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

Cognitive Fatigue Influences Time-On-Task during Bodyweight Resistance Training Exercise

Prior investigations have shown measurable performance impairments on continuous physical performance tasks when preceded by a cognitively fatiguing task. However, the effect of cognitive fatigue on bodyweight resistance training exercise task performance is unknown. In the current investigation 18 amateur athletes completed a full body exercise task preceded by either a cognitive fatiguing or control intervention. In a randomized repeated measure design, each participant completed the same exercise task preceded by a 52 minute cognitively fatiguing intervention (vigilance) or control intervention (video). Data collection sessions were separated by 1 week. Participants rated the fatigue intervention as being significantly more mentally demanding than the control intervention (p .05). There was no statistical difference for heart rate or metabolic expenditure as a function of fatigue intervention during exercise. Cognitively fatigued athletes have decreased time-on-task in bodyweight resistance training exercise tasks

Combining proton or photon irradiation with epothilone B : An in vitro study of cytotoxicity in human cancer cells

Recently, the use of proton beams in cancer therapy is becoming widespread, and tumour treatment modalities combining radiosensitizing chemical agents with irradiation are under investigation in order to achieve greater tumour local control and reduce the probability of distant failures. The combined treatment modality of radiation and the clinically relevant microtubule-stabilizing compound epothilone B is a promising approach for anticancer therapy. In the present study, we investigated the cytotoxicity of a spread out Bragg peak (SOBP) proton beam, as well as of 6 MV photons, in human glioblastoma (U251 MG) and lung adenocarcinoma (A549) cells pretreated for 24 h, or not, with epothilone B at concentrations of 0.125 and 0.075 nM respectively. Proton irradiation was performed at the middle position of an actively modulated SOBP (12\u201318 cm depth in water) and cell survival was evaluated by a colony forming assay. For both cell lines, survival curves after proton or photon irradiation alone showed linear quadratic behaviour with proton RBE (relative biological effectiveness), compared with photons at 10% survival, of 1.5 \ub1 0.2. Treatment of cells with epothilone B at subnanomolar concentration has an anticlonogenic effect. Furthermore, differently from the results found with radiation alone, the survival curves for the combined treatment epothilone B\u2013radiation showed a linear trend and analysis of the interaction of the two cytotoxic agents indicated a slight synergism. These data provide a radiobiological basis for further experiments, as well as clinical studies

High resolution melting analysis for the detection of EMS induced mutations in wheat SBEIIa genes

Background Manipulation of the amylose-amylopectin ratio in cereal starch has been identified as a major target for the production of starches with novel functional properties. In wheat, silencing of starch branching enzyme genes by a transgenic approach reportedly caused an increase of amylose content up to 70% of total starch, exhibiting novel and interesting nutritional characteristics. In this work, the functionality of starch branching enzyme IIa (SBEIIa) has been targeted in bread wheat by TILLING. An EMS-mutagenised wheat population has been screened using High Resolution Melting of PCR products to identify functional SNPs in the three homoeologous genes encoding the target enzyme in the hexaploid genome. Results This analysis resulted in the identification of 56, 14 and 53 new allelic variants respectively for SBEIIa-A, SBEIIa-B and SBEIIa-D. The effects of the mutations on protein structure and functionality were evaluated by a bioinformatic approach. Two putative null alleles containing non-sense or splice site mutations were identified for each of the three homoeologous SBEIIa genes; qRT-PCR analysis showed a significant decrease of their gene expression and resulted in increased amylose content. Pyramiding of different single null homoeologous allowed to isolate double null mutants showing an increase of amylose content up to 21% compared to the control. Conclusion TILLING has successfully been used to generate novel alleles for SBEIIa genes known to control amylose content in wheat. Single and double null SBEIIa genotypes have been found to show a significant increase in amylose content

Studio degli effetti dell&#8217;uso combinato di fasci terapeutici di particelle cariche e radiosensibilizzanti in cellule tumorali umane coltivate in vitro

Nell\u2019ambito delle terapie oncologiche \ue8 crescente l\u2019interesse per trattamenti che abbinino alle radiazioni l\u2019utilizzo di agenti chimici potenziali radiosensibilizzanti. L\u2019obiettivo di questo tipo di trattamenti \ue8 quello di ottenere un miglior controllo locale del tumore e di ridurre la probabilit\ue0 di formazione di metastasi. In letteratura sono stati pubblicati numerosi studi sull\u2019uso concomitante di fasci terapeutici di fotoni e diversi agenti chimici, ma attualmente ben pochi dati sono disponibili sull\u2019azione combinata di adroni e agenti chimici radiosensibilizzanti. In questo lavoro si descrivono i risultati di uno studio radiobiologico condotto su fasci terapeutici di fotoni e di protoni, ed alcuni dati preliminari relativi a fasci di ioni Carbonio, utilizzando cellule umane tumorali coltivate in vitro, trattate o meno, durante le 24 ore precedenti l\u2019irraggiamento, con il chemioterapico Epothilone B, un agente microtubulo-stabilizzante. I principali obiettivi dello studio sono: - determinare l\u2019efficacia dei fasci di adroni rispetto ai fotoni, per le linee cellulari studiate ( RBE) - studiare la modalit\ue0 di interazione delle diverse radiazioni con Epothilone B per valutare se sia additiva o sinergica. Sono stati utilizzati i fasci terapeutici di adroni di CNAO (Pavia) e di fotoni presso l\u2019Istituto Tumori di Milano. Lo studio \ue8 stato condotto su linee cellulari umane di glioblastoma multiforme (U251MG), adenocarcinoma polmonare a cellule non piccole (A549) e medulloblastoma pediatrico (DAOY). I principali effetti biologici studiati sono il mantenimento della capacit\ue0 proliferativa (sopravvivenza clonogenica), la crescita e l\u2019invasivit\ue0 cellulare. I risultati ottenuti hanno mostrato che: - l\u2019RBE dei protoni dipende fortemente dalla linea cellulare e, per glioblastoma multiforme e per adenocarcinoma polmonare, \ue8 risultata maggiore di 1.1, valore attualmente usato nella pratica clinica. - Il chemioterapico Epothilone B aumenta la citotossicit\ue0 dei fasci di protoni e di fotoni e l\u2019effetto del trattamento combinato \ue8 pi\uf9 che additivo

Combined use of gliadins and SSRs to analyse the genetic variability of the Spanish collection of cultivated diploid wheat (Triticum monococcum L. ssp. monococcum)

This work studied the combined use of gliadins and SSRs to analyse inter- and intra-accession variability of the Spanish collection of cultivated einkorn (Triticum monococcum L. ssp. monococcum) maintained at the CRF-INIA. In general, gliadin loci presented higher discrimination power than SSRs, reflecting the high variability of the gliadins. The loci on chromosome 6A were the most polymorphic with similar PIC values for both marker systems, showing that these markers are very useful for genetic variability studies in wheat. The gliadin results indicated that the Spanish einkorn collection possessed high genetic diversity, being the differentiation large between varieties and small within them. Some associations between gliadin alleles and geographical and agro-morphological data were found. Agro-morphological relations were also observed in the clusters of the SSRs dendrogram. A high concordance was found between gliadins and SSRs for genotype identification. In addition, both systems provide complementary information to resolve the different cases of intra-accession variability not detected at the agro-morphological level, and to identify separately all the genotypes analysed. The combined use of both genetic markers is an excellent tool for genetic resource evaluation in addition to agro-morphological evaluation

Costs of Inducible Defence along a Resource Gradient

In addition to having constitutive defence traits, many organisms also respond to predation by phenotypic plasticity. In order for plasticity to be adaptive, induced defences should incur a benefit to the organism in, for example, decreased risk of predation. However, the production of defence traits may include costs in fitness components such as growth, time to reproduction, or fecundity. To test the hypothesis that the expression of phenotypic plasticity incurs costs, we performed a common garden experiment with a freshwater snail, Radix balthica, a species known to change morphology in the presence of molluscivorous fish. We measured a number of predator-induced morphological and behavioural defence traits in snails that we reared in the presence or absence of chemical cues from fish. Further, we quantified the costs of plasticity in fitness characters related to fecundity and growth. Since plastic responses may be inhibited under limited resource conditions, we reared snails in different densities and thereby levels of competition. Snails exposed to predator cues grew rounder and thicker shells, traits confirmed to be adaptive in environments with fish. Defence traits were consistently expressed independent of density, suggesting strong selection from predatory molluscivorous fish. However, the expression of defence traits resulted in reduced growth rate and fecundity, particularly with limited resources. Our results suggest full defence in predator related traits regardless of resource availability, and costs of defence consequently paid in traits related to fitness

Wheat Seed Proteins: Factors Influencing Their Content, Composition, and Technological Properties, and Strategies to Reduce Adverse Reactions

Wheat is the primary source of nutrition for many, especially those living in developing countries, and wheat proteins are among the most widely consumed dietary proteins in the world. However, concerns about disorders related to the consumption of wheat and/or wheat gluten proteins have increased sharply in the last 20 years. This review focuses on wheat gluten proteins and amylase trypsin inhibitors, which are considered to be responsible for eliciting most of the intestinal and extraintestinal symptoms experienced by susceptible individuals. Although several approaches have been proposed to reduce the exposure to gluten or immunogenic peptides resulting from its digestion, none have proven sufficiently effective for general use in coeliac-safe diets. Potential approaches to manipulate the content, composition, and technological properties of wheat proteins are therefore discussed, as well as the effects of using gluten isolates in various food systems. Finally, some aspects of the use of gluten-free commodities are discussed