Antibodies to Toxoplasma gondii in Eurasian badgers

Antibodies to Toxoplasma gondii were detected in samples collected from 90 live-trapped adult Eurasian badgers (Meles meles) sampled at three sites (two agricultural and one woodland) in southern England. Serum was tested using a qualitative latex agglutination test procedure and 63 of 90 (70%) badgers tested positive for T. gondii antibodies. Antibody prevalence varied between the sites; 67% and 77% of badgers from agricultural sites and 39% from a nonagricultural site tested positive.     

Insulin degrading enzyme is a cellular receptor mediating varicella-zoster virus infection and cell-to-cell spread

Varicella-zoster virus (VZV) causes chickenpox and shingles. While varicella is likely spread as cell-free virus to susceptible hosts, the virus is transmitted by cell-to-cell spread in the body and in vitro. Since VZV glycoprotein E (gE) is essential for virus infection, we postulated that gE binds to a cellular receptor. We found that insulin-degrading enzyme (IDE) interacts with gE through its extracellular domain. Downregulation of IDE by siRNA, or blocking of IDE with antibody, with soluble IDE protein extracted from liver, or with bacitracin inhibited VZV infection. Cell-to-cell spread of virus was also impaired by blocking IDE. Transfection of cell lines impaired for VZV infection with a plasmid expressing human IDE resulted in increased entry and enhanced infection with cell-free and cell-associated virus. These studies indicate that IDE is a cellular receptor for both cell-free and cell-associated VZV.     

Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro

Human embryonic stem cells (hESCs) have enormous potential as a source of cells for cell replacement therapies and as a model for early human development. In this study we examined the differentiating potential of hESCs into hepatocytes in two- and three-dimensional (2D and 3D) culture systems. Embryoid bodies (EBs) were inserted into a collagen scaffold 3D culture system or cultured on collagen-coated dishes and stimulated with exogenous growth factors to induce hepatic histogenesis. Immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK-18). The differentiated cells in 2D and 3D culture system displayed several characteristics of hepatocytes, including expression of transthyretin, alpha-1-antitrypsin, cytokeratin 8, 18, 19, tryptophan-2,3-dioxygenase, tyrosine aminotransferase, glucose-6-phosphatase (G6P), cytochrome P450 subunits 7a1 and secretion of alpha-fetoprotein (AFP) and ALB and production of urea. In 3D culture, ALB and G6P were detected earlier and higher levels of urea and AFP were produced, when compared with 2D culture. Electron microscopy of differentiated hESCs showed hepatocyte-like ultrastructure, including glycogon granules, well-developed Golgi apparatuses, rough and smooth endoplasmic reticuli and intercellular canaliculi. The differentiation of hESCs into hepatocyte-like cells within 3D collagen scaffolds containing exogenous growth factors, gives rise to cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes and may provide a source of differentiated cells for treatment of liver diseases.     

The plasticins: membrane adsorption, lipid disorders, and biological activity

The present study investigates the relationships between structural polymorphism, adsorption onto membrane mimetic support, lipid disturbance, and biological activity of bactericidal 23-residue, glycine-leucine-rich dermaseptin orthologues from the Phyllomedusinae frog skin, the "plasticins". Biological activities were evaluated using the membrane models DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) for prokaryotic membranes and DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine) for eukaryotic membranes. We performed a conformational analysis of plasticins by molecular simulations and spectroscopic methods and analyzed phospholipid perturbations by infrared spectroscopy. Adsorption onto synthetic model membranes was quantified by surface plasmon resonance. Biological assays including antimicrobial and membrane potential-dissipating activities, together with hemolytic tests and imaging analysis of cytotoxicity, were carried out to clarify the peptide-membrane interactions. Two major groups were distinguished: (i) Neutral plasticins revealed the presence of strong beta-structures with the zwitterionic or anionic phospholipid vesicles. They were weakly adsorbed in the range of antibacterial activity concentrations (micromolar). Nevertheless, for millimolar concentrations, they caused perturbations at the interface peptide-DMPG vesicles and in the bilayer alkyl chains, suggesting insertion into bacterial membranes. (ii) Cationic plasticins revealed multiple conformational transitions, including destabilized helix states, beta-structures, and disordered states. Peptide-lipid complex densities depended on hydrophobic bond strengths. The most soluble cationic plasticins were strongly adsorbed, with stable peptide-lipid interactions inducing noticeable perturbations of bilayer alkyl chains, pointing out possible insertion into bacterial membranes. In contrast, cytotoxic plasticins were less adsorbed, with less stable peptide-lipid interactions causing membrane dehydration, formation of peptide-membrane hydrogen bonds, and little disturbances of lipid alkyl chains. These characteristics could be compatible with their putative action on intracellular targets leading to apoptosis.     

Protein refolding assisted by molecular tube based α-cyclodextrin as an artificial chaperone

In this study, we evaluated, for the first time, the application of molecular tube based alpha-cyclodextrin for improving the refolding yields of two different enzymes: carbonic anhydrase and alkaline phosphatase. Our results indicate that under the optimal developed refolding environments, the denatured carbonic anhydrase and alkaline phosphatase were refolded with a yield of 51 and 61% using 15 and 5 mg/ml of the molecular tube, respectively. Regardless of lower refolding yields compared with liquid-phase artificial chaperone assisted approach, the new technique (solid-phase artificial chaperone assisted refolding) benefits from easier and faster separation of the refolded product from the refolding environment, recycling of the stripping agent, and finally, significantly less environmental effect at the industrial levels. However, further improvements in solid-phase artificial chaperone assisted technique are needed either through synthesizing better stripping agents or by optimizing and defining better refolding environments.     

Epithelial-mesenchymal transition occurs after epidermal development in mouse skin

In the present study, we studied epithelial-mesenchymal transition (EMT) with fetal and postnatal serial skin sections. E-cadherin, occludin and zonula occludens 1 (ZO-1)-expressing cells appear in the dermal area from E18.5 to postnatal day 9 (P9), with highest expression from P2 to P5. The co-expression of mesenchymal marker alpha-smooth muscle (alpha-SMA), fibronectin and vimentin with E-cadherin in these dermal cells was further examined. Almost no dermal cells express alpha-SMA before P0. From P2 to P6, cells expressing both E-cadherin and alpha-SMA appear in the dermis. In contrast, fibronectin-releasing cells were detected in the dermis as early as on E15.5, although on P5, some dermal cells was found weakly expressing both fibronectin and E-cadherin, most cells strongly expressing fibronectin did not express E-cadherin. Vimentin was mainly expressed in both endothelial and blood-derived cells and did not show co-expression with E-cadherin. Confocal microscopy studies further found that during EMT, E-cadherin appears intracellularly, while the expression of alpha-SMA starts from the membrane area and moves to the cytosol of the cells. Our data are the first in vivo evidence that EMT occurs during mouse skin development. Dermal cells are derived from EMT and other origins, including blood, during skin development.     

H2AX prevents DNA breaks from progressing to chromosome breaks and translocations

Histone H2AX promotes DNA double-strand break (DSB) repair and immunoglobulin heavy chain (IgH) class switch recombination (CSR) in B-lymphocytes. CSR requires activation-induced cytidine deaminase (AID) and involves joining of DSB intermediates by end joining. We find that AID-dependent IgH locus chromosome breaks occur at high frequency in primary H2AX-deficient B cells activated for CSR and that a substantial proportion of these breaks participate in chromosomal translocations. Moreover, activated B cells deficient for ATM, 53BP1, or MDC1, which interact with H2AX during the DSB response, show similarly increased IgH locus breaks and translocations. Thus, our findings implicate a general role for these factors in promoting end joining and thereby preventing DSBs from progressing into chromosomal breaks and translocations. As cellular p53 status does not markedly influence the frequency of such events, our results also have implications for how p53 and the DSB response machinery cooperate to suppress generation of lymphomas with oncogenic translocations.     

Antimicrobial bioassay of Colchicum luteum Baker

The methanolic extract of the corms of Colchicum luteum Baker (Liliaceae) and its subsequent fractions in different solvent systems were screened for antibacterial and antifungal activities. The crude extract and all the fractions demonstrated moderate to excellent antifungal activities against tested pathogens in antifungal bioassay. Excellent antifungal activity was shown against trichophyton longifusus, up to 75%, and microsporum canis, up to 85%, while the crude extract and subsequent fractions showed mild to moderate activities in an antibacterial bioassay with maximum antibacterial activity 58% against Bacillus subtilis.