Development and validation of the Axiom®Apple480K SNP genotyping array

Cultivated apple (Malus × domestica Borkh.) is one of the most important fruit crops in temperate regions, and has great economic and cultural value. The apple genome is highly heterozygous and has undergone a recent duplication which, combined with a rapid linkage disequilibrium decay, makes it difficult to perform genome‐wide association (GWA) studies. Single nucleotide polymorphism arrays offer highly multiplexed assays at a relatively low cost per data point and can be a valid tool for the identification of the markers associated with traits of interest. Here, we describe the development and validation of a 487K SNP Affymetrix Axiom^® genotyping array for apple and discuss its potential applications. The array has been built from the high‐depth resequencing of 63 different cultivars covering most of the genetic diversity in cultivated apple. The SNPs were chosen by applying a focal points approach to enrich genic regions, but also to reach a uniform coverage of non‐genic regions. A total of 1324 apple accessions, including the 92 progenies of two mapping populations, have been genotyped with the Axiom^®Apple480K to assess the effectiveness of the array. A large majority of SNPs (359 994 or 74%) fell in the stringent class of poly high resolution polymorphisms. We also devised a filtering procedure to identify a subset of 275K very robust markers that can be safely used for germplasm surveys in apple. The Axiom^®Apple480K has now been commercially released both for public and proprietary use and will likely be a reference tool for GWA studies in apple.     

The relevance of malondialdehyde as a biochemical index of lipid peroxidation of postischemic tissues in the rat and human beings

By using a recently developed ion-pairing high-performance liquid chromatographic method for the direct determination of malondialdehyde (MDA) and several other acid-soluble low-mol-wt compounds (ascorbate, oxypurines, nucleosides, nicotinic coenzymes, high-energy phosphates), the variations of tissue and plasma MDA as a function of ischemia and reperfusion were determined in the rat (isolated Langendorff-perfused hearts and short-term incomplete cerebral ischemia) and in human beings (patients suffering from acute myocardial infarction subjected to fibrinolysis). In the rat, the data obtained indicate that, contrary to what had been previously reported in literature, MDA is not present either in control heart or in control brain. Oxygen deprivation induces the production of a low, but detectable amount of MDA in both heart and brain, whereas reperfusion causes a marked increase of MDA in both tissues. In human beings, plasma MDA was deeply affected only in patients suffering from acute myocardial infarction with successful thrombolysis, thus indicating the occurrence of oxygen radical-mediated tissue injury also in humans. On the whole, these results suggest that MDA is a valid biochemical marker of lipid peroxidation of postischemic tissues, which however needs a reliable analytical technique for its determination.     

A new genomic tool for walnut (Juglans regia L.): development and validation of the high‐density Axiom™ J. regia 700K SNP genotyping array

Over the last 20 years, global production of Persian walnut (Juglans regia L.) has grown enormously, likely reflecting increased consumption due to its numerous benefits to human health. However, advances in genome‐wide association (GWA) studies and genomic selection (GS) for agronomically important traits in walnut remain limited due to the lack of powerful genomic tools. Here, we present the development and validation of a high‐density 700K single nucleotide polymorphism (SNP) array in Persian walnut. Over 609K high‐quality SNPs have been thoroughly selected from a set of 9.6 m genome‐wide variants, previously identified from the high‐depth re‐sequencing of 27 founders of the Walnut Improvement Program (WIP) of University of California, Davis. To validate the effectiveness of the array, we genotyped a collection of 1284 walnut trees, including 1167 progeny of 48 WIP families and 26 walnut cultivars. More than half of the SNPs (55.7%) fell in the highest quality class of ‘Poly High Resolution’ (PHR) polymorphisms, which were used to assess the WIP pedigree integrity. We identified 151 new parent‐offspring relationships, all confirmed with the Mendelian inheritance test. In addition, we explored the genetic variability among cultivars of different origin, revealing how the varieties from Europe and California were differentiated from Asian accessions. Both the reconstruction of the WIP pedigree and population structure analysis confirmed the effectiveness of the Applied Biosystems? Axiom? J. regia 700K SNP array, which initiates a novel genomic and advanced phase in walnut genetics and breeding.     

Ischemia and Reperfusion: Effect of Fructose-1,6-Bisphosphate

Several lines of evidence indicating a close relationship among ischemia, concentration of high-energy metabolites and onset of the “oxygen paradox” in reperfused tissues have been published. In this framework, we have recently studied the effects of exogenous fructose-1,6-bisphosphate on energy metabolism and on oxygen free radical damages of isolated rat heart subjected to anoxia and reoxygenation. In comparison with control groups, hearts perfused in the presence of 5mM fructose-1,6-bisphosphate throughout the different perfusion conditions showed higher concentrations of energy metabolites at the end of anoxia, most of which were normalized after reperfusion. Furthermore, in comparison with control hearts, a reduction of tissue malondialdehyde and of lactate dehydrogenase release in the perfusate was observed in fructose-1,6-bisphosphate-perfused hearts. In this article we review most of the available data concerning the ability of fructose-1,6-bisphosphate to protect from ischemia and reperfusion damage outlining those recent findings which contributed both to clarify the pharmacological profile of the drug and to give an insight in its probable mechanism of action.     

Malondialdehyde production and ascorbate decrease are associated to the reperfusion of the isolated postischemic rat heart.

Isolated Langendorff-perfused rat hearts after 20 min of normoxic perfusion in the presence of 2.5 mM Ca++ and 11 mM glucose were subjected to 30 min of global normothermic ischemia followed by 30 min of normoxic reperfusion with the starting buffer. At the end of each perfusion condition, hearts were freeze-clamped and deproteinized by 0.6 M HClO4. Two-hundred microL of the neutralized tissue extracts were analyzed by a recently developed high-performance liquid chromatography (HPLC) method for the simultaneous determination of malondialdehyde (MDA), ascorbic acid, and adenine nucleotides. By means of this analytical technique, it was possible to demonstrate that MDA is undetectable in control hearts. In contrast, 30 min of ischemia induced a modest production of MDA (0.012 mumol/g dw), while a large amount of MDA (0.103 mumol/g dw) was observed in reperfused hearts. Values referring to ascorbic acid showed that the concentration of this antioxidant progressively decreased from 1.190 (control hearts) to 0.837 (ischemic hearts) and to 0.595 mumol/g dw (reperfused hearts). The overall conclusions of this study are that reperfusion induces an oxidative stress to the isolated myocardium, a decrease of ascorbate, and an increase of lipid peroxidation. Therefore, by means of a proper analytical method, MDA may represent a valid biochemical parameter to demonstrate the relationship between myocardial reperfusion and a detectable tissue damage.     

Regulatory Mode and Risk-Taking: The Mediating Role of Anticipated Regret

We propose that decision maker’s regulatory mode affects risk-taking through anticipated regret. In the Study 1 either a locomotion or an assessment orientation were experimentally induced, and in the Studies 2 and 3 these different orientations were assessed as chronic individual differences. To assess risk-taking we used two behavioral measures of risk: BART and hot-CCT. The results show that experimentally induced assessment orientation–compared to locomotion–leads to decreased risk-taking through increased anticipated regret (Study 1). People chronically predisposed to be in the assessment state take less risk through increased anticipated regret (Study 2 and Study 3). Study 2 results also show a marginally non-significant indirect effect of chronic locomotion mode on BART through anticipated regret. Differently, Study 3 shows that people chronically predisposed to be in the locomotion state take greater risk through decreased anticipated regret, when play a dynamic risk task triggering stronger emotional arousal. Through all three studies, the average effect size for the relationship of assessment with anticipated regret was in the moderate-large range, whereas for risk-taking was in the moderate range. The average effect size for the relationship of locomotion with anticipated regret was in the moderate range, whereas for risk-taking was in the small-moderate range. These results increase our understanding of human behavior under conditions of risk obtaining novel insights into regulatory mode theory and decision science.     

Prognostic Impact of Reduced Connexin43 Expression and Gap Junction Coupling of Neoplastic Stromal Cells in Giant Cell Tumor of Bone

Missense mutations of the GJA1 gene encoding the gap junction channel protein connexin43 (Cx43) cause bone malformations resulting in oculodentodigital dysplasia (ODDD), while GJA1 null and ODDD mutant mice develop osteopenia. In this study we investigated Cx43 expression and channel functions in giant cell tumor of bone (GCTB), a locally aggressive osteolytic lesion with uncertain progression. Cx43 protein levels assessed by immunohistochemistry were correlated with GCTB cell types, clinico-radiological stages and progression free survival in tissue microarrays of 89 primary and 34 recurrent GCTB cases. Cx43 expression, phosphorylation, subcellular distribution and gap junction coupling was also investigated and compared between cultured neoplastic GCTB stromal cells and bone marow stromal cells or HDFa fibroblasts as a control. In GCTB tissues, most Cx43 was produced by CD163 negative neoplastic stromal cells and less by CD163 positive reactive monocytes/macrophages or by giant cells. Significantly less Cx43 was detected in α-smooth muscle actin positive than α-smooth muscle actin negative stromal cells and in osteoclast-rich tumor nests than in the adjacent reactive stroma. Progressively reduced Cx43 production in GCTB was significantly linked to advanced clinico-radiological stages and worse progression free survival. In neoplastic GCTB stromal cell cultures most Cx43 protein was localized in the paranuclear-Golgi region, while it was concentrated in the cell membranes both in bone marrow stromal cells and HDFa fibroblasts. In Western blots, alkaline phosphatase sensitive bands, linked to serine residues (Ser369, Ser372 or Ser373) detected in control cells, were missing in GCTB stromal cells. Defective cell membrane localization of Cx43 channels was in line with the significantly reduced transfer of the 622 Da fluorescing calcein dye between GCTB stromal cells. Our results show that significant downregulation of Cx43 expression and gap junction coupling in neoplastic stromal cells are associated with the clinical progression and worse prognosis in GCTB.     

Transglutaminase-catalyzed site-specific glycosidation of catalase with aminated dextran

An enzymatic approach, based on a transglutaminase-catalyzed coupling reaction, was investigated to modify bovine liver catalase with an end-group aminated dextran derivative. We demonstrated that catalase activity increased after enzymatic glycosidation and that the conjugate was 3.8-fold more stable to thermal inactivation at 55 degrees C and 2-fold more resistant to proteolytic degradation by trypsin. Moreover, the transglutaminase-mediated modification also improved the pharmacokinetics behavior of catalase, increasing 2.5-fold its plasma half-life time and reducing 3-fold the total clearance after its i.v. administration in rats.