La regolamentazione delle attività di lobbying: esperienze internazionali a confronto

L’attività di lobbying s’inscrive nel contesto più ampio della “rappresentanza di interessi” e più specificamente agli aspetti legislativi ed esecutivi della stessa. Questo lavoro propone una rassegna comparativa delle legislazioni sul lobbying in vigore nel panorama internazionale. In particolare sono state analizzate e classificate le legislazioni di una serie di paesi UE ed extra UE. Per l’Italia il lavoro è stato fatto utilizzando la proposta di legge più recente. La maggioranza dei paesi UE con leggi in materia di rappresentanza di interessi hanno sistemi fondati sul sistema di civil law, con l’eccezione di Regno Unito e Irlanda. È stato quindi possibile delineare tre sottogruppi: Paesi UE di common law, Paesi extra UE di common law e Paesi UE di civil law. In base a questa suddivisione vengono presentate la diverse normative evidenziandone gli elementi omogenei che permettono un raffronto delle caratteristiche, dei punti qualificanti e degli aspetti critici delle leggi vigenti nei singoli paesi.The activity of lobbying is part of the wider context of “representation of interests” and more specifically the legislative and executive aspects thereof. This work aims at a comparative review of the lobbying legislation in force on the international scene. In particular, the legislation of a number of EU and non-EU countries has been analysed and classified. For Italy, the work was done using the most recent draft law. Most EU countries with laws on the representation of interests have systems based on civil law, with the exception of the United Kingdom and Ireland. It was therefore possible to outline three sub-groups: EU common law countries, non-EU common law countries and EU civil law countries. On the basis of this subdivision the different regulations are presented highlighting the homogeneous elements that allow a comparison of the characteristics, the qualifying points, and the critical aspects of the laws in force in the individual countries

Absolute and relative quantitation of amylase/trypsin-inhibitors by LC-MS/MS from wheat lines obtained by CRISPR-Cas9 and RNAi

Quantitation of wheat proteins is still a challenge, especially regarding amylase/trypsin-inhibitors (ATIs). A selection of ATIs was silenced in the common wheat cultivar Bobwhite and durum wheat cultivar Svevo by RNAi and gene editing, respectively, in order to reduce the amounts of ATIs. The controls and silenced lines were analyzed after digestion to peptides by LC-MS/MS with different approaches to evaluate changes in composition of ATIs. First, a targeted method with stable isotope dilution assay (SIDA) using labeled peptides as internal standards was applied. Additionally, four different approaches for relative quantitation were conducted, in detail, iTRAQ labeled and label free quantitation (LFQ) combined with data dependent acquisition (DDA) and data independent acquisition (DIA). Quantitation was performed manually (Skyline and MASCOT) and with different proteomics software tools (PLGS, MaxQuant, and PEAKS X Pro). To characterize the wheat proteins on protein level, complementary techniques as high-performance liquid chromatography (HPLC) and gel electrophoresis were performed. The targeted approach with SIDA was able to quantitate all ATIs, even at low levels, but an optimized extraction is necessary. The labeled iTRAQ approach revealed an indistinct performance. LFQ with low resolution equipment (IonTrap) showed similar results for major ATIs, but low abundance ATIs as CM1, were not detectable. DDA measurements with an Orbitrap system and evaluation using MaxQuant showed that the relative quantitation was dependent on the wheat species. The combination of manual curation of the MaxQuant search with Skyline revealed a very good performance. The DIA approach with analytical flow found similar results compared to absolute quantitation except for some minor ATIs, which were not detected. Comparison of applied methods revealed that peptide selection is a crucial step for protein quantitation. Wheat proteomics faces challenges due to the high genetic complexity, the close relationship to other cereals and the incomplete, redundant protein database requiring sensitive, precise and accurate LC-MS/MS methods

Production and characterization of wheat lines silenced in alpha amylase/trypsin inhibitor genes involved in adverse reactions to wheat

Although wheat is the most consumed crop worldwide, it is also the main factor triggering different adverse reactions, among which celiac disease, true allergies and Non Celiac Wheat Sensitivity (NCWS). Among allergies, the so called \u201cbaker\u2019s asthma\u201d, is the most common professional asthma in Europe and is caused mainly by proteins present in the soluble fraction, especially alpha-amylase/trypsin inhibitors (ATI). Recent findings indicate in this class of proteins also the main factor triggering NCWS, that at present affects people with a frequency around 1:80, higher than celiac disease (1:100), but this is still a matter of debate. On this basis, we have produced RNAi wheat plants (both durum and bread wheat) in which different ATI genes have been silenced, to be used as a proof of concept, in order to test if they have a minor impact on adverse reactions, by using in vitro tests. We have silenced CM3, CM16 and 0.28 genes and have now available several lines in T4 generation. ELISA tests and immunoblotting analysis, by using a monoclonal antibody against ATI proteins, have shown that RNAi silenced wheat kernels present a lower amount of ATI proteins. Moreover, we are characterizing these lines in relation to respiratory allergies. Protein extracts from silenced plants are being tested by using human sera of allergic patients in order to verify if a lower amount of immunogenic polypeptides is recognized in comparison to wild type untransformed plants. If this is the case, the realization of new wheat genotypes expressing a lower amount of ATI proteins can be a realistic target to be reached by classical breeding procedures

A novel wheat variety with elevated content of amylose increases resistant starch formation and may beneficially influence glycaemia in healthy subjects

Previous studies indicate that elevated amylose content in products from rice, corn, and barley induce lower postprandial glycaemic responses and higher levels of resistant starch (RS). Consumption of slowly digestible carbohydrates and RS has been associated with health benefits such as decreased risk of diabetes and cardiovascular disease.To evaluate the postprandial glucose and insulin responses in vivo to bread products based on a novel wheat genotype with elevated amylose content (38%).Bread was baked from a unique wheat genotype with elevated amylose content, using baking conditions known to promote amylose retrogradation. Included test products were bread based on whole grain wheat with elevated amylose content (EAW), EAW with added lactic acid (EAW-la), and ordinary whole grain wheat bread (WGW). All test breads were baked at pumpernickel conditions (20 hours, 120°C). A conventionally baked white wheat bread (REF) was used as reference. Resistant starch (RS) content was measured in vitro and postprandial glucose and insulin responses were tested in 14 healthy subjects.The results showed a significantly higher RS content (on total starch basis) in breads based on EAW than in WGW (p<0.001). Lactic acid further increased RS (p<0.001) compared with both WGW and EAW. Breads baked with EAW induced lower postprandial glucose response than REF during the first 120 min (p<0.05), but there were no significant differences in insulin responses. Increased RS content per test portion was correlated to a reduced glycaemic index (GI) (r= − 0.571, p<0.001).This study indicates that wheat with elevated amylose content may be preferable to other wheat genotypes considering RS formation. Further research is needed to test the hypothesis that bread with elevated amylose content can improve postprandial glycaemic response

Interaction between Sulfate and Selenate in Tetraploid Wheat (Triticum turgidum L.) Genotypes

Selenium (Se) is an essential micronutrient of fundamental importance to human health and the main Se source is from plant-derived foods. Plants mainly take up Se as selenate (SeO42−), through the root sulfate transport system, because of their chemical similarity. The aims of this study were (1) to characterize the interaction between Se and S during the root uptake process, by measuring the expression of genes coding for high-affinity sulfate transporters and (2) to explore the possibility of increasing plant capability to take up Se by modulating S availability in the growth medium. We selected different tetraploid wheat genotypes as model plants, including a modern genotype, Svevo (Triticum turgidum ssp. durum), and three ancient Khorasan wheats, Kamut, Turanicum 21, and Etrusco (Triticum turgidum ssp. turanicum). The plants were cultivated hydroponically for 20 days in the presence of two sulfate levels, adequate (S = 1.2 mM) and limiting (L = 0.06 mM), and three selenate levels (0, 10, 50 μM). Our findings clearly showed the differential expression of genes encoding the two high-affinity transporters (TdSultr1.1 and TdSultr1.3), which are involved in the primary uptake of sulfate from the rhizosphere. Interestingly, Se accumulation in shoots was higher when S was limited in the nutrient solution

Whole-exome sequencing of selected bread wheat recombinant inbred lines as a useful resource for allele mining and bulked segregant analysis

Although wheat (Triticum aestivum L.) is the main staple crop in the world and a major source of carbohydrates and proteins, functional genomics and allele mining are still big challenges. Given the advances in next-generation sequencing (NGS) technologies, the identification of causal variants associated with a target phenotype has become feasible. For these reasons, here, by combining sequence capture and target-enrichment methods with high-throughput NGS re-sequencing, we were able to scan at exome-wide level 46 randomly selected bread wheat individuals from a recombinant inbred line population and to identify and classify a large number of single nucleotide polymorphisms (SNPs). For technical validation of results, eight randomly selected SNPs were converted into Kompetitive Allele-Specific PCR (KASP) markers. This resource was established as an accessible and reusable molecular toolkit for allele data mining. The dataset we are making available could be exploited for novel studies on bread wheat genetics and as a foundation for starting breeding programs aimed at improving different key agronomic traits

Silencing of ATI genes involved in adverse reactions to wheat by RNAi and CRISPR-Cas9 technologies

Although wheat is consumed worldwide as a staple food, it can give rise to different adverse reactions, some of which have not been deeply characterized. They are caused mainly by wheat proteins, both gluten and non-gluten proteins. Structural and metabolic proteins, like \u3b1amylase/trypsin inhibitors (ATI) are involved in the onset of wheat allergies (bakers\u2019 asthma) and probably non-coeliac wheat sensitivity (NCWS). The ATI are encoded by a multigene family dispersed over several. Notably, WTAI-CM3 and WTAI-CM16 subunits are involved in the onset of bakers\u2019 asthma and are likely to contribute to NCWS. In this study we report the RNAi silencing of WTAI-CM3, WTAI-CM16 and WMAI-0.28 genes in the bread wheat cultivar Bobwhite and the CRISPR/Cas9 mediated gene knockout of WTAI-CM3 and WTAI-CM16 in the durum wheat cultivar Svevo. We have obtained different RNAi transgenic lines showing an effective decrease in the expression in the targeted genes. These lines do not show differences in terms of yield, but have unintended effects on the accumulation of the high molecular weight glutenin subunits which play a crucial role in the technological performances of wheat flour. Furthermore, the editing of WTAI-CM3 and WTAI-CM16 genes was obtained through a CRISPR-Cas9 multiplexing strategy in the Italian durum wheat cultivar Svevo with a marker-free approach. The regeneration of plants without selection agents allowed T0 homozygous mutant plants to be obtained without the integration in the wheat genome of CRISPR/Cas9 vectors, demonstrating the capability of CRISPR technology to produce wheat lines in a reduced time compared to conventional breeding approaches. The possibility to develop new wheat genotypes accumulating a lower amount of proteins effectively involved in such pathologies, not only offers the possibility to use them as a basis for the creation of wheat varieties with a lower impact on adverse reactions, but also to test if these proteins are actually implicated in those pathologies for which the triggering factor has yet to be established

Increasing the amylose content of durum wheat through silencing of the SBEIIa genes

<p>Abstract</p> <p>Background</p> <p>High amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food. RS plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. In particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. In this work, genes encoding starch branching enzymes of class II (SBEIIa) were silenced using the RNA interference (RNAi) technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium). Expression of RNAi transcripts was targeted to the seed endosperm using a tissue-specific promoter.</p> <p>Results</p> <p>Amylose content was markedly increased in the durum wheat transgenic lines exhibiting <it>SBEIIa </it>gene silencing. Moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. Two novel granule bound proteins, identified by SDS-PAGE in SBEIIa RNAi lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. RVA analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. Finally, pleiotropic effects on other starch genes were found by semi-quantitative and Real-Time reverse transcription-polymerase chain reaction (RT-PCR).</p> <p>Conclusion</p> <p>We have found that the silencing of <it>SBEIIa </it>genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. Results obtained with two different methods of transformation and in two durum wheat cultivars were comparable.</p

Association between work related stress and health related quality of life: the impact of socio-demographic variables. A cross sectional study in a region of central Italy

The aim of this work is investigate relationship between health-related quality of life and work-related stress and the impact of gender, education level, and age on this relationship. A cross-sectional study was conducted among workers of various setting in Rome and Frosinone. Work-related stress was measured with a demand-control questionnaire and health-related functioning by SF (short form)-12 health survey. There were 611 participants. Men reported high mental composite summary (MCS) and physical composite summary (PCS). In multivariate analysis age, gender (p < 0.001) and job demand (0.045) predicted low PCS. Low MCS predicted poor PCS. Job demand and educational level resulted negatively associated with MCS. In an analysis stratified for age, gender, and educational level, gender and age resulted effect modifier for MCS, gender and education level for PCS. In women increase of decision latitude predict (p = 0.001) an increase in MCS; a low job demand predict high MCS in male (p ≤ 0.001). In younger workers, a lower level of job demand predicted high MCS (<0.001). For PCS, gender and education level resulted effect modifier. In women, high decision latitude predicted higher PCS (p = 0.001) and lower level of job demand results in higher PCS (p ≤ 0.001). Higher educational level resulted predictor of low PCS. Management of risk about work-related stress should consider socio-demographic factors