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Allergy to freshly consumed apple fruits is often associated to pollinosis and manifested as oral allergy syndrome (OAS). The allergenic properties of apple varieties differ greatly, spanning from low allergenic to high allergenic varieties. The knowledge of the genetic determinants for allergenicity has been of great interest in scientific community for several years, but the molecular mechanisms involved are still little understood. Here, factors putatively involved in allergenicity were investigated at biochemical and molecular level in pollen and in fruits of apple varieties differing in their allergenic potential. Among putative sensitizing factors, transglutaminase (TGase) and phospholipase A 2 (PLA 2 ) were considered together with reactive oxygen species (ROS) and known apple allergen genes, with particular attention devoted to the Mal d 1 gene family, the most important one in sensitization. We found that the expression of some allergen genes and the activities of TGase, PLA 2 and ROS producing enzyme are lower in the hypo-allergenic variety 'Durello di Forlì' in comparison with the high-allergenic genotypes 'Gala' and 'Florina'. These results highlight correlations among allergen expressions, enzymatic activities and apple cultivars; these data underline the possibility that some of them could be used in the future as markers for allergenicity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The availability of the peach genome sequence has fostered relevant research in peach and related Prunus species enabling the identification of genes underlying important horticultural traits as well as the development of advanced tools for genetic and genomic analyses. The first release of the peach genome (Peach v1.0) represented a high-quality WGS (Whole Genome Shotgun) chromosome-scale assembly with high contiguity (contig L50 214.2 kb), large portions of mapped sequences (96%) and high base accuracy (99.96%). The aim of this work was to improve the quality of the first assembly by increasing the portion of mapped and oriented sequences, correcting misassemblies and improving the contiguity and base accuracy using high-throughput linkage mapping and deep resequencing approaches., Results: Four linkage maps with 3,576 molecular markers were used to improve the portion of mapped and oriented sequences (from 96.0% and 85.6% of Peach v1.0 to 99.2% and 98.2% of v2.0, respectively) and enabled a more detailed identification of discernible misassemblies (10.4 Mb in total). The deep resequencing approach fixed 859 homozygous SNPs (Single Nucleotide Polymorphisms) and 1347 homozygous indels. Moreover, the assembled NGS contigs enabled the closing of 212 gaps with an improvement in the contig L50 of 19.2%., Conclusions: The improved high quality peach genome assembly (Peach v2.0) represents a valuable tool for the analysis of the genetic diversity, domestication, and as a vehicle for genetic improvement of peach and related Prunus species. Moreover, the important phylogenetic position of peach and the absence of recent whole genome duplication (WGD) events make peach a pivotal species for comparative genomics studies aiming at elucidating plant speciation and diversification processes.

Freshly consumed apples (Malus domestica L. Borkh) can cause allergic reactions because of the presence of four classes of allergens. Knowledge of the genetic factors affecting the allergenic potential of apples would provide important information for the selection of hypoallergenic genotypes, which can be combined with the adoption of new agronomical practices to produce fruits with a reduced amount of allergens. In the present research, a multiple analytical approach was adopted to characterize the allergenic potential of 24 apple varieties released at different ages (pre- and post-green revolution). A specific workflow was set up including protein quantification by means of polyclonal antibodies, immunological analyses with sera of allergic subjects, enzymatic assays, clinical assessments on allergic patients, and gene expression assays on fruit samples. Taken as a whole, the results indicate that most of the less allergenic genotypes were found among those deriving from selection processes carried out prior to the so-called "green revolution".

Quantitative trait loci (QTL) mapping approaches rely on the correct ordering of molecular markers along the chromosomes, which can be obtained from genetic linkage maps or a reference genome sequence. For apple ( Malus domestica Borkh), the genome sequence v1 and v2 could not meet this need; therefore, a novel approach was devised to develop a dense genetic linkage map, providing the most reliable marker-loci order for the highest possible number of markers. The approach was based on four strategies: (i) the use of multiple full-sib families, (ii) the reduction of missing information through the use of HaploBlocks and alternative calling procedures for single-nucleotide polymorphism (SNP) markers, (iii) the construction of a single backcross-type data set including all families, and (iv) a two-step map generation procedure based on the sequential inclusion of markers. The map comprises 15 417 SNP markers, clustered in 3 K HaploBlock markers spanning 1 267 cM, with an average distance between adjacent markers of 0.37 cM and a maximum distance of 3.29 cM. Moreover, chromosome 5 was oriented according to its homoeologous chromosome 10. This map was useful to improve the apple genome sequence, design the Axiom Apple 480 K SNP array and perform multifamily-based QTL studies. Its collinearity with the genome sequences v1 and v3 are reported. To our knowledge, this is the shortest published SNP map in apple, while including the largest number of markers, families and individuals. This result validates our methodology, proving its value for the construction of integrated linkage maps for any outbreeding species.

Background: Genetic modification of allergenic foods such as apple has the potential to reduce their clinical allergenicity, but this has never been studied by oral challenges in allergic individuals., Methods: We performed oral food challenges in 21 apple-allergic individuals with Elstar apples which had undergone gene silencing of the major allergen of apple, Mal d 1, by RNA interference. Downregulation of Mal d 1 gene expression in the apples was verified by qRT-PCR. Clinical responses to the genetically modified apples were compared to those seen with the wild-type Elstar using a visual analogue scale (VAS)., Results: Gene silencing produced two genetically modified apple lines expressing Mal d 1.02 and other Mal d 1 gene mRNA levels which were extensively downregulated, that is only 0.1-16.4% (e-DR1) and 0.2-9.9% (e-DR2) of those of the wild-type Elstar, respectively. Challenges with these downregulated apple lines produced significantly less intense maximal symptoms to the first dose (Vmax1) than with Elstar (Vmax1 Elstar 3.0 mm vs 0.0 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043), as well as significantly less intense mean symptoms per dose (meanV/d) than with Elstar (meanV/d Elstar 2.2 mm vs 0.2 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043). Only one subject (5%) remained symptom-free when challenged with the Elstar apple, whereas 43% did so with e-DR1 and 63% with e-DR2., Conclusion: These data show that mRNA silencing of Mal d 1 results in a marked reduction of Mal d 1 gene expression in the fruit and reduction of symptoms when these apples are ingested by allergic subjects. Approximately half of the subjects developed no symptoms whatsoever, and virtually all subjects wished to consume the apple again in the future., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Breeders in the allo-octoploid strawberry currently make little use of molecular marker tools. As a first step of a QTL discovery project on fruit quality traits and resistance to soil-borne pathogens such as Phytophthora cactorum and Verticillium we built a genome-wide SSR linkage map for the cross Holiday x Korona. We used the previously published MADCE method to obtain full haplotype information for both of the parental cultivars, facilitating in-depth studies on their genomic organisation., Results: The linkage map incorporates 508 segregating loci and represents each of the 28 chromosome pairs of octoploid strawberry, spanning an estimated length of 2050 cM. The sub-genomes are denoted according to their sequence divergence from F. vesca as revealed by marker performance. The map revealed high overall synteny between the sub-genomes, but also revealed two large inversions on LG2C and LG2D, of which the latter was confirmed using a separate mapping population. We discovered interesting breeding features within the parental cultivars by in-depth analysis of our haplotype data. The linkage map-derived homozygosity level of Holiday was similar to the pedigree-derived inbreeding level (33% and 29%, respectively). For Korona we found that the observed homozygosity level was over three times higher than expected from the pedigree (13% versus 3.6%). This could indicate selection pressure on genes that have favourable effects in homozygous states. The level of kinship between Holiday and Korona derived from our linkage map was 2.5 times higher than the pedigree-derived value. This large difference could be evidence of selection pressure enacted by strawberry breeders towards specific haplotypes., Conclusion: The obtained SSR linkage map provides a good base for QTL discovery. It also provides the first biologically relevant basis for the discernment and notation of sub-genomes. For the first time, we revealed genomic rearrangements that were verified in a separate mapping population. We believe that haplotype information will become increasingly important in identifying marker-trait relationships and regions that are under selection pressure within breeding material. Our attempt at providing a biological basis for the discernment of sub-genomes warrants follow-up studies to streamline the naming of the sub-genomes among different octoploid strawberry maps.

Background: A considerable number of individuals suffer from oral allergy syndrome (OAS) to apple, resulting in the avoidance of apple consumption. Apple cultivars differ greatly in their allergenic properties, but knowledge of the causes for such differences is incomplete. Mal d 1 is considered the major apple allergen. For Mal d 1, a wide range of isoallergens and variants exist, and they are encoded by a large gene family. To identify the specific proteins/genes that are potentially involved in the allergy, we developed a PCR assay to monitor the expression of each individual Mal d 1 gene. Gene-specific primer pairs were designed for the exploitation of sequence differences among Mal d 1 genes. The specificity of these primers was validated using both in silico and in vitro techniques. Subsequently, this assay was applied to the peel and flesh of fruits from the two cultivars 'Florina' and 'Gala'., Results: We successfully developed gene-specific primer pairs for each of the 31 Mal d 1 genes and incorporated them into a qRT-PCR assay. The results from the application of the assay showed that 11 genes were not expressed in fruit. In addition, differential expression was observed among the Mal d 1 genes that were expressed in the fruit. Moreover, the expression levels were tissue and cultivar dependent., Conclusion: The assay developed in this study facilitated the first characterisation of the expression levels of all known Mal d 1 genes in a gene-specific manner. Using this assay on different fruit tissues and cultivars, we obtained knowledge concerning gene relevance in allergenicity. This study provides new perspectives for research on both plant breeding and immunotherapy.

European populations exhibit progressive sensitisation to food allergens, and apples are one of the foods for which sensitisation is observed most frequently. Apple cultivars vary greatly in their allergenic characteristics, and a better understanding of the genetic basis of low allergenicity may therefore allow allergic individuals to increase their fruit intake. Mal d 1 is considered to be a major apple allergen, and this protein is encoded by the most complex allergen gene family. Not all Mal d 1 members are likely to be involved in allergenicity. Therefore, additional knowledge about the existence and characteristics of the different Mal d 1 genes is required. In the present study, we investigated the genomic organisation of the Mal d 1 gene cluster in linkage group 16 of apple through the sequencing of two bacterial artificial chromosome clones. The results provided new information on the composition of this family with respect to the number and orientation of functional and pseudogenes and their physical distances. The results were compared with the apple and peach genome sequences that have recently been made available. A broad analysis of the whole apple genome revealed the presence of new genes in this family, and a complete list of the observed Mal d 1 genes is supplied. Thus, this study provides an important contribution towards a better understanding of the genetics of the Mal d 1 family and establishes the basis for further research on allelic diversity among cultivars in relation to variation in allergenicity. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9588-4) contains supplementary material, which is available to authorized users.

Increases in temperature and air pollution influence pollen allergenicity, which is responsible for the dramatic raise in respiratory allergies. To clarify possible underlying mechanisms, an anemophilous pollen (hazel, Corylus avellana), known to be allergenic, and an entomophilous one (apple, Malus domestica), the allergenicity of which was not known, were analysed. The presence also in apple pollen of known fruit allergens and their immunorecognition by serum of an allergic patient were preliminary ascertained, resulting also apple pollen potentially allergenic. Pollens were subjected to simulated stressful conditions, provided by changes in temperature, humidity, and copper and acid rain pollution. In the two pollens exposed to environmental criticalities, viability and germination were negatively affected and different transglutaminase (TGase) gel bands were differently immunodetected with the polyclonal antibody AtPng1p. The enzyme activity increased under stressful treatments and, along with its products, was found to be released outside the pollen with externalisation of TGase being predominant in C. avellana, whose grain presents a different cell wall composition with respect to that of M. domestica. A recombinant plant TGase (AtPng1p) stimulated the secreted phospholipase A(2) (sPLA(2)) activity, that in vivo is present in human mucosa and is involved in inflammation. Similarly, stressed pollen, hazel pollen being the most efficient, stimulated to very different extent sPLA(2) activity and putrescine conjugation to sPLA(2). We propose that externalised pollen TGase could be one of the mediators of pollen allergenicity, especially under environmental stress induced by climate changes.

In environmental epidemiology research, decisions about when and how to intervene requires adequate ethical reflection. In fact, different kinds of issues may arise about: research methods and knowledge production; management of the results in terms of their overall assessments or for the implementation of preventive actions; reclamation intervention. In this contribution we propose to consider three topics we regard as crucial to this ethical debate: the reporting of conclusive research data; the correct application of the precautionary principle; and the environmental equity issues.

Objective: Nonalcoholic fatty liver disease (NAFLD) is a very frequent disease in Western countries. NAFLD shares with metabolic syndrome the same etiologic factors, such as obesity, diabetes, and dyslipidemia, which are also major cardiovascular risk factors. Cardio-GOOSE (Cardio-Gambettola ObservatOry liver Steatosis Estimation) is a population-based cohort study finalized to evaluate the relationship between NAFLD, subclinical vascular damage, and arterial stiffness., Methods: The study population consisted of 220 participants (123 women), aged between 30 and 70 years, who participated in the GOOSE study. Arterial stiffness was determined by measuring the carotid-femoral pulse wave velocity (PWV) by means of the PulsePen device. Preclinical atherosclerosis was detected by carotid intima-media thickness (IMT) measurement., Results: NAFLD was associated with metabolic syndrome in 48% of cases. IMT values were strongly related to metabolic syndrome factors. No significant differences in IMT were found between controls and patients with isolated NAFLD (0.77 +/- 0.15 mm versus 0.76 +/- 0.14 mm). Conversely, in patients with NAFLD associated with metabolic syndrome, IMT values were significantly higher than in patients with NAFLD alone (0.85 +/- 0.16 mm, P < 0.005). PWV values were significantly lower in controls compared to patients with isolated NAFLD (7.40 +/- 1.47 versus 7.98 +/- 1.51 m/s, P < 0.05) as well as patients with both NAFLD and metabolic syndrome (8.29 +/- 2.2 m/s, P < 0.001). The prevalence in NAFLD was increased in patients with the highest PWV values, and persisted after adjustment for factors determining metabolic syndrome (P < 0.05)., Conclusions: This study has shown a possible independent role of NAFLD in determining arterial stiffness.

From March 1990 to July 1991, 110 patients (44 males, 66 females; mean age 54.44 +/- 8.8 years) underwent hearth valve replacement with a CarboMedics bileaflet prosthesis (CarboMedics Inc, Texas, USA). Preoperative pathophysiologic conditions were: aortic stenosis in 32 patients, aortic regurgitation in 22 patients; mitral stenosis in 27 patients and mitral regurgitation in 14 patients. Mitroaortic disease was present in 14 patients but 1 had triple valve disease. NYHA class was III or IV in 91 patients (83%). Operative mortality rate was 0.91% (1 patients). Actuarial survival rate is 95% at 58 months. Actuarial freedom from thromboembolic events in 95% at 58 months. Actuarial freedom from hemorrhage is 100%. Endocarditis befell in 2 patients; actuarial freedom from this complication at 58 months is 95%. Actuarial freedom from reoperation in 99%. We conclude that the low incidence of valve related events and the low mortality supports the use of the bileaflet valve CarboMedics.