Analysis of DNA-damage response to ionizing radiation in serum-shock synchronized human fibroblasts

Many aspects of cellular physiology, including cellular response to genotoxic stress, are related to the circadian rhythmicity induced by the molecular clock. The current study investigated if the cellular response to DNA damage is in relation to endogenous expression levels of the PER2 protein, a key component of the molecular regulatory system that confers rhythmicity in mammalian cells. Human normal fibroblasts (CCD-34Lu) were subjected to serum shock to induce circadian oscillations of the PER2 protein and then irradiated with γ- rays at times corresponding to the trough and peak expression of the PER2 protein. To better examine cellular response to DNA damage, the experiments performed in this study were carried out in non-proliferating CCD-34Lu fibroblasts in order to maintain the cell and circadian cycles separated while they were being exposed to genotoxic stress. Study results demonstrated that clonogenic cell survival, double-strand break repair kinetics, and TP53 protein levels were affected in the cells irradiated at the trough than in those irradiated at peak expression of the PER2 protein.     

Design and synthesis of a novel inhibitor of T. Viride chitinase through an in silico target fishing protocol

In the last ten years, we identified and developed a new therapeutic class of antifungal agents, the macrocyclic amidinoureas. These compounds are active against several Candida species, including clinical isolates resistant to currently available antifungal drugs. The mode of action of these molecules is still unknown. In this work, we developed an in-silico target fishing procedure to identify a possible target for this class of compounds based on shape similarity, inverse docking procedure and consensus score rank-by-rank. Chitinase enzyme emerged as possible target. To confirm this hypothesis a novel macrocyclic derivative has been produced, specifically designed to increase the inhibition of the chitinase. Biological evaluation highlights a stronger enzymatic inhibition for the new derivative, while its antifungal activity drops probably because of pharmacokinetic issues. Collectively, our data suggest that chitinase represent at least one of the main target of macrocyclic amidinoureas.     

Landscape simplification weakens the association between terrestrial producer and consumer diversity in Europe

Land‐use change is one of the primary drivers of species loss, yet little is known about its effect on other components of biodiversity that may be at risk. Here, we ask whether, and to what extent, landscape simplification, measured as the percentage of arable land in the landscape, disrupts the functional and phylogenetic association between primary producers and consumers. Across seven European regions, we inferred the potential associations (functional and phylogenetic) between host plants and butterflies in 561 seminatural grasslands. Local plant diversity showed a strong bottom‐up effect on butterfly diversity in the most complex landscapes, but this effect disappeared in simple landscapes. The functional associations between plant and butterflies are, therefore, the results of processes that act not only locally but are also dependent on the surrounding landscape context. Similarly, landscape simplification reduced the phylogenetic congruence among host plants and butterflies indicating that closely related butterflies become more generalist in the resources used. These processes occurred without any detectable change in species richness of plants or butterflies along the gradient of arable land. The structural properties of ecosystems are experiencing substantial erosion, with potentially pervasive effects on ecosystem functions and future evolutionary trajectories. Loss of interacting species might trigger cascading extinction events and reduce the stability of trophic interactions, as well as influence the longer term resilience of ecosystem functions. This underscores a growing realization that species richness is a crude and insensitive metric and that both functional and phylogenetic associations, measured across multiple trophic levels, are likely to provide additional and deeper insights into the resilience of ecosystems and the functions they provide.     

Proteomic analysis of the pathophysiological process involved in the antisnake venom effect of Mucuna pruriens extract

Previously, we reported the antisnake venom properties of a Mucuna pruriens seed extract (MPE) and tested its in vivo efficacy against Echis carinatus venom (EV) in short- (1 injection) and long-term (three weekly injections) treatments. The aim of the present study was to investigate plasma proteome changes associated with MPE treatments and identify proteins responsible for survival of envenomated mice (CHALLENGED mice). Six treatment groups were studied. Three control groups: one saline, one short-term and one long-term MPE treatment. One group received EV alone. Two test groups received EV with either a short-term or long-term MPE treatment (CHALLENGED mice). The plasma from each group was analysed by 2-DE/MALDI-TOF MS. The most significant changes with treatment were: albumin, haptoglobin, fibrinogen, serum amyloid A and serum amyloid P. Most of these changes were explained by EV effects on coagulation, inflammation and haemolysis. However, MPE treatments prevented the EV-induced elevation in HPT. Consequently, HPT levels were similar to controls in the plasma of CHALLENGED mice. The plasma of CHALLENGED mice showed substantial proteomic modifications. This suggests the mechanism of MPE protection involves the activation of counterbalancing processes to compensate for the imbalances caused by EV.     

Beta-catenin triggers nuclear factor kappaB-dependent up-regulation of hepatocyte inducible nitric oxide synthase

Disruption of cell-to-cell contacts, as observed in many pathophysiological conditions, prime hepatocytes for compensatory hyperplastic response that involves induction of several genes, including proto-oncogenes and other gene targets of beta-catenin signaling pathway. By using cultured hepatocytes and experimental models of adherens junction disruption we have investigated changes in beta-catenin subcellular localization and their relationships with inducible nitric oxide synthase (iNOS) expression. Two experimental models were employed: (a) rat hepatocytes obtained by collagenase liver perfusion within the first 48 h of culture; (b) 48-h old cultured hepatocytes, transiently transfected or not with a plasmid encoding for dominant/negative inhibitory kappa B-alpha, exposed to ethylene glycol-bis-(2-aminoethylether)-N,N,N',N'-tetraacetic acid/LiCl treatment. beta-Catenin signaling and cellular localization, iNOS expression and nuclear factor kappaB involvement, were investigated using morphological, cell and molecular biology techniques. E-cadherin-mediated disruption of cell-to-cell contacts induces early beta-catenin translocation from membrane to cytoplasm and nuclear compartments, events that are followed by up-regulation of c-myc, cyclin D1 and beta-transducin repeat-containing protein expression. This, in turn, resulted eventually in iNOS induction that was mechanistically related to nuclear factor kappaB activation, as unequivocally shown in cells expressing dominant negative inhibitory kappa B-alpha. Our data indicate that E-cadherin disassembly and concomitant inactivation of glycogen synthase kinase-3beta result in nuclear factor kappaB-dependent induction of iNOS in hepatocytes.     

Biotic, Abiotic, and Management Controls on the Net Ecosystem CO2 Exchange of European Mountain Grassland Ecosystems

The net ecosystem carbon dioxide (CO₂) exchange (NEE) of nine European mountain grassland ecosystems was measured during 2002–2004 using the eddy covariance method. Overall, the availability of photosynthetically active radiation (PPFD) was the single most important abiotic influence factor for NEE. Its role changed markedly during the course of the season, PPFD being a better predictor for NEE during periods favorable for CO₂ uptake, which was spring and autumn for the sites characterized by summer droughts (southern sites) and (peak) summer for the Alpine and northern study sites. This general pattern was interrupted by grassland management practices, that is, mowing and grazing, when the variability in NEE explained by PPFD decreased in concert with the amount of aboveground biomass (BM_ag). Temperature was the abiotic influence factor that explained most of the variability in ecosystem respiration at the Alpine and northern study sites, but not at the southern sites characterized by a pronounced summer drought, where soil water availability and the amount of aboveground biomass were more or equally important. The amount of assimilating plant area was the single most important biotic variable determining the maximum ecosystem carbon uptake potential, that is, the NEE at saturating PPFD. Good correspondence, in terms of the magnitude of NEE, was observed with many (semi-) natural grasslands around the world, but not with grasslands sown on fertile soils in lowland locations, which exhibited higher maximum carbon gains at lower respiratory costs. It is concluded that, through triggering rapid changes in the amount and area of the aboveground plant matter, the timing and frequency of land management practices is crucial for the short-term sensitivity of the NEE of the investigated mountain grassland ecosystems to climatic drivers.     

Large Differences in Aging Phenotype between Strains of the Short-Lived Annual Fish Nothobranchius furzeri

Background

A laboratory inbred strain of the annual fish Nothobranchius furzeri shows exceptionally short life expectancy and accelerated expression of age markers. In this study, we analyze new wild-derived lines of this short-lived species.

Methodology/Principal Findings

We characterized captive survival and age-related traits in F1 and F2 offspring of wild-caught N. furzeri. Wild-derived N. furzeri lines showed expression of lipofuscin and neurodegeneration at age 21 weeks. Median lifespan in the laboratory varied from to 20 to 23 weeks and maximum lifespan from 25 to 32 weeks. These data demonstrate that rapid age-dependent decline and short lifespan are natural characteristics of this species. The N. furzeri distribution range overlaps with gradients in altitude and aridity. Fish from more arid habitats are expected to experience a shorter survival window in the wild. We tested whether captive lines stemming from semi-arid and sub-humid habitats differ in longevity and expression of age-related traits. We detected a clear difference in age-dependent cognitive decline and a slight difference in lifespan (16% for median, 15% for maximum lifespan) between these lines. Finally, we observed shorter lifespan and accelerated expression of age-related markers in the inbred laboratory strain compared to these wild-derived lines.

Conclusions/Significance

Owing to large differences in aging phenotypes in different lines, N. furzeri could represent a model system for studying the genetic control of life-history traits in natural populations.