ANALISI MORFOLOGICA DELLA MUCOSA ORALE CHERATINIZZATA UMANA NORMALE DOPO ESPOSIZIONE A STIMOLI ESOGENI

Cigarette smoke and alendronate are two different exogenous stimuli involved in the pathogenesis of oral diseases, but their actual role in altering the epithelial barrier and function has not been thoroughly investigated, yet. To evaluate the morphological chronic effect of both agents, biopsies of normal human keratinized oral mucosa are collected respectively from smoking women (n = 5) and from osteoporotic women undergoing chronic oral therapy with alendronate (n = 6). Both groups are compared to age and sex-matched controls. The acute effects of smoke are investigated in a three-dimensional model of human oral mucosa organotypic cultures (n = 5) after exposure to the mainstream smoke coming from one single cigarette. Morphological analysis by light and transmission electron microscopy is performed on all considered samples. Chronic smoke and chronic alendronate treatment affect keratinocyte terminal differentiation and intercellular adhesion impairing desmosomal molecular composition and morphology, in a stress specific and time exposure related manner. Desmoglein 3 and desmoglein 1 distribution are altered respectively after chronic smoke and chronic alendronate treatment. Epithelial proliferation is also impaired in the alendronate treated group. On the contrary, after three hours from cigarette smoke exposure, the first significant response of the oral epithelium comes from the immediately suprabasal keratinocytes, without impairment of the epithelial junctional apparatus and apoptosis induction. The collected data highlight differences in the acute and chronic response of the oral epithelium to cigarette smoke. Moreover, reported results support the crucial signaling role of desmosomal cadherins in the oral epithelium and introduce a new issue in oral biology: the specific response of human oral mucosa to different physico-chemical stresses

Knowledge, attitudes, and practices of influenza and pneumococcal vaccines among agricultural workers: Results of an Italian a cross-sectional study

Background: Working age is increasing across Europe. Seasonal influenza (SID) and pneumococcal disease (PND) immunization programmes might be successfully implemented at the workplace. We conducted a cross-sectional survey among to assess SID and PND vaccine status, as well as knowledge, attitudes and practices (KAP) in a representative sample of agricultural workers (AWs) aged ≥55 years in North-Eastern Italy. Methods: A structured questionnaire was administered in person by trained personnel. Bivariate and multivariate logistic regression analyses were carried out to identify behavioral and work-related factors associated with SID and PND vaccine uptake. Results: Among 707 participants, 238 were aged 55 years or more (33.7% of total). Of them, 39.1% had an up-to-date immunization status towards influenza, and 17.6% towards pneumococcus. Factors associated with inadequate immunization were doubts about influenza vaccine safety (40.0%) and the confidence in natural immunity towards pneumococcus (30.8%). Attitude towards vaccinations was somehow favorable in 44.5% of participants for SID, and 37.8% for PND. Overall, 37.4% and 21.8% workers were aware of national recommendations on SID and PND immunization, respectively. This factor was characterized as a significant predictor for SID vaccination (multivariated Odds Ratio, OR 32.688 95%CI 12.015-88.930), as well as the perception of SID as a severe disease (OR 7.539 95%CI 3.312-17.164), and the perceived value of preventing new infections (OR 3.215 95%CI 1.205-8.578). A somehow favorable attitude towards vaccinations was the main predictor (OR 39.214 95%CI 10.179-151.1) for PND vaccination. Conclusions: Our study indicates that older workers lack appropriate knowledge of national recommendations and correct risk perception of SID and PND infections, but also vaccines’ side effects. As the latter has been recognized as predictive factor for SID vaccination, our results stress the importance for tailored informative interventions in the workplaces aimed to increase risk perception and vaccine acceptance. (www.actabiomedica.it)

Molecular dissection of translation initiation factor IF2. Evidence for two structural and functional domains.

By means of limited proteolysis of Bacillus stearothermophilus initiation factor IF2 and genetic manipulation of its structural gene, infB, we have been able to produce (or hyperproduce) and purify two polypeptide fragments corresponding to two structurally and functionally separate domains of the protein. The first is the G-domain (approximately 41 kDa), which makes up the central part of the molecule and contains the conserved structural elements found in all GTP/GDP-binding sites of G-proteins. This domain is resistant to proteolysis in the presence of GTP or GDP, retains the capacity to interact with the 50 S subunit, binds weakly to the 30 S subunit, and displays ribosome-dependent GTPase activity with an approximately 2-fold higher Km for GTP and the same Vmax as compared with intact IF2. The second is the C-domain (approximately 24 kDa), which corresponds to the COOH-terminal part of IF2 and constitutes an extraordinarily compact domain containing the fMet-tRNA binding site of IF2. In spite of its negligible affinity for the ribosomes, the C-domain weakly stimulates the ribosomal binding of fMet-tRNA, presumably by affecting the conformation of the initiator tRNA molecule

Sofosbuvir-based therapies in genotype 2 hepatitis C virus cirrhosis: A real-life experience with focus on ribavirin dose

This study aimed to investigate the efficacy and safety of sofosbuvir-based therapies for the treatment of cirrhosis from hepatitis C virus (HCV) genotype 2 infection. Data of all consecutive HCV genotype 2 cirrhotic patients who started sofosbuvir-based treatments between January 2015 and March 2017 in eight Italian tertiary hospitals were collected retrospectively. Overall, 273 patients (Child A: 94.5%) were enrolled. In the 194 subjects treated with sofosbuvir/ribavirin, median initial ribavirin dosage was 13.9 mg/kg/day, and therapy duration was 16 weeks. Sustained virological response (SVR) rates were 93.8% in intention-to-treat (ITT) and 95.3% in per-protocol (PP) analyses for the 129 treatment-naïve patients, and 96.9% (ITT) and 98.4% (PP) for the 65 treatment-experienced subjects. Adverse events were reported in 142 patients (73.2%), but only 1.5% discontinued treatment. Eighty-eight subjects with treatment-induced anemia (mild: 34.5%, moderate: 7.7%, severe: 3.1%) had to reduce ribavirin dosage, but SVR rates were comparable to the weight-based dose group, both in ITT (95.4% and 94.3%) and PP (97.7% and 95.2%) analyses, respectively. Moreover, ITT and PP SVR rates were similar between shorter (<20 weeks) (94.1% and 96.0%, respectively) and prolonged (≥20 weeks) regimens (95.7% and 96.7%, respectively). SVR rates in the 79 subjects treated with sofosbuvir/daclatasvir (without ribavirin) were similar (ITT: 96.2%; PP: 97.4%, respectively), without de novo/worsening anemia. In conclusion, in a real-life study centered on genotype 2 patients with well-compensated cirrhosis, sofosbuvir-based regimens were associated with good SVR and tolerability rates, regardless of previous antiviral treatments, without a significant impact of on treatment ribavirin dose reductions

Molecular dissection of translation initiation factor IF2. Evidence for two structural and functional domains.

By means of limited proteolysis of Bacillus stearothermophilus initiation factor IF2 and genetic manipulation of its structural gene, infB, we have been able to produce (or hyperproduce) and purify two polypeptide fragments corresponding to two structurally and functionally separate domains of the protein. The first is the G-domain (approximately 41 kDa), which makes up the central part of the molecule and contains the conserved structural elements found in all GTP/GDP-binding sites of G-proteins. This domain is resistant to proteolysis in the presence of GTP or GDP, retains the capacity to interact with the 50 S subunit, binds weakly to the 30 S subunit, and displays ribosome-dependent GTPase activity with an approximately 2-fold higher Km for GTP and the same Vmax as compared with intact IF2. The second is the C-domain (approximately 24 kDa), which corresponds to the COOH-terminal part of IF2 and constitutes an extraordinarily compact domain containing the fMet-tRNA binding site of IF2. In spite of its negligible affinity for the ribosomes, the C-domain weakly stimulates the ribosomal binding of fMet-tRNA, presumably by affecting the conformation of the initiator tRNA molecule

Identification of a cold shock transcriptional enhancer of the Escherichia coli gene encoding nucleoid protein H-NS

The hns (27 min) gene encoding the 15.4-kDa nucleoid protein H-NS was shown to belong to the cold shock regulon of Escherichia coli, its expression being enhanced 3- to 4-fold during the growth lag that follows a shift from 37 degrees C to 10 degrees C. A 110-base-pair (bp) DNA fragment containing the promoter of hns fused to a promoterless cat gene (hns-cat fusion) conferred a similar cold shock response to the expression of chloramphenicol acetyltransferase (CAT) activity in vivo and in coupled transcription-translation systems prepared with extracts of cold-shocked cells. Extracts of the same cells produce a specific gel shift of the 110-bp DNA fragment and this fragment, immobilized on a solid support, specifically retains a single 7-kDa protein present only in cold-shocked cells that was found to be identical to F10.6 (CS7.4), the product of cspA. This purified protein, which is homologous to human DNA-binding protein YB-1, recognizes some feature of the 110-bp promoter region of hns and acts as a cold shock transcriptional activator of this gene since it stimulates the expression of CAT activity and of cat transcription in in vitro systems programmed with plasmid DNA carrying the hns-cat fusion

Site-directed mutagenesis and NMR spectroscopic approaches to the elucidation of the structure-function relationships in translation initiation factors IF1 and IF3.

The recent developments in the knowledge of the structure and structure-function relationships of prokaryotic initiation factors IF1 and IF3 obtained in our laboratory by site-directed mutagenesis, biochemical and NMR-spectroscopic approaches are discussed

The Antibiotics Dityromycin and GE82832 Bind Protein S12 and Block EF-G-Catalyzed Translocation

SummaryThe translocation of mRNA and tRNA through the ribosome is catalyzed by elongation factor G (EF-G), a universally conserved guanosine triphosphate hydrolase (GTPase). The mechanism by which the closely related decapeptide antibiotics dityromycin and GE82832 inhibit EF-G-catalyzed translocation is elucidated in this study. Using crystallographic and biochemical experiments, we demonstrate that these antibiotics bind to ribosomal protein S12 in solution alone as well as within the small ribosomal subunit, inducing long-range effects on the ribosomal head. The crystal structure of the antibiotic in complex with the 70S ribosome reveals that the binding involves conserved amino acid residues of S12 whose mutations result in in vitro and in vivo antibiotic resistance and loss of antibiotic binding. The data also suggest that GE82832/dityromycin inhibits EF-G-catalyzed translocation by disrupting a critical contact between EF-G and S12 that is required to stabilize the posttranslocational conformation of EF-G, thereby preventing the ribosome-EF-G complex from entering a conformation productive for translocation