Phytic acid is considered as one of the major antinutritional compounds in cereal and legume seeds. The development of lpa (low phytic acid) grains, resulting in increased mineral cation availability, is considered a major goal in the improvement of the nutritional quality of seed crops, especially those largely consumed in developing countries. From a mutagenized population of common bean we isolated a homozygous lpa mutant line (lpa-280-10) showing, compared to wild type, a 90% reduction of phytic acid, a 25% reduction of raffinosaccharides and a much higher amount of free or weakly bound iron cations in the seed. Genetic analysis showed that the lpa character is due to a recessive mutation that segregates in a monogenic, Mendelian fashion. Germination tests performed using varying ageing or stress conditions, clearly showed that the bean line lpa-280-10 has a better germination response than the wild type. These data, together with those obtained from 2 years of agronomic trials showing that the mutant seed yield is close to that of its parents and other evidence, indicate that the new lpa-280-10 mutation might be the first devoid of visible macroscopic negative effects in plants, pods and seeds.

Key words: inositol 1,4,5-tris-phosphate kinase, low phytic acid, myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP 6 ), myo-inositol-phosphate monophosphatase (IMP), myo-inositol-3-phosphate synthase (MIPS), multidrug resistance protein (MRP) type ATP-binding cassette (ABC) transporter, Phaseolus vulgaris (common bean). Summary• We previously identified the lpa1 (low phytic acid) 280-10 line that carries a mutation conferring a 90% reduction in phytic acid (InsP 6 ) content. In contrast to other lpa mutants, lpa1(280-10) does not display negative pleiotropic effects. In the present paper, we have identified the mutated gene and analysed its impact on the phytic acid pathway.• Here, we mapped the lpa1(280-10) mutation by bulk analysis on a segregating F 2 population, an then, by comparison with the soybean genome, we identified and sequenced a candidate gene. The InsP 6 pathway was analysed by gene expression and quantification of metabolites.• The mutated Pvmrp1(280-10) cosegregates with the lpa1(280-10) mutation, and the expression level of several genes of the InsP 6 pathway are reduced in the lpa1(280-10) mutant as well as the inositol and raffinosaccharide content. PvMrp2, a very similar paralogue of PvMrp1 was also mapped and sequenced.• The lpa1 mutation in beans is likely the result of a defective Mrp1 gene (orthologous to the lpa genes AtMRP5 and ZmMRP4), while its Mrp2 paralog is not able to complement the mutant phenotype in the seed. This mutation appears to down-regulate the InsP 6 pathway at the transcriptional level, as well as altering inositol-related metabolism and affecting ABA sensitivity.Abbreviations: ABC, ATP-binding cassette; MRP, multidrug resistance-associated protein; InsP 6, myo-inositol-1,2,3,4,5,6-hexakisphosphate; IMP, myo-inositolphosphate monophosphatase; MIPS, myo-inositol-3-phosphate synthase; MIK, myo-inositol kinase; IPK2, inositol 1,4,5-tris-phosphate kinase; ITPK, inositol 1,3,4-triphosphate 5 ⁄ 6-kinase; IPK1, inositol 1,3,4,5,6 pentakisphosphate 2-kinase.

BACKGROUND: Quantification of patient effort during spontaneous breathing is important to tailor ventilatory assistance. Because a correlation between inspiratory muscle pressure (P mus ) and electrical activity of the diaphragm (EA di ) has been described, we aimed to assess the reliability of surface electromyography (EMG) of the respiratory muscles for monitoring diaphragm electrical activity and subject effort during assisted ventilation. METHODS: At a general ICU of a single university-affiliated hospital, we enrolled subjects who were intubated and on pressure support ventilation (PSV) and were on mechanical ventilation for > 48 h. The subjects were studied at 3 levels of pressure support. Airway flow and pressure; esophageal pressure; EA di ; and surface EMG of the diaphragm (surface EA di ), intercostal, and sternocleidomastoid muscles were recorded. Respiratory cycles were sampled for off-line analysis. The P mus /EA di index (PEI) was calculated by relying on EA di and surface EA di (surface PEI) from an airway pressure drop during end-expiratory occlusions performed every minute. RESULTS: surface EA di well correlated with EA di and P mus , in particular, after averaging breaths into deciles (R ‫؍‬ 0.92 and R ‫؍‬ 0.84). When surface PEI was used with surface EA di , it provided a reliable estimation of P mus (R ‫؍‬ 0.94 in comparison with measured P mus ). CONCLUSIONS: During assisted mechanical ventilation, EA di can be reliably monitored by both EA di and surface EMG. The measurement of P mus based on the calibration of EA di was also feasible by the use of surface EMG. Key words: mechanical ventilation; pressuresupport ventilation; electrical activity of the diaphragm; surface electromyography; esophageal pressure.

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BackgroundAntibodies raised against selected antigens over-expressed at the cell surface of malignant cells have been chemically conjugated to protein toxin domains to obtain immunotoxins (ITs) able to selectively kill cancer cells. Since latest generation immunotoxins are composed of a toxic domain genetically fused to antibody fragment(s) which confer on the IT target selective specificity, we rescued from the hydridoma 4KB128, a recombinant single-chain variable fragment (scFv) targeting CD22, a marker antigen expressed by B-lineage leukaemias and lymphomas. We constructed several ITs using two enzymatic toxins both able to block protein translation, one of bacterial origin (a truncated version of Pseudomonas exotoxin A, PE40) endowed with EF-2 ADP-ribosylation activity, the other being the plant ribosome-inactivating protein saporin, able to specifically depurinate 23/26/28S ribosomal RNA. PE40 was selected because it has been widely used for the construction of recombinant ITs that have already undergone evaluation in clinical trials. Saporin has also been evaluated clinically and has recently been expressed successfully at high levels in a Pichia pastoris expression system. The aim of the present study was to evaluate optimal microbial expression of various IT formats.ResultsAn anti-CD22 scFv termed 4KB was obtained which showed the expected binding activity which was also internalized by CD22+ target cells and was also competed for by the parental monoclonal CD22 antibody. Several fusion constructs were designed and expressed either in E. coli or in Pichia pastoris and the resulting fusion proteins affinity-purified. Protein synthesis inhibition assays were performed on CD22+ human Daudi cells and showed that the selected ITs were active, having IC50 values (concentration inhibiting protein synthesis by 50% relative to controls) in the nanomolar range.ConclusionsWe undertook a systematic comparison between the performance of the different fusion constructs, with respect to yields in E. coli or P. pastoris expression systems and also with regard to each constructs specific killing efficacy. Our results confirm that E. coli is the system of choice for the expression of recombinant fusion toxins of bacterial origin whereas we further demonstrate that saporin-based ITs are best expressed and recovered from P. pastoris cultures after yeast codon-usage optimization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0202-z) contains supplementary material, which is available to authorized users.