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.     

Three-dimensional model for the isolated recombinant influenza virus polymerase heterotrimer

The genome of influenza A virus is organized into eight ribonucleoprotein complexes (RNPs), each containing one RNA polymerase complex. This RNA polymerase has also been found non-associated to RNPs and is possibly involved in distinct functions in the infection cycle. We have expressed the virus RNA polymerase complex by co-tranfection of the PB1, PB2 and PA genes in mammalian cells and the heterotrimer was purified by the TAP tag procedure. Its 3D structure was determined by electron microscopy and single-particle image processing. The model obtained resembles the structure previously reported for the polymerase complex associated to viral RNPs but appears to be in a more open conformation. Detailed model comparison indicated that specific areas of the complex show important conformational changes as compared to the structure for the RNP-associated polymerase, particularly in regions known to interact with the adjacent NP monomers in the RNP. Also, the PB2 subunit seems to undergo a substantial displacement as a result of the association of the polymerase to RNPs. The structural model presented suggests that a core conformation of the polymerase in solution exists but the interaction with other partners, such as proteins or RNA, will trigger distinct conformational changes to activate new functional properties.     

Liver injury after an aggressive encounter in male mice

Acute and intense psychological stressors induce cell damage in several organs, including the heart and the liver. Much less is known about social stress. In male mice, aggressive behavior is the most common social stressor. It is remarkable that upon fighting, submandibular salivary glands release a number of peptides into the bloodstream including epidermal growth factor (EGF). We showed previously that released EGF protects the heart from cell damage in this particular stressful situation. Here, we studied the effect of an aggressive encounter on the liver and whether EGF has a similar effect on this organ. An aggressive encounter in male mice caused inflammatory response and a transient increase in plasma alanine and aspartate transaminase activities. At 3 h, focal infiltration of neutrophils was observed in liver parenchyma. These cells accumulate on eosinophilic hepatocytes, which may correspond to dying cells. A few hours later, evidence of necrotic lesion was observed. Surgical excision of submandibular glands, sialoadenectomy, did not prevent the rise in plasma EGF concentration and did not affect the increase in plasma transaminase activities. Neither did the administration of tyrphostin AG-1478 (inhibitor of EGF receptor kinase) alter the increase in plasma alanine transaminase activity. However, it did enhance the rise in both aspartate transaminase and creatine kinase activity, suggesting heart damage. We conclude that an aggressive encounter causes mild liver damage and that released EGF does not protect this organ, in contrast to its effect on the heart.     

Re-engineering specificity in 1,3-1, 4-β-glucanase to accept branched xyloglucan substrates

Family 16 carbohydrate active enzyme members Bacillus licheniformis 1,3-1,4-β-glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase (XET16-34) are highly structurally related but display different substrate specificities. Although the first binds linear gluco-oligosaccharides, the second binds branched xylogluco-oligosaccharides. Prior engineered nucleophile mutants of both enzymes are glycosynthases that catalyze the condensation between a glycosyl fluoride donor and a glycoside acceptor. With the aim of expanding the glycosynthase technology to produce designer oligosaccharides consisting of hybrids between branched xylogluco- and linear gluco-oligosaccharides, enzyme engineering on the negative subsites of 1,3-1,4-β-glucanase to accept branched substrates has been undertaken. Removal of the 1,3-1,4-β-glucanase major loop and replacement with that of XET16-34 to open the binding cleft resulted in a folded protein, which still maintained some β-glucan hydrolase activity, but the corresponding nucleophile mutant did not display glycosynthase activity with either linear or branched glycosyl donors. Next, point mutations of the 1,3-1,4-β-glucanase β-sheets forming the binding site cleft were mutated to resemble XET16-34 residues. The final chimeric protein acquired binding affinity for xyloglucan and did not bind β-glucan. Therefore, binding specificity has been re-engineered, but affinity was low and the nucleophile mutant of the chimeric enzyme did not show glycosynthase activity to produce the target hybrid oligosaccharides. Structural analysis by X-ray crystallography explains these results in terms of changes in the protein structure and highlights further engineering approaches toward introducing the desired activity.     

Recent trends in butterfly populations from north-east Spain and Andorra in the light of habitat and climate change

Although butterfly declines have been reported across Europe, no assessment based on detailed quantitative data has ever been made for any extensive area in the Mediterranean Basin. In 1994, a Butterfly Monitoring Scheme was launched in Catalonia (NE Spain), and in 2005 a similar, albeit much smaller, scheme started in the neighbouring Pyrenean country of Andorra. Here we provide a first thorough assessment of butterfly trends in both areas for the last 15 years. Several patterns emerged, above all a worrying decline of a substantial part of the fauna. It was also evident that habitat specialists are experiencing greater declines than habitat generalists, thereby butterfly communities becoming progressively dominated by common species. However, habitat indicators based on characteristic species also revealed that trends are actually associated with habitat types, grassland and scrub specialists declining strongly but woodland specialists showing a marginal increase. These differences are certainly related to profound landscape changes, mainly a dramatic reduction of semi-natural grasslands and open Mediterranean scrub, and a major increase in woodlands. The general effect of climatic warming on butterfly populations was investigated by using the temperature community index (CTI) approach. The thermal structure of butterfly communities remained very stable over time, except in one case where, contrary to the expectations, a significant negative trend in the CTI was noted. However, this surprising result can be explained by taking into account the above-reported pattern of butterfly communities becoming dominated by common species, characterized by low thermal indices in comparison with declining Mediterranean specialists.     

Capture myopathy in little bustards after trapping and marking

Four little bustards (Tetrax tetrax) (one adult and three juvenile males), captured with leg nooses and fitted with a backpack radiotag, died after capture. The first bird was found after 16 days with its left foot caught in the harness and died after 1 day. The other birds showed symptoms of capture myopathy after release, such as the difficulty or inability to fly and/or walk. They died after 5, 6, and 8 days, respectively. At necropsy, muscles affected in all cases were those from the legs, and these were diffusely pale and dull, with a soft friable texture. Microscopically these muscles had multiple foci of myofiber fragmentation, loss of striation, and necrosis; a mononuclear cell infiltrate was observed in muscle from two birds. These findings suggest the little bustard is susceptible to capture myopathy and that caution should be exercised during its capture and handling.     

S 23521 Decreases Food Intake and Body Weight Gain in Diet‐Induced Obese Rats

Objective: To investigate the effect of S 23521, a new glucagon‐like peptide‐1‐(7‐36) amide analogue, on food intake and body weight gain in obese rats, as well as on gene expression of several proteins involved in energy homeostasis. Research Methods and Procedures: Lean and diet‐induced obese rats were treated with either S 23521 or vehicle. S 23521 was given either intraperitoneally (10 or 100 μg/kg) or subcutaneously (100 μg/kg) for 14 and 20 days, respectively. Because the low‐dose treatment did not affect food intake and body weight, the subcutaneous treatment at high dose was selected to test the effect on selected end‐points. Results: Treated obese rats significantly decreased their cumulative energy intake in relation to vehicle‐treated counterparts (3401 ± 65 vs. 3898 ± 72 kcal/kg per 20 days; p < 0.05). Moreover, their body weight gain was reduced by 110%, adiposity was reduced by 20%, and plasma triglyceride levels were reduced by 38%. The treatment also improved glucose tolerance and insulin sensitivity of obese rats. Regarding gene expression, no changes in uncoupling protein‐1, uncoupling protein‐3, leptin, resistin, and peroxisome proliferator‐activated receptor (PPAR)‐γ were observed. Discussion: S 23521 is an effective glucagon‐like peptide‐1‐(7‐36) amide analogue, which induced a decrease in energy intake, body weight, and adiposity in a rat model of diet‐induced obesity. In addition, the treatment also improved glucose tolerance and insulin sensitivity of obese rats. These results strongly support S 23521 as a putative molecule for the treatment of obesity.     

Targeting Dyrk1A with AAVshRNA attenuates motor alterations in TgDyrk1A, a mouse model of Down syndrome

Genetic-dissection studies carried out with Down syndrome (DS) murine models point to the critical contribution of Dyrk1A overexpression to the motor abnormalities and cognitive deficits displayed in DS individuals. In the present study we have used a murine model overexpressing Dyrk1A (TgDyrk1A mice) to evaluate whether functional CNS defects could be corrected with an inhibitory RNA against Dyrk1A, delivered by bilateral intrastriatal injections of adeno-associated virus type 2 (AAVshDyrk1A). We report that AAVshDyrk1A efficiently transduced HEK293 cells and primary neuronal cultures, triggering the specific inhibition of Dyrk1A expression. Injecting the vector into the striata of TgDyrk1A mice resulted in a restricted, long-term transduction of the striatum. This gene therapy was found to be devoid of toxicity and succeeded in normalizing Dyrk1A protein levels in TgDyrk1A mice. Importantly, the behavioral studies of the adult TgDyrk1A mice treated showed a reversal of corticostriatal-dependent phenotypes, as revealed by the attenuation of their hyperactive behavior, the restoration of motor-coordination defects, and an improvement in sensorimotor gating. Taken together, the data demonstrate that normalizing Dyrk1A gene expression in the striatum of adult TgDyrk1A mice, by means of AAVshRNA, clearly reverses motor impairment. Furthermore, these results identify Dyrk1A as a potential target for therapy in DS.