Peptidoglycan and Bacterial DNA Induce Inflammation and Coagulation Markers in Synergy

Bacterial compounds signal the presence of foreign pathogens in the innate immune system. These microbial components are key players in infectious diseases and implicate toll-like receptors in the activation of inflammation and coagulation. Nevertheless, the existence of a synergistic relationship between peptidoglycan and bacterial DNA on these two physiological responses has not been investigated. The present study reports new findings on the regulation of tumor necrosis factor alpha and tissue factor in peripheral blood mononuclear cells by peptidoglycan and bacterial DNA. These were found to induce tumor necrosis factor alpha and tissue factor simultaneously and in a synergistic manner. These findings provide a new proinflammatory and procoagulant mechanism likely to play a role in sepsis pathogenesis

Bubble technique for Descemet membrane endothelial keratoplasty tissue preparation in an eye bank: Air or liquid?

Purpose To compare the big-bubble method using air and liquid as medium of separation for Descemet membrane endothelial keratoplasty (DMEK) lenticule preparation in an eye bank. Methods Donor corneas (n = 20) were immersed in liquid [tissue culture medium (TCM)]. Air and liquid was injected using a 25-gauge needle in the posterior stroma or as near to the stroma-Descemet membrane (DM) phase as possible to create a complete bubble of larger diameter. The endothelial cell density and mortality were checked pre- and postbubble after deflating the tissue. Four pairs of tissues were used to analyse the intracellular tight junctions and three pairs for histological examination and DNA integrity studies, respectively. Results The yield obtained using air was 80%, whereas that with liquid was 100%. Single injection was required in six cases; twice in two cases; three and four times in one case each with air bubble, whereas seven cases required single injection; twice in two cases; and thrice in just one case with liquid bubble. The average diameter of the final lenticule was 9.12 (± 1.71) mm for air bubble and 9.78 (± 1.75) mm for liquid bubble with p = 0.4362 (no statistical significance). Endothelial cell mortality postbubble preparation was 8.9 (± 12.38) % for air and 6.25 (± 9.57) % for liquid (p = 0.6268). Conclusions DM and endothelium could be separated exclusively using air or liquid bubble. However, liquid bubble seems to have certain advantages over air such as the generation of yield, larger diameter and higher maintenance of endothelial cell density and integrity

NCAM180 Regulates Ric8A Membrane Localization and Potentiates β-Adrenergic Response

Cooperation between receptors allows integrated intracellular signaling leading to appropriate physiological responses. The Neural Cell Adhesion Molecule (NCAM) has three main isoforms of 120, 140 and 180 kDa, with adhesive and signaling properties, but their respective functions remains to be fully identified. Here we show that the human NCAM180 intracellular domain is a novel interactor of the human guanosine exchange factor (GEF) Ric8A using the yeast two hybrid system and immunoprecipitation. Furthermore, NCAM, Ric8A and Gαs form a tripartite complex. Colocalization experiments by confocal microscopy revealed that human NCAM180 specifically induces the recruitment of Ric8A to the membrane. In addition, using an in vitro recombinant system, and in vivo by comparing NCAM knock-out mouse brain to NCAM heterozygous and wild type brains, we show that NCAM expression dose dependently regulates Ric8A redistribution in detergent resistent membrane microdomains (DRM). Previous studies have demonstrated essential roles for Ric8 in Gα protein activity at G protein coupled receptors (GPCR), during neurotransmitter release and for asymmetric cell division. We observed that inhibition of Ric8A by siRNA or its overexpression, decreases or increases respectively, cAMP production following β-adrenergic receptor stimulation. Furthermore, in human HEK293T recombinant cells, NCAM180 potentiates the Gαs coupled β-adrenergic receptor response, in a Ric8A dependent manner, whereas NCAM120 or NCAM140 do not. Finally, in mouse hippocampal neurons expressing endogenously NCAM, NCAM is required for the agonist isoproterenol to induce cAMP production, and this requirement depends on Ric8A. These data illustrate a functional crosstalk between a GPCR and an IgCAM in the nervous system

Ric8A is not involved in NCAM induced cortical neurite outgrowth.

<p>Embryonic mice cortical neurons were plated on poly-D-lysine alone or in combination with PSA-NCAM-Fc. Non transfected neurons, or neurons transfected with siCtrl or siRic8A were either not treated or PSA-NCAM-Fc treated. After 3 days in culture, neurons were stained with calcein as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032216#s2" target="_blank">Materials and Methods</a>, and neurite length was quantified using the Metamorph software. Data are from a representative experiment (out of 5 performed) and are expressed as mean +/− SEM. One-way ANOVA for more than 2 groups was performed. The effect of PSA-NCAM-Fc treatment, was then analysed by with the Bonferroni post-hoc test.</p

NCAM180 regulates β-adrenergic response via Ric8A in HEK293T cells and neurons.

<p>A- Representative experiment (out of 3 performed) of isoproterenol (10 µM) induced cAMP production in HEK293T cells transfected or not with NCAM180, in the presence (siCtrl) or absence (siRic8A) of Ric8A. Data are expressed as mean +/− SEM of 3 independent wells and analyzed by ANOVA and Bonferroni post-hoc test. B- Increase of cAMP production by Ric8A overexpression in HEK293T cells transfected for 48 h with Ric8A-GFP (▪) or PmaxGFP (⧫). Data are expressed as mean +/− SEM of 3 independent wells and analyzed by the t-test. C- HEK293T cells were transfected with NCAM180, and control or Ric8A siRNA for 24 h. Anti-NCAM beads were used for immunoprecipitation of cell extracts, A Western blot with anti-NCAM (first box) indicates that NCAM180 has been immunoprecipitated. A Western blot was also performed with anti-G_αs on each extract before immunoprecipitation to check the amount of loaded G_αs (second box). Anti-G_αs (third box) and anti-Ric8A (fourth box) were used on NCAM180 immunoprecipitated extracts and show that Ric8A is significantly decreased by siRic8A compared to siCtrl, and that G_αs is immunoprecipitated when Ric8A is present (siCtrl).. D- Amount of cAMP generated by isoproterenol (10 µM) that led to maximal efficacy in HEK293T, or HEK293T cells transfected with the different NCAM isoforms in the presence (siCtrl) or absence (siRic8A) of Ric8A. Data are expressed as % of siCtrl transfected cells and as mean +/− SEM of at least 3 independent experiments. Data were analyzed by ANOVA for more than 2 groups and Bonferroni post-hoc test. E- P-ERK1/2 induction by isoproterenol (10 µM) in hippocampal neurons from NCAM +/+ and −/− E15.5 embryos, in the presence (siCtrl) or absence (siRic8A) of Ric8A. Actin was used as constitutive protein. F- Amount of cAMP produced by NCAM^+/+ and NCAM^−/− non treated hippocampal neurons or after treatment with 1 µM isoproterenol in the presence (siCtrl) or absence (siRic8A) of Ric8A. Data are expressed as percentage of non-treated neurons (mean +/− SEM of 3 independent cultures). One-way ANOVA for more than 2 groups was performed. The effect of the single factor ‘isoproterenol treament’ was then analysed by the Bonferroni post-hoc test.</p

NCAM affects Ric8A relative distribution in DSM and DRM fractions <i>in vitro</i> and <i>in vivo</i>.

<p>A- Western blot of overexpressed Ric8A-V5 in cytosol, DSM and DRM. B, C- Quantitative analysis of Western blots in A. D- Western blot of Ric8A in DSM and DRM from brain extracts prepared from NCAM null (−/−), heterozygous (+/−) and wild type (+/+) mice. Caveolin-3, a marker for enrichment of lipid rich domains was detected only in DRM. E- Quantitative analysis of Western blots presented in D (n = 3 per genotype). Data are expressed as mean +/− SEM; *p<0,05, **p<0,01, ***p<0,001. One-way ANOVA for more than 2 groups was performed. The effect of the single factor ‘genotype’ was then analysed by the Bonferroni post-hoc test.</p

NCAM180 induces Ric8A-GFP recruitement at the membrane where NCAM and Ric8A colocalize.

<p>A- Ric8A-GFP co-transfected with NCAM120, NCAM140 or NCAM180 in HEK293T cells co-localizes specifically with the NCAM180 isoform. Scale bar: 10 µm. B- Ric8A-GFP or PmaxGFP plasmids were transfected in cortical neurons for 24 h. Images were taken focussing at the membrane plane with a 20× objective. NCAM staining is clearly associated to the cell membrane (B, E, K). Ric8A-GFP is strongly localized at the membrane at the level of the cell body and processes (A, D). PmaxGFP control plasmid is mostly found in the cytoplasm (J). Ric8A-GFP, but not PmaxGFP colocalizes with NCAM at the membrane (Ric8A/NCAM merge: C, F versus Pmax/NCAM merge: L). (G–I): higher magnification pictures (dotted line box D–F) using a 63× oil immersion objective indicate a strong co-localization of Ric8A-GFP with NCAM in the growth cone (I). Scale bars: 10 µm (A–F, J–O); 1 µm (G–I).</p