Diverse reductive dehalogenases are associated with Clostridiales-enriched microcosms dechlorinating 1,2-dichloroethane

The achievement of successful biostimulation of active microbiomes for the cleanup of a polluted site is strictly dependent on the knowledge of the key microorganisms equipped with the relevant catabolic genes responsible for the degradation process. In this work, we present the characterization of the bacterial community developed in anaerobic microcosms after biostimulation with the electron donor lactate of groundwater polluted with 1,2-dichloroethane (1,2-DCA). Through a multilevel analysis, we have assessed (i) the structural analysis of the bacterial community; (ii) the identification of putative dehalorespiring bacteria; (iii) the characterization of functional genes encoding for putative 1,2-DCA reductive dehalogenases (RDs). Following the biostimulation treatment, the structure of the bacterial community underwent a notable change of the main phylotypes, with the enrichment of representatives of the order Clostridiales. Through PCR targeting conserved regions within known RD genes, four novel variants of RDs previously associated with the reductive dechlorination of 1,2-DCA were identified in the metagenome of the Clostridiales-dominated bacterial community

Hypoxia and hydrogen sulfide differentially affect normal and tumor-derived vascular endothelium

Background: endothelial cells play a key role in vessels formation both under physiological and pathological conditions. Their behavior is influenced by blood components including gasotransmitters (H2S, NO and CO). Tumor cells are subjected to a cyclic shift between pro-oxidative and hypoxic state and, in this scenario, H2S can be both cytoprotective and detrimental depending on its concentration. H2S effects on tumors onset and development is scarcely studied, particularly concerning tumor angiogenesis. We previously demonstrated that H2S is proangiogenic for tumoral but not for normal endothelium and this may represent a target for antiangiogenic therapeutical strategies. Methods: in this work, we investigate cell viability, migration and tubulogenesis on human EC derived from two different tumors, breast and renal carcinoma (BTEC and RTEC), compared to normal microvascular endothelium (HMEC) under oxidative stress, hypoxia and treatment with exogenous H2S. Results: all EC types are similarly sensitive to oxidative stress induced by hydrogen peroxide; chemical hypoxia differentially affects endothelial viability, that results unaltered by real hypoxia. H2S neither affects cell viability nor prevents hypoxia and H2O2-induced damage. Endothelial migration is enhanced by hypoxia, while tubulogenesis is inhibited for all EC types. H2S acts differentially on EC migration and tubulogenesis. Conclusions: these data provide evidence for a great variability of normal and altered endothelium in response to the environmental conditions. Keywords: Hydrogen sulfide, Human microvascular endothelial cells, Human breast carcinoma-derived EC, Human renal carcinoma-derived EC, Tumor angiogenesi

Digging into protein metalation differences triggered by fluorine containing-dirhodium tetracarboxylate analogues

The catalytic and biological properties of dirhodium tetracarboxylates ([Rh2(μ-O2CR)4L2], L = axial ligand and R = CH3-, CH3CH2-, etc.) largely depend on the nature of bridging carboxylate equatorial μ-O2CR ligands, which can be easily exchanged by solvent molecules when R is CF3 (i.e. μ-O2CR is trifluoroacetate, tfa). Here, we prepared the [Rh2(OAc)(tfa)3] compound and investigated its interaction with bovine pancreatic ribonuclease and lysozyme under the same conditions used to study the reactivity of these proteins with [Rh2(OAc)4] and [cis-Rh2(OAc)2(tfa)2]. UV-vis absorption spectroscopy and 19F nuclear magnetic resonance studies indicate that [Rh2(OAc)(tfa)3] rapidly loses tfa ligands and interacts with the proteins. Crystallographic data demonstrate that the reaction of [Rh2(OAc)(tfa)3] with proteins can lead to products that are significantly different when compared to those obtained with [Rh2(OAc)4] and [cis-Rh2(OAc)2(tfa)2]: the dirhodium centre can bind the side chain of His residues at both axial and equatorial sites, at variance with what is found in the case of [Rh2(OAc)4] and [cis-Rh2(OAc)2(tfa)2]. These data indicate that the hydrolysis of dirhodium tetracarboxylates plays a significant role in defining their reaction with proteins allowing the formation of unexpected reaction products. These results suggest that [cis-Rh2(OAc)2(tfa)2] and [Rh2(OAc)(tfa)3] can be used to obtain different dirhodium/peptide and dirhodium/protein adducts with distinct catalytic properties and can explain the different cytotoxicity exhibited by tfa-containing dirhodium tetracarboxylates

Reactivity of a fluorine-containing dirhodium tetracarboxylate compound with proteins

: Dirhodium complexes of general formula [Rh2(O2CR)4]L2 are a well-known class of bimetallic compounds that are used as efficient catalysts for a variety of reactions and have been shown to be potent antibacterial and anticancer agents. The catalytic and biological properties of these complexes largely depend on the nature of the bridging carboxylate ligands. Trifluoroacetate (tfa)-containing dirhodium compounds have been used to build artificial metalloenzymes upon reaction with peptides and have been shown to be more cytotoxic than dirhodium tetraacetate. However, there is no structural information on the interaction between these compounds and proteins. Here, cis-Rh2(μ-O2CCH3)2(μ-O2CCF3)2 ([cis-Rh2(OAc)2(tfa)2]) has been synthesized and its reaction with bovine pancreatic ribonuclease (RNase A) and hen egg white lysozyme (HEWL) was analyzed using a combination of different techniques, including Fluorine-19 nuclear magnetic resonance spectroscopy and macromolecular X-ray crystallography, with the aim to unveil the differences in the reactivity of tfa-containing dihrodium complexes with proteins when compared to [Rh2(OAc)4]. [cis-Rh2(OAc)2(tfa)2] and [Rh2(OAc)4] bind the N atoms of His side chains of RNase A at the axial position; however the fluorine-containing compound rapidly loses its tfa ligands, while [Rh2(OAc)4] can retain the acetate ligands upon protein binding. The reactivity of [cis-Rh2(OAc)2(tfa)2] with HEWL is slightly distinct when compared to that of [Rh2(OAc)4] under the same experimental conditions; however, both [cis-Rh2(OAc)2(tfa)2] and [Rh2(OAc)4] degrade when soaked within HEWL crystals. These results provide a structural-based guide for the design of new heterogenous chiral dirhodium/peptide and dirhodium/protein adducts with application in the fields of organic synthesis and asymmetric catalysis