Biogeochemical characterization of four depleted gas reservoirs for conversion into underground hydrogen storage

Depleted gas reservoirs are a valuable option for underground hydrogen storage (UHS). However, different classes of microorganisms, which are capable of using free H₂ as a reducing agent for their metabolism, inhabit deep underground formations and can potentially affect the storage. This study integrates metagenomics based on Illumina-NGS sequencing of bacterial and archaeal 16S rRNA and dsrB and mcrA functional genes to unveil the composition and the variability of indigenous microbial populations of four Italian depleted reservoirs. The obtained mcrA sequences allow us to implement the existing taxonomic database for mcrA gene sequences with newly classified sequences obtained from the Italian gas reservoirs. Moreover, the KEGG and COG predictive functional annotation was used to highlight the metabolic pathways potentially associated with hydrogenotrophic metabolisms. The analyses revealed the specificity of each reservoir microbial community, and taxonomic and functional data highlighted the presence of an enriched number of taxa, whose activity depends on both reservoir hydrochemical composition and nutrient availability, of potential relevance in the context of UHS. This study is the very first to address the profiling of the microbial population and allowed us to perform a preliminary assessment of UHS feasibility in Italy.     

Transfected Poly(I:C) Activates Different dsRNA Receptors,Leading to Apoptosis or Immunoadjuvant Response in Androgen-independent Prostate Cancer Cells

Despite the effectiveness of surgery or radiation therapy for the treatment of early-stage prostate cancer (PCa), there is currently no effective strategy for late-stage disease. New therapeutic targets are emerging; in particular, dsRNA receptors Toll-like receptor 3 (TLR3) and cytosolic helicases expressed by cancer cells, once activated, exert a pro-apoptotic effect in different tumors. We previously demonstrated that the synthetic analog of dsRNA poly(I:C) induces apoptosis in the androgen-dependent PCa cell line LNCaP in a TLR3-dependent fashion, whereas only a weak apoptotic effect is observed in the more aggressive and androgen-independent PCa cells PC3 and DU145. In this paper, we characterize the receptors and the signaling pathways involved in the remarkable apoptosis induced by poly(I:C) transfected by Lipofectamine (in-poly(I:C)) compared with the 12-fold higher free poly(I:C) concentration in PC3 and DU145 cells. By using genetic inhibition of different poly(I:C) receptors, we demonstrate the crucial role of TLR3 and Src in in-poly(I:C)-induced apoptosis. Therefore, we show that the increased in-poly(I:C) apoptotic efficacy is due to a higher binding of endosomal TLR3. On the other hand, we show that in-poly(I:C) binding to cytosolic receptors MDA5 and RIG-I triggers IRF3-mediated signaling, leading uniquely to the up-regulation of IFN-β, which likely in turn induces increased TLR3, MDA5, and RIG-I proteins. In summary, in-poly(I:C) activates two distinct antitumor pathways in PC3 and DU145 cells: one mediated by the TLR3/Src/STAT1 axis, leading to apoptosis, and the other one mediated by MDA5/RIG-I/IRF3, leading to immunoadjuvant IFN-β expression.     

Quantitative analysis of the impact of a human pathogenic mutation on the CCT5 chaperonin subunit using a proxy archaeal ortholog

The human chaperonin complex is a ~ 1 MDa nanomachine composed of two octameric rings formed from eight similar but non-identical subunits called CCT. Here, we are elucidating the mechanism of a heritable CCT5 subunit mutation that causes profound neuropathy in humans. In previous work, we introduced an equivalent mutation in an archaeal chaperonin that assembles into two octameric rings like in humans but in which all subunits are identical. We reported that the hexadecamer formed by the mutant subunit is unstable with impaired chaperoning functions. This study quantifies the loss of structural stability in the hexadecamer due to the pathogenic mutation, using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The disassembly of the wild type complex, which is tightly coupled with subunit denaturation, was decoupled by the mutation without affecting the stability of individual subunits. Our results verify the effectiveness of the homo-hexadecameric archaeal chaperonin as a proxy to assess the impact of subtle defects in heterologous systems with mutations in a single subunit.     

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.

     

Synthesis and in vitro antimicrobial activities of new (cyano-NNO-azoxy)pyrazole derivatives

The antibacterial and antifungal activity of a series of products, in which the 1,5-dimethyl-4-(cyano-NNO-azoxy)pyrazol-3-yl and 1,3-dimethyl-4-(cyano-NNO-azoxy)pyrazol-5-yl moieties were linked to pyridine, pyrazole, isoxazole, thiophene and the furan ring, were examined. No molecule displayed activity against the Gram-negative bacteria tested. Conversely, some compounds displayed activity against two Staphylococcus aureus strains, including the methicillin resistant strain. All compounds displayed interesting antifungal activity, the most active compound of the series being the thiophene derivative 7a. This compound’s activity against Candidakrusei and Candidaglabrata (MIC = 0.25 and 0.5 μg/mL, respectively), two fungal species resistant to azoles, is noteworthy. The presence of the cyano function appeared essential for activity.     

Sensitivity to cisplatin in primary cell lines derived from human glioma correlates with levels of EGR-1 expression

Background  

Less than 30% of malignant gliomas respond to adjuvant chemotherapy. Here, we have asked whether variations in the constitutive expression of early-growth response factor 1 (EGR-1) predicted acute cytotoxicity and clonogenic cell death in vitro, induced by six different chemotherapics.     

Activation of proteinase-activated receptor-1 inhibits neurally evoked chloride secretion in the mouse colon in vitro

The proteinase-activated thrombin receptor-1 (PAR-1) belongs to a unique family of G protein-coupled receptors activated by proteolytic cleavage. We studied the effect of PAR-1 activation in the regulation of ion transport in mouse colon in vitro. Expression of PAR-1 in mouse colon was assessed by RT-PCR and immunohistochemistry. To study the role of PAR-1 activation in chloride secretion, mouse colon was mounted in Ussing chambers. Changes in short-circuit current (Isc) were measured in tissues exposed to either thrombin, saline, the PAR-1-activating peptide TFLLR-NH2, or the inactive reverse peptide RLLFT-NH2, before electrical field stimulation (EFS). Experiments were repeated in the presence of either a PAR-1 antagonist or in PAR-1-deficient mice to assess receptor specificity. In addition, studies were conducted in the presence of chloride-free buffer or the muscarinic antagonist atropine to assess chloride dependency and the role of cholinergic neurons in the PAR-1-induced effect. PAR-1 mRNA was expressed in full-thickness specimens and mucosal scrapings of mouse colon. PAR-1 immunoreactivity was found on epithelial cells and on neurons in submucosal ganglia where it was colocalized with both VIP and neuropeptide Y. After PAR-1 activation by thrombin or TFLLR-NH2, secretory responses to EFS but not those to forskolin or carbachol were significantly reduced. The reduction in the response to EFS was not observed in the presence of the PAR-1 antagonist, in PAR-1-deficient mice, when chloride was excluded from the bathing medium, or when atropine was present. PAR-1 is expressed in submucosal ganglia in the mouse colon and its activation leads to a decrease in neurally evoked epithelial chloride secretion.     

Glycine residues appear to be evolutionarily conserved for their ability to inhibit aggregation

Six glycine residues of human muscle acylphosphatase (AcP) are evolutionarily conserved across the three domains of life. We have generated six variants of AcP, each having a glycine substituted by an alanine (G15A, G19A, G37A, G45A, G53A, and G69A). Three additional variants had Gly45 replaced by serine, glutamate, and arginine, respectively. The mutational variants do not, on average, have a lower conformational stability than other variants with substitutions of nonconserved residues. In addition, only the G15A variant is enzymatically inactive. However, all variants, with the exception of the G15A mutant, form amyloid aggregates more rapidly than the wild-type. Dynamic light-scattering experiments carried out under conditions close to physiological confirm that aggregate formation is generally more pronounced for the glycine-substituted variants. Apart from the glycine at position 15, all other conserved glycine residues in this protein could have been maintained during evolution because of their ability to inhibit aggregation.