Silent mutations at the 5'-end of the cDNA of actinoporins from the sea anemone Stichodactyla helianthus allow their heterologous overproduction in Escherichia coli

Wild-type actinoporins StnI and StnII from the sea anemone Stichodactyla helianthus, as well as their NH(2)-terminal six-His tagged versions, have been overproduced in Escherichia coli. Overproduction of both wild-type proteins was only possible after introducing silent mutations within the 5'-end of their original cDNA sequences. These mutations would prevent the formation of RNA secondary structures blocking the ribosome-binding site and the initiation codon. The four recombinant proteins were purified to homogeneity in milligrams amount and characterized from spectroscopic and functional points of view. All the isolated proteins behaved as the corresponding natural ones although the six-His tagged variants exhibited a decreased lytic activity. The strategy described will be useful to allow the production of mutant variants of these proteins and probably of other actinoporins.     

Development of an Analytical Method for the Metaproteomic Investigation of Bioaerosol from Work Environments

The metaproteomic analysis of air particulate matter provides valuable information about the properties of bioaerosols in the atmosphere and their influence on climate and public health. In this work, a new method for the extraction and analysis of proteins in airborne particulate matter from quartz microfiber filters is developed. Different protein extraction procedures are tested to select the best extraction protocol based on protein recovery. The optimized method is tested for the extraction of proteins from spores of ubiquitous bacteria species and used for the metaproteomic characterization of filters from three work environments. In particular, ambient aerosol samples are collected in a composting plant, in a wastewater treatment plant, and in an agricultural holding. A total of 179, 15, 205, and 444 proteins are identified in composting plant, wastewater treatment plant, and agricultural holding, (cow stable and blending plant), respectively. In agreement with the major categories of primary biological aerosol particles, all identified proteins originated primarily from fungi, bacteria, and plants. The paper is the first metaproteomic study applied to bioaerosol samples collected in occupationally relevant environmental sites and, even though not aimed at monitoring the risk exposure of workers, it provides information on the possible exposure in the working environmental sites.     

Origin, diffusion, and differentiation of Y-chromosome haplogroups E and J: inferences on the neolithization of Europe and later migratory events in the Mediterranean area

The phylogeography of Y-chromosome haplogroups E (Hg E) and J (Hg J) was investigated in >2400 subjects from 29 populations, mainly from Europe and the Mediterranean area but also from Africa and Asia. The observed 501 Hg E and 445 Hg J samples were subtyped using 36 binary markers and eight microsatellite loci. Spatial patterns reveal that (1). the two sister clades, J-M267 and J-M172, are distributed differentially within the Near East, North Africa, and Europe; (2). J-M267 was spread by two temporally distinct migratory episodes, the most recent one probably associated with the diffusion of Arab people; (3). E-M81 is typical of Berbers, and its presence in Iberia and Sicily is due to recent gene flow from North Africa; (4). J-M172(xM12) distribution is consistent with a Levantine/Anatolian dispersal route to southeastern Europe and may reflect the spread of Anatolian farmers; and (5). E-M78 (for which microsatellite data suggest an eastern African origin) and, to a lesser extent, J-M12(M102) lineages would trace the subsequent diffusion of people from the southern Balkans to the west. A 7%-22% contribution of Y chromosomes from Greece to southern Italy was estimated by admixture analysis.     

Generation of functional neutrophils from a mouse model of X-linked chronic granulomatous disorder using induced pluripotent stem cells

Murine models of human genetic disorders provide a valuable tool for investigating the scope for application of induced pluripotent stem cells (iPSC). Here we present a proof-of-concept study to demonstrate generation of iPSC from a mouse model of X-linked chronic granulomatous disease (X-CGD), and their successful differentiation into haematopoietic progenitors of the myeloid lineage. We further demonstrate that additive gene transfer using lentiviral vectors encoding gp91(phox) is capable of restoring NADPH-oxidase activity in mature neutrophils derived from X-CGD iPSC. In the longer term, correction of iPSC from human patients with CGD has therapeutic potential not only through generation of transplantable haematopoietic stem cells, but also through production of large numbers of autologous functional neutrophils.     

Selectivity to Translational Egomotion in Human Brain Motion Areas

The optic flow generated when a person moves through the environment can be locally decomposed into several basic components, including radial, circular, translational and spiral motion. Since their analysis plays an important part in the visual perception and control of locomotion and posture it is likely that some brain regions in the primate dorsal visual pathway are specialized to distinguish among them. The aim of this study is to explore the sensitivity to different types of egomotion-compatible visual stimulations in the human motion-sensitive regions of the brain. Event-related fMRI experiments, 3D motion and wide-field stimulation, functional localizers and brain mapping methods were used to study the sensitivity of six distinct motion areas (V6, MT, MST+, V3A, CSv and an Intra-Parietal Sulcus motion [IPSmot] region) to different types of optic flow stimuli. Results show that only areas V6, MST+ and IPSmot are specialized in distinguishing among the various types of flow patterns, with a high response for the translational flow which was maximum in V6 and IPSmot and less marked in MST+. Given that during egomotion the translational optic flow conveys differential information about the near and far external objects, areas V6 and IPSmot likely process visual egomotion signals to extract information about the relative distance of objects with respect to the observer. Since area V6 is also involved in distinguishing object-motion from self-motion, it could provide information about location in space of moving and static objects during self-motion, particularly in a dynamically unstable environment.     

GLT-1 down-regulation induced by clozapine in rat frontal cortex is associated with synaptophysin up-regulation

In rat frontal cortex, extracellular levels of glutamate are raised by the anti-psychotic drug clozapine. We have recently shown that a significant reduction in the levels of the glutamate transporter GLT-1 may be one of the mechanisms responsible for this elevation. Here we studied whether GLT-1 down-regulation induced by chronic clozapine treatment is associated with changes in the expression of synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25) and vesicular glutamate transporter 1 (VGLUT1), three major presynaptic proteins involved in neurotransmitter release. Quantitative high-resolution confocal microscopy studies in vivo showed that GLT-1 down-regulation is closely associated with a significant increase in synaptophysin, but not SNAP-25 and VGLUT1, expression. This was confirmed in vitro studies, and in western blotting studies of synaptophysin, SNAP-25 and VGLUT1. In addition, our results show that, following clozapine treatment, synaptophysin expression increases in the very cortical regions in which GLT-1 expression is down-regulated. These findings suggest that part of the effects of clozapine may be exerted via an action on the presynaptic machinery involved in neurotransmitter release.     

Systemic Inflammation in Non-Demented Elderly Human Subjects: Brain Microstructure and Cognition

The purpose of this study was to test the hypothesis that higher levels of systemic inflammation in a community sample of non-demented subjects older than seventy years of age are associated with reduced diffusion anisotropy in brain white matter and lower cognition. Ninety-five older persons without dementia underwent detailed clinical and cognitive evaluation and magnetic resonance imaging, including diffusion tensor imaging. Systemic inflammation was assessed with a composite measure of commonly used circulating inflammatory markers (C-reactive protein and tumor necrosis factor-alpha). Tract-based spatial statistics analyses demonstrated that diffusion anisotropy in the body and isthmus of the corpus callosum was negatively correlated with the composite measure of systemic inflammation, controlling for demographic, clinical and radiologic factors. Visuospatial ability was negatively correlated with systemic inflammation, and diffusion anisotropy in the body and isthmus of the corpus callosum was shown to mediate this association. The findings of the present study suggest that higher levels of systemic inflammation may be associated with lower microstructural integrity in the corpus callosum of non-demented elderly individuals, and this may partially explain the finding of reduced higher-order visual cognition in aging.     

Minimalism in radiation synthesis of biomedical functional nanogels

A scalable, single-step, synthetic approach for the manufacture of biocompatible, functionalized micro- and nanogels is presented. In particular, poly(N-vinyl pyrrolidone)-grafted-(aminopropyl)methacrylamide microgels and nanogels were generated through e-beam irradiation of PVP aqueous solutions in the presence of a primary amino-group-carrying monomer. Particles with different hydrodynamic diameters and surface charge densities were obtained at the variance of the irradiation conditions. Chemical structure was investigated by different spectroscopic techniques. Fluorescent variants were generated through fluorescein isothiocyanate attachment to the primary amino groups grafted to PVP, to both quantify the available functional groups for bioconjugation and follow nanogels localization in cell cultures. Finally, a model protein, bovine serum albumin, was conjugated to the nanogels to demonstrate the attachment of biologically relevant molecules for targeting purposes in drug delivery. The described approach provides a novel strategy to fabricate biohybrid nanogels with a very promising potential in nanomedicine.