The diet of otters (Lutra lutra) on the Agri river system, one of the most important presence sites in Italy: A molecular approach

Background. The Eurasian otter (Lutra lutra) underwent a strong decline in large areas of the Central-Western part of its distribution range, during the second half of the twentieth century. In Italy, only residual fragmented nuclei survive in the Central- Southern part of the peninsula. Nowadays, the otter is one of the most endangered mammals in Italy, and increasing the knowledge about the ecology of this species is a key step in defining fitting management strategies. Here we provide information about the diet of otter on the Agri river system, one of the most important presence sites in Italy, to understand both the species' food requirements and the impact on fish communities. Methods. DNA metabarcoding and High Throughput Sequencing were used on DNA extracted from spraints. We amplified DNA with a primer set for vertebrates, focusing efforts on the bulk of the otter's diet (fishes and amphibians). Results. Our findings showed that the diet of the otter was dominated by cyprinids (97.77%, and 99.14% of fishes), while amphibians represented 0.85% of the sequences analyzed. Results are in general accordance with previous studies based on morphological characterization; however, molecular analyses allow the resolving of some morphological uncertainties. Although the study area offers a very wide range of available prey, the diet of the otters shows marked selectivity. We highlighted a variation in prey consumed, in accordance with the typology of water system (i.e., river, lake, tributary). Some of the preys found in the diet were alien species introduced by man for sport fishing. Our findings could help define strategies useful for the conservation of the otter population in Southern Italy, suggesting management actions directed at avoiding fish community alterations through illegal stockings without severe controls on their taxonomic status. These introductions could result in a general reduction in the diversity of the otter's preys, affecting its predatory behavior

Component Interactions and Electron Transfer in Toluene/o-Xylene Monooxygenase

The multicomponent protein toluene/o-xylene monooxygenase (ToMO) activates molecular oxygen to oxidize aromatic hydrocarbons. Prior to dioxygen activation, two electrons are injected into each of two diiron(III) units of the hydroxylase, a process that involves three redox active proteins: the ToMO hydroxylase (ToMOH), Rieske protein (ToMOC), and an NADH oxidoreductase (ToMOF). In addition to these three proteins, a small regulatory protein is essential for catalysis (ToMOD). Through steady state and pre-steady state kinetics studies, we show that ToMOD attenuates electron transfer from ToMOC to ToMOH in a concentration-dependent manner. At substoichiometric concentrations, ToMOD increases the rate of turnover, which we interpret to be a consequence of opening a pathway for oxygen transport to the catalytic diiron center in ToMOH. Excess ToMOD inhibits steady state catalysis in a manner that depends on ToMOC concentration. Through rapid kinetic assays, we demonstrate that ToMOD attenuates formation of the ToMOC–ToMOH complex. These data, coupled with protein docking studies, support a competitive model in which ToMOD and ToMOC compete for the same binding site on the hydroxylase. These results are discussed in the context of other studies of additional proteins in the superfamily of bacterial multicomponent monooxygenases.National Institute of General Medical Sciences (U.S.) (5-R01-GM032134)United States. National Institutes of Health (T32GM008334

Patterns of Gene Flow Define Species of Thermophilic Archaea

A genomic view of speciation in Archaea shows higher rates of gene flow within coexisting microbial species than between them

Atomic picture of ligand migration in toluene 4-monooxygenase

Computational modeling combined with mutational and activity assays was used to underline the substrate migration pathways in toluene 4-monooxygenase, a member of the important family of bacterial multicomponent monooxygenases (BMMs). In all structurally defined BMM hydroxylases, several hydrophobic cavities in the α-subunit map a preserved path from the protein surface to the diiron active site. Our results confirm the presence of two pathways by which different aromatic molecules can enter/escape the active site. While the substrate is observed to enter from both channels, the more hydrophilic product is withdrawn mainly from the shorter channel ending at residues D285 and E214. The long channel ends in the vicinity of S395, whose variants have been seen to affect activity and specificity. These mutational effects are clearly reproduced and rationalized by the in silico studies. Furthermore, the combined computational and experimental results highlight the importance of residue F269, which is located at the intersection of the two channels.This work has been funded by the EU projects INDOX (KBBE20137613549) and ERC 2009Adg25027PELE (to V.G) and the Spanish Ministry of Education and Science project CTQ201348287 (to V.G).Peer ReviewedPostprint (author's final draft

Novel promising Linezolid analogues: rational design and biological evaluation

The rising incidence of multidrug resistance in Gram-positive pathogen bacteria represents one of the most significant challenge for scientific communities worldwide. Linezolid, an oxazolidinone antibiotic, is highly effective for treatment of serious infections caused by Gram-positive pathogens resistant to other antibiotics including meticillin-resistant S. aureus (MRSA), vancomycin-resistant enterococci (VRE) and penicillin-resistant S. pneumonia. Linezolid has a unique mechanism of action which targets protein synthesis. Unfortunately, the emergence of Linezolid-resistant of MRSA and other bacteria has already been observed highlighting the need for finding novel oxazolidinone-type drugs with improved potency. In our study we describe the design, the synthesis and biological evaluation of previously unreported Linezolid analogues bearing the urea and thiourea functionality at the C-5 position. We used a computational approach in order to find new derivatives with improved activity. We have found a Linezolid analogoue showing good anti-microbial activity. This compound is effective against S. aureus, also meticillin-resistant, vancomycin-resistant enterococci and penicillin-resistant S. pneumonia. To understand the mechanism of action of this analogue, resistant mutants of S. aureus were generated. They contain mutations in the L3 gene coding for the L3 riboprotein

Mining for encrypted peptide antibiotics in the human proteome

The emergence of drug-resistant bacteria calls for the discovery of new antibiotics. Yet, for decades, traditional discovery strategies have not yielded new classes of antimicrobial. Here, by mining the human proteome via an algorithm that relies on the sequence length, net charge, average hydrophobicity and other physicochemical properties of antimicrobial peptides, we report the identification of 2,603 encrypted peptide antibiotics that are encoded in proteins with biological function unrelated to the immune system. We show that the encrypted peptides kill pathogenic bacteria by targeting their membrane, modulate gut and skin commensals, do not readily select for bacterial resistance, and possess anti-infective activity in skin abscess and thigh infection mouse models. We also show, in vitro and in the two mouse models of infection, that encrypted antibiotic peptides from the same biogeographical area display synergistic antimicrobial activity. Our algorithmic strategy allows for the rapid mining of proteomic data and opens up new routes for the discovery of candidate antibiotics

The First Report on the Ecology and Distribution of the Wolf Population in Cilento, Vallo di Diano and Alburni National Park

The Apennine wolf (Canis lupus italicus, Altobello, 1921) is currently experiencing a period of great population change. Any information that helps to understand this transformation will be valuable for manag- ing it. Here we provide, for the first time, quantitative data (habitat suitability, segregation, seasonal activity, daily time budget, social structure and sex-ratio) on the population of Italian wolf from the Cilento, Vallo di Diano and Alburni National Park, the largest protected area in Italy, that houses a rising population. Using transects to locate signs and traces, we have identified 224 presence points, useful for elaborating the ecolog- ical suitability map for the wolf, in and around the Park. The suitable area expands up to 1042.65 km2, with elevation being the strongest predictive variable affecting the species. The model predicts two main suitable patches (Alburni Mountains and Cervati/Motola Mountains), and at least seven remote areas, differently linked to each other by ecological corridors. Camera-trapping information was collected in the two suitable patches (Alburni and Cervati/Motola) for the wolf and revealed some characteristics of this population. The highest number of wolves was recorded in May and December, and during the night, depending on the repro- ductive behavior and dynamic of dispersion. Single individuals and couples (at least 3 recorded) are observed more frequently than groups (maximum of 3 groups that varied from three to eight individuals). The sex-ratio in the population, inferred by camera-trapping, showed a balanced population, with males found to be group leader in 91% of the cases. Furthermore, genetic analysis revealed that the males were in charge of marking the territory in 86% of cases. Our data increases the knowledge of the wolf population from Cilento, which seemed to have stopped at the 1990s, until now. Our contribution could be helpful in defining a wolf manage- ment strategy in the National Park, as well as in the other regions of the Apennine mountains and Alps, where the wolf is expanding, aiming also at coexistence with local human communities

Novel bioactive peptides from PD-L1/2, a type 1 ribosome inactivating protein from Phytolacca dioica L. Evaluation of their antimicrobial properties and anti-biofilm activities

Antimicrobial peptides, also called Host Defence Peptides (HDPs), are effectors of innate immune response found in all living organisms. In a previous report, we have identified by chemical fragmentation, and characterized the first cryptic antimicrobial peptide in PD-L4, a type 1 ribosome inactivating protein (RIP) from leaves of Phytolacca dioica L. We applied a recently developed bioinformatic approach to a further member of the differently expressed pool of type 1 RIPs from P. dioica (PD-L1/2), and identified two novel putative cryptic HDPs in its N-terminal domain. These two peptides, here named IKY31 and IKY23, exhibit antibacterial activities against planktonic bacterial cells and, interestingly, significant anti-biofilm properties against two Gram-negative strains. Here, we describe that PD-L1/2 derived peptides are able to induce a strong dose-dependent reduction in biofilm biomass, affect biofilm thickness and, in the case of IKY31, interfere with cell-to-cell adhesion, likely by affecting biofilm structural components. In addition to these findings, we found that both PD-L1/2 derived peptides are able to assume stable helical conformations in the presence of membrane mimicking agents (SDS and TFE) and intriguingly beta structures when incubated with extracellular bacterial wall components (LPS and alginate). Overall, the data collected in this work provide further evidence of the importance of cryptic peptides derived from type 1 RIPs in host/pathogen interactions, especially under pathophysiological conditions induced by biofilm forming bacteria. This suggests a new possible role of RIPs as precursors of antimicrobial and anti-biofilm agents, likely released upon defensive proteolytic processes, which may be involved in plant homeostasis

Pyrene and chrysene tolerance and biodegradation capability of pleurotus sajor-caju

Introduction: The present work focused on the biodegradation capability of a white-rot fungus, the Pleurotus sajor-caju, when exposed to polycyclic aromatic hydrocarbons. Methods: The research was carried out by using in vitro systems developed on Petri dishes, to evaluate the fungal tolerance to pyrene and chrysene, followed by experiments in liquid medium. The first experimental campaign was necessary to evaluate the conditions promoting fungal growth and tolerance (presence of surfactants, peptone, copper sulphate and lecithin) and it was designed and analysed using statistical techniques. Results: It was found that the fungal population growth is strongly inhibited by chrysene presence. On the other hand, pyrene had a mild negative impact on the mycelia growth, which seemed to be positively influenced by the presence of Tween 80 and copper sulphate. Starting from these results, the behaviour of Pleurotus sajor-caju in presence of pyrene was investigated in liquid medium. Results showed that the depletion of pyrene was evident during a period of 20 days, and removal efficiency was greater than 90%