Listeriaphages and coagulin C23 act synergistically to kill Listeria monocytogenes in milk under refrigeration conditions

© 2015 Elsevier B.V. Bacteriophages and bacteriocins are promising biocontrol tools in food. In this work, two Listeria bacteriophages, FWLLm1 and FWLLm3, were assessed in combination with the bacteriocin coagulin C23 to inhibit Listeria monocytogenes. Preliminary results under laboratory conditions demonstrated that both antimicrobials act synergistically when they were applied in suboptimal concentrations. The combined approach was further assessed in milk contaminated with 5×104CFU/ml L. monocytogenes 2000/47 and stored at 4°C for 10days. When used alone, phage FWLLm1 added at 5×106PFU/ml, FWLLm3 at 5×105PFU/ml and coagulin C23 at 584AU/ml kept L. monocytogenes 2000/47 counts lower than the untreated control throughout storage. However, when used in combination, inhibition was enhanced and in the presence of FWLLm1 and coagulin C23, L. monocytogenes 2000/47 counts were under the detection limits (less than 10CFU/ml) from day 4 until the end of the experiment. Resistant mutants towards phages and coagulin C23 could be obtained, but cross-resistance was not detected. Mutants resistant to FWLLm3 and coagulin C23 were also recovered from surviving colonies after cold storage in milk which may explain the failure of this combination to inhibit L. monocytogenes. Remarkably, the fraction of resistant mutants isolated from the combined treatment was lower than that from each antimicrobial alone, suggesting that synergy between bacteriocins and phages could be due to a lower rate of resistance development and the absence of cross-resistance.This research study was supported by the mobility grant PRI-AIBNZ-2011-1043 (Ministry of Science and Innovation, Spain) to BM and SPN12-01 (Royal Society of New Zealand) to CB. BM, PG and AR also acknowledge funding by grants BIO2010-17414 and AGL2012-40194-C02-01 (Ministry of Science and Innovation, Spain), and Plan de Ciencia, Tecnología e Innovación 2013–2017 (Principado de Asturias, Spain) and FEDER EU funds GRUPIN14-139. PG, BM and AR are members of the FWO Vlaanderen funded “Phagebiotics” research community (WO.016.14).Peer Reviewe

Extracellular Monomeric and Aggregated Tau Efficiently Enter Human Neurons through Overlapping but Distinct Pathways.

In Alzheimer's disease, neurofibrillary tangle pathology appears to spread along neuronal connections, proposed to be mediated by the release and uptake of abnormal, disease-specific forms of microtubule-binding protein tau MAPT. It is currently unclear whether transfer of tau between neurons is a toxic gain-of-function process in dementia or reflects a constitutive biological process. We report two entry mechanisms for monomeric tau to human neurons: a rapid dynamin-dependent phase typical of endocytosis and a second, slower actin-dependent phase of macropinocytosis. Aggregated tau entry is independent of actin polymerization and largely dynamin dependent, consistent with endocytosis and distinct from macropinocytosis, the major route for aggregated tau entry reported for non-neuronal cells. Anti-tau antibodies abrogate monomeric tau entry into neurons, but less efficiently in the case of aggregated tau, where internalized tau carries antibody with it into neurons. These data suggest that tau entry to human neurons is a physiological process and not a disease-specific phenomenon

TREM2 shedding by cleavage at the H157-S158 bond is accelerated for the Alzheimer’s disease-associated H157Y variant

We have characterised the proteolytic cleavage events responsible for the shedding of Triggering Receptor Expressed on Myeloid cells 2 (TREM2) from primary cultures of human macrophages, murine microglia and TREM2-expressing human embryonic kidney (HEK293) cells. In all cell types, a soluble 17 kDa N-terminal cleavage fragment was shed into the conditioned media in a constitutive process that is inhibited by G1254023X and metalloprotease inhibitors and siRNA targeting ADAM10. Inhibitors of serine proteases and matrix metalloproteinases 2/9, and ADAM17 siRNA did not block TREM2 shedding. Peptidomimetic protease inhibitors highlighted a possible cleavage site and mass spectrometry confirmed that shedding occurred predominantly at the H157-S158 peptide bond for both wild type and H157Y human TREM2 and for the wild type murine orthologue. Crucially, we also show that the Alzheimer diseaseassociated H157Y TREM2 variant was shed more rapidly than wild type from HEK293 cells, possibly by a novel, batimastat- and ADAM10-siRNA-independent, sheddase activity. These insights offer new therapeutic targets for modulating the innate immune response in Alzheimer’s and other neurological diseases.Funding from the Wellcome Trust and the Canadian Institutes of Health Research contributed to the support of this study

Exploring the role of phages in food preservation by hurdle technology

Trabajo presentado en la EMBO Conference "Viruses of microbes: Structure and function, from molecules to communities", celebrada en Zurich del 14 al 18 de julio de 2014.Peer reviewe

GABA(B) receptor isoforms GBR1a and GBR1b, appear to be associated with pre- and post-synaptic elements respectively in rat and human cerebellum

1. Metabotropic γ-aminobutyric acid (GABA) receptors, GABA(B), are coupled through G-proteins to K(+) and Ca(2+) channels in neuronal membranes. Cloning of the GABA(B) receptor has not uncovered receptor subtypes, but demonstrated two isoforms, designated GBR1a and GBR1b, which differ in their N terminal regions. In the rodent cerebellum GABA(B) receptors are localized to a greater extent in the molecular layer, and are reported to exist on granule cell parallel fibre terminals and Purkinje cell (PC) dendrites, which may represent pre- and post-synaptic receptors. 2. The objective of this study was to localize the mRNA splice variants, GBR1a and GBR1b for GABA(B) receptors in rat cerebellum, for comparison with the localization in human cerebellum using in situ hybridization. 3. Receptor autoradiography was performed utilizing [(3)H]-CGP62349 to localize GABA(B) receptors in rat and human cerebellum. Radioactively labelled oligonucleotide probes were used to localize GBR1a and GBR1b, and by dipping slides in photographic emulsion, silver grain images were obtained for quantification at the cellular level. 4. Binding of 0.5 nM [(3)H]-CGP62349 demonstrated significantly higher binding to GABA(B) receptors in the molecular layer than the granule cell (GC) layer of rat cerebellum (molecular layer binding 200±11% of GC layer; P<0.0001). GBR1a mRNA expression was found to be predominantly in the GC layer (PC layer grains 6±6% of GC layer grains; P<0.05), and GBR1b expression predominantly in PCs (PC layer grains 818±14% of GC layer grains; P<0.0001). 5. The differential distribution of GBR1a and GBR1b mRNA splice variants for GABA(B) receptors suggests a possible association of GBR1a and GBR1b with pre- and post-synaptic elements respectively