Scanning Electron Microscope (SEM) is a powerful research tool, but since it requires high vacuum conditions, the wet materials and biological samples must undergo a complex preparation that limits the application of SEM on this kind of specimen and often causes the introduction of artifacts. The introduction of Environmental Scanning Electron Microscope (ESEM), working in gaseous atmosphere, represented a new perspective in biological research. Despite the fact that many biological applications have demonstrated the convenience of ESEM, the full potentialities of this technology are still under investigation. In this review, the exploration of the recent literature data confronted with the first results obtained in our experimental work suggest that ESEM represents an important extension of conventional scanning microscopy.

The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a β-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape.IMPORTANCE Hepatitis C virus (HCV) remains a major health care burden, affecting almost 3% of the global population. The conserved epitope comprising residues 412 to 423 of the viral E2 glycoprotein is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a β-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit anti-HCV antibodies. MAbs that specifically recognize a cyclic variant of the epitope bind to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such MAb and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing MAbs in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines.

Recent studies, on cells cultured in 3D collagen gels, have shown that, beside from their well known biochemical role, fibronectin (FN) and laminin (LM) affect cell functions via a modification of mechanical and structural properties of matrix due to interaction with collagen molecules. Though biochemical properties of FN and LM have been widely studied, little is known about their role in collagen matrix assembly. The aim of this work was to characterize FN- and LM-based collagen semi-interpenetrating polymer networks (semi-IPNs), in order to understand how these biomacromolecular species can affect collagen network assembly and properties. Morphology, viscoelasticity and diffusivity of collagen gels and FN- and LM-based collagen semi-IPNs were analysed by Confocal Laser Scanning microscopy (CLSM), Environmental Scanning Electron microscopy (ESEM), Transmission Electron microscopy (TEM), Rheometry and Fluorescence Recovery After Photobleaching (FRAP) techniques. It was found that FN and LM were organized in aggregates, interspersed in collagen gel, and in thin fibrils, distributed along collagen fibres. In addition, high FN and LM concentrations affected collagen fibre assembly and structure and induced drastic effects on rheological and transport properties.

Abstract. This paper provides an analysis of genetic\ud variability in local populations of the clonal\ud moss Pleurochaete squarrosa, a Mediterranean\ud moss typical of post-fire recovery, and characterised\ud by asexual reproduction. ISSR (Internal\ud Simple Sequence Repeats) primers and trnLUAA\ud (intron of plastid gene for Leu tRNA) length\ud polymorphism were employed to evaluate genetic\ud structure in five southern Italy populations of this\ud moss. Both molecular tools highlight high values\ud of genetic diversity with geographic structure of\ud sampled populations and a low gene flow among\ud the investigated sites. Gene diversity is significant\ud at every hierarchical level of sampling, and generally\ud increases from lower to higher hierarchical\ud levels. Among other factors, the ubiquitous genetic\ud variability detected in P. squarrosa can be\ud related to continuous, occasionally massive, shortrange\ud recruitment of propagules, and to the high\ud degree of intermingling, both favoured by the\ud modality of vegetative reproduction and growth\ud occurring in the species

We used combined plasma-deposition process to deposit smooth and nanostructured fluorocarbon coatings on polyethylenethereftalate (PET) substrates, to obtain surfaces with identical chemical composition and different roughness, and investigate the effect of surface nanostructures on adhesion and proliferation of 3T3 Swiss Albino Mouse fibroblasts. Untreated PET and polystyrene (PS) were used as controls for cell culture. We have found that the statistically significant increase of cell proliferation rate and FAK (a nonreceptor tyrosine kinase) activation detected on ROUGH fluorocarbon surfaces is due to the presence of nanostructures. Changes in cytoskeletal organization and phospho FAK (tyr 397) localization were evident after 60 min on cells adhering to ROUGH surfaces. This change was characterized by the formation of actin stress fibers along lamellar membrane protrusion instead of usual focal contacts. Also the morphology of the adhering fibroblasts (60 min) adhering on ROUGH surfaces was found quite different compared to cells adhering on smooth ones.

Bryophytes seem particularly suitable to investigate genetic diversity in relation to habitat disturbance due to their large employment as bioindicators and to the recent application of molecular markers to moss population studies. Genetic variation and structure were analysed in seven urban, extraurban and remote populations of Leptodon smithii, an epiphytic moss of Quercus ilex, a phanerogamic species of Mediterranean climax vegetation. A total of 210 individual shoots were DNA extracted and amplified with internal simple sequence repeat (ISSR) primers, and 54 haplotypes were identified. An uneven distribution of haplotype number and frequencies was observed among sites, with a higher number of haplotypes and more homogeneous haplotype frequencies in the extraurban/remote populations. Molecular diversity indices were overall higher in the extraurban sites than in the urban ones. Multilocus linkage disequilibrium values were in line with the occurrence of sexual/asexual reproduction in the seven populations. The isolation-by-distance model was not supported by Mantel test among sites; however, within-population fixation index (F(ST)) highlighted a clear relation between genetic and physic distances among trees, suggesting a limited dispersal range for L. smithii's spores. The genetic structure was mainly affected by population size, wood structure and extent, and genetic drift consequent to habitat fragmentation and human-induced disturbance.

In this work we used a combination of immunogold labelling (IGL) and environmental scanning electron microscopy (ESEM) to detect the presence of a protein on the cell surface. To achieve this purpose we chose as experimental system 3T3 Swiss Albino Mouse Fibroblasts and galectin-3. This protein, whose sub-cellular distribution is still under discussion, is involved in a large number of cell physiological and pathological processes. IGL technique has been utilised by many authors in combination with SEM and TEM to obtain the identification/localisation of receptors and antigens, both in cells and tissues. ESEM represents an important tool in biomedical research, since it does not require any severe processing of the sample, lowering the risk of generating artefacts and interfere with IGL procedure. The absence of metal coating could yield further advantages for our purpose as the labelling detection is based on the atomic number difference between Nanogold spheres and the biological material. Using the gaseous secondary electron detector (GSED) compositional contrast is easily revealed by the backscattered electrons component of the signal. In spite of this fact, only few published papers present a combination of ESEM and IGL. Hereby we present our method, optimised to improve the intensity and the specificity of the labelling signal, in order to obtain a semi-quantitative evaluation of the labelling signal.

An aqueous extract of wild rocket (Diplotaxis tenuifolia DC) was tested for its allelopathic activity in vitro on radish germination and seedling growth in light and darkness. The extract caused a delay in the onset and a significant decrease in the rate of germination (40%) in the light. The photo-inhibition was accompanied by an inhibition of water uptake into the seed, and a decrease of protein content as well as an increase of peroxidase activity into the seedlings. Microscopic observations suggest that the extract markedly changes radish radicle development inducing a decreased imbibition and distension of seed cells. Consistent results were obtained with some species such as purslane, lambsquarter and tree of heaven present in the cultivated wild rocket field and with cultivated lettuce and barley. Finally, a potential allelochemical, biologically active, was isolated from the extract: S -glucopyranosyl thiohydroximate at concentration of 6.3 )/10 (4 M.