposure to Ultra Violet (UV) R. Microencapsulated Spore Crystal Aggregate (SCA) formulations were prepared by the emulsion gelling method. The protective effect of polymers was evaluated by measuring spore viability. Bioassay and release tests were done on the microencapsulated formulations. Sodium alginate (5% w/w) showed the highest viabilities of 90 and 86% after exposure to Ultra Violet in long term (UVB 385 nm) and Ultra Violet in short term (UVC 254 nm) radiation, respectively, while viabilities of non-microencapsulated spores under these conditions were 40 and 50%, respectively. The crystal activity (mortality) of irradiated and non-irradiated free spore formulations on second-instar larvae of Ephestia kuehniella were 15 and 93%. However, the mortalities caused by irradiated and non-irradiated microencapsulated formulations were 70 and 80% on the 10th day of the experiment. The size range of the microcapsules was 7–20 µm while the microcapsulation efficiency was 86%. The release behaviour of microspheres conformed best to Korsmeyer–Peppas semi-empirical model with the correlation of R² = 0.98.     

Magneto-structural correlations and DFT calculations in two rare tetranuclear copper(II)-clusters with doubly phenoxo and end-on azido bridges: Syntheses, structural variations and EPR studies

By slightly changing the synthetic conditions, we have prepared two closely related linear tetranuclear Cu^II complexes with the symmetrical ONNO donor tetradentate Schiff-base ligand [H₂L = (OH)C₆H₄(CH₃)CN(CH₂)₃NC(CH₃)C₆H₄(OH)] and with azide ions. These two distinctly coloured crystalline products were characterized by elemental analysis, IR and UV-Vis spectroscopy, CV, EPR spectra and variable temperature magnetic measurements. Single crystal X-ray diffraction studies of the green [Cu₄(μ-L)₂(μ_1,1-N₃)₂(N₃)₂] (1) and the red [Cu₄(μ-L)₂(μ_1,1-N₃)₂(N₃)₂(H₂O)₂] (2) crystals show that the coordination environment of the two μ-phenoxo and μ_1,1-azido bridged isomorphous tetranuclear Cu^II complexes are slightly different. Thus, both complexes are formed by very similar building units, although with a significant variation in the bridging Cu-O(phenoxo)-Cu and Cu-N(azido)-Cu bond angles. The consequences of these structural variations on the magnetic properties have been investigated from both the experimental and theoretical points of view by variable temperature magnetic measurements and DFT calculations.     

Graves' ophthalmopathy: a review of immunogenetics

Graves' disease (GD) is the most common cause of thyrotoxicosis and often involves the orbits. Graves' ophthalmopathy (GO), also known as Thyroid Eye Disease (TED), can be clinically significant and advance to sight-threatening stages. Our knowledge of the immunogenetic pathophysiology of GO is rapidly expanding. The present review is an attempt to summarize the current state of knowledge on the immunogenetics of GO. First we briefly review the epidemiology and clinical importance of GO, and then we describe in detail the macromolecular pathogenesis and finally immunogenetics of GO. Discrepancies between the results from various reports and the limitations of the available data are discussed. In particular, there is a scarcity of data from non-Asian populations. While several studies have demonstrated significant associations between polymorphisms in certain genes (especially CTLA-4, HLA-DRB-1, and TNF-α), there is a need for studies that investigate the relationship between polymorphisms and both serum and local concentrations of the resulting proteins. A complete understanding of GO susceptibility and pathogenesis has not been yet possible due to a number of important knowledge gaps that need to be filled by future research.     

A comparison of three strategies for biopanning of phage-scFv library against diphtheria toxin

The biopanning process is a critical step in phage display for isolating peptides or proteins with specific binding properties. Conventional panning methods are sometimes not so effective and may result in nonspecific or low-yield positive results. In this study, three different strategies including soluble antibody-capturing, pH-stepwise elution, and conventional panning were used for enrichment of specific clones against diphtheria toxoid. The reactivity of the selected clones was evaluated using an indirect enzyme-linked immunosorbent assay. The positive clones were screened using Vero cell viability assay. The neutralizing clones were expressed in HB2151 strain of Escherichia coli and soluble single-chain fragment variable (scFv) fragments were purified by nickel-nitrilotriacetic acid affinity chromatography. Finally, the ability of scFv fragments for neutralizing diphtheria toxin (DT) were evaluated again using Vero cell viability assay. After four rounds of panning, the soluble antibody-capturing method yielded 15 positive phage-scFv clones against diphtheria toxoid. Conventional panning and pH-stepwise elution model resulted from nine and five positive phage-scFv clones, respectively. Among all positive clones, three clones were able to neutralize DT in Vero cell viability assay. Two of these clones belonged to a soluble antibody-capturing method and one of them came from conventional panning. Three neutralizing clones were used for soluble expression and purification of scFvs fragments. It was found that these soluble scFv fragments possessed neutralizing activity ranging from 0.15 to 0.6 µg against two-fold cytotoxic dose 99% of DT. In conclusion, the results of our study indicate that soluble antibody-capturing method is an efficient method for isolation of specific scFv fragments.     

Histochemical analyses of altered fetal lung development following single vs multiple courses of antenatal steroids.

A single course of antenatal steroids is widely used during preterm labor to promote fetal lung maturation. However, little is known regarding efficacy and safety of multiple courses of antenatal steroids. In animal models and clinical trials, treatment with glucocorticoids can inhibit growth. The present study of single- vs multiple-course steroids in pregnant ewes analyzes the effects of steroids vs placebo on fetal lung histopathology. Single-course groups received dexamethasone (Dex) 6 mg or normal saline every 12 hr for 48 hr at 104-106 days of gestation (term = 150 days). Multiple-course groups received the first course at 76-78 days; this was repeated weekly for 5 weeks. At 108 days, lungs were analyzed using immunohistochemistry for alpha-smooth muscle actin, a myofibroblast marker and proliferating cell nuclear antigen. Cell injury/death was evaluated using TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) analysis. Although fetal growth was restricted by either single or multiple courses of Dex, alveolar development was accelerated as measured by mean linear intercepts. Alveolar walls were thinner, developing septa were longer, and septal myofibroblasts were increased for both Dex groups compared with controls. Cell proliferation increased following multiple steroid courses, especially in the distal parenchyma, with a corresponding decrease in apoptosis. These observations suggest that Dex promotes alveolarization, whether given in single or multiple courses.     

Enzyme-Linked Immunosorbent Spot (ELISpot) monitoring of cytokine-producing cells for the prediction of acute rejection in renal transplant patients

The purpose of this study was to evaluate T-cell immunity markers using serial post-transplantation monitoring of cytokine-producing cells during the first post-transplant months for the prediction of acute rejection and potentially chronic rejection of kidney allograft. We followed 57 kidney allograft recipients for meanly 3 years post-transplantation. Blood samples were collected pre-transplant, 2, 4 and 12 weeks post-transplant. The frequencies of IL-10-, IL-17- and IFN-γ-producing cells were determined in all time-points using ELISPOT assay. The results of ELISpot monitoring and levels of IL-23 and TGF-β were compared between recipients with acute (n = 12) or chronic rejection episodes and patients with stable graft function (n = 45). In all post-transplant time-points, significantly high frequencies of IFN-γ- and IL-17-producing cells and low frequency of IL-10-producing cells were observed in rejection group versus patients with stable graft function (P<0.0001). TheROCcurve analysis for determining the reliability of cytokine-producing cells for the prediction of acute rejection revealed that AUC was 0.046 for IL-10 (P<0.001), 0.927 for IL-17 (P<0.001) and 0.929 for INF-γ-producing cells (P<0.001). Our results indicate that analyzing the frequencies of INF-γ/IL-10/IL-17-producing cells may define a reliable panel for the prediction of acute rejection within the first post-transplant year which could also be applicable for the prediction of chronic rejection episodes.     

Intergenic spacer (IGS) polymorphism: a new genetic marker for differentiation of Toxoplasma gondii strains and Neospora caninum

The region between the 28S and 18S rRNA genes, including the intergenic spacer (IGS) region and the 5S rRNA gene, from 32 strains of Toxoplasma gondii and the NC1 strain of Neospora caninum was amplified and used for DNA sequencing and/or restriction fragment length polymorphism (RFLP) analysis. The 5S rDNA sequences from 20 strains of T. gondii were identical. The IGS region between the 5S and 18S rRNA genes (nontranscribed spacer 2 or NTS 2) showed 10 nucleotide variations. Six of the 10 variant positions correlated with the murine virulence of the strains. Intraspecific polymorphisms distinguished the virulent strains of zymodemes 5, 6, and 8 from other virulent strains (in zymodeme 1). RFLP methods (IGS-RFLP) were developed and used to characterize the virulent and avirulent patterns among 29 T. gondii strains. Sequence diversity of 19.8% was found between T. gondii and N. caninum when comparing a region of 919 bp at the 3' end of NTS 2. The sequence variation in ribosomal IGS could therefore be a useful marker for Toxoplasma strain identification and for distinguishing N. caninum from T. gondii.     

Ultraviolet protection of nucleopolyhedrovirus through microencapsulation with different polymers

A microencapsulated formulation of Helicoverpa armigera nuclear polyhedrosis virus (HaNPV) was produced by emulsion technique to improve its stability under ultraviolet (UV) radiation. The polymers used include sodium alginate, gelatin and starch at concentration levels of 3% and 5% w/v. Except for the starch microencapsulated formulation (3% w/v), the difference in mortality of treated insects between microencapsulated and non-microencapsulated suspensions before irradiation was not significant according to variance analysis (Duncan test, P < 0.05, df = 6). This indicates that microencapsulation of HaNPV does not affect viral activity. Among the three polymers, gelatin performed the best and provided the most stable formulation. The Original Activity Remaining (OAR) percentage for the gelatin formulations did not change from its initial value after 24 h of irradiation. There was no significant difference between the OAR percentage values of 3% and 5% gelatin formulations after 72 h of UVA exposure (90 and 94, respectively; Duncan test, P < 0.05, df = 6). The OAR percentage for the gelatin microencapsulated formulation was 90 after 30 minutes of exposure to UVC radiation. However, for the non-microencapsulated virus suspension, the OAR percentage value declined sharply to 16 after 30 minutes of exposure to UVC radiation. Concerning the in vitro release behaviour of gelatin microparticles (MPs), virus release initiated quickly, but continued at a slower rate until it reached 100% after 1 h of exposure to the release media. The experimental data for the gelatin MPs showed good correlations with the Korsmeyer–Peppas semi-empirical model, indicating that the transport mechanism is primarily consistent with Fickian diffusion.