Evolutionary and Functional Analysis of Celiac Risk Loci Reveals SH2B3 as a Protective Factor against Bacterial Infection

Celiac disease (CD) is an intolerance to dietary proteins of wheat, barley, and rye. CD may have substantial morbidity, yet it is quite common with a prevalence of 1%–2% in Western populations. It is not clear why the CD phenotype is so prevalent despite its negative effects on human health, especially because appropriate treatment in the form of a gluten-free diet has only been available since the 1950s, when dietary gluten was discovered to be the triggering factor. The high prevalence of CD might suggest that genes underlying this disease may have been favored by the process of natural selection. We assessed signatures of selection for ten confirmed CD-associated loci in several genome-wide data sets, comprising 8154 controls from four European populations and 195 individuals from a North African population, by studying haplotype lengths via the integrated haplotype score (iHS) method. Consistent signs of positive selection for CD-associated derived alleles were observed in three loci: IL12A, IL18RAP, and SH2B3. For the SH2B3 risk allele, we also show a difference in allele frequency distribution (F_st) between HapMap phase II populations. Functional investigation of the effect of the SH2B3 genotype in response to lipopolysaccharide and muramyl dipeptide revealed that carriers of the SH2B3 rs3184504^∗A risk allele showed stronger activation of the NOD2 recognition pathway. This suggests that SH2B3 plays a role in protection against bacteria infection, and it provides a possible explanation for the selective sweep on SH2B3, which occurred sometime between 1200 and 1700 years ago.     

Isolation of an <Emphasis Type="Italic">Escherichia coli</Emphasis> K4 <Emphasis Type="Italic">kfoC</Emphasis> mutant over-producing capsular chondroitin

Background  

Chondroitin sulphate is a complex polysaccharide having important structural and protective functions in animal tissues. Extracted from animals, this compound is used as a human anti-inflammatory drug. Among bacteria, Escherichia coli K4 produces a capsule containing a non-sulphate chondroitin and its development may provide an efficient and cheap fermentative production of the polysaccharide.     

Perturbation of cytochrome P450, generation of oxidative stress and induction of DNA damage in Cyprinus carpio exposed in situ to potable surface water

Epidemiological evidence suggests a link between consumption of chlorinated drinking water and various cancers. Chlorination of water rich in organic chemicals produces carcinogenic organochlorine by-products (OBPs) such as trihalomethanes and haloacetic acids. Since the discovery of the first OBP in the 1970s, there have been several investigations designed to determine the biological effects of single chemicals or small artificial OBP combinations. However, there is still insufficient information regarding the general biological response to these compounds, and further studies are still needed to evaluate their potential genotoxic effects. In the current study, we evaluated the effect of three drinking water disinfectants on the activity of cytochrome P450 (CYP)-linked metabolizing enzymes and on the generation of oxidative stress in the livers of male and female Cyprinus carpio fish (carp). The fish were exposed in situ for up 20 days to surface water obtained from the Trasmene lake in Italy. The water was treated with 1-2 mg/L of either sodium hypochlorite (NaClO) or chlorine dioxide (ClO2) as traditional disinfectants or with a relatively new disinfectant product, peracetic acid (PAA). Micronucleus (MN) frequencies in circulating erythrocytes from the fish were also analysed as a biomarker of genotoxic effect. In the CYP-linked enzyme assays, a significant induction (up to a 57-fold increase in the deethylation of ethoxyresorufin with PAA treatment) and a notable inactivation (up to almost a 90% loss in hydroxylation of p-nitrophenol with all disinfectants, and of testosterone 2beta-hydroxylation with NaClO) was observed in subcellular liver preparations from exposed fish. Using the electron paramagnetic resonance (EPR) spectroscopy radical-probe technique, we also observed that CYP-modulation was associated with the production of reactive oxygen species (ROS). In addition, we found a significant increase in MN frequency in circulating erythrocytes after 10 days of exposure of fish to water treated with ClO2, while a non-significant six-fold increase in MN frequency was observed with NaClO, but not with PAA. Our data suggest that the use of ClO2 and NaClO to disinfect drinking water could generate harmful OBP mixtures that are able to perturb CYP-mediated reactions, generate oxidative stress and induce genetic damage. These data may provide a mechanistic explanation for epidemiological studies linking consumption of chlorinated drinking water to increased risk of urinary, gastrointestinal and bladder cancers.     

Effects of TGF-beta and glucocorticoids on map kinase phosphorylation, IL-6/IL-11 secretion and cell proliferation in primary cultures of human lung fibroblasts

Transforming growth factor-beta1 (TGF-beta1) is crucially involved in the fibrotic events characterizing interstitial lung diseases (ILDs), as well as in the airway remodeling process typical of asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal and fibrotic human lung fibroblasts (HLFs), the effects of TGF-beta1 on mitogen-activated protein kinase (MAPK) phosphorylation, cell proliferation, and production of interleukins 6 (IL-6) and 11 (IL-11), in the presence or absence of a pretreatment with budesonide (BUD). MAPK phosphorylation was detected by Western blotting, cell viability and proliferation were evaluated using Trypan blue staining and [(3)H]-thymidine incorporation assay, respectively, and the release of IL-6 and IL-11 into cell culture supernatants was assessed by ELISA. TGF-beta1 (10 ng/ml) significantly stimulated MAPK phosphorylation (P < 0.01), and also enhanced cell proliferation as well as the secretion of both IL-6 and IL-11, which reached the highest increases at the 72nd h of cell exposure to this growth factor. All such effects were prevented by BUD (10(-8) M) and, with the exception of IL-6 release, also by a mixture of MAPK inhibitors. Therefore, our findings suggest that the fibrotic action exerted by TGF-beta1 in the lung is mediated at least in part by MAPK activation and by an increased synthesis of the profibrogenic cytokines IL-6 and IL-11; all these effects appear to be prevented by corticosteroids via inhibition of MAPK phosphorylation.     

Two different pathways are involved in peroxynitrite-induced senescence and apoptosis of human erythrocytes

CO(2) changes the biochemistry of peroxynitrite basically in two ways: (i) nitrating species is the CO(3)(-) / ()NO(2) radical pair, and (ii) peroxynitrite diffusion distance is significantly reduced. For peroxynitrite generated extracellularly this last effect is particularly dramatic at low cell density because CO(3)(-) and ()NO(2) are short-lived and decay mostly in the extracellular space or at the cell surface/membrane. This study was aimed to distinguish between peroxynitrite-induced extra- and intracellular modifications of red blood cells (RBC). Our results show that at low cell density and in the presence of CO(2) peroxynitrite induced the oxidation of surface thiols, the formation of 3-nitrotyrosine and DMPO-RBC adducts, and the down-regulation of glycophorins A and C (biomarkers of senescence). Reactivation of glycolysis reversed only the oxidation of surface thiols. Without CO(2) peroxynitrite also induced the oxidation of hemoglobin and glutathione, the accumulation of lactate, a decrease in ATP, the clustering of band 3, the externalization of phosphatidylserine, and the activation of caspases 8 and 3 (biomarkers of apoptosis). The latter biomarkers were all reversed by reactivation of glycolysis. We hypothesize that cell senescence could (generally) be derived by irreversible radical-mediated oxidation of membrane targets, while the appearance of apoptotic biomarkers could be bolstered by oxidation of intracellular targets. These results suggest that, depending on extracellular homolysis or diffusion to the intracellular space, peroxynitrite prompts RBCs toward either senescence or apoptosis through different oxidation mechanisms.     

The primordial metabolism: an ancestral interconnection between leucine,arginine, and lysine biosynthesis

Background

It is generally assumed that primordial cells had small genomes with simple genes coding for enzymes able to react with a wide range of chemically related substrates, interconnecting different metabolic routes. New genes coding for enzymes with a narrowed substrate specificity arose by paralogous duplication(s) of ancestral ones and evolutionary divergence. In this way new metabolic pathways were built up by primordial cells. Useful hints to disclose the origin and evolution of ancestral metabolic routes and their interconnections can be obtained by comparing sequences of enzymes involved in the same or different metabolic routes. From this viewpoint, the lysine, arginine, and leucine biosynthetic routes represent very interesting study-models. Some of the lys, arg and leu genes are paralogs; this led to the suggestion that their ancestor genes might interconnect the three pathways. The aim of this work was to trace the evolutionary pathway leading to the appearance of the extant biosynthetic routes and to try to disclose the interrelationships existing between them and other pathways in the early stages of cellular evolution.

Results

The comparative analysis of the genes involved in the biosynthesis of lysine, leucine, and arginine, their phylogenetic distribution and analysis revealed that the extant metabolic "grids" and their interrelationships might be the outcome of a cascade of duplication of ancestral genes that, according to the patchwork hypothesis, coded for unspecific enzymes able to react with a wide range of substrates. These genes belonged to a single common pathway in which the three biosynthetic routes were highly interconnected between them and also to methionine, threonine, and cell wall biosynthesis. A possible evolutionary model leading to the extant metabolic scenarios was also depicted.

Conclusion

The whole body of data obtained in this work suggests that primordial cells synthesized leucine, lysine, and arginine through a single common metabolic pathway, whose genes underwent a set of duplication events, most of which can have predated the appearance of the last common universal ancestor of the three cell domains (Archaea, Bacteria, and Eucaryotes). The model proposes a relative timing for the appearance of the three routes and also suggests a possible evolutionary pathway for the assembly of bacterial cell-wall.

     

Hypoxia enhances proliferation and stemness of human adipose-derived mesenchymal stem cells

The aim of the study was to obtain the highest number of multipotent adipose-derived mesenchymal stem cells (ADMSCs) by using culture conditions which favour cell expansion without loss of mesenchymal stem cells (MSC)-like properties. Based on the assumption that stem cells reside in niches characterized by hypoxic condition, we investigated if the low oxygen tension may improve the proliferation and stemness of ADMSCs. Intact adipose tissue was resected from eight subjects, and the stromal vascular fraction was obtained by using type II collagenase. The heterogeneity of cellular lineages was confirmed by immunophenotypic analysis that showed the presence of leukocytes (CD45+), endothelial cells (CD34+), and pericytes (CD140+). The immunophenotype of confluent ADMSCs was similar to that of bone marrow-derived MSCs, except for the expression of CD34, which was variable (donor-dependent) and inversely correlated to the CD36 expression. ADMSCs showed a high clonal efficiency (94.5 ± 1 %) and were able to generate osteoblastic, chondrocytic and adipocytic lineages. ADMSCs were cultured under normoxic (21 % O₂) and hypoxic (1 % O₂) conditions, and we found that hypoxia significantly favoured ADMSC proliferation and preserved the expression of stemness genes, i.e. Nanog and Sox2. Since hypoxia reflects the microenvironment in which ADMSCs must proliferate and differentiate, the culture in hypoxic condition allows to better understand the biology of these cells and their regenerative potential. Low oxygen concentrations promote cell proliferation and stemness, thus enriching the pool of cells potentially able to differentiate into multi-lineages, and extending the possibility of a long-term expansion.