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Objective Black stain (BS) is an extrinsic dental discoloration particularly difficult to treat. Although its etiology is not fully clear yet, chromogenic bacteria inside the oral cavity seem to be involved. In this pilot study, we evaluated whether a toothpaste containing enzymes and salivary proteins could improve oral health and reduce the presence of periodontal pathogens in subjects predisposed to BS discoloration. Methods Twenty-six subjects were enrolled in the study: 10 subjects without BS; 16 subjects with BS, randomly assigned in two groups: test (n = 8) and control (n = 8). The test group used a toothpaste containing sodium fluoride, enzymes, and salivary proteins. The control group used a toothpaste with amine fluoride. At enrollment and after 14 weeks, participants were subjected to professional oral hygiene, evaluation of BS (through Shourie index) and oral health status, collection of saliva and dental plaque samples. The presence of periodontal pathogens in plaque and saliva of all subjects was investigated by molecular analysis (PCR). Statistical Analysis The prevalence of investigated microbial species in patients with/without BS was performed by Chi-squared test. The variation in the prevalence of the investigated species after treatment in test and control group was analyzed by t-test. Results Clinical evaluation showed that 86% of participants with BS had a reduction in the Shourie index, independently from the toothpaste used. In particular, a greater reduction in the Shourie index was observed in subjects using an electric toothbrush. We did not observe an effect of the fluoride toothpaste containing enzymes and salivary proteins on the composition of the oral microbiota of the test subjects in comparison with controls. When comparing all subjects with BS (n = 16) and without BS (n = 10), P. gingivalis detection was significantly higher in saliva samples collected from subjects with BS (p = 0.0129). Conclusion We verified that the use of an enzyme-containing toothpaste alone is not sufficient to prevent the formation of BS dental pigmentation in subjects predisposed to this discoloration. Mechanical cleaning, especially using electrical toothbrushes, seems to be useful to counteract BS formation. Moreover, our results suggest a possible association between BS and the presence of P. gingivalis at the salivary level.

The recovery and characterization of a multidrug-resistant, KPC-3-producing Klebsiella michiganensis that was obtained from Venus clam samples is reported in this study. A whole-genome sequencing (WGS) analysis using Illumina and Nanopore technologies of the

Achromobacter spp. can establish occasional or chronic lung infections in patients with cystic fibrosis (CF). Chronic colonization has been associated with worse prognosis highlighting the need to identify markers of bacterial persistence. To this purpose, we analyzed phenotypic features of 95 Achromobacter spp. isolates from 38 patients presenting chronic or occasional infection. Virulence was tested in Galleria mellonella larvae, cytotoxicity was tested in human bronchial epithelial cells, biofilm production in static conditions was measured by crystal violet staining and susceptibility to selected antibiotics was tested by the disk diffusion method. The presence of genetic loci associated to the analyzed phenotypic features was evaluated by a genome-wide association study. Isolates from occasional infection induced significantly higher mortality of G. mellonella larvae and showed a trend for lower cytotoxicity than chronic infection isolates. No significant difference was observed in biofilm production among the two groups. Additionally, antibiotic susceptibility testing showed that isolates from chronically-infected patients were significantly more resistant to sulfonamides and meropenem than occasional isolates. Candidate genetic biomarkers associated with antibiotic resistance or sensitivity were identified. Achromobacter spp. strains isolated from people with chronic and occasional lung infection exhibit different virulence and antibiotic susceptibility features, which could be linked to persistence in CF lungs. This underlines the possibility of identifying predictive biomarkers of persistence that could be useful for clinical purposes.

SARS-CoV-2 serological tests are used to assess the infection seroprevalence within a population. This study aims at assessing potential biases in estimating infection prevalence amongst healthcare workers (HCWs) when different diagnostic criteria are considered. A multi-site cross-sectional study was carried out in April–September 2020 amongst 1.367 Italian HCWs. SARS-CoV-2 prevalence was assessed using three diagnostic criteria: RT-PCR on nasopharyngeal swab, point-of-care fingerprick serological test (POCT) result and COVID-19 clinical pathognomonic presentation. A logistic regression model was used to estimate the probability of POCT-positive result in relation to the time since infection (RT-PCR positivity). Among 1.367 HCWs, 69.2% were working in COVID-19 units. Statistically significant differences in age, role and gender were observed between COVID-19/non-COVID-19 units. Prevalence of SARS-CoV-2 infection varied according to the criterion considered: 6.7% for POCT, 8.1% for RT-PCR, 10.0% for either POCT or RT-PCR, 9.6% for infection pathognomonic clinical presentation and 17.6% when at least one of the previous criteria was present. The probability of POCT-positive result decreased by 1.1% every 10 days from the infection. This study highlights potential biases in estimating SARS-CoV-2 point-prevalence data according to the criteria used. Although informative on infection susceptibility and herd immunity level, POCT serological tests are not the best predictors of previous COVID-19 infections for public health monitoring programmes.

Background: Blood culturing remains the mainstream tool to inform an appropriate treatment in hospital-acquired bloodstream infections and to diagnose any bacteremia. Methods: A retrospective investigation on the prevalence of Gram-negative bacteria (GNB) and their resistance in hospitalized patients by age, sex, and units from blood cultures (BCs) was conducted from January 2018 to April 2020 at Sant’Elia hospital, Caltanissetta, southern Italy. We divided the patient age range into four equal intervals. Results: Multivariate demographic and microbiological variables did not show an association between bacteria distributions and gender and age. The distribution by units showed a higher prevalence of Klebsiella pneumoniae and Acinetobacter baumannii in the intensive care unit (ICU) and Escherichia coli in the non-intensive care units (non-ICUs). The analysis of antibiotic resistance showed that E. coli was susceptible to a large class of antibiotics such as carbapenem and trimethoprim-sulfamethoxazole. K. pneumoniae showed a significant susceptibility to colistin, tigecycline, and trimethoprim-sulfamethoxazole. From the survival analysis, patients with E. coli had a higher survival rate. Conclusions: The authors stress the importance of the implementation of large community-level programs to prevent E. coli bacteremia. K. pneumoniae and E. coli susceptibility patterns to antibiotics, including in the prescription patterns of general practitioners, suggest that the local surveillance and implementation of educational programs remain essential measures to slow down the spread of resistance and, consequently, increase the antibiotic lifespan.

We report a novel IncHI2 plasmid coharboring bla VIM-1 , two copies of bla KPC-3 and mcr-9.1 resistance genes in a human Escherichia coli of the new serogroup O188. The bla VIM-1 gene was included in a class 1 integron, mcr-9.1 in a cassette bracketed by IS 903 and ΔIS1R, and bla KPC-3 in two copies within a new composite Tn 4401 -like transposon. The emergence of carbapenem and colistin resistance genes in a single plasmid is of great concern for upcoming clinical therapies.

Soil salinity is one of the main abiotic stresses affecting yield in major crop plants, including legumes. Research carried out on model legumes such as barrel medic (Medicago truncatula Gaertn.) showed that the Tyrosyl-DNA phosphodiesterase 2α (MtTdp2α) DNA repair gene, involved in the removal of topoisomerase-DNA covalent complexes, play a key role in the plant response to osmotic and copper stresses. However, no informations are currently available about the involvement of MtTdp2α in response to salt stress and salt shock. In the present study we investigated the role of MtTdp2α under salinity (0, 50, 100, 150 and 200 mM NaCl) stress conditions in transgenic M. truncatula overexpressing the MtTdp2α gene. The level of salt tolerance of Tdp2-28 selected transgenic line was significantly higher than control as measured by the increase in shoot fresh weight, shoot dry weight and salt sensitivity index, in response to 100 mM NaCl. After salt stress, Tdp2-28 transgenic line showed significantly higher chlorophyll and carotenoid total contents, 2,2-Diphenyl-1-Picrylhydrazyl radical scavenging activity, and significantly lower levels of oxidative DNA damage than the control line. Interestingly, the expression levels of several genes, including genes linked to genome maintenance and regulation of DNA topology (MtTdp1α, MtTop2), base excision repair pathway (MtOGG1) and double strand break sensing/repair (MtMRE11) were enhanced in Tdp2-28 transgenic shoots under salt stress conditions as compared to their controls. These findings suggest that MtTdp2α play an important role in plant tolerance to salt stress. Overexpression of MtTdp2α gene in Medicago truncatula confers salt stress tolerance through better plant growth performances, higher chlorophyll and carotenoid contents, increased antioxidant capacity, reduced oxidative DNA damage, and by up-regulation of several genes involved in DNA metabolism.

Germplasm collections of major crops include thousands of accessions whose agronomic evaluation is prevented by high costs, while their molecular characterization is getting increasingly affordable. This study aimed to assess the consistency between alfalfa (Medicago sativa L.) landrace diversity measures relative to adaptation pattern, eleven morphophysiological traits, and four genotyping strategies using SSR or genotyping-by-sequencing (GBS) SNP markers recorded on either five individual plants or three bulks of 100 plants each per accession. We focused on eleven landraces from Northern Italy featuring large genotype × environment interaction for biomass yield across environments with contrasting level of summer drought. We recorded 34 SSR and 237 SNP polymorphic markers for individual plants, and 41 SSR and 1274 SNP markers for bulked plants. Landrace diversity based on SNP data from bulked plants was the only molecular diversity measure associated with both adaptive diversity as expressed by pattern analysis (r = 0.34) and drought stress at collecting sites (r = 0.50). No molecular diversity measure was associated with overall morphophysiological diversity. Molecular diversity was highly consistent only between SSR and SNP data from bulked plants (r = 0.67). SSR-based diversity from individual plants exhibited marked inconsistency with other molecular diversity measures but was useful for genetic structure assessment, indicating much smaller among-population variation relative to within-population variation (7.5% in terms of variance ratio; 7.8% as global FST). Moderate inferences on germplasm adaptive diversity may be achieved by molecular characterization that maximizes the sampling of genomic areas and genotypes within accession through GBS applied to bulked plants.

In this study, we describe a duplex real-time PCR assay for the simultaneous detection of KIPyV and WUPyV polyomaviruses based on TaqMan probes. This assay detected 500 copies/mL both for KIPyV and WUPyV in 100% of tested positive samples. We assessed this technique on 482 nasopharyngeal aspirate specimens from hospitalized pediatric patients with respiratory symptoms, previously analyzed with commercial multiplex assay for 16 major respiratory viruses. Our assay detected KIPyV genome in 15 out of 482 samples (3.1%) and WUPyV genome in 24 out of 482 samples (4.9%), respectively, and in three samples the coinfection of the two viruses was found. Interestingly, 29 out of 36 of samples with KIPyV and/or WUPyV infection exhibited a co-infection with one or more respiratory viruses confirming that KIPyV and WUPyV were often detected in association to other viral infections. Of note, KIPyV and WUPyV were detected singularly in 4 out of 15 cases and 3 out of 24 cases, respectively, suggesting a possible direct role of these viruses in the respiratory diseases. In conclusion, this method could be taken into account as an alternative technical approach to detect KIPyV and/or WUPyV in respiratory samples for epidemiological and diagnostic analyses., Highlights • Duplex real-time PCR assay for the detection of human KIPyV and WUPyV was assessed. • This assay was evaluated on nasopharyngeal aspirate samples from pediatric patients. • KIPyV and WUPyV were detected in 3.1% and 4.9% of samples, respectively.

This observational study aimed to: (i) assess the presence of periodontal disease among patients requiring aortic valve replacement, (ii) investigate the presence of oral pathogens in aortic valve specimens and compare them with the microorganisms detected in the oral cavity. Twenty-six patients (15 men and 11 women) were scheduled to be visited the day before the cardiac surgery: periodontal conditions were accurately registered through clinical and radiographic examinations, dental plaque or salivary samples were collected. Valve specimens were collected during surgical aortic valve replacement and analyzed for pathogens detection through microbiological 16SrRna gene sequencing. Bacteria found in plaque samples and valve specimens were assessed according to oral and periodontal conditions. A qualitative comparison between oral and cardiac profiles of the microorganisms detected was performed. The overall number of patients examined for soft tissues conditions was 19, as 7 patients were edentulous. Twelve and three individuals, respectively, presented moderate and severe periodontitis. Nine valves were found to be positive for the presence of oral and periodontopathic bacterial DNA. The microbial species found in valve samples of patients with periodontitis suggest that the presence of these microorganisms in valvular tissue seems to be not coincidental.

Current knowledge on genes regulating saponin synthesis is reported for the model species Medicago truncatula. A focus on synthesis on different plant organs is reported.

IFI16, member of the IFN-inducible PYHIN-200 gene family, modulates proliferation, survival and differentiation of different cell lineages. In particular, IFI16 expression, which is regulated during the differentiation of B cells, was recently studied in B-CLL as well. Here, we compared IFI16 expression in several lymphomas including Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma and mantle cell lymphoma with respect to normal cell counterparts. We observed that IFI16 expression was significantly deregulated only in mantle cell lymphoma (p < 0.05). Notably, IFI16 was associated with the expression of genes involved in interferon response, cell cycle, cell death and proliferation and, interestingly, lipid and glucose metabolism, suggesting that IFI16 deregulation might be associated with relevant changes in cell biology. In our group of mantle cell lymphoma samples a correlation between patient survival and IFI16 expression was not detected even though mantle cell lymphoma prognosis is known to be associated with cell proliferation. Altogether, these results suggest a complex relationship between IFI16 expression and MCL which needs to be analyzed in further studies., Molecular biology; Cell biology; Biochemistry; Oncology; Pathology; Cancer research; Mantle cell lymphoma; Immunohistochemistry; IFI16; Proliferation; Gene expression

In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmaceutics, agrochemicals and in the food and cosmetic industries has raised interest in identifying the enzymes involved in their synthesis. We have identified a cytochrome P450 (CYP72A67) involved in hemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethylmethanesulfonate (EMS) mutagenized collection. Genetic and biochemical analyses, mutant complementation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible for hydroxylation at the C-2 position downstream of oleanolic acid synthesis. The affinity of CYP72A67 for substrates with different substitutions at multiple carbon positions was investigated in the same in vitro yeast system, and in relation to two other CYP450s (CYP72A68) responsible for the production of medicagenic acid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants were compared for their sapogenin profile, expression patterns of the genes involved in sapogenin synthesis, and response to inoculation with Sinorhizobium meliloti. The results obtained allowed us to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities (leaves/roots) of sapogenin synthesis.

White lupin (Lupinus albus L.) is a valuable source of seed protein, carbohydrates and oil, but requires genetic improvement to attain its agronomic potential. This study aimed to (i) develop a new high-density consensus linkage map based on new, transcriptome-anchored markers; (ii) map four important agronomic traits, namely, vernalization requirement, seed alkaloid content, and resistance to anthracnose and Phomopsis stem blight; and, (iii) define regions of synteny between the L. albus and narrow-leafed lupin (L. angustifolius L.) genomes. Mapping of white lupin quantitative trait loci (QTLs) revealed polygenic control of vernalization responsiveness and anthracnose resistance, as well as a single locus regulating seed alkaloid content. We found high sequence collinearity between white and narrow-leafed lupin genomes. Interestingly, the white lupin QTLs did not correspond to previously mapped narrow-leafed lupin loci conferring vernalization independence, anthracnose resistance, low alkaloids and Phomopsis stem blight resistance, highlighting different genetic control of these traits. Our suite of allele-sequenced and PCR validated markers tagging these QTLs is immediately applicable for marker-assisted selection in white lupin breeding. The consensus map constitutes a platform for synteny-based gene cloning approaches and can support the forthcoming white lupin genome sequencing efforts.

Cultivar registration agencies typically require morphophysiological trait-based distinctness of candidate cultivars. This requirement is difficult to achieve for cultivars of major perennial forages because of their genetic structure and ever-increasing number of registered material, leading to possible rejection of agronomically valuable cultivars. This study aimed to explore the value of molecular markers applied to replicated bulked plants (three bulks of 100 independent plants each per cultivar) to assess alfalfa ( L. subsp. ) cultivar distinctness. We compared genotyping-by-sequencing information based on 2902 polymorphic single-nucleotide polymorphism (SNP) markers (>30 reads per DNA sample) with morphophysiological information based on 11 traits and with simple-sequence repeat (SSR) marker information from 41 polymorphic markers for their ability to distinguish 11 alfalfa landraces representative of the germplasm from northern Italy. Three molecular criteria, one based on cultivar differences for individual SSR bands and two based on overall SNP marker variation assessed either by statistically significant cultivar differences on principal component axes or discriminant analysis, distinctly outperformed the morphophysiological criterion. Combining the morphophysiological criterion with either molecular marker method increased discrimination among cultivars, since morphophysiological diversity was unrelated to SSR marker-based diversity ( = 0.04) and poorly related to SNP marker-based diversity ( = 0.23, < 0.15). The criterion based on statistically significant SNP allele frequency differences was less discriminating than morphophysiological variation. Marker-based distinctness, which can be assessed at low cost and without interactions with testing conditions, could validly substitute for (or complement) morphophysiological distinctness in alfalfa cultivar registration schemes. It also has interest in sui generis registration systems aimed at marketing alfalfa landraces.

Saponins, a group of glycosidic compounds present in several plant species, have aglycone moieties that are formed using triterpenoid or steroidal skeletons. In spite of their importance as antimicrobial compounds and their possible benefits for human health, knowledge of the genetic control of saponin biosynthesis is still poorly understood. In the Medicago genus, the hemolytic activity of saponins is related to the nature of their aglycone moieties. We have identified a cytochrome P450 gene (CYP716A12) involved in saponin synthesis in Medicago truncatula using a combined genetic and biochemical approach. Genetic loss-of-function analysis and complementation studies showed that CYP716A12 is responsible for an early step in the saponin biosynthetic pathway. Mutants in CYP716A12 were unable to produce hemolytic saponins and only synthetized soyasaponins, and were thus named lacking hemolytic activity (lha). In vitro enzymatic activity assays indicate that CYP716A12 catalyzes the oxidation of β-amyrin and erythrodiol at the C-28 position, yielding oleanolic acid. Transcriptome changes in the lha mutant showed a modulation in the main steps of triterpenic saponin biosynthetic pathway: squalene cyclization, β-amyrin oxidation, and glycosylation. The analysis of CYP716A12 expression in planta is reported together with the sapogenin content in different tissues and stages. This article provides evidence for CYP716A12 being a key gene in hemolytic saponin biosynthesis.

We have established mutant collections of the model species Medicago truncatula according to current protocols. In particular, we used a transposon (Tnt1) tagging method and an ethyl methanesulfonate (EMS) mutagenesis approach (TILLING). The collections were subjected to both forward and reverse genetics screenings, and several mutants were isolated that affect plant traits (e.g. shoot, root developments, flower morphology, etc.) and also biosynthetic pathways of secondary compounds (saponins and tannins). Genes responsible for some of the mutations were cloned and further characterized.

Double ‘free-hybrids’ (DH) in alfalfa were obtained by crossing in a diallelic scheme, six multiplied simple hybrids (SH) derived from four partly inbred (S2) lines. Analysis of the specific combining ability demonstrated that the main source of variation was for dry matter yield (DMY) in DHs and supported heterosis values of DHs versus the best parent of an average +45% (ranging from +5 to +76%). Investigation at the molecular level was carried out by analysis of simple sequence repeat markers on the six parental SHs and 15 DH progenies and by comparison of gene expression profiles using microarrays of a single DH line to its parental lines. The variation of heterozygosity estimates of the DHs explained a small part (about 20%) of their variation in DMY, while the number of alleles was significantly related to DM performance (r = 0.61; P

Summary In the Medicago genus, saponins are a complex mixture of triterpene glycosides showing a broad spectrum of biological properties. Here we analyzed the variation in the sapogenin content and composition of inter-specific hybrid Medicago sativa × Medicago arborea derivatives to highlight the pattern of this variation in plant organs (leaves/roots) and the possible mechanisms underlying it. In Sativa Arborea Cross (SAC) leaves and roots, saponins and sapogenins were evaluated using chromatographic methods. Phenotypic correlations between sapogenin content and bio-agronomic traits were examined. Expression studies on β-amyrin synthase and four cytochromes P450 (CYPs) involved in sapogenin biosynthesis and sequence analysis of the key gene of the hemolytic sapogenin pathway (CYP716A12) were performed. Chromatographic analyses revealed a different pattern of among-family variation for hemolytic and nonhemolytic sapogenins and saponins and for the two organs/tissues. Different correlation patterns of gene expression in roots and leaves were found. Diachronic analysis revealed a relationship between sapogenin content and gene transcriptional levels in the early stages of the productive cycle. The results suggest that there are different control mechanisms acting on sapogenin biosynthesis for leaves and roots, which are discussed. A key role for medicagenic acid in the control of sapogenin content in both the tissues is proposed and discussed.

Medicago truncatula is one of the model species for legume molecular genetics. In the last decade different types of mutant populations have been created in this species that can be screened by forward and reverse-genetic approaches to identify and functionally characterize genes of interest. TILLING is a reverse-genetic method combining random chemical mutagenesis and a PCR-based screen to identify point mutations in regions of interest. The different steps of the TILLING analysis are described in a mutant collection of ~2,300 M2 individuals for which genomic DNA and M3 seed were obtained. A two-dimensional DNA pooling strategy was adopted to reduce the number of PCR reactions necessary to screen the collection and to unambigously identify the individual M2 plant carrying the mutation. The genotypic and phenotypic analyses of the mutant M3 progeny provide the possibility to study the gene function. In spite of its reduced size, this mutant collection has proved valid in the study of the biosynthetic pathway of a class of secondary metabolites present in the genus Medicago, the triterpenic saponins.

Medicago truncatula is one of the model species for legume molecular genetics. In the last decade different types of mutant populations have been created in this species that can be screened by forward and reverse-genetic approaches to identify and functionally characterize genes of interest. TILLING is a reverse-genetic method combining random chemical mutagenesis and a PCR-based screen to identify point mutations in regions of interest. The different steps of the TILLING analysis are described in a mutant collection of ~2,300 M2 individuals for which genomic DNA and M3 seed were obtained. A two-dimensional DNA pooling strategy was adopted to reduce the number of PCR reactions necessary to screen the collection and to unambigously identify the individual M2 plant carrying the mutation. The genotypic and phenotypic analyses of the mutant M3 progeny provide the possibility to study the gene function. In spite of its reduced size, this mutant collection has proved valid in the study of the biosynthetic pathway of a class of secondary metabolites present in the genus Medicago, the triterpenic saponins.

The effect of peripheral benzodiazepine receptor ligands: PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)isoquinoline-3-carboxamid e), Ro 5-4864 (4-chlorodiazepam), hemin, N-methyl protoporphyrin IX and protoporphyrin IX on liver mitochondrial 27-hydroxylation of cholesterol was studied by adding them together with [4-14C]cholesterol. N-Methyl protoporphyrin IX, PK11195 and protoporphyrin IX stimulated mitochondrial 27-hydroxylation of [4-14C] cholesterol in vitro, the first two being the most potent (2-3-fold increase). Ro 5-4864 and hemin were not active. 27-Hydroxylation of [4-14C]cholesterol was reduced to below control levels (respectively 40 and 56% decrease compared to control, P0.01) when PK11195, N-methyl protoporphyrin IX or protoporphyrin IX were allowed to equilibrate in vitro with mitochondria for 20 min at 37 degrees C. Hepatic protoporphyria was induced using 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) (100 mg/kg, i.p.) to study the effect of in vivo accumulation of large amounts of dicarboxylic porphyrins, i.e. endogenous peripheral benzodiazepine receptor ligands, on cholesterol 27-hydroxylation. DDC treatment caused an increase in total porphyrin content in liver homogenate (10-fold) and mitochondria (2-fold). Mitochondrial 27-hydroxylation of [4-14C]cholesterol was depressed after treatment (60% decrease, P0.01). We suggest that peripheral benzodiazepine receptor ligands act on liver mitochondrial 27-hydroxylation of cholesterol by a mechanism coupled to these receptors and that the time of exposure of peripheral benzodiazepine receptors to ligands is a major factor. The modulation of 27-hydroxycholesterol production may have a physiological role in liver and possibly in other tissues.

Aims/Methods: Interferon β is used as a therapeutic agent, but its effects on the hepatic cytochrome P-450-dependent drug metabolizing system have not yet been characterized. We investigated the effect of interferon β on cytochrome P-450 in mice. Results: Interferon β (2×10 5 units/mouse) significantly reduced total hepatic cytochrome P-450 (20%) and the activity of NADPH cytochrome C reductase (12%) 24 h after administration; lower doses had no such effect. Various monooxygenase activities were slightly reduced, the one most affected being 7-ethoxycoumarin O-deethylase (29%). In phenobarbital-treated mice, interferon β reduced the induction of total cytochrome P-450 (22%), the activities of pentoxyresorufin O-dealkylase (38%), benzyloxyresorufin O-dealkylase (30%), erythromycin N-demethylase (30%), 7-ethoxycoumarin O-deethylase (16%) and cytochrome P-450 2B1 (33%) and 3A (45%) proteins. In β-naphthoflavone-treated mice, interferon β lowered the induction of total cytochrome P-450 (18%), the activities of ethoxyresorufin O-deethylase (31%) and of 7-ethoxycoumarin O-deethylase (25%) and of cytochrome P-450 1A1 protein (31%). Conclusions: Thus it appears that induced cytochrome(s) P-450 were susceptible to interferon β, this effect not being influenced by the type of inducer. Since various members of the same cytochrome P-450 subfamilies catalyze oxidation of drugs in humans, our findings have potential significance as regards the fate of drugs or exogenous compounds given to patients receiving interferon β.

Ciliary neurotrophic factor (CNTF) supports the survival of ciliary ganglion neurons and was shown to induce the synthesis of acute-phase proteins and fever. We studied the effect of CNTF, alone or in association with IL-1, on levels of corticosterone (CS), glucose, serum amyloid A (SAA), and IL-6. We also compared the effect of CNTF with that of IL-6, since the gp130 receptor subunit for CNTF is shared with that of IL-6. A single intravenous injection of CNTF induced hypoglycaemia and SAA and potentiated IL-1-induced CS and IL-6. Chronic CNTF, but not IL-6, resulted in decreased food intake and body weight up to days 6-7. After this time, body weight and food intake recovered even if CNTF treatment was continued, indicating that a phenomenon of tolerance occurred. Finally, CNTF (unlike IL-1) was not toxic in adrenalectomized mice. Therefore the similarities of CNTF activities with those of other cytokines, particularly IL-6, might go beyond the activation of the same receptor-signal transduction pathway of IL-6.

In in vitro systems haem oxygenase-1 (HO-1) mRNA increases after exposure to agents causing oxidative stress. We lowered cellular antioxidant defence systems in vivo by giving mice increasing doses (0.15 g/kg-1.6 g/kg) of DL-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of glutathione synthesis. Maximum glutathione depletion (80%) coincided with maximum hepatic HO-1 mRNA accumulation (about 20 times), whereas with 50% depletion, accumulation was only doubled. It has been suggested that reactive oxygen and nitrogen intermediates are involved in hepatic toxicity of endotoxin (lipopolysaccharide, LPS); LPS even at low doses [0.1 mg/kg, intraperitoneally (i.p.)] induces HO-1 mRNA about 25-fold after 1 h. Hepatic glutathione depletion (respectively 40% and 80%) after a low (0.3 g/kg) or a high (1.6 g/kg) BSO dose, resulted in potentiation of the HO-1 mRNA accumulation induced by LPS (0.1 mg/kg, i.p.). In the absence of BSO, N-acetylcysteine (NAC) (1 g/kg orally) reduced LPS-induced HO-1 mRNA accumulation to one fourth. Under the same experimental conditions S-adenosylmethionine (SAM) was not effective. NAC also reduced HO-1 mRNA accumulation when administered to mice in which glutathione was depleted and its synthesis blocked by BSO (1.6 g/kg). Thus reactive oxygen intermediates are likely mediators of LPS-induced HO-1 mRNA accumulation, and glutathione content appears to be one of the factors regulating this accumulation in the liver. Our findings are compatible with the theory that HO-1 induction might have a protective function in vivo when defence mechanisms against oxidants are challenged.

Interleukin (IL)-2 is known to induce vascular leak syndrome (VLS), which was suggested to be mediated by immune system-derived cytokines, including tumor necrosis factor (TNF). To characterize the role of TNF in IL-2 toxicity in C3H/HeN mice, we used two approaches to downregulate TNF production in vivo: treatment with dexamethasone (DEX) and induction of endotoxin (lipopolysaccharide) (LPS) tolerance by a 4-day pretreatment with LPS (35 micrograms/mouse/day). Mice were then treated with IL-2 for 5 days (1.8 x 10(5) IU/mouse, twice daily). Both DEX and LPS tolerance blocked development of hydrothorax in IL-2-treated mice and inhibited TNF induction. DEX and LPS tolerance also ameliorated IL-2 toxicity in terms of decrease in food intake and inhibited the increase of the acute-phase protein, serum amyloid A (SAA). The IL-2 activation of splenic natural killer (NK) cell activity was also diminished by DEX and, to a lesser extent, by LPS-tolerance. Treatment with IL-2 also caused induction of the superoxide-generating enzyme xanthine oxidase (XO) in tissues and serum and induced bacterial translocation in the mesenteric lymph nodes (MLN). These data suggest that multiple mechanisms, including NK cell activity, cytokines, and reactive oxygen intermediates, might be important in the vascular toxicity of IL-2.

Background Medicago truncatula is a model species for legumes. Its functional genomics have been considerably boosted in recent years due to initiatives based both in Europe and US. Collections of mutants are becoming increasingly available and this will help unravel the genetic control of important traits for many species of legumes. Findings Our report is on the production of three complementary mutant collections of the model species Medicago truncatula produced in Italy in the frame of a national genomic initiative. Well established strategies were used: Tnt1 mutagenesis, TILLING and activation tagging. Both forward and reverse genetics screenings proved the efficiency of the mutagenesis approaches adopted, enabling the isolation of interesting mutants which are in course of characterization. We anticipate that the reported collections will be complementary to the recently established functional genomics tools developed for Medicago truncatula both in Europe and in the United States.

When teachers want to introduce a discussion about the use of cigarette to a group of teenagers, it can be interesting to know what this group thinks about it in order to contextualize the approach to their profile. It is proposed here a framework to instance web game supported by common sense knowledge to approach the called “transversal themes” of the school curriculum, like healthcare, sexual education and ethic. The quiz game framework called “What is it?” is presented as a support for teachers in contextualizing the content to the students’ local culture, promoting a more effective and significant learning, where a new knowledge is related to a previous one in the student’s cognitive structure. Teachers can set up a web quiz game based on common sense knowledge in the prototype presented.

Treatment with a single low dose (80 to 800 ng) of interleukin-1 (IL-1) 24 h before a lethal bacterial challenge of granulocytopenic and normal mice enhances nonspecific resistance. Since IL-1 induces secretion of acute-phase proteins, liver proteins which possess several detoxifying effects, we investigated the role of these proteins in the IL-1-induced protection. Inhibition of liver protein synthesis with D-galactosamine (GALN) completely inhibited the IL-1-induced synthesis of acute-phase proteins. GALN pretreatment abolished the protective effect of IL-1 on survival completely (neutropenic mice infected with Pseudomonas aeruginosa) or partially (nonneutropenic mice infected with Klebsiella pneumoniae). Pretreatment with IL-6, a cytokine induced by IL-1, did not reproduce the protection offered after IL-1 pretreatment, nor did it enhance or deteriorate the IL-1-enhanced resistance to infection. A protective effect of IL-1 via effects on glucose homeostasis during the acute-phase response was investigated by comparing plasma glucose levels in IL-1-treated mice and control mice before and during infection. Although glucose levels in IL-1-pretreated mice were somewhat higher in the later stages of infection, no significant differences from levels in control mice were present, and the glucose levels in control-treated animals never fell to hypoglycemic values. We conclude that the IL-1-induced nonspecific resistance is mediated neither by the induction of IL-6 nor by the effects of IL-1 on glucose homeostasis. Acute-phase proteins generated after IL-1 pretreatment, however, seem to play a critical role in the IL-1-induced protection to infection.

Systemic and localized inflammation elicit a number of host responses which include fever, cachexia, hypoglycemia, and major changes in the concentration of liver plasma proteins. Interleukin 6 (IL-6) is considered an important mediator of the inflammatory response, together with IL-1 and tumor necrosis factor alpha (TNF-alpha). The purpose of this study was to unequivocally determine the role of IL-6 in these phenomena making use of IL-6-deficient mice that we have recently generated by gene targeting. We report here that in the absence of IL-6, mice are unable to mount a normal inflammatory response to localized tissue damage generated by turpentine injection. The induction of acute phase proteins is dramatically reduced, mice do not lose body weight and only suffer from mild anorexia and hypoglycemia. In contrast, when systemic inflammation is elicited through the injection of bacterial lipopolysaccharide (LPS), these parameters are altered to the same extent both in wild-type and IL-6-deficient mice, demonstrating that under these conditions IL-6 function is dispensable. Moreover, we show that LPS-treated IL-6-deficient mice produce three times more TNF-alpha than wild-type controls, suggesting that increased TNF-alpha production might be one of the compensatory mechanisms through which a normal response to LPS is achieved in the absence of IL-6. We also show that corticosterone is normally induced in IL-6-deficient mice, demonstrating that IL-6 is not required for the activation of the hypothalamic-pituitary-adrenal axis. Our results reinforce the idea that different patterns of cytokines are involved in systemic and localized tissue damage, and identify IL-6 as an essential mediator of the inflammatory response to localized inflammation.