Are periodontitis and psoriasis associated? A pre-clinical murine model.

AIM: To investigate the bidirectional influence between periodontitis and psoriasis, using the respective experimental models of ligature- and imiquimod-induced diseases on murine models. MATERIALS AND METHODS: Thirty-two C57/BL6J mice were randomly allocated to four experimental groups: control (P- Pso-), ligature-induced periodontitis (P+ Pso-), imiquimod-induced psoriasis (P- Pso+) and periodontitis and psoriasis (P+ Pso+). Samples (maxilla, dorsal skin and blood) were harvested immediately after death. Measures of periodontitis (distance between the cemento-enamel junction and alveolar bone crest [CEJ-ABC] and the number of osteoclasts) and psoriasis (epidermal thickness and infiltrate cell [/0.03mm2]) severity as well as systemic inflammation (IL-6, IL-17A, TNF-α) were collected. RESULTS: The P+ Pso+ group exhibited the most severe experimental periodontitis and psoriasis, with the highest values of CEJ-ABC, number of osteoclasts, epidermal thickness and infiltrate cells in the dorsal skin, as well as the highest blood cytokine concentration. The P+ Pso- group presented with higher cell infiltrate (/0.03mm2) compared to the control group (p <.05), while the P- Pso+ group showed substantially higher alveolar bone loss (CEJ-ABC) than the control group (p <.05). CONCLUSIONS: Experimental periodontitis may initiate and maintain psoriasiform skin inflammation and, vice versa, experimental psoriasis may contribute to the onset of periodontitis. In a combined model of the diseases, we propose a bidirectional association between periodontitis and psoriasis via systemic inflammation.

Biological responses to heavy metal stress in the moss Leptodictyum riparium (Hedw.) Warnst.

Leptodictyum riparium, a widely distributed aquatic moss, can both tolerate and accumulate very high concentrations of toxic heavy metals, with only slight apparent damage. Here we report the effects on photosynthetic yield, glutathione (GSH), phytochelatin (PCn) synthesis, nitrogen metabolism and cellular localization of molecules rich in SH groups in L. riparium exposed in vitro to heavy metals. We simulated the concentrations of Cu, Zn, Cd, Pb detected in Regi Lagni, Italy, one of the most contaminated freshwater sites in Southern Europe, in the laboratory to test how the moss responds to heavy metal contamination. There was a steady decrease of photosynthetic efficiency correlated with the heavy metal concentrations and ultrastructural organization. All PCn levels increased significantly as the concentration of heavy metals increased, while the GSH levels did not appear to be particularly affected. A significant increase of GDH and NADH-GOGAT activities increased with increasing heavy metal concentration. Immunoblotting analysis revealed an increase of the chl-GS2 while no significant increase was detected in the cyt-GS1. These results give insight into the molecular events underlying the metal-tolerance of the aquatic moss L. riparium exposed to environmental heavy metal concentrations.

Ancestral function of the phytochelatin synthase C-terminal domain in inhibition of heavy metal-mediated enzyme overactivation.

Phytochelatin synthases (PCSs) play essential roles in detoxification of a broad range of heavy metals in plants and other organisms. Until now, however, no PCS gene from liverworts, the earliest branch of land plants and possibly the first one to acquire a PCS with a C-terminal domain, has been characterized. In this study, we isolated and functionally characterized the first PCS gene from a liverwort, Marchantia polymorpha (MpPCS). MpPCS is constitutively expressed in all organs examined, with stronger expression in thallus midrib. The gene expression is repressed by Cd2+ and Zn2+. The ability of MpPCS to increase heavy metal resistance in yeast and to complement cad1-3 (the null mutant of the Arabidopsis ortholog AtPCS1) proves its function as the only PCS from M. polymorpha. Site-directed mutagenesis of the most conserved cysteines of the C-terminus of the enzyme further uncovered that two twin-cysteine motifs repress, to different extents, enzyme activation by heavy metal exposure. These results highlight an ancestral function of the PCS elusive C-terminus as a regulatory domain inhibiting enzyme overactivation by essential and non-essential heavy metals. The latter finding may be relevant for obtaining crops with decreased root to shoot mobility of cadmium, thus preventing its accumulation in the food chain.

The Moss Leptodictyum riparium Counteracts Severe Cadmium Stress by Activation of Glutathione Transferase and Phytochelatin Synthase, but Slightly by Phytochelatins.

In the present work, we investigated the response to Cd in Leptodictyum riparium, a cosmopolitan moss (Bryophyta) that can accumulate higher amounts of metals than other plants, even angiosperms, with absence or slight apparent damage. High-performance liquid chromatography followed by electrospray ionization tandem mass spectrometry of extracts from L. riparium gametophytes, exposed to 0, 36 and 360 µM Cd for 7 days, revealed the presence of γ-glutamylcysteine (γ-EC), reduced glutathione (GSH), and traces of phytochelatins. The increase in Cd concentrations progressively augmented reactive oxygen species levels, with activation of both antioxidant (catalase and superoxide dismutase) and detoxifying (glutathione-S-transferase) enzymes. After Cd treatment, cytosolic and vacuolar localization of thiol peptides was performed by means of the fluorescent dye monochlorobimane and subsequent observation with confocal laser scanning microscopy. The cytosolic fluorescence observed with the highest Cd concentrations was also consistent with the formation of γ-EC-bimane in the cytosol, possibly catalyzed by the peptidase activity of the L. riparium phytochelatin synthase. On the whole, activation of phytochelatin synthase and glutathione-S-transferase, but minimally phytochelatin synthesis, play a role to counteract Cd toxicity in L. riparium, in this manner minimizing the cellular damage caused by the metal. This study strengthens previous investigations on the L. riparium ability to efficiently hinder metal pollution, hinting at a potential use for biomonitoring and phytoremediation purposes.

Evolution and functional differentiation of recently diverged phytochelatin synthase genes from Arundo donax L.

Phytochelatin synthases (PCSs) play pivotal roles in the detoxification of heavy metals and metalloids in plants; however, little information on the evolution of recently duplicated PCS genes in plant species is available. Here we characterize the evolution and functional differentiation of three PCS genes from the giant reed (Arundo donax L.), a biomass/bioenergy crop with remarkable resistance to cadmium and other heavy metals. Phylogenetic reconstruction with PCS genes from fully sequenced monocotyledonous genomes indicated that the three A. donax PCSs, namely AdPCS1-3, form a monophyletic clade. The AdPCS1-3 genes were expressed at low levels in many A. donax organs and displayed different levels of cadmium-responsive expression in roots. Overexpression of AdPCS1-3 in Arabidopsis thaliana and yeast reproduced the phenotype of functional PCS genes. Mass spectrometry analyses confirmed that AdPCS1-3 are all functional enzymes, but with significant differences in the amount of the phytochelatins synthesized. Moreover, heterogeneous evolutionary rates characterized the AdPCS1-3 genes, indicative of relaxed natural selection. These results highlight the elevated functional differentiation of A. donax PCS genes from both a transcriptional and an enzymatic point of view, providing evidence of the high evolvability of PCS genes and of plant responsiveness to heavy metal stress.

Trichormus variabilis (Cyanobacteria) Biomass: From the Nutraceutical Products to Novel EPS-Cell/Protein Carrier Systems.

A native strain of the heterocytous cyanobacterium Trichormus variabilis VRUC 168 was mass cultivated in a low-cost photobioreactor for a combined production of Polyunsaturated Fatty Acids (PUFA) and Exopolymeric Substances (EPS) from the same cyanobacterial biomass. A sequential extraction protocol was optimized leading to high yields of Released EPS (REPS) and PUFA, useful for nutraceutical products and biomaterials. REPS were extracted and characterized by chemical staining, Reversed Phase-High-Performance Liquid Chromatography (RP-HPLC), Fourier Transform Infrared Spectroscopy (FT-IR) and other spectroscopic techniques. Due to their gelation property, REPS were used to produce a photo-polymerizable hybrid hydrogel (REPS-Hy) with addition of polyethylene glycol diacrylated (PEGDa). REPS-Hy was stable over time and resistant to dehydration and spontaneous hydrolysis. The rheological and functional properties of REPS-Hy were studied. The enzyme carrier ability of REPS-Hy was assessed using the detoxification enzyme thiosulfate:cyanide sulfur transferase (TST), suggesting the possibility to use REPS-Hy as an enzymatic hydrogel system. Finally, REPS-Hy was used as a scaffold for culturing human mesenchymal stem cells (hMSCs). The cell seeding onto the REPS-Hy and the cell embedding into 3D-REPS-Hy demonstrated a scaffolding property of REPS-Hy with non-cytotoxic effect, suggesting potential applications of cyanobacteria REPS for producing enzyme- and cell-carrier systems.

Photosynthetic Efficiency of Marchantia polymorpha L. in Response to Copper, Iron, and Zinc.

Metal micronutrients are essential for plant nutrition, but their toxicity threshold is low. In-depth studies on the response of light-dependent reactions of photosynthesis to metal micronutrients are needed, and the analysis of chlorophyll a fluorescence transients is a suitable technique. The liverwort Marchantia polymorpha L., a model organism also used in biomonitoring, allowed us to accurately study the effects of metal micronutrients in vivo, particularly the early responses. Gametophytes were treated with copper (Cu), iron (Fe) or zinc (Zn) for up to 120 h. Copper showed the strongest effects, negatively affecting almost the entire light phase of photosynthesis. Iron was detrimental to the flux of energy around photosystem II (PSII), while the acceptor side of PSI was unaltered. The impact of Fe was milder than that of Cu and in both cases the structures of the photosynthetic apparatus that resisted the treatments were still able to operate efficiently. The susceptibility of M. polymorpha to Zn was low: although the metal affected a large part of the electron transport chain, its effects were modest and short-lived. Our results may provide a contribution towards achieving a more comprehensive understanding of response mechanisms to metals and their evolution in plants, and may be useful for supporting the development of biomonitoring techniques.

Anti-Tumor Efficacy of In Situ Vaccination Using Bacterial Outer Membrane Vesicles.

In situ vaccination (ISV) is a promising cancer immunotherapy strategy that consists of the intratumoral administration of immunostimulatory molecules (adjuvants). The rationale is that tumor antigens are abundant at the tumor site, and therefore, to elicit an effective anti-tumor immune response, all that is needed is an adjuvant, which can turn the immunosuppressive environment into an immunologically active one. Bacterial outer membrane vesicles (OMVs) are potent adjuvants since they contain several microbe-associated molecular patterns (MAMPs) naturally present in the outer membrane and in the periplasmic space of Gram-negative bacteria. Therefore, they appear particularly indicted for ISV. In this work, we first show that the OMVs from E. coli BL21(DE3)Δ60 strain promote a strong anti-tumor activity when intratumorally injected into the tumors of three different mouse models. Tumor inhibition correlates with a rapid infiltration of DCs and NK cells. We also show that the addition of neo-epitopes to OMVs synergizes with the vesicle adjuvanticity, as judged by a two-tumor mouse model. Overall, our data support the use of the OMVs in ISV and indicate that ISV efficacy can benefit from the addition of properly selected tumor-specific neo-antigens.

Outer Membrane Vesicles From The Gut Microbiome Contribute to Tumor Immunity by Eliciting Cross-Reactive T Cells.

A growing body of evidence supports the notion that the gut microbiome plays an important role in cancer immunity. However, the underpinning mechanisms remain to be fully elucidated. One attractive hypothesis envisages that among the T cells elicited by the plethora of microbiome proteins a few exist that incidentally recognize neo-epitopes arising from cancer mutations ("molecular mimicry (MM)" hypothesis). To support MM, the human probiotic Escherichia coli Nissle was engineered with the SIINFEKL epitope (OVA-E.coli Nissle) and orally administered to C57BL/6 mice. The treatment with OVA-E.coli Nissle, but not with wild type E. coli Nissle, induced OVA-specific CD8+ T cells and inhibited the growth of tumors in mice challenged with B16F10 melanoma cells expressing OVA. The microbiome shotgun sequencing and the sequencing of TCRs from T cells recovered from both lamina propria and tumors provide evidence that the main mechanism of tumor inhibition is mediated by the elicitation at the intestinal site of cross-reacting T cells, which subsequently reach the tumor environment. Importantly, the administration of Outer Membrane Vesicles (OMVs) from engineered E. coli Nissle, as well as from E. coli BL21(DE3)ΔompA, carrying cancer-specific T cell epitopes also elicited epitope-specific T cells in the intestine and inhibited tumor growth. Overall, our data strengthen the important role of MM in tumor immunity and assign a novel function of OMVs in host-pathogen interaction. Moreover, our results pave the way to the exploitation of probiotics and OMVs engineered with tumor specific-antigens as personalized mucosal cancer vaccines.

Responses to Cadmium in Early-Diverging Streptophytes (Charophytes and Bryophytes): Current Views and Potential Applications.

Several transition metals are essential for plant growth and development, as they are involved in various fundamental metabolic functions. By contrast, cadmium (Cd) is a metal that can prove extremely toxic for plants and other organisms in a dose-dependent manner. Charophytes and bryophytes are early-diverging streptophytes widely employed for biomonitoring purposes, as they are able to cope with high concentrations of toxic metal(loid)s without showing any apparent heavy damage. In this review, we will deal with different mechanisms that charophytes and bryophytes have evolved to respond to Cd at a cellular level. Particular attention will be addressed to strategies involving Cd vacuolar sequestration and cell wall immobilization, focusing on specific mechanisms that help achieve detoxification. Understanding the effects of metal(loid) pollution and accumulation on the morpho-physiological traits of charophytes and bryophytes can be in fact fundamental for optimizing their use as phytomonitors and/or phytoremediators.

Eukaryotic and Prokaryotic Phytochelatin Synthases Differ Less in Functional Terms Than Previously Thought: A Comparative Analysis of Marchantia polymorpha and Geitlerinema sp. PCC 7407.

This paper reports functional studies on the enzyme phytochelatin synthase in the liverwort Marchantia polymorpha and the cyanobacterium Geitlerinema sp. strain PCC 7407. In vitro activity assays in control samples (cadmium-untreated) showed that phytochelatin synthase was constitutively expressed in both organisms. In the presence of 100 µM cadmium, in both the liverwort and the cyanobacterium, the enzyme was promptly activated in vitro, and produced phytochelatins up to the oligomer PC4. Likewise, in vivo exposure to 10-36 µM cadmium for 6-120 h induced in both organisms phytochelatin synthesis up to PC4. Furthermore, the glutathione (GSH) levels in M. polymorpha were constitutively low (compared with the average content in higher plants), but increased considerably under cadmium stress. Conversely, the GSH levels in Geitlerinema sp. PCC 7407 were constitutively high, but were halved under metal treatments. At odds with former papers, our results demonstrate that, as in M. polymorpha and other plants, the cyanobacterial phytochelatin synthase exposed to cadmium possesses manifest transpeptidasic activity, being able to synthesize phytochelatins with a degree of oligomerization higher than PC2. Therefore, prokaryotic and eukaryotic phytochelatin synthases differ less in functional terms than previously thought.

Ascorbic Acid and Ozone: Novel Perspectives to Explain an Elusive Relationship.

A huge amount of studies highlighted the importance of high ascorbic acid (AA) content in ozone tolerance, yet the relationship between them appears more complex than a simple direct correlation. Sometimes the connection is clear, for example, two Arabidopsis mutants defective in the main AA biosynthetic pathway (vtc mutants) were identified by means of their ozone sensitivity. However, some low-AA containing mutants are relatively tolerant, suggesting that AA location/availability could be more relevant than total content. A clear distinction should also be made between ozone tolerance obtained when AA content is increased by experimental supplementation (exogenous AA), and the physiological role of plant-synthesized AA (endogenous AA), whose amount is apparently subjected to tight regulation. Recent findings about the role of AA in signal transduction and epigenetic regulation of gene expression open new routes to further research.

Characterization and quantification of thiol-peptides in Arabidopsis thaliana using combined dilution and high sensitivity HPLC-ESI-MS-MS.

Although thiol-peptide compounds, such as reduced glutathione (GSH), γ-glutamylcysteine (γ-EC), and phytochelatins, play fundamental roles in plants, their analytical determination and characterization is still somewhat problematic, mainly due to their high polarity and oxidation propensity. Thus, in this work a reliable and sensitive HPLC-ESI-MS-MS method was developed, in order to simultaneously assay, within 14-min instrumental runs, γ-EC, GSH, and phytochelatins up to phytochelatin 4. This analytical method was validated in shoot and root extracts of the model plant Arabidopsis thaliana (Brassicaceae) and guaranteed accurate quantification by using specific isotope labelled-internal standards for both GSH and phytochelatins, as well as standards for external calibration. Good linearities in the method performance were observed (R > 0.99), with a dynamic range over three orders of magnitude in thiol-peptide concentrations. In MRM mode, the detection sensitivity of the thiol-peptides was equal to approximately 16, 6, 7, 13, 10 fmol for γ-EC, GSH, phytochelatin 2, phytochelatin 3, and phytochelatin 4, respectively (20 µl injection each). The reproducibility of the method was confirmed by high intra- and inter-day accuracy and precision values. The recovery rates were estimated approximately in the range of 73.8-91.0% and the matrix effect evaluation revealed that all analytes exhibited ionization suppression. The use of stable isotope-labelled analogs of the thiol-peptides as internal standards was particularly worthy of note: it offered the considerable advantage of overcoming the consequences of matrix effect and thiol-peptide loss through sample preparation, by normalizing the analyte signal during the quantification process. Thus, by validating the method's sensitivity, accuracy, precision, reproducibility, stability, recovery, and matrix effect, data reliability and robustness were ensured.

The phytochelatin synthase from Nitella mucronata (Charophyta) plays a role in the homeostatic control of iron(II)/(III).

Although some charophytes (sister group to land plants) have been shown to synthesize phytochelatins (PCs) in response to cadmium (Cd), the functional characterization of their phytochelatin synthase (PCS) is still completely lacking. To investigate the metal response and the presence of PCS in charophytes, we focused on the species Nitella mucronata. A 40 kDa immunoreactive PCS band was revealed in mono-dimensional western blot by using a polyclonal antibody against Arabidopsis thaliana PCS1. In two-dimensional western blot, the putative PCS showed various spots with acidic isoelectric points, presumably originated by post-translational modifications. Given the PCS constitutive expression in N. mucronata, we tested its possible involvement in the homeostasis of metallic micronutrients, using physiological concentrations of iron (Fe) and zinc (Zn), and verified its role in the detoxification of a non-essential metal, such as Cd. Neither in vivo nor in vitro exposure to Zn resulted in PCS activation and PC significant biosynthesis, while Fe(II)/(III) and Cd were able to activate the PCS in vitro, as well as to induce PC accumulation in vivo. While Cd toxicity was evident from electron microscopy observations, the normal morphology of cells and organelles following Fe treatments was preserved. The overall results support a function of PCS and PCs in managing Fe homeostasis in the carophyte N. mucronata.

The role of psychological support in cardiac surgery: initial experience.

The scientific literature has pointed out several predictors of negative outcome after surgery such as pain and depression, negatively affecting the postoperative outcome in cardiac surgery. From January 2009 until June 2010, 15 patients scheduled for cardiac surgery were enrolled. The patients were assessed by psychological evaluation either in the hospital stay either in the rehabilitation period with the aim of identifying their emotional condition (sentiments about the onset of the disease, support received from family and friends) even by means of preformed tests for anxiety and depression (tests of Stay and Back). Thus, in our preliminary experience, the psychological evaluation failed to detect the occurrence of postoperative complications. Conversely, the psychological evaluation is very effective in detecting a poor emotional state and the psychological support decreases the degree of anxiety and depression with positive effects on postoperative outcome. In conclusion, a standardize test for anxiety and depression should be used for patients at hospital admission to detect who may benefits by psychological support.