Earthquake forecasting in Italy, before and after Umbria-Marche seismic sequence 1997. A review of the earthquake occurrence modeling at different spatio-temporal-magnitude scales.

The main goal of this work is to review the scientific researches carried out before and after the Umbria-Marche sequence related to the earthquake forecasting/prediction in Italy. In particular, I focus the attention on models that aim addressing three main practical questions: was (is) Umbria-Marche a region with high probability of occurrence of a destructive earthquake? Was a precursory activity recorded before the mainshock(s)? What was our capability to model the spatio-temporal-magnitude evolution of that seismic sequence? The models are reviewed pointing out what we have learned after the Umbria-Marche earthquakes, in terms of physical understanding of earthquake occurrence process, and of improving our capability to forecast earthquakes and to track in real-time seismic sequences

Comparative genotypic and phenotypic analysis of human peripheral blood monocytes and surrogate monocyte-like cell lines commonly used in metabolic disease research

<div><p>Monocyte-like cell lines (MCLCs), including THP-1, HL-60 and U-937 cells, are used routinely as surrogates for isolated human peripheral blood mononuclear cells (PBMCs). To systematically evaluate these immortalised cells and PBMCs as model systems to study inflammation relevant to the pathogenesis of type II diabetes and immuno-metabolism, we compared mRNA expression of inflammation-relevant genes, cell surface expression of cluster of differentiation (CD) markers, and chemotactic responses to inflammatory stimuli. Messenger RNA expression analysis suggested most genes were present at similar levels across all undifferentiated cells, though notably, <i>IDO1</i>, which encodes for indoleamine 2,3-dioxygenase and catabolises tryptophan to kynureninase (shown to be elevated in serum from diabetic patients), was not expressed in any PMA-treated MCLC, but present in GM-CSF-treated PBMCs. There was little overall difference in the pattern of expression of CD markers across all cells, though absolute expression levels varied considerably and the correlation between MCLCs and PBMCs was improved upon MCLC differentiation. Functionally, THP-1 and PBMCs migrated in response to chemoattractants in a transwell assay, with varying sensitivity to MCP-1, MIP-1α and LTB-4. However, despite similar gene and CD expression profiles, U-937 cells were functionally impaired as no migration was observed to any chemoattractant. Our analysis reveals that the MCLCs examined only partly replicate the genotypic and phenotypic properties of human PBMCs. To overcome such issues a universal differentiation protocol should be implemented for these cell lines, similar to those already used with isolated monocytes. Although not perfect, in our hands the THP-1 cells represent the closest, simplified surrogate model of PBMCs for study of inflammatory cell migration.</p></div

Effects of differentiation treatment on cluster of differentiation (CD) markers expression on human peripheral blood monocytes (PBMCs; grey bars) and monocyte-like cell lines (MCLCs; U-937; red bars and THP-1; blue bars) and concentration-response curves to proinflammatory chemoattractants using a transwell migration assay using PBMCs (grey circles), U-937 (red circles) and THP-1 (blue circles) cells.

<p>Data are expressed as % cells expressing (%PE+ population) for (<b>A</b>) CD11c, (<b>B</b>) CD163, (<b>C</b>) CD14, (<b>D</b>) CD68, (<b>E</b>) CD80, (<b>F</b>) CD86 and (<b>G</b>) HLA Class II, or as the relative level of expression (mean PE) for (<b>H</b>) CD11c, (<b>I</b>) CD163, (<b>J</b>) CD14, (<b>K</b>) CD68, (<b>L</b>) CD80, (<b>M</b>) CD86 and (<b>N</b>) HLA Class II. Grouped data are shown as mean ± SEM (n = 3–5). Data are expressed on a Log2 scale as the chemotactic index compared to basal levels following 3–4 h treatment with the various chemoattractants using (<b>O</b>) MCP-1, (<b>P</b>) fMLP, (<b>Q</b>) LTB4, (<b>R</b>) MIP1α and (<b>S</b>) 10% FBS. Grouped data are shown ± SEM (n = 3–22). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test compared to undifferentiated cells for the CD markers, and two-way ANOVA with Dunnett’s multiple comparison tests compared to vehicle control for chemotaxis, with *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 and ****<i>P</i><0.0001 deemed significant.</p

Significant upregulation or downregulation in the relative gene expression levels compared to the un-differentiated cell of monocyte-like cells lines and human macrophages upon activation by either 20 nM PMA for 24 h for the cell lines, and 10 ng/mL GM-CSF for 6 days with or without polarization using 10 ng/mL LPS and 20 ng/mL IFNγ.

<p>Summary data used to generate the Venn diagrams as shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197177#pone.0197177.g003" target="_blank">Fig 3</a></b>.</p

Relative expression analysis of chemokines, cytokines, receptors, enzymes, and regulatory factors for undifferentiated, differentiated and polarized human peripheral blood monocytes (PBMCs; A and E; white, grey and black bars respectively), and monocyte-like cell lines (MCLCs) including U-937 (B and F; red bars), THP-1 (C and G; blue bars), and HL-60 (D and H; yellow bars).

<p>PBMCs were differentiated using 10 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF) for 6 days to give M(GC) and activated using 100 ng/mL LPS and 20 μg/mL IFNγ for 24 h to generate M(GC)LPS/IFNγ. MCLCs were differentiated using 16 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 h. Grouped data are shown as mean ± SEM (n = 3–10). Relative expression data including the mean ± SEM are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197177#pone.0197177.s003" target="_blank">S3 Table</a></b>. Genes not detected were marked as <i>n/d</i>. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test compared to undifferentiated cells for the PBMCs, or by Student’s t-test for the MCLCs, with *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 and ****<i>P</i><0.0001 deemed significant.</p

Hierarchical clustered heat map of mean relative expression values for undifferentiated and differentiated CD14⁺ human peripheral blood mononuclear cells (PBMC, M(GC) and M(GC)LPS/IFNγ) and monocyte-like cell lines (MCLCs ± PMA) including THP-1, U-937 and HL-60s.

<p>PBMCs were differentiated using 10 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF) for 6 days to give M(GC) and activated using 100 ng/mL LPS and 20 μg/mL IFNγ for 24 h to generate M(GC)LPS/IFNγ. MCLCs were differentiated using 16 ng/mL phorbol-12-myristate-13-acetate (PMA) for 48 h. Data represent n = 3–10; raw data, including the relative expression ± SEM used to generate this heat map, are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197177#pone.0197177.s002" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197177#pone.0197177.s003" target="_blank">S3</a> Tables</b>. All qPCR data were normalized against both housekeeper genes <i>GAPDH</i> and <i>ACTB2</i> and the values calculated as described in the Methods. Relative expression values were then calculated versus the relevant non-differentiated cell type. Where no expression was detected a value of 0 was used. Thirty-five genes that encode for inflammatory chemokines, cytokines, adipokines and their relevant receptors were selected as they are associated with inflammation or have been implicated in the development and/or progression of obesity-induced insulin resistance. In addition, small subsets of genes encoding for regulatory factors and enzymatic processes that have been implicated in the pathogenesis of T2DM were profiled. Differential gene expression data were analysed using R version 3.4.1 and hierarchical clustering was performed using complete linkage method with Euclidean distance measure.</p

Murine GPRC6A Mediates Cellular Responses to L-Amino Acids, but Not Osteocalcin Variants

<div><p>Phenotyping of <i>Gprc6a</i> KO mice has shown that this promiscuous class C G protein coupled receptor is variously involved in regulation of metabolism, inflammation and endocrine function. Such effects are described as mediated by extracellular calcium, L-amino acids, the bone-derived peptide osteocalcin (OCN) and the male hormone testosterone, introducing the concept of a bone-energy-metabolism-reproduction functional crosstalk mediated by GPRC6A. However, whilst the calcium and L-amino acid-sensing properties of GPRC6A are well established, verification of activity of osteocalcin at both human and mouse GPRC6A <i>in vitro</i> has proven somewhat elusive. This study characterises the <i>in vitro</i> pharmacology of mouse GPRC6A in response to its putative ligands in both recombinant and endogenous GPRC6A-expressing cells. Using cell signalling, and glucagon-like peptide (GLP)-1 and insulin release assays, our results confirm that basic L-amino acids act as agonists of the murine GPRC6A receptor in both recombinant cells and immortalised entero-endocrine and pancreatic β-cells. In contrast, our studies do not support a role for OCN as a direct ligand for mouse GPRC6A, suggesting that the reported <i>in vivo</i> effects of OCN that require GPRC6A may be indirect, rather than <i>via</i> direct activation of the receptor.</p></div

Modulation of cAMP levels and ERK1/2 phosphorylation in FlpIn-TREx-HEK293-mGPRC6A cells.

<p>(A) OCN variants do not stimulate cAMP accumulation in FlpIn-TREx-HEK293 cells stably expressing mGPRC6A (open circles). Neither L-ornithine nor OCN variants inhibit forksolin (3 μM)-stimulated cAMP accumulation in the same cell line (filled circles; OCN variants as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146846#pone.0146846.t001" target="_blank">Table 1</a>). (B) Neither L-ornithine (1 mM) nor OCN variants (40 ng/ml) stimulate ERK1/2 phosphorylation in FlpIn-TREx-HEK293-mGPRC6A cells; (C) a similar lack of activity was observed in cells co-expressing Gα_qG66D.</p

Effect of OCN on inositol phosphate accumulation in FlpIn-TREx-HEK293-mGPRC6A cells.

<p>(A) Variants of the putative GPRC6A peptide ligand osteocalcin (OCN) fail to induce inositol phosphate accumulation in FlpIn-TREx-HEK293-mGPRC6A cells co-expressing Gα_qG66D. (B) OCN variants (40 ng/ml) do not modulate L-ornithine-stimulated inositol phosphate accumulation at mGPRC6A (OCN variants as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146846#pone.0146846.t001" target="_blank">Table 1</a>).</p

Homology modelling of the ρ-Da1a binding site in the α_1A-AR and the MT7 toxin.

<p>Views from the side of the TM bundle (Panel A), and from the top of the extracellular space (Panel B). F187^5.41, F193^5.47, F281^6.44, M292^6.55, F308^7.35 in green. D106^3.32 and the double S188^5.42/S192^5.46 in orange. F86^2.64, F288^6.51 and F312^7.39 in red. Panel C :3D structure of the three-finger fold MT7 toxin (2vlw) with the four conserved disulfide bridges in red.</p