First survey of the wheat chromosome 5A composition through a next generation sequencing approach

Wheat is one of the world's most important crops and is characterized by a large polyploid genome. One way to reduce genome complexity is to isolate single chromosomes using flow cytometry. Low coverage DNA sequencing can provide a snapshot of individual chromosomes, allowing a fast characterization of their main features and comparison with other genomes. We used massively parallel 454 pyrosequencing to obtain a 2x coverage of wheat chromosome 5A. The resulting sequence assembly was used to identify TEs, genes and miRNAs, as well as to infer a virtual gene order based on the synteny with other grass genomes. Repetitive elements account for more than 75% of the genome. Gene content was estimated considering non-redundant reads showing at least one match to ESTs or proteins. The results indicate that the coding fraction represents 1.08% and 1.3% of the short and long arm respectively, projecting the number of genes of the whole chromosome to approximately 5,000. 195 candidate miRNA precursors belonging to 16 miRNA families were identified. The 5A genes were used to search for syntenic relationships between grass genomes. The short arm is closely related to Brachypodium chromosome 4, sorghum chromosome 8 and rice chromosome 12; the long arm to regions of Brachypodium chromosomes 4 and 1, sorghum chromosomes 1 and 2 and rice chromosomes 9 and 3. From these similarities it was possible to infer the virtual gene order of 392 (5AS) and 1,480 (5AL) genes of chromosome 5A, which was compared to, and found to be largely congruent with the available physical map of this chromosome.     

Obestatin regulates cardiovascular function and promotes cardioprotection through the nitric oxide pathway

Patients with ischaemic heart disease or chronic heart failure show altered levels of obestatin, suggesting a role for this peptide in human heart function. We have previously demonstrated that GH secretagogues and the ghrelin gene‐derived peptides, including obestatin, exert cardiovascular effects by modulating cardiac inotropism and vascular tone, and reducing cell death and contractile dysfunction in hearts subjected to ischaemia/reperfusion (I/R), through the Akt/nitric oxide (NO) pathway. However, the mechanisms underlying the cardiac actions of obestatin remain largely unknown. Thus, we suggested that obestatin‐induced activation of PI3K/Akt/NO and PKG signalling is implicated in protection of the myocardium when challenged by adrenergic, endothelinergic or I/R stress. We show that obestatin exerts an inhibitory tone on the performance of rat papillary muscle in both basal conditions and under β‐adrenergic overstimulation, through endothelial‐dependent NO/cGMP/PKG signalling. This pathway was also involved in the vasodilator effect of the peptide, used both alone and under stress induced by endothelin‐1. Moreover, when infused during early reperfusion, obestatin reduced infarct size in isolated I/R rat hearts, through an NO/PKG pathway, comprising ROS/PKC signalling, and converging on mitochondrial ATP‐sensitive potassium [mitoK(ATP)] channels. Overall, our results suggest that obestatin regulates cardiovascular function in stress conditions and induces cardioprotection by mechanisms dependent on activation of an NO/soluble guanylate cyclase (sGC)/PKG pathway. In fact, obestatin counteracts exaggerated β‐adrenergic and endothelin‐1 activity, relevant factors in heart failure, suggesting multiple positive effects of the peptide, including the lowering of cardiac afterload, thus representing a potential candidate in pharmacological post‐conditioning.     

Homology,limbs, and genitalia

SUMMARY Similarities in genetic control between the main body axis and its appendages have been generally explained in terms of genetic co-option. In particular, arthropod and vertebrate appendages have been explained to invoke a common ancestor already provided with patterned body outgrowths or independent recruitment in limb patterning of genes or genetic cassettes originally used for purposes other than axis patterning. An alternative explanation is that body appendages, including genitalia, are evolutionarily divergent duplicates (paramorphs) of the main body axis. However, are all metazoan limbs and genitalia homologous? The concept of body appendages as paramorphs of the main body axis eliminates the requirement for the last common ancestor of limb-bearing animals to have been provided with limbs. Moreover, the possibility for an animal to express complex organs ectopically demonstrates that positional and special homology may be ontogenetically and evolutionarily uncoupled. To assess the homology of animal genitalia, we need to take into account three different sets of mechanisms, all contributing to their positional and/or special homology and respectively involved (1) in the patterning of the main body axis, (2) in axis duplication, followed by limb patterning mechanisms diverging away from those still patterning the main body axis (axis paramorphism), and (3) in controlling the specification of sexual/genital features, which often, but not necessarily, come into play by modifying already developed and patterned body appendages. This analysis demonstrates that a combinatorial approach to homology helps disentangling phylogenetic and ontogenetic layers of homology.     

If pemphigus vulgaris IgG are the cause of acantholysis, new IgG-independent mechanisms are the concause

Pemphigus vulgaris (PV) is a disease of epidermal adhesion. Its pathogenesis is currently traced back to the action of autoantibodies against antigens located within the intercellular substance of keratinocytes, such as desmogleins and acetylcholine receptors. In the present paper, we sought to elucidate the non-IgG-mediated effects of PV sera on keratinocytes. Results showed that PV sera depleted of IgG were able to induce well-defined changes on keratinocyte morphology and metabolic activity. Indeed, PV IgG-free sera determined marked alterations on cell shape, accompanied by partial loss of keratinocyte-keratinocyte interactions within 48 h after treatment. Furthermore, PV IgG-depleted sera caused a sharp reduction of cell viability along with a less sustained weakening of intercellular adhesion strength. In light of the above findings, loss of cell-cell adhesion in PV occurs as a result of the cooperating action of both IgG and non-IgG-mediated mechanisms. These data have remarkable consequences on experimental models of PV and might open new "biological" approaches to its therapy. Thus, researchers are well advised that PV pathophysiology cannot be faithfully reproduced by leaving non-IgG serum factors out of consideration.     

Epigenetic chromatin modifiers in barley: IV. The study of barley Polycomb group (PcG) genes during seed development and in response to external ABA

Background  

Epigenetic phenomena have been associated with the regulation of active and silent chromatin states achieved by modifications of chromatin structure through DNA methylation, and histone post-translational modifications. The latter is accomplished, in part, through the action of PcG (Polycomb group) protein complexes which methylate nucleosomal histone tails at specific sites, ultimately leading to chromatin compaction and gene silencing. Different PcG complex variants operating during different developmental stages have been described in plants. In particular, the so-called FIE/MEA/FIS2 complex governs the expression of genes important in embryo and endosperm development in Arabidopsis. In our effort to understand the epigenetic mechanisms regulating seed development in barley (Hordeum vulgare), an agronomically important monocot plant cultivated for its endosperm, we set out to characterize the genes encoding barley PcG proteins.     

Relationship Between Basal Metabolic Rate,Gender, Age,and Body Composition in 8,780 White Obese Subjects

The objective of the present study was to explore the relationship between basal metabolic rate (BMR), gender, age, anthropometric characteristics, and body composition in severely obese white subjects. In total, 1,412 obese white children and adolescents (BMI > 97° percentile for gender and age) and 7,368 obese adults (BMI > 30 kg/m²) from 7 to 74 years were enrolled in this study. BMR was measured using an indirect calorimeter equipped with a canopy and fat free mass (FFM) were obtained using tetrapolar bioelectrical impedance analysis (BIA). Using analysis of covariance, we tested the effect of gender on the relationship between BMR, age, anthropometry, and body composition. In children and adolescents, the predictor × gender interaction was significant in all cases except for FFM × gender. In adults, all predictor × gender interactions were significant. A prediction equation based on body weight (BW), age, and gender had virtually the same accuracy of the one based on FFM, age, and gender to predict BMR in both children and adults (R²_adj = 0.59 and 0.60, respectively). In conclusion, gender was a significant determinant of BMR in children and adolescents but not in adults. Our results support the hypothesis that the age‐related decline in BMR is due to a reduction in FFM. Finally, anthropometric predictors of BMR are as accurate as body composition estimated by BIA.     

The membrane environment of endogenous cellular prion protein in primary rat cerebellar neurons

We studied the membrane environment of cellular prion protein in primary cultured rat cerebellar neurons differentiated in vitro. In these cells, about 45% of total cellular prion protein (corresponding to a 35-fold enrichment) is associated with a low-density, sphingolipid- and cholesterol-enriched membrane fraction, that can be separated by flotation on sucrose gradient. Biotinylation experiments indicated that almost all prion protein recovered in this fraction was exposed at the cell surface. Prion protein was efficiently separated from this fraction by a monoclonal antibody immuno-separation procedure. Under conditions designed to preserve lipid-mediated membrane organization, several proteins were found in the prion protein-enriched membrane domains (i.e. the non-receptor tyrosine kinases Lyn and Fyn and the neuronal glycosylphosphatidylinositol-anchored protein Thy-1). The prion protein-rich membrane domains contained, as well, about 50% of the sphingolipids, cholesterol and phosphatidylcholine present in the sphingolipid-enriched membrane fraction. All main sphingolipids, including sphingomyelin, neutral glycosphingolipids and gangliosides, were similarly enriched in the prion protein-rich membrane domains. Thus, prion protein plasma membrane environment in differentiated neurons resulted to be a complex entity, whose integrity requires a network of lipid-mediated non-covalent interactions.     

Influence of formulation and process parameters on pellet production by powder layering technique

The goal of the present study was to evaluate the influence of the formulation and operating conditions on pellet preparation by pan technique. To this end, a new pelletization process, typified by the application of powdered drug on sugar-based cores using the GS coating system was studied. Inert cores were intermittently treated with micronized drug powder and adhesive solution. This treatment led to the formation of multiple layers of drug particles around an inert core resulting in the production of pellets that can further be coated by different polymers to obtain modified release formulations. Different procedures have been used to evaluate a series of important parameters such as initial core weight; speed of powder application; speed, type, and position of the atomizers; atomization degree: temperature; and air cap. Good yield of drug layering was obtained by adjusting the quantity of both the drug powder to apply and the binder solution. Pellets obtained following the optimal operating conditions (defined in a pre-formulation study) were film coated with the acrylic polymer Eudragit L30D in order to produce a model formulation consisting of enteric polymer-coated pellets containing ibuprofen. During its preparation, the formulation showed no degradation of the drug, moreover, a low percentage of residual humidity was obtained, indicating that this system is very efficient for the production of highly stable formulations. This study showed the good performance of the GS automated pan-coating system in obtaining enteric coated pellets prepared by powder layering technique using aqueous solutions.     

Identification of pathogenicity‐related genes in Fusarium oxysporum f. sp. cepae

Pathogenic isolates of Fusarium oxysporum, distinguished as formae speciales (f. spp.) on the basis of their host specificity, cause crown rots, root rots and vascular wilts on many important crops worldwide. Fusarium oxysporum f. sp. cepae (FOC) is particularly problematic to onion growers worldwide and is increasing in prevalence in the UK. We characterized 31 F. oxysporum isolates collected from UK onions using pathogenicity tests, sequencing of housekeeping genes and identification of effectors. In onion seedling and bulb tests, 21 isolates were pathogenic and 10 were non‐pathogenic. The molecular characterization of these isolates, and 21 additional isolates comprising other f. spp. and different Fusarium species, was carried out by sequencing three housekeeping genes. A concatenated tree separated the F. oxysporum isolates into six clades, but did not distinguish between pathogenic and non‐pathogenic isolates. Ten putative effectors were identified within FOC, including seven Secreted In Xylem (SIX) genes first reported in F. oxysporum f. sp. lycopersici. Two highly homologous proteins with signal peptides and RxLR motifs (CRX1/CRX2) and a gene with no previously characterized domains (C5) were also identified. The presence/absence of nine of these genes was strongly related to pathogenicity against onion and all were shown to be expressed in planta. Different SIX gene complements were identified in other f. spp., but none were identified in three other Fusarium species from onion. Although the FOC SIX genes had a high level of homology with other f. spp., there were clear differences in sequences which were unique to FOC, whereas CRX1 and C5 genes appear to be largely FOC specific.     

Evidence of the crucial role of the linker domain on the catalytic activity of human topoisomerase I by experimental and simulative characterization of the Lys681Ala mutant

The functional and structural-dynamical properties of the Lys681Ala mutation in the human topoisomerase IB linker domain have been investigated by catalytic assays and molecular dynamics simulation. The mutant is characterized by a comparable cleavage and a strongly reduced religation rate when compared to the wild type protein. The mutant also displays perturbed linker dynamics, as shown by analysis of the principal components of the motion, and a reduced electrostatic interaction with DNA. Inspection of the inter atomic distances in proximity of the active site shows that in the mutant the distance between the amino group of Lys532 side chain and the 5′ OH of the scissile phosphate is longer than the wild type enzyme, providing an atomic explanation for the reduced religation rate of the mutant. Taken together these results indicate the existence of a long range communication between the linker domain and the active site region and points out the crucial role of the linker in the modulation of the catalytic activity.