CRISPR-Cas9 Multiplex Editing of the α-Amylase/Trypsin Inhibitor Genes to Reduce Allergen Proteins in Durum Wheat

Wheat and its derived foods are widespread, representing one of the main food sources globally. During the last decades, the incidence of disorders related to wheat has become a global issue for the human population, probably linked to the spread of wheat-derived foods. It has been ascertained that structural and metabolic proteins, like \u3b1-amylase/trypsin inhibitors (ATI), are involved in the onset of wheat allergies (bakers' asthma) and probably Non-Coeliac Wheat Sensitivity (NCWS). The ATI are a group of exogenous protease inhibitors, which are encoded by a multigene family dispersed over several chromosomes in durum and bread wheat. WTAI-CM3 and WTAI-CM16 subunits are considered among the main proteins involved in the onset of bakers' asthma and probably NCWS. A CRISPR-Cas9 multiplexing strategy was used to edit the ATI subunits WTAI-CM3 and WTAI-CM16 in the grain of the Italian durum wheat cultivar Svevo with the aim to produce wheat lines with reduced amount of potential allergens involved in adverse reactions. Using a marker gene-free approach, whereby plants are regenerated without selection agents, homozygous mutant plants without the presence of CRISPR vectors were obtained directly from T0 generation. This study demonstrates the capability of CRISPR technology to knock out immunogenic proteins in a reduced time compared to conventional breeding programmes. The editing of the two target genes was confirmed either at molecular (sequencing and gene expression study) or biochemical (immunologic test) level. Noteworthy, as a pleiotropic effect, is the activation of the ATI 0.28 pseudogene in the edited lines

Efficacy and safety of honeybee and wasp tyrosine-adsorbed venom immunotherapy

It is acknowledged that any claim of efficacy of allergen immunotherapy must be done for each specific product, and this remains true also for venom immunotherapy (VIT). Thus, we evaluated the efficacy and safety of a specific tyrosine-adsorbed VIT for vespula spp. and honeybee in real-life

Antagonistic Transcription Factor Complexes Modulate the Floral Transition in Rice

Plants measure day or night lengths to coordinate specific developmental changes with a favorable season. In rice (Oryza sativa), the reproductive phase is initiated by exposure to short days when expression of HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1) is induced in leaves. The cognate proteins are components of the florigenic signal and move systemically through the phloem to reach the shoot apical meristem (SAM). In the SAM, they form a transcriptional activation complex with the bZIP transcription factor OsFD1 to start panicle development. Here, we show that Hd3a and RFT1 can form transcriptional activation or repression complexes also in leaves and feed back to regulate their own transcription. Activation complexes depend on OsFD1 to promote flowering. However, additional bZIPs, including Hd3a BINDING REPRESSOR FACTOR1 (HBF1) and HBF2, form repressor complexes that reduce Hd3a and RFT1 expression to delay flowering. We propose that Hd3a and RFT1 are also active locally in leaves to fine-tune photoperiodic flowering response

Kinetics of cation ordering in synthetic Mg(Al, Fe3+)2O4 spinels

The kinetics of cation ordering (quench method) for two synthetic Mg(Al2-yFe3+y )O4 spinels (y 0.39 and 0.54, samples F39 and F54, respectively) were studied by means of X-ray single-crystal diffraction. The equilibrium distribution of Mg\u2013Al\u2013Fe3+ between tetrahedral and octahedral sites was investigated at 750, 650 and 550 C (in ordering run) starting from an equilibrium ordering state corresponding to 1000 C, through several time-steps in order to monitor the rate of change of cation distribution. The cation distribution between T and M sites was calculated from the bond lengths using the Minuit program. The Mueller kinetic model, applied to the Mg\u2013Al cation exchange, allowed the calculation of the kinetic ordering constants K, linearly related to temperature by the Arrhenius equations. The Fe3+ content influences the kinetics of the ordering process. The equilibrium for the three isotherms at 750, 650 and 550 C was reached at different elapsed times for the F39 and F54 spinels. In particular, the time for the F39 sample was, in all the ordering runs, higher than that of the F54 sample. Consequently, the activation energy (274 and 265 kJ/mol for low- and high-Fe3+ samples, respectively) for the intra-crystalline Mg\u2013Al ordering slightly decreases with increasing Fe3\ufe content. The intracrystalline closure temperature (Tc) seems to be only marginally affected by the different Fe3+ content. As magnesiochromite spinels are among the most abundant phases in diamonds such results could have implications for diamond thermometry because magnesiochromite inclusions can contain as much as 45 % total iron as Fe3+

Kinetics of cation ordering in synthetic Mg(Al, Fe3+)2O4 spinels

4The kinetics of cation ordering (quench method) for two synthetic Mg(Al2-yFe3+y )O4 spinels (y 0.39 and 0.54, samples F39 and F54, respectively) were studied by means of X-ray single-crystal diffraction. The equilibrium distribution of Mg–Al–Fe3+ between tetrahedral and octahedral sites was investigated at 750, 650 and 550 C (in ordering run) starting from an equilibrium ordering state corresponding to 1000 C, through several time-steps in order to monitor the rate of change of cation distribution. The cation distribution between T and M sites was calculated from the bond lengths using the Minuit program. The Mueller kinetic model, applied to the Mg–Al cation exchange, allowed the calculation of the kinetic ordering constants K, linearly related to temperature by the Arrhenius equations. The Fe3+ content influences the kinetics of the ordering process. The equilibrium for the three isotherms at 750, 650 and 550 C was reached at different elapsed times for the F39 and F54 spinels. In particular, the time for the F39 sample was, in all the ordering runs, higher than that of the F54 sample. Consequently, the activation energy (274 and 265 kJ/mol for low- and high-Fe3+ samples, respectively) for the intra-crystalline Mg–Al ordering slightly decreases with increasing Fe3þ content. The intracrystalline closure temperature (Tc) seems to be only marginally affected by the different Fe3+ content. As magnesiochromite spinels are among the most abundant phases in diamonds such results could have implications for diamond thermometry because magnesiochromite inclusions can contain as much as 45 % total iron as Fe3+.nonemixedPRINCIVALLE F.; MARTIGNAGO F.; NESTOLA F.; DAL NEGRO A.Princivalle, Francesco; Martignago, F.; Nestola, F.; DAL NEGRO, A

Thermodynamics and kinetics of cation ordering in natural and synthetic Mg(Al,Fe3+)(2)O-4 spinels from in situ high-temperature X-ray diffraction

One natural and two synthetic spinels with compositions Ma(Al1-zFez3+)(2)O-4 (with 2z = 0.078, 0.138 and 0.200, respectively) were studied by in situ, high-temperature, single-crystal X-ray diffraction. All samples were heated from room temperature to 1050 degrees C. Thermal expansion was monitored by measuring the cell edge variation. Cation disorder was monitored by measuring the variation of the oxygen positional parameter it, which is closely correlated with the inversion parameter i. All samples showed extensive Al reordering at the M site between 550 and 650 degrees C, followed by an increase of disorder at T > 650 degrees C due to both Mg-Al and Mg-Fe3+ intersite exchanges. The measured cation distributions were compared with those calculated using the general thermodynamic model for spinel binary solid-solutions of O'Neill and Navrotsky (1984). Measured and calculated inversion parameters compare satisfactorily at T > 650 degrees C, i.e. at conditions under which equilibrium was achieved at any temperature. In fact, at T > 650 degrees C, both (IV)Fe(3+)and Al-IV increase with increasing T, following the equilibrium path. The reproducibility of Al-IV occupancies was very high, whereas Fe-IV(3+) occupancies were not satisfactorily matched. The cation distribution relaxation observed between 550 and 650 degrees C was interpreted on the basis of kinetic considerations. In this temperature ran,,e, inversion decreases to a minimum because the amounts of Al that reorder are far more abundant than those of Fe3+ that disorder. The Mg-Fe3+ exchange was confirmed to proceed at a faster rate than the Mg-Al exchange. Moreover, the Mg-Fe3+ exchange wits found to be active at laboratory times at about 550 degrees C, whereas the Mg-Al exchange was hard to monitor below 600 degrees C

Cadmium, lead and metallothionein contents in tissues of the sea bream Sparus aurata from three different fish farming systems

Heavy metals in marine environment have a particular significance in ecotoxicology, since they are highly persistent and can be toxic in traces. Metals such as Pb and Cd are potentially toxic and pose a serial risk for human health when they enter the food chain. Fish are exposed to metals at different intensities through two major routes: metal ions dissolved in the water can be absorbed through the gills; metals bound to solid particles can be ingested and absorbed through the gut epithelium. For farmed fish, growing conditions (food and water chemistry) may determine metal composition of fish tissues as well as the response that these fish exhibit to metal toxicity. The distribution and bioaccumulation of dietary and waterborne cadmium and lead in tissues of sea bream Sparus aurata was studied in relation to three different fish farming systems. Metallothionein levels in fish tissues were also evaluated