Metabolites from the Euryhaline Ciliate Pseudokeronopsis erythrina

TThree new secondary metabolites (named erythrolactones A2, B2 and C2), that are characterized by a central 4‐hydroxy‐unsaturated δ lactone ring bearing an alkyl saturated chain at C(2) and a butyl‐benzenoid group at C(5), together with their respective sulfate esters (erythrolactones A1, B1 and C1), have been isolated from cell cultures of Pseudokeronopsis erythrina, clone TL‐1. The structures are assigned on the basis of extensive spectroscopic measurements (1D and 2D NMR, UV, IR and HR‐MALDI‐TOF). A plausible biogenetic route for their formation is also suggested. Cold‐shock treatment was performed in order to induce the discharge of the metabolites contained in pigment granules lying on the ciliary organelles of this microorganism. HPLC‐ESI‐MS analysis of this granule discharge reveals that erythrolactones A2–C2 are actually therein contained, strongly suggesting a possible role for these metabolites in the chemical defence strategy of P. erythrina

Genetic relationships in bipolar species of the protist ciliate, Euplotes

Protists thrive in polar oceans, where they represent a major driving force for globally important biogeochemical cycles and a key food-web component. Their biogeography is frequently associated to bipolar patterns of distribution. Although conceptually well supported by apparently unrestricted migration rates, the experimental certification of these patterns copes with the protist paucity of morphological characters with taxonomic value and difficulties in applying conventional species concepts. We studied three marine species of the ciliate Euplotes, E. euryhalinus, E. nobilii, and E. petzi, for their bipolar distribution by comparing the SSU-rRNA gene sequences and mating interactions of Antarctic, Patagonian, and Arctic strains. Each species was analogously found not to carry significantly varied SSU-rRNA gene sequences, implying a common occurrence of trans-equatorial genetic mixing. However, mating analyses revealed significant inter-species differences. Scarce Antarctic x Arctic strain mating compatibility distinguished E. petzi from E. euryhalinus and E. nobilii, in which mating pairs between Antarctic and Arctic strains were successfully induced. Yet, E. nobilii was the only one of the two species to show cross-fertilizing and fertile mating pairs. Taking the biological concept of species as discriminatory, it was thus concluded that only E. nobilii warrants the definition of genuine bipolar species

Investigating the role of Acanthamoeba polyphaga in protecting Human Adenovirus from water disinfection treatment

Human adenoviruses are responsible for a wide range of clinical infections and are present in aquatic environments, includingriver, seawater, drinking-water and sewage. Free-living amoebae (Acanthamoeba) in the same environments may internalizethem and other microorganisms can act as a reservoir for the internalized viruses. In this study, we studied the interactionbetween Acanthamoeba polyphaga and Human Adenovirus type 5 (HAdV 5) to determine whether the amoeba played a rolein protecting the internalized viruses from chemical disinfection. The efficacy of sodium hypochlorite disinfection against A.polyphaga and HAdV 5 either singly or in combination was assessed at three different concentrations. Individually, the amoebawere more resistant to chemical disinfection than HAdV 5 and remained alive after exposure to 5 mg/l of sodium hypochlorite.In contrast, HAdV 5 lost infectivity following exposure to 2.5 mg/l of sodium hypochlorite. When the amoeba and HAdV 5were co-cultured, infectious virus was found in the cytoplasm of the amoeba at 5 mg/l disinfectant concentration. These findingssuggest that the A. polyphaga is providing protection for the HAdV 5

Test Generation Based on CLP

Functional ATPGs based on simulation are fast, but generally, they are unable to cover corner cases, and they cannot prove untestability. On the contrary, functional ATPGs exploiting formal methods, being exhaustive, cover corner cases, but they tend to suffer of the state explosion problem when adopted for verifying large designs. In this context, we have defined a functional ATPG that relies on the joint use of pseudo-deterministic simulation and Constraint Logic Programming (CLP), to generate high-quality test sequences for solving complex problems. Thus, the advantages of both simulation-based and static-based verification techniques are preserved, while their respective drawbacks are limited. In particular, CLP, a form of constraint programming in which logic programming is extended to include concepts from constraint satisfaction, is well-suited to be jointly used with simulation. In fact, information learned during design exploration by simulation can be effectively exploited for guiding the search of a CLP solver towards DUV areas not covered yet. The test generation procedure relies on constraint logic programming (CLP) techniques in different phases of the test generation procedure. The ATPG framework is composed of three functional ATPG engines working on three different models of the same DUV: the hardware description language (HDL) model of the DUV, a set of concurrent EFSMs extracted from the HDL description, and a set of logic constraints modeling the EFSMs. The EFSM paradigm has been selected since it allows a compact representation of the DUV state space that limits the state explosion problem typical of more traditional FSMs. The first engine is randombased, the second is transition-oriented, while the last is fault-oriented. The test generation is guided by means of transition coverage and fault coverage. In particular, 100% transition coverage is desired as a necessary condition for fault detection, while the bit coverage functional fault model is used to evaluate the effectiveness of the generated test patterns by measuring the related fault coverage. A random engine is first used to explore the DUV state space by performing a simulation-based random walk. This allows us to quickly fire easy-to-traverse (ETT) transitions and, consequently, to quickly cover easy-to-detect (ETD) faults. However, the majority of hard-to-traverse (HTT) transitions remain, generally, uncovered. Thus, a transition-oriented engine is applied to cover the remaining HTT transitions by exploiting a learning/backjumping-based strategy. The ATPG works on a special kind of EFSM, called SSEFSM, whose transitions present the most uniformly distributed probability of being activated and can be effectively integrated to CLP, since it allows the ATPG to invoke the constraint solver when moving between EFSM states. A constraint logic programming-based (CLP) strategy is adopted to deterministically generate test vectors that satisfy the guard of the EFSM transitions selected to be traversed. Given a transition of the SSEFSM, the solver is required to generate opportune values for PIs that enable the SSEFSM to move across such a transition. Moreover, backjumping, also known as nonchronological backtracking, is a special kind of backtracking strategy which rollbacks from an unsuccessful situation directly to the cause of the failure. Thus, the transition-oriented engine deterministically backjumps to the source of failure when a transition, whose guard depends on previously set registers, cannot be traversed. Next it modifies the EFSM configuration to satisfy the condition on registers and successfully comes back to the target state to activate the transition. The transition-oriented engine generally allows us to achieve 100% transition coverage. However, 100% transition coverage does not guarantee to explore all DUV corner cases, thus some hard-to-detect (HTD) faults can escape detection preventing the achievement of 100% fault coverage. Therefore, the CLP-based fault-oriented engine is finally applied to focus on the remaining HTD faults. The CLP solver is used to deterministically search for sequences that propagate the HTD faults observed, but not detected, by the random and the transition-oriented engine. The fault-oriented engine needs a CLP-based representation of the DUV, and some searching functions to generate test sequences. The CLP-based representation is automatically derived from the S2EFSM models according to the defined rules, which follow the syntax of the ECLiPSe CLP solver. This is not a trivial task, since modeling the evolution in time of an EFSM by using logic constraints is really different with respect to model the same behavior by means of a traditional HW description language. At first, the concept of time steps is introduced, required to model the SSEFSM evolution through the time via CLP. Then, this study deals with modeling of logical variables and constraints to represent enabling functions and update functions of the SSEFSM. Formal tools that exhaustively search for a solution frequently run out of resources when the state space to be analyzed is too large. The same happens for the CLP solver, when it is asked to find a propagation sequence on large sequential designs. Therefore we have defined a set of strategies that allow to prune the search space and to manage the complexity problem for the solver

Natural Products among Brown Algae: The Case of Cystoseira schiffneri HAMEL (Sargassaceae, Phaeophyceae)

A chemotaxonomic study on the marine brown alga Cystoseira schiffneri collected from the Tunisian marine coast allowed us to identify kjellmanianone (1) and a new isololiolide derivative named schiffnerilolide (2). The structu re elucidati on and the assignment of relative con figurations of the isolated natur al products were based on advanced mass spectrometric and nuclear magnetic resonance techniques. This outcome suggested a close phylogenetic relationship of C. schiffneri with brown algae bel onging to genus Sargassum C. A GARDH. Molecular characterization using the nuclear small subunit rRNA (SSU rRNA) gene (18S) sequence as genetic marker was made. Pigme nt analysis showed a significant seasonal change of carotenoids, in particular of fucoxanthin and fucoxanthinol. Also galactolipids, the main constituents of the thylakoid membranes, showed remarkable seasonal changes

Cytotoxic effects and apoptotic signalling mechanisms of the sesquiterpenoid euplotin C, a secondary metabolite of the marine ciliate Euplotes crassus, in tumour cells

Most antitumour agents with cytotoxic properties induce apoptosis. The lipophilic compound euplotin C, isolated from the ciliate Euplotes crassus, is toxic to a number of different opportunistic or pathogenic microorganisms, although its mechanism of action is currently unknown. We report here that euplotin C is a powerful cytotoxic and pro-apoptotic agent in mouse AtT-20 and rat PC12 tumour-derived cell lines. In addition, we provide evidence that euplotin C treatment results in rapid activation of ryanodine receptors, depletion of Ca2+ stores in the endoplasmic reticulum (ER), the release of cytochrome c from the mitochondria, activation of caspase-12, and activation of caspase-3, leading to apoptosis. Intracellular Ca2+ overload is an early event which induces apoptosis and is parallelled by ER stress and the release of cytochrome c, whereas caspase-12 may be activated by euplotin C at a later stage in the apoptosis pathway. These events, either independently or concomitantly, lead to the activation of the caspase-3 and its downstream effectors, triggering the cell to undergo apoptosis. These results demonstrate that euplotin C may be considered for the design of cytotoxic and pro-apoptotic new drugs.L'articolo è disponibile sul sito dell'editore http://www.springerlink.com

Molecular mechanisms of euplotin C-induced apoptosis: involvement of mitochondrial dysfunction, oxidative stress and proteases

The metabolite euplotin C (EC), isolated from the marine ciliate Euplotes crassus, is a powerful cytotoxic and pro-apoptotic agent in tumour cell lines. For instance, EC induces the rapid depletion of ryanodine Ca2+ stores, the release of cytochrome c from the mitochondria, and the activation of caspase-3, leading to apoptosis. The purpose of this study was to gain further insight into the mechanisms of EC-induced apoptosis in rat pheochromocytoma PC12 cells. We found that EC increases Bax/Bcl-2 ratio and that Bax is responsible of the EC-induced dissipation of the mitochondrial membrane potential (Δψm). In addition, EC induces the generation of reactive oxygene species (ROS) without involvement of p53. The inhibition of ROS generation prevents, at least in part, the pro-apoptotic effects of EC as well as the effects of EC on Bax, Δψm and intracellular free Ca2+, indicating a cross-talk between different pathways. However, definition of the effector cascade turns out to be more complex than expected and caspase-independent mechanisms, acting in parallel with caspases, should also be considered. Among them, EC increases the expression/activity of calpains downstream of ROS generation, although calpains seem to exert protective effects.L'articolo è disponibile sul sito dell'editore http://www.springerlink.co

Pole-to-Pole Gene Flow in Protozoan Ciliates

Microorganisms represent the smallest but arguably most important component of the ocean life. They are essential to all nutrient cycles because they form the bottom of the marine food chain and outnumber all other marine species by orders of magnitude. Sampling of remote and inaccessible habitats and large-scale genomic analysis have shown how little we know about the microbial life in the oceans, and how our poor knowledge of the marine chemistry and biology is preventing us from foreseeing the detrimental effects that a too rapidly changing world has on the oceans’ ecosystems. In this context, polar microorganisms are attracting particular interest because of their role in global-scale biogeochemical cycles, in particular the carbon dioxide exchange with the atmosphere (Falkowski et al. 2008). Considering this interest, planktonic and benthic microbial communities from Arctic and Antarctic areas have become the focus of more systematic samplingand rigorous analyses for their taxonomic, prokaryotic (bacterial) and eukaryotic (protist), biodiversity. A relevant result of these analyses was the finding of microbial species that, like a diverse range of plant and animal species (Lindberg 1991; Crame 1993), warrant the definition ‘‘bipolar’’ (or ‘‘anti-tropical’’), i.e. species represented by high-latitude populations physically separated in distribution across the tropics (Darling et al. 2000; Montresor et al. 2003; Brandt et al. 2007; Pawlowski et al. 2007). This concept of species bipolarity has inherently raised the intriguing question whether co-specific Antarctic and Arctic populations evolved independently since the effective separation (approximately 10–15 million years ago) between the Arctic and Antarctic cold-water provinces, or whether a trans-tropical gene flow ensures that these polar populations maintain genetic continuity (Darling et al. 2000). Morphological studies alone are clearly insufficient to address this question, due to recurrent phenomena of parallel or convergent morphological evolution that take place under similar environmental forces. Therefore, more solid grounds supporting the concept of species bipolarity have been obtained in some species of foraminifera and dinozoans from analysis of genetic variation in sequences of the small subunit (SSU) rRNA nuclear gene (Darling et al. 2000; Montresor et al. 2003; Brandt et al. 2007; Pawlowski et al. 2007). Nevertheless, unless the calibration of a molecular clock is supported by abundance in fossil records, as is the case in foraminifera (Pawlowski et al. 1997), also this genetic approach is impaired by the fact that the same DNA regions may evolve at different rates among closely related organisms. A solution to overcoming this challenge is provided by the well-defined, monophyletic group of ciliates which are ideal organisms for the analysis of the breeding structure of natural microbial populations and, therefore, for obtaining data which satisfy the interbreeding criterion on which the biological (Darwinian) concept of species is founded. Ciliates govern their gene exchanges through a unique sexual phenomenon, known as conjugation (or mating), in which two functionally hermaphroditic individuals unite temporarily in pairs for a mutual exchange of a ‘‘migratory’’ (‘‘male’’) gametic nucleus destined to fuse into a synkaryon with a ‘‘resident’’ (‘‘female’’) gametic nucleus. In addition, ciliates more than any other group of polar microorganisms can readily provide living laboratory material in virtually unlimited amounts, since every aquatic polar habitat hosts a large variety of ciliate species that are relatively easy to collect and expand into permanent clonal cultures (i.e., descendants from single wild-type specimens). Here we review results that have been obtained from breeding analyses of Antarctic, Fuegian and Arctic populations of the ciliate Euplotes nobilii. These results have provided compelling evidence that these populations are genetically interconnected by gene flow and form a unique interbreeding biological species. The description of these results is preceded by an outline of our current knowledge of ciliate biodiversity in the polar regions, and by a rationalization of the genetic mechanism of the mating types that represents the major driving force of ciliate breeding strategies