Energy estimation in protein design

The progress achieved by several groups in the field of computational protein design shows that successful design methods include two major features: efficient algorithms to deal with the combinatorial exploration of sequence space and optimal energy functions to rank sequences according to their fitness for the given fold.     

Whole-Genome Analysis of Diversity and SNP-Major Gene Association in Peach Germplasm

Peach was domesticated in China more than four millennia ago and from there it spread world-wide. Since the middle of the last century, peach breeding programs have been very dynamic generating hundreds of new commercial varieties, however, in most cases such varieties derive from a limited collection of parental lines (founders). This is one reason for the observed low levels of variability of the commercial gene pool, implying that knowledge of the extent and distribution of genetic variability in peach is critical to allow the choice of adequate parents to confer enhanced productivity, adaptation and quality to improved varieties. With this aim we genotyped 1,580 peach accessions (including a few closely related Prunus species) maintained and phenotyped in five germplasm collections (four European and one Chinese) with the International Peach SNP Consortium 9K SNP peach array. The study of population structure revealed the subdivision of the panel in three main populations, one mainly made up of Occidental varieties from breeding programs (POP1OC_B), one of Occidental landraces (POP2OC_T) and the third of Oriental accessions (POP3OR). Analysis of linkage disequilibrium (LD) identified differential patterns of genome-wide LD blocks in each of the populations. Phenotypic data for seven monogenic traits were integrated in a genome-wide association study (GWAS). The significantly associated SNPs were always in the regions predicted by linkage analysis, forming haplotypes of markers. These diagnostic haplotypes could be used for marker-assisted selection (MAS) in modern breeding programs.     

Trehalose favors a cutinase compact intermediate off-folding pathway

The folding of cutinase, an enzyme displaying lipolytic activity, has been studied in the presence of trehalose. Equilibrium unfolding data show that trehalose increases the free energy change between folded and unfolded states. Unfolding kinetics reveal the presence of an intermediate which is ca. 60% folded in terms of solvent exposure. Trehalose stabilizes this intermediate relative to the folded state. In contrast, the intermediate revealed by folding kinetics is more compact than the transition state, as shown by the positive slope observed at low denaturant concentration in the chevron plot, as well as the decrease in the observable rate constant for folding with the increase in trehalose concentration. This intermediate displays more than 50% of area buried from the solvent (relative to the native state) compared to around 40% for the transition state for folding and therefore appears to be off the folding pathway. Trehalose stabilizes and guanidine hydrochloride destabilizes this compact intermediate. Both unfolding and folding kinetics show that compact conformational states are stabilized by trehalose, in agreement with current models on the effect of compatible solutes. This effect occurs even for compact states that decelerate the folding as in the case of the intermediate revealed by folding kinetics.     

Structural and functional implications of the instability of the ADP/ATP transporter purified from mitochondria as revealed by FTIR spectroscopy

The ADP/ATP transporter shows a high instability when solubilized, making it difficult to obtain functional protein with sufficient purity for long-term spectroscopic studies. When solubilized in the detergent dodecyl maltoside the protein is in equilibrium between the so-called CATR and BA conformations and in a few hours it becomes nonfunctional, unable to bind either its inhibitors or its substrates. By Fourier transform infrared spectroscopy, we studied the structural changes involved in this denaturation process. To do so, the carboxyatractyloside-inhibited protein was used as a structural model for the protein in the CATR conformation and its spectrum was compared with that of the unliganded time-inactivated protein. From the difference spectra of the amide I, amide II, and amide A bands combined with dichroism spectra of the carboxyatractyloside-inhibited protein, we concluded that few structural differences exist between both states, affecting as few as 11 amino acids (3.5% of the protein); the structural changes consisted in the disappearance of large loop structure and the appearance of aggregated strands. We hypothesize that some mitochondrial loop (tentatively loop M1) shows a high tendency to aggregate, being responsible for the observed features. The functional consequences of this hypothesis are discussed.     

Seroprevalence of antibodies to Neospora caninum in dogs from Spain

The prevalence of Neospora caninum antibodies was determined in sera of 139 dogs from Catalonia (northeastern Spain) using the indirect immunofluorescence antibody test (IFAT). Antibodies in the IFAT were found in 17 of 139 dogs (12.2%) with titers ranging from 1:50 to 1: 1,600. Seroprevalence was higher in dogs over 1 yr old compared with dogs younger than 1 yr (P < 0.05). No statistical difference was observed when sex, breed, purpose, or modus vivendi was compared with seropositivity. Most dogs had low antibody titers, which indicated subclinical infection in the area studied. No neosporosis-related disease was reported from any dog, although a German shepherd with an antibody titer of 1:800 showed pododermatitis. All sera were also screened using a commercial direct agglutination test (DAT). The DAT showed a similar specificity but a lower sensitivity when compared with IFAT as a reference technique.     

Physiological regulation of the expression of a GLUT4 homolog in fish skeletal muscle

We have recently cloned a glucose transporter from brown trout muscle (btGLUT) with high sequence homology to mammalian GLUT4 that is predominantly expressed in red and white skeletal muscle, the two major sites of glucose uptake in trout. To study the physiological regulation of this putative fish GLUT4, we have investigated the expression of btGLUT in red and white skeletal muscle of trout in which blood insulin levels have been altered experimentally. The expression of btGLUT in red muscle increased significantly when insulin plasma levels were elevated by either insulin or arginine treatment and decreased significantly when insulin plasma levels were reduced either by fasting or by feeding a low-protein, high-carbohydrate diet. In contrast, the expression of btGLUT in white muscle was not affected by changes in the plasma levels of insulin. These results strongly suggest that insulin could be regulating the expression of btGLUT in trout red muscle in vivo and set the ground to test the hypothesis that btGLUT may be considered a GLUT4 homolog in fish.     

The effects of deer exclusion on the development of a Mediterranean plant community affected by a wildfire

Despite the resilience of Mediterranean ecosystems to fire, the ecological restoration of burned plant communities can be hindered by ungulate herbivores, particularly in areas with high population densities. This study compares the postfire development of a shrub community with and without deer, after a wildfire occurred in 2003 in a protected area in Central Portugal. We monitored 12 fenced and 12 unfenced plots 2, 3, 4, and 8 years after fire. Within each plot, we established a linear transect and measured the monospecific canopy projections (plant patches). Five plant community indicators (patch number, average patch length, average patch height, patch cover, and patch phytovolume per square meter) were obtained. The diameter and height of individuals of the most abundant shrub species (Cistus salvifolius, Erica scoparia, Myrtus communis, Pistacia lentiscus, Rubus ulmifolius, and Ulex jussiaei) were also measured. These measurements were used as response variables in generalized linear mixed models in order to assess the effects of time‐after‐fire and fencing, on the development of the plant community. Patch height and phytovolume had a significantly higher growth in fenced plots. At the species level, C. salvifolius, M. communis, R. ulmifolius, and U. jussiaei showed a higher growth across time both in height and in diameter, in the absence of herbivory. This work shows that deer exclusion needs to be considered when aiming at the postfire restoration of Mediterranean shrub communities.     

Impacts on Coralligenous Outcrop Biodiversity of a Dramatic Coastal Storm

Extreme events are rare, stochastic perturbations that can cause abrupt and dramatic ecological change within a short period of time relative to the lifespan of organisms. Studies over time provide exceptional opportunities to detect the effects of extreme climatic events and to measure their impacts by quantifying rates of change at population and community levels. In this study, we show how an extreme storm event affected the dynamics of benthic coralligenous outcrops in the NW Mediterranean Sea using data acquired before (2006–2008) and after the impact (2009–2010) at four different sites. Storms of comparable severity have been documented to occur occasionally within periods of 50 years in the Mediterranean Sea. We assessed the effects derived from the storm comparing changes in benthic community composition at sites exposed to and sheltered from this extreme event. The sites analyzed showed different damage from severe to negligible. The most exposed and impacted site experienced a major shift immediately after the storm, represented by changes in the species richness and beta diversity of benthic species. This site also showed higher compositional variability immediately after the storm and over the following year. The loss of cover of benthic species resulted between 22% and 58%. The damage across these species (e.g. calcareous algae, sponges, anthozoans, bryozoans, tunicates) was uneven, and those with fragile forms were the most impacted, showing cover losses up to 50 to 100%. Interestingly, small patches survived after the storm and began to grow slightly during the following year. In contrast, sheltered sites showed no significant changes in all the studied parameters, indicating no variations due to the storm. This study provides new insights into the responses to large and rare extreme events of Mediterranean communities with low dynamics and long-lived species, which are among the most threatened by the effects of global change.