Highlights from the 5th Annual Meeting of the Italian Society of Virology

The 5th National Congress of the Italian Society of Virology (SIV) was attended by junior- and senior-level virologists to promote interactions and scientific collaborations among the different areas of Virology and allied sciences. The invited and selected lecturers covered the following topics: General Virology and Viral Genetics; Virus-host Interaction and Pathogenesis; Viral Oncogenesis; Viral Immunology and Vaccines; Anti-viral Therapy; Innovative Diagnostics; Viral Biotechnologies and Cell and Gene Therapy. As in the previous editions (Salata and Palù, 2004; Salata et al., 2005), a specific topic was thoroughly covered in a roundtable. This year the elected subject was "HIV: determinants of pathogenicity and clinical implications." The final program and the abstract book can be found at the web site http://www.siv-virologia.it. This report summarizes the lessons learned from the plenary lectures and the selected oral presentations of the 2005 meeting.     

Activation of smooth muscle and myenteric plexus cells of jejunum via Toll-like receptor 4

The cell types of the gut expressing Toll-like receptor 4, which recognizes specifically bacterial lipopolysaccharides, as well as the functionality of this receptor, have remained controversial. We aimed to clarify these issues. Mouse and human intestinal specimens were stained immunohistochemically to detect Toll-like receptor 4 expression. Smooth muscle and myenteric plexus cells but not enterocytes revealed receptor expression. Murine intestinal smooth muscle and myenteric plexus cells but not enterocytes showed nuclear translocation of nuclear factor-kappaB after in vivo stimulation with lipopolysaccharide. Moreover, lipopolysaccharide added to human jejunum biopsies free of epithelial cells induced release of interleukin-8 (IL-8). We can conclude that Toll-like receptor 4 is not expressed in epithelial layer, but rather on smooth muscle and myenteric plexus cells and that expression is functional. The expression of Toll-like receptor 4 on smooth muscle and myenteric plexus cells is consistent with the possibility that these cells are involved in intestinal immune defense; the low or absent expression of Toll-like receptor 4 on enterocytes might explain the intestinal epithelium hyporesponsiveness to the abundance of LPS in the intestinal lumen.     

Degradation of Low-Ethoxylated Nonylphenols by a Stenotrophomonas Strain and Development of New Phylogenetic Probes for Stenotrophomonas spp. Detection

An aerobic bacterium (BCc6), isolated from nonylphenol polyethoxylates (NPEOs)-contaminated sludge, was shown to be capable of degrading low-ethoxylated NPEO mixtures. Sequencing of 16S rRNA gene (rDNA) showed that it clustered with Stenotrophomonas nitritireducens. Fluorescent in situ hybridization (FISH), performed on BCc6 strain and on the previously isolated Stenotrophomonas BCaL2, also involved in NPEO degradation but clustering with S. maltophilia, showed that strain BCc6 did not hybridize with the S. maltophilia-specific probe, and neither of the two strains hybridized with probes targeted to the Gammaproteobacteria site, rDNA analyses performed on the two strains evidenced two new polymorphisms, the first one at the 23S rRNA Gammaproteobacteria site, characterizing the known members of the Stenotrophomonas genus, and the other one at the 16S rRNA level, characteristic of S. nitritireducens. Two new FISH probes were designed accordingly, tested on control bacterial cultures, and employed for in situ monitoring of Stenotrophomonas representatives.     

Molecular characterization of a glycerophosphoinositol transporter in mammalian cells

The glycerophosphoinositols are ubiquitous phosphoinositide metabolites involved in the control of several cell functions. They exert their actions both intracellularly and by rapidly equilibrating across the plasma membrane when added to cells, implying the existence of a transporter for their membrane permeation. Such a transporter, GIT1, has been cloned in yeast. By PSI-BLAST analysis, we have identified the Glut2 transporter as a human-genome candidate ortholog of GIT1. This was supported directly through the use of inhibitors, siRNAs and competition studies of specific uptake of GroPIns in HeLa cells over-expressing human Glut2. These data identify Glut2 as a GroPIns transporter in mammals, and define a physiologically relevant cell-permeation mechanism.     

Structural insights into the interaction between prion protein and nucleic acid

The infectious agent of transmissible spongiform encephalopathies (TSE) is believed to comprise, at least in part, the prion protein (PrP). Other molecules can modulate the conversion of the normal PrP(C) into the pathological conformer (PrP(Sc)), but the identity and mechanisms of action of the key physiological factors remain unclear. PrP can bind to nucleic acids with relatively high affinity. Here, we report small-angle X-ray scattering (SAXS) and nuclear magnetic resonance spectroscopy measurements of the tight complex of PrP with an 18 bp DNA sequence. This double-stranded DNA sequence (E2DBS) binds with nanomolar affinity to the full-length recombinant mouse PrP. The SAXS data show that formation of the rPrP-DNA complex leads to larger values of the maximum dimension and radius of gyration. In addition, the SAXS studies reveal that the globular domain of PrP participates importantly in the formation of the complex. The changes in NMR HSQC spectra were clustered in two major regions: one in the disordered portion of the PrP and the other in the globular domain. Although interaction is mediated mainly through the PrP globular domain, the unstructured region is also recruited to the complex. This visualization of the complex provides insight into how oligonucleotides bind to PrP and opens new avenues to the design of compounds against prion diseases.     

Molten globule formation in apomyoglobin monitored by the fluorescent probe Nile Red

The interaction of nile red (NR) with apomyoglobin (ApoMb) in the native (pH 7) and molten globule (pH 4) states was investigated using experimental and computational methods. NR binds to hydrophobic locations in ApoMb with higher affinity (K(d) = 25 +/- 5 microM) in the native state than in the molten globule state (K(d) = 52 +/- 5 microM). In the molten globule state, NR is located in a more hydrophobic environment. The dye does not bind to the holoprotein, suggesting that the binding site is located at the heme pocket. In addition to monitoring steady-state properties, the fluorescence emission of NR is capable of tracking submillisecond, time-resolved structural rearrangements of the protein, induced by a nanosecond pH jump. Molecular dynamics simulations were run on ApoMb at neutral pH and at pH 4. The structure obtained for the molten globule state is consistent with the experimentally available structural data. The docking of NR with the crystal structure shows that the ligand binds into the binding pocket of the heme group, with an orientation bringing the planar ring system of NR to overlap with the position of two of the heme porphyrin rings in Mb. The docking of NR with the ApoMb structure at pH 4 shows that the dye binds to the heme pocket with a slightly less favorable binding energy, in keeping with the experimental K(d) value. Under these conditions, NR is positioned in a different orientation, reaching a more hydrophobic environment in agreement with the spectroscopic data.     

Low resolution structure and stability studies of human GrpE#2, a mitochondrial nucleotide exchange factor

GrpE acts as a nucleotide exchange factor for the Hsp70 chaperone system. Only one GrpE isoform is present in Escherichia coli, but for reasons not yet well understood, two GrpE isoforms have been found in mammalian mitochondria.Therefore, studies aimed at evaluating the physico-chemical characteristics of these proteins are important for the comprehension of the function of the Hsp70 chaperone system in different organisms. Here we report biophysical studies on human mitochondrial GrpE isoform 2. Small angle X-ray scattering measurements of human GrpE isoform 2 showed that this protein has a quaternary structure which is similar to those of human GrpE isoform 1 and E. coli GrpE: a dimer with a cruciform elongated shape. However, mitochondrial isoforms differed from each other regarding chemical and thermal denaturation profiles. This fact, combined with results of distinct expression patterns previously reported, point out that these proteins may have different response to external stimuli. Our results also indicate that human GrpE isoform 2 is more similar to the GrpE from E. coli than to human GrpE isoform 1. These results are relevant because differences in the conformation of Hsp70 co-chaperones are considered to be one of the reasons for functional diversity of this system.     

Impact of preanalytical handling and timing for peripheral blood mononuclear cells isolation and RNA studies: the experience of the Interinstitutional Multidisciplinary BioBank (BioBIM)

Multicenter studies and biobanking projects require blood transportation from the participating center to a central collection or diagnostic laboratory. The impact of time delays between venous blood collection and peripheral blood mononuclear cells (PBMC) isolation prior to RNA extraction may affect the quality and quantity of isolated nucleic acids for genomic applications. Thus, standard operating procedure (SOP) optimization for the treatment of biological samples before RNA extraction is crucial in a biological repository. In order to define SOPs for whole blood preservation prior to RNA extraction, we sought to determine whether different blood storage times (0, 3, 6, 10, 24, and 30 hours) prior to PBMCs isolation and storage at -80°C, could affect the quality and quantity of extracted RNA. After spectrophotometric quantification, the quality and integrity of RNA were assessed by agarose gel electrophoresis, RNA integrity number and real time-PCR (RT-PCR).?Across the different time points we did not observe significant differences within the first 24 hours of blood storage at room temperature, while a significant loss in RNA yield and integrity was detected between 24 and 30 hours. We conclude that time delays before PBMCs isolation prior to RNA extraction may have a significant impact on downstream molecular biological applications.     

Hypoxia modulation of peroxisome proliferator-activated receptors (PPARs) in human glioblastoma stem cells. Implications for therapy

Gliobastoma (GB), the most common adult brain tumor, infiltrates normal brain area rendering impossible the complete surgical resection, resulting in a poor median survival (14–15 months), despite the aggressive multimodality treatments post‐surgery, such as radiation and chemo‐therapy. GB is characterized by hypoxic and necrotic regions due to a poorly organized tumor vascularization, leading to inadequate blood supply and consequently to hypoxic and necrotic areas. We have previously shown that, under hypoxia GB primary cells increased the expression of stemness markers as well as the expression of the nuclear receptor peroxisome proliferator‐activated receptor α (PPARα) and also the crucial role played by PPARs in mouse neural stem cells maintenance and differentiation. Due to the importance of lipid signaling in cell proliferation and differentiation, in this work, we analyzed the expression of PPARs in GB neurospheres both in normoxic and hypoxic conditions. The results obtained suggest a differential regulation of the three PPARs by hypoxia, thus indicating a possible therapeutic strategy to counteract GB recurrencies. J. Cell. Biochem. 113: 3342–3352, 2012. © 2012 Wiley Periodicals, Inc.