D-amino acid oxidase from Trigonopsis variabilis: immobilisation of whole cells in natural polymeric gels for glutaryl-7-aminocephalosporanic acid production

The enzymatic oxidation of cephalosporin C to glutaryl-7-amminocephalosporanic acid (glutaryl-7-ACA) was carried out utilizing permeabilized whole cells of the yeast Trigonopsis variabilis entrapped in Ca-alginate beads. The biomass, cultured in a rich medium containing D,L methionine and harvested after 72 h of growth, exhibited high levels of D-aminoacid oxidase activity. Prior to use, thewho,lke cells were permeabilized with four freeze-thawing cycles and immobilized in polysaccharide matrices, such as Ca-alginate and K- carrageenan, and in an insolubilised gelatin gel. The best results in terms of activity yield and storage stability were obtained with cells entrapped in Ca-alginate beads. These cells were utilized for glutaryl-7-ACA production in a continuous stirred batch reactor (CSTR) and in a packed bed reactor working in a plug flow reactor (PFR), using 50 mm Cephalosporin C as substrate. The performances of the two systems were compared. The overall on a void volume basis) were 1.63 g and 255 mg of glutaryl-7-ACA h-1 in the PFR and in CSTR, respectively

Conjugated linoleic acid enhances glutathione synthesis and attenuates pathological signs in MRL/MpJ-Faslpr mice

Conjugated linoleic acid (CLA), a naturally occurring peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand, exhibits proapoptotic, immunomodulatory, and anticancer properties. In this study, we examined the biological effects of CLA administration in the MRL/MpJ-Fas(lpr) mouse, an animal model of systemic lupus erythematosus (SLE). We found that CLA exerted apparently opposed activities in in vitro experiments, depending on its concentration: 100 microM CLA downregulated IFN gamma synthesis and cell proliferation of splenocytes, in association with apoptosis induction and a decrease of intracellular thiols (GSH + GSSG), whereas 25 microM CLA did not significantly influence cell proliferation but enhanced the expression of gamma-glutamylcysteine ligase catalytic subunit (GCLC) and intracellular GSH concentration. Interestingly, the antiproliferative effect at 100 microM was not inhibited by the PPAR gamma antagonist GW9662. In vivo, CLA administration drastically reduced SLE signs (splenomegaly, autoantibodies, and cytokine synthesis), a condition paralleled by the enhancement of GCLC expression and intracellular GSH content. Moreover, CLA administration significantly downregulated nuclear factor kappaB activity independent of PPAR gamma activation and apoptosis induction. In conclusion, enhanced GSH content and GCLC expression in CLA-treated mice suggest a novel biochemical mechanism underlying its immunomodulatory activity and the beneficial effects on murine SLE signs

Costruire processi partecipativi attorno al cibo: le esperienze di Bergamo e Trento

This contribution offers a first reflection on two field research experiences carried out in the context of two projects aiming to create urban food policies in two medium-sized cities: Bergamo and Trento. In particular, it reflects on the factors that favour and hinder the spread of good practices of responsible consumption and production \u2013 the so-called Alternative Food Networks or AFNs \u2013 and on the collaboration between civil society actors and institutions. After a first introductory part and before outlining the characteristics of the two contexts taken into examination, the chapter discusses the methodology adopted called \u201cresearch for action\u201d, a particular approach building upon participatory action research traditions in the human and social sciences which aims to support the exchange and the collaboration among actors involved in the local food system to facilitate collective action through the use of different tools including digital maps, in-depth interviews and focus groups. In conclusion, it reflects on which approaches and tools may be utilized to encourage the participation of social actors with institutions to address the problems of the current system of production, consumption and distribution of food (starting from the local scale), in order to better utilize the opportunities for dissemination and coordination of good environmental and social practices in the development of participatory and sustainable food policies

Characterization of the Anti-Tissue Transglutaminase Antibody Response in Nonobese Diabetic Mice

Abstract Type 1 diabetes mellitus is an autoimmune disorder characterized by destruction of insulin-producing pancreatic β cells by T lymphocytes. In nonobese diabetic (NOD) mice, a role has been hypothesized for dietary gluten proteins in the onset of diabetes, and because gluten dependence is the major feature of celiac disease, together with production of Abs to the autoantigen tissue transglutaminase (tTG), we looked for the presence of anti-tTG Abs in the serum of NOD mice and, to establish their origin, analyzed the Ab repertoire of NOD mice using phage display Ab libraries. We found significant levels of serum anti-tTG Abs and were able to isolate single-chain Ab fragments to mouse tTG mainly from the Ab libraries made from intestinal lymphocytes and to a lesser extent from splenocytes. Data from NOD mice on a gluten-free diet suggest that the anti-tTG response is not gluten-dependent. The intestinal Ab response to tTG is a feature of NOD mice, but the underlying mechanisms remain obscure

Gliadin-Mediated Proliferation and Innate Immune Activation in Celiac Disease Are Due to Alterations in Vesicular Trafficking

Background and Objectives: Damage to intestinal mucosa in celiac disease (CD) is mediated both by inflammation due to adaptive and innate immune responses, with IL-15 as a major mediator of the innate immune response, and by proliferation of crypt enterocytes as an early alteration of CD mucosa causing crypts hyperplasia. We have previously shown that gliadin peptide P31-43 induces proliferation of cell lines and celiac enterocytes by delaying degradation of the active epidermal growth factor receptor (EGFR) due to delayed maturation of endocytic vesicles. IL-15 is increased in the intestine of patients affected by CD and has pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells belonging to the innate and adaptive branches of the immune response. Aims of this study were to investigate the role of P31-43 in the induction of cellular proliferation and innate immune activation. Methods/Principal Findings: Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation both in CaCo-2 cells and in biopsies from active CD cases and controls. We used real-time PCR to evaluate IL-15 mRNA levels and FACS as well as ELISA and Western Blot (WB) analysis to measure protein levels and distribution in CaCo-2 cells. Gliadin and P31-43 induce a proliferation of both CaCo-2 cells and CD crypt enterocytes that is dependent on both EGFR and IL-15 activity. In CaCo-2 cells, P31-43 increased IL-15 levels on the cell surface by altering intracellular trafficking. The increased IL-15 protein was bound to IL15 receptor (IL-15R) alpha, did not require new protein synthesis and functioned as a growth factor. Conclusion: In this study, we have shown that P31-43 induces both increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by altering the trafficking of the vesicular compartments as well as proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR

Population- and individual-specific regulatory variation in Sardinia

Genetic studies of complex traits have mainly identified associations with noncoding variants. To further determine the contribution of regulatory variation, we combined whole-genome and transcriptome data for 624 individuals from Sardinia to identify common and rare variants that influence gene expression and splicing. We identified 21,183 expression quantitative trait loci (eQTLs) and 6,768 splicing quantitative trait loci (sQTLs), including 619 new QTLs. We identified high-frequency QTLs and found evidence of selection near genes involved in malarial resistance and increased multiple sclerosis risk, reflecting the epidemiological history of Sardinia. Using family relationships, we identified 809 segregating expression outliers (median z score of 2.97), averaging 13.3 genes per individual. Outlier genes were enriched for proximal rare variants, providing a new approach to study large-effect regulatory variants and their relevance to traits. Our results provide insight into the effects of regulatory variants and their relationship to population history and individual genetic risk.M.P. is supported by the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement 633964 (ImmunoAgeing). Z.Z. is supported by the National Science Foundation (NSF) GRFP (DGE- 114747) and by the Stanford Center for Computational, Evolutionary, and Human Genomics (CEHG). Z.Z., J.R.D., and G.T.H. also acknowledge support from the Stanford Genome Training Program (SGTP; NIH/NHGRI T32HG000044). J.R.D. is supported by the Stanford Graduate Fellowship. K.R.K. is supported by Department of Defense, Air Force Office of Scientific Research, National Defense Science and Engineering Graduate (NDSEQ) Fellowship 32 CFR 168a. S.J.S. is supported by the NIHR Cambridge Biomedical Research Centre. The SardiNIA project is supported in part by the intramural program of the National Institute on Aging through contract HHSN271201100005C to the Consiglio Nazionale delle Ricerche of Italy. The RNA sequencing was supported by the PB05 InterOmics MIUR Flagship grant; by the FaReBio2011 “Farmaci e Reti Biotecnologiche di Qualità” grant; and by Sardinian Autonomous Region (L.R. no. 7/2009) grant cRP3-154 to F. Cucca, who is also supported by the Italian Foundation for Multiple Sclerosis (FISM 2015/R/09) and by the Fondazione di Sardegna (ex Fondazione Banco di Sardegna, Prot. U1301.2015/AI.1157.BE Prat. 2015-1651). S.B.M. is supported by the US National Institutes of Health through R01HG008150, R01MH101814, U01HG007436, and U01HG009080. All of the authors would like to thank the CRS4 and the SCGPM for the computational infrastructure supporting this project

Mucosal immunity induced by gliadin-presenting spores of Bacillus subtilis in HLA-DQ8-transgenic mice

The induction of mucosal immunity requires efficient antigen delivery and adjuvant systems. Probiotic bacterial strains are considered to be very promising tools to address both of these needs. In particular, Bacillus subtilis spores are currently under investigation as a long-lived, protease-resistant adjuvant system for different antigens. Furthermore, a non-recombinant approach has been developed based on the stable adsorption of antigen on the spore surface. In the present study, we explored this strategy as a means of modulating the immune response to wheat gliadin, the triggering agent of celiac disease (CD), an enteropathy driven by inflammatory CD4+ T cells. Gliadin adsorption was tested on untreated or autoclaved wild-type (wt) and mutant (cotH or cotE) spores. We found that gliadin was stably and maximally adsorbed by autoclaved wt spores. We then tested the immune properties of the spore-adsorbed gliadin in HLA-DQ8-transgenic mice, which express one of the two HLA heterodimers associated with CD. In vitro, spore-adsorbed gliadin was efficiently taken up by mouse dendritic cells (DCs). Interestingly, gliadin-pulsed DCs efficiently stimulated splenic CD4+ T cells from mice immunised with spore-adsorbed gliadin. Nasal pre-dosing with spore-adsorbed gliadin failed to down-regulate the ongoing cellular response in gliadin-sensitised DQ8 mice. Notably, naïve mice inoculated intranasally with multiple doses of spore-adsorbed gliadin developed an intestinal antigen-specific CD4+ T cell-mediated response. In conclusion, our data highlight the ability of spore-adsorbed gliadin to elicit a T-cell response in the gut that could be exploitable for developing immune strategies in CD

L’Acquedotto Romano ipogeo e la Fontana di Helvius a Sant’Egidio del Monte Albino (Salerno, Campania)

Le ricerche speleologiche condotte negli ultimi anni stanno approfondendo le conoscenze su alcuni piccoli ma interessanti acquedotti ipogei diffusi sul territorio campano. Queste opere sebbene caratterizzate da modesti sviluppi, rappresentavano importanti fonti di approvvigionamento idropotabile per piccole comunità rurali. In questa nota si riporta lo studio e il rilievo topografico di una galleria drenante ubicata nel comune di Sant’Egidio del Monte Albino, in provincia di Salerno, situato alle falde del settore settentrionale dei Monti Lattari, non lontano dalle note città romane di Pompei, Nocera e Stabia. L’acquedotto è stato interamente scavato in depositi di conoide costituiti da alternanze di livelli di ghiaie e piroclastiti pedogenizzate per complessivi 478 m di sviluppo. Con riferimento ai soli condotti idraulici, il sistema di gallerie risale nella fascia pedemontana per circa 16 m al fine di drenare le acque di falda che si raccolgono nel materasso della conoide detritico-alluvionale che si sviluppa allo sbocco del Vallone del Lupo. L’opera è composta da un ramo principale e tre rami laterali minori. Le tracce più antiche dell’abitato in cui si trova l’acquedotto, facente parte dell’antica Nuceria (odierna Nocera), sono rappresentate dai resti di una villa rustica del I sec. a.C. - I sec. d.C., successivamente inglobati nella cripta dell’abbazia di Santa Maria Maddalena in Armillis. Alla stessa epoca appartiene anche un blocco marmoreo con raffigurazioni del dio Sarno, in cui è realizzata la “fontana di San Nicola” o “fontana di Helvius” (dal nome del pretore di Nuceria, Publius Helvius, che la fece realizzare). Il dio viene raffigurato sui diversi lati del blocco nelle due versioni iconografiche di giovane e di uomo maturo, riferibili ai vari tratti del fiume Sarno che vanno dalla sorgente alla foce. La fontana in marmo, connessa all’acquedotto, fornisce una datazione indiretta anche sull’epoca di realizzazione del sistema di gallerie