Exploring the Structure-Function Loop Adaptability of a (β/α)(8)-Barrel Enzyme through Loop Swapping and Hinge Variability

Cellulosomes are large extracellular multi-enzyme complexes that exhibit elevated activity on plant cell-wall polysaccharides. In the present study, the relationships between the conformational flexibility and efficacy of cellulosomes, and the inter-modules linkers of their scaffold protein were investigated. For this purpose, the length of the intrinsically disordered Ser/Thr-rich 50-residue linker connecting a Clostridium thermocellum and a Clostridium cellulolyticum cohesin in a hybrid scaffoldin (Scaf4) was changed by sequences ranging from 4 to 128 residues. The composition was also modified and new linkers composed of series of N, S or repeats of the EPPV motif were generated. Two model cellulases (Cel48F and Cel9G) appended with appropriate dockerins were subsequently bound to the engineered scaffoldins. All the resulting minicomplexes displayed the same activity on crystalline cellulose as the complex based on the initial Scaf4, and were found to be 2-fold more active than Cel48F and Cel9G bound to separate cohesins. Small-angle X-ray scattering assays of the engineered scaffoldins confirmed, however, that the size and the conformational flexibility of some of the new inter-cohesins linkers differed significantly from that of the initial 50 residue linker displayed by the parental Scaf4. Our data suggest that the synergy induced by proximity does not require a specific inter-cohesins sequence or distance. The present study reveals that complexation onto the hybrid scaffoldins modifies the type of soluble sugars released from crystalline cellulose by the selected cellulases, compared to the free enzyme system.     

Novel histone H3 binding protein ORF158L from the Singapore grouper iridovirus

Singapore grouper iridovirus (SGIV), a major pathogen of concern for grouper aquaculture, has a double-stranded DNA (dsDNA) genome with 162 predicted open reading frames, for which a total of 62 SGIV proteins have been identified. One of these, ORF158L, bears no sequence homology to any other known protein. Knockdown of orf158L using antisense morpholino oligonucleotides resulted in a significant decrease in virus yield in grouper embryonic cells. ORF158L was observed in nuclei and virus assembly centers of virus-infected cells. This observation led us to study the structure and function of ORF158L. The crystal structure determined at 2.2-Å resolution reveals that ORF158L partially exhibits a structural resemblance to the histone binding region of antisilencing factor 1 (Asf1), a histone H3/H4 chaperon, despite the fact that there is no significant sequence identity between the two proteins. Interactions of ORF158L with the histone H3/H4 complex and H3 were demonstrated by isothermal titration calorimetry (ITC) experiments. Subsequently, the results of ITC studies on structure-based mutants of ORF158L suggested Arg67 and Ala93 were key residues for histone H3 interactions. Moreover, a combination of approaches of ORF158L knockdown and isobaric tags/mass spectrometry for relative and absolute quantifications (iTRAQ) revealed that ORF158L may be involved in both the regulation and the expression of histone H3 and H3 methylation. Our present studies suggest that ORF158L may function as a histone H3 chaperon, enabling it to control host cellular gene expression and to facilitate viral replication.     

Phylogeny of the Mexican coastal leopard frogs of the Rana berlandieri group based on mtDNA sequences

Phylogenetic relationships among specimens from 25 different locations for the six Mexican coastal leopard frog species of the Rana berlandieri species group were investigated using 797 bp of the mitochondrial 12S rDNA gene. Relationships among the haplotypes obtained were recovered using maximum parsimony and Bayesian analyses. Most of the clades recovered by both tree building methods are strongly supported, but conflicting clades recovered by each analysis are generally poorly supported. Both analyses reject the previously proposed subgroupings of the R. berlandieri species group. Based on the strongly supported relationships, genetic differentiation, and geographic distribution of the haplotypes examined, nine independent lineages appear to comprise the group of study. However, confirmation of the new proposed lineages will require further analyses based on other genetic markers and additional samples that cover their entire geographic distribution. Concordance was noted between Miocene-Pliocene geological and climatic events in Mexico and the relationships recovered among the lineages proposed and their geographic distribution.     

Variation in Trill Characteristics in Tree Pipit Songs: Different Trills for Different Use?

Transmitting information about singer's quality is an important function of song in many bird species, and this information should be useful in territorial interactions. Fast trills, being physically demanding song structures, are particularly suitable candidates for signalling of quality or aggressive motivation. We have evaluated trill characteristics in songs within a population of the Tree Pipit, a common European songbird with no sexual dimorphism, in which song apparently plays a key role in territory defence as well as mate choice. Two types of relatively fast trills (each of them in multiple variants differing in complexity) were commonly observed in repertoires of Tree Pipit males. Trill rates significantly differed among individuals, suggesting that these song structures may carry information about male quality in this species. We tested by playback experiments whether both trill types are used in territorial encounters. Only one of the trill types was sung by males in response to playback, regardless on the trill type played to them. In an immediate response to playback, they increased the frequency of use of this trill, and also significantly increased the trill rate in comparison with spontaneous songs. This confirmed field observations, suggesting that this trill is important in male–male interactions. On the contrary, the use of the fastest, apparently more demanding, trill type actually decreased after the simulated territorial intrusion. We hypothesize that the latter one is more directed towards females, and that while performance of both trill types may reflect male quality, they are primarily used in different contexts.     

Intra- and interannual variation in the diet of the Magellanic penguin (Spheniscus magellanicus) at Martillo Island,Beagle Channel

Knowledge of seabirds’ diet at each breeding site and its temporal variation is key to understanding and evaluating how changes in marine resources affect each seabird population. In this study, we determined the diet of Magellanic penguins (MP, Spheniscus magellanicus) at Martillo Island, accounting for sex, breeding stage and year. We analyzed a total of 144 stomach contents during three consecutive breeding seasons (2006–2007, 2007–2008 and 2008–2009) and stages (incubation, early and late chick-rearing). MP fed mainly on fuegian sprat (Sprattus fuegensis), which represented 75 % of the biomass consumed by birds during the entire study. The next important prey was squat lobster (Munida gregaria), followed by Patagonian squid (Loligo gahi). Both sexes consumed similar prey items. We observed variation in diet relative composition among breeding years and stages. Fuegian sprat consumption decreased throughout the years whereas squat lobster increased. Penguins consumed a higher proportion of squat lobster and Patagonian squid during the incubation stage than in the chick-rearing stages, whereas fuegian sprat was almost the only prey item consumed during the late chick-rearing stage. MPs show certain flexibility in the use of resources probably as a response to changes in prey populations. Variability in the diet among different reproductive stages could be related to changes in the distribution and abundance of their main prey near the colony during the breeding season together with changes in the energy requirements of seabirds.     

French broom (Teline monspessulana) invasion in south-central Chile depends on factors operating at different spatial scales

The interaction between plant attributes, environmental factors and the history of human intervention determines which species show a more invasive behavior and which areas are more likely to be invaded. Identifying which factors favor the invasion and which constraint it, remains an essential goal for understanding the process of invasion and to provide a stronger scientific basis for designing management actions to reduce susceptibility to invasion. In this paper we analyze the relative role of different environmental factors in plant invasion, in particular the comparison between human versus climatic and biotic variables at different spatial scales. Furthermore, we show how these factors interact to influence the distribution and abundance of an invasive plant. We have built mixed models to explain the presence and abundance of a renowned invasive species in the south-central area of Chile, Teline monspessulana (French broom, Fabaceae). We recorded the presence and cover of this invasive shrub in a series of transects along the main roads of the study area. For each transect we recorded variables at different spatial scales related to climate and land use. We found that the presence and cover of T. monspessulana are strongly related to proximity to roads, urban areas and locations with higher rainfall. Although this shrub is present in most of the landscape, it is most abundant in shrublands and forest plantations. In a complex human-dominated landscape matrix, shrublands and forest plantations act as a source of propagules from which new areas can be invaded. Interestingly, the presence of this species can be better predicted by combining predictors taken at two hierarchical levels, the so-called transect and plot scales, whereas the species abundance only depends on factors at the plot level. The presence and abundance of this species is highly dependent on human disturbance, and the occurrence of certain landscape elements closely linked to land management is critical to the performance of this species. Maintaining a dense tree canopy cover and reducing the use of fire could help reduce the rate of expansion of this species across the landscape.     

Thiol redox biochemistry: insights from computer simulations

Thiol redox chemical reactions play a key role in a variety of physiological processes, mainly due to the presence of low-molecular-weight thiols and cysteine residues in proteins involved in catalysis and regulation. Specifically, the subtle sensitivity of thiol reactivity to the environment makes the use of simulation techniques extremely valuable for obtaining microscopic insights. In this work we review the application of classical and quantum–mechanical atomistic simulation tools to the investigation of selected relevant issues in thiol redox biochemistry, such as investigations on (1) the protonation state of cysteine in protein, (2) two-electron oxidation of thiols by hydroperoxides, chloramines, and hypochlorous acid, (3) mechanistic and kinetics aspects of the de novo formation of disulfide bonds and thiol−disulfide exchange, (4) formation of sulfenamides, (5) formation of nitrosothiols and transnitrosation reactions, and (6) one-electron oxidation pathways.     

Using Unplanned Fires to Help Suppressing Future Large Fires in Mediterranean Forests

Despite the huge resources invested in fire suppression, the impact of wildfires has considerably increased across the Mediterranean region since the second half of the 20^th century. Modulating fire suppression efforts in mild weather conditions is an appealing but hotly-debated strategy to use unplanned fires and associated fuel reduction to create opportunities for suppression of large fires in future adverse weather conditions. Using a spatially-explicit fire–succession model developed for Catalonia (Spain), we assessed this opportunistic policy by using two fire suppression strategies that reproduce how firefighters in extreme weather conditions exploit previous fire scars as firefighting opportunities. We designed scenarios by combining different levels of fire suppression efficiency and climatic severity for a 50-year period (2000–2050). An opportunistic fire suppression policy induced large-scale changes in fire regimes and decreased the area burnt under extreme climate conditions, but only accounted for up to 18–22% of the area to be burnt in reference scenarios. The area suppressed in adverse years tended to increase in scenarios with increasing amounts of area burnt during years dominated by mild weather. Climate change had counterintuitive effects on opportunistic fire suppression strategies. Climate warming increased the incidence of large fires under uncontrolled conditions but also indirectly increased opportunities for enhanced fire suppression. Therefore, to shift fire suppression opportunities from adverse to mild years, we would require a disproportionately large amount of area burnt in mild years. We conclude that the strategic planning of fire suppression resources has the potential to become an important cost-effective fuel-reduction strategy at large spatial scale. We do however suggest that this strategy should probably be accompanied by other fuel-reduction treatments applied at broad scales if large-scale changes in fire regimes are to be achieved, especially in the wider context of climate change.     

Decreased Serum Level of miR-146a as Sign of Chronic Inflammation in Type 2 Diabetic Patients

Background

There is increasing evidence that chronic inflammation is an important determinant in insulin resistance and in the pathogenesis of type 2 diabetes (T2D). MicroRNAs constitute a newly discovered system of cell regulation and in particular two microRNAs (miR-146a and miR-155) have been described as regulators and biomarkers of inflammation.

Aim

To determine a putative association between the levels of miR-146a and miR-155 in serum of T2D patients, clinical parameters and serological indicators of inflammation.

Methods

We performed quantitative Real Time PCR (qPCR) of microRNAs from serum (56 Ecuadorian T2D ambulatory patients and 40 non-diabetic controls). In addition, we evaluated T2D-related serum cytokines.chemokines and growth factors using a commercially available multi-analyte cytometric bead array system. We correlated outcomes to clinical parameters, including BMI, HbA1c and lipid state.

Results

The Ecuadorian non-diabetic controls appeared as overweight (BMI>25: patients 85%, controls 82.5%) and as dyslipidemic (hypercholesterolemia: patients 60.7%, controls 67.5%) as the patients.

• The serum levels of miR-146a were significantly reduced in T2D patients as compared to these non-diabetic, but obese/dyslipidemic control group (mean patients 0.61, mean controls set at 1; p = 0.042), those of miR-155 were normal.
• The serum levels of both microRNAs correlated to each other (r = 0.478; p<0.001) and to leptin levels. The microRNAs did not correlate to BMI, glycemia and dyslipidemia.
• From the tested cytokines, chemokines and growth factors, we found IL-8 and HGF significantly raised in T2D patients versus non-diabetic controls (p = 0.011 and 0.023 respectively).

Conclusions

This study shows decreased serum anti-inflammatory miR-146a, increased pro-inflammatory IL-8 and increased HGF (a vascular/insular repair factor) as discriminating markers of failure of glucose control occurring on the background of obesity and dyslipidemia.