Low‐gluten,nontransgenic wheat engineered with CRISPR/Cas9

Coeliac disease is an autoimmune disorder triggered in genetically predisposed individuals by the ingestion of gluten proteins from wheat, barley and rye. The α‐gliadin gene family of wheat contains four highly stimulatory peptides, of which the 33‐mer is the main immunodominant peptide in patients with coeliac. We designed two sgRNAs to target a conserved region adjacent to the coding sequence for the 33‐mer in the α‐gliadin genes. Twenty‐one mutant lines were generated, all showing strong reduction in α‐gliadins. Up to 35 different genes were mutated in one of the lines of the 45 different genes identified in the wild type, while immunoreactivity was reduced by 85%. Transgene‐free lines were identified, and no off‐target mutations have been detected in any of the potential targets. The low‐gluten, transgene‐free wheat lines described here could be used to produce low‐gluten foodstuff and serve as source material to introgress this trait into elite wheat varieties.     

Plant‐expressed Fc‐fusion protein tetravalent dengue vaccine with inherent adjuvant properties

Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi Pasteur Dengvaxia vaccine does not protect children under the age of nine, and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant expression to produce a humanized and highly immunogenic poly‐immunoglobulin G scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor‐targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralizing antibodies and evidence of cell‐mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue ex vivo as evidenced by antigen‐specific CD4⁺ and CD8⁺ T‐cell proliferation, IFN‐γ and antibody production. The purified polymeric fraction of dengue PIGS (D‐PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low‐affinity Fcγ receptors on antigen‐presenting cells. These results show that the plant‐expressed D‐PIGS have the potential for translation towards a safe and easily scalable single antigen‐based tetravalent dengue vaccine.     

Caffeine-induced Release of Intracellular Ca2+ from Chinese Hamster Ovary Cells Expressing Skeletal Muscle Ryanodine Receptor : Effects on Full-Length and Carboxyl-Terminal Portion of Ca2+Release Channels

The ryanodine receptor (RyR)/Ca²⁺ release channel is an essential component of excitation–contraction coupling in striated muscle cells. To study the function and regulation of the Ca²⁺ release channel, we tested the effect of caffeine on the full-length and carboxyl-terminal portion of skeletal muscle RyR expressed in a Chinese hamster ovary (CHO) cell line. Caffeine induced openings of the full length RyR channels in a concentration-dependent manner, but it had no effect on the carboxyl-terminal RyR channels. CHO cells expressing the carboxyl-terminal RyR proteins displayed spontaneous changes of intracellular [Ca²⁺]. Unlike the native RyR channels in muscle cells, which display localized Ca²⁺ release events (i.e., “Ca²⁺ sparks” in cardiac muscle and “local release events” in skeletal muscle), CHO cells expressing the full length RyR proteins did not exhibit detectable spontaneous or caffeine-induced local Ca²⁺ release events. Our data suggest that the binding site for caffeine is likely to reside within the amino-terminal portion of RyR, and the localized Ca²⁺ release events observed in muscle cells may involve gating of a group of Ca²⁺ release channels and/or interaction of RyR with muscle-specific proteins.     

Field study of beech (t Fagus sylvatica L.) and melojo oak (t Quercus pyrenaica Willd) leaf litter decomposition in the centre of the Iberian Peninsula

The effect of the canopy on leaf decomposition of beech (t Fagus sylvatica) and melojo oak (t Quercus pyrenaica) was studied during a period of 660 days in a mountain forest of central Spain; response of leaves to leaching was also studied to determine the effects of rainfall after leaf fall. Beech leaves lost 5.8% of their weight by leaching, whereas melojo oak leaves lost 13%. Under both types of canopy, beech leaves showed almost no difference in their decomposition patterns, with decay constants of 0.31 and 0.32 respectively. Melojo oak leaves showed quite a different behaviour under both canopies; decay constant was 0.47 under t Quercus pyrenaica and 0.77 under beech canopy. Total immobilization of nitrogen was less in the melojo oak forest. Effects of summer dryness were sharper in the melojo oak forest, where decomposition stopped during the summer. This delay in the decomposition might have been due to the lower canopy density in the melojo oak forest. The decomposition patterns of the leaves of both species, under the canopy of the other species, suggests what might happen in mixed stands. Thus, the presence of melojo oaks in beech forest would increase decay and decrease nitrogen immobilization. The presence of beech trees in melojo oak forests would improve microclimatic conditions and increase decay, whereas beech litter on the soil would immobilizate more nitrogen.     

Lectin-induced alterations on the proliferation of three human prostatic cancer cell lines

Summary While lectins are known to influence the cell growth of several types of normal and neoplastic tissues, their roles in the case of prostatic cancer cells remain relatively unexplored. In the present work, we report thein vitro influence of five lectins, namely peanut (PNA), wheat germ (WGA), Concanavalin A (Con A),Griffonia simplicifolia (GSA-IA₄), andPhaseolus vulgaris (PHA-L) agglutinins, on the cell proliferation of one androgen-sensitive (LNCaP) and two androgen-insensitive (PC-3 and DU 145) human prostatic cancer cell lines cultured in either 10% or 1% fetal bovine serum (FBS)-supplemented media. The cell proliferation was assessed by means of the colorimetric 3-(4,5-dimethythiazol-2-yle)2,5-diphenyltetrazolium bromide. (MTT) assay. Four lectin concentrations were tested (i.e., 0.1, 1, 10, and 100 μg/ml) at five experimental states (i.e., 2, 3, 5, 7, and 9 d following the addition of each lectin to the culture media). Our results demonstrated that the five lectins under study had a globally significant dose-dependent toxic effect on prostatic cancer cell proliferation. Nevertheless, low doses of GSA-IA₄ and PHA-L significantly (P<0.05 toP<0.001) increased the cell proliferation of confluent PC-3 cells. Increasing the FBS from 1% to 10% in the culture media significantly antagonized lectin-induced toxicity in the three prostatic cell lines. In conclusion, the present data strongly suggest that some lectins might influence the proliferation of prostatic carcinoma cells. In addition, because lectins are present in our diet, and are able to pass into the systemic circulation and thus reach the prostate, the present results suggest that some lectins might exert an influence on prostate cancer growth under clinical conditions.     

Contribution to the determination of the chemotherapeutic drug G1 in biological fluids

A sensitive gas chromatographic method for the quantitative determination of the new antibacterial and antifungal drug G1, 1-(5-bromofuran-2-yl)-2-bromo-2-nitroethene, has been optimized. The method involves a fast and single extraction step from spiked serum and urine samples. The G1 drug was quantified using an internal standard method and by means of a nitrogen-selective detector. The results are statistically significant and show that mean levels of G1 as low as 1 μg ml⁻¹ can be measured accurately.     

Role of thyroid hormones in renal tubule acidification

Renal tubule acidification was studied in thyroparathyroidectomized rats which had the parathyroids reimplanted into cervical muscle tissue, by stopped-flow microperfusion using ion-exchange resin microelectrodes. Hypothyroid rats had decreased rates of proximal and late distal bicarbonate reabsorption. This reduction occurred in the absence of changes in pH gradients, and was due mostly to decreases in acidification half-times, that is, of the rate of bicarbonate exit from the tubule lumen. H⁺ back-flux from the lumen measured during luminal perfusion with solutions at pH 6 (below stationary pH) was decreased in proximal tubule of hypothyroid rats, showing that the acidification defect was not due to an increased H⁺ shunt across the epithelium. These data indicate that in hypothyroid rats the proximal tubule luminal density of Na⁺/H⁺ exchangers or their turnover is decreased in the absence of alterations in the driving force (H⁺ and Na⁺ gradients across the luminal membrane) for H⁺ secretion. The effect observed in distal tubule may be due to action on Na⁺/H⁺ exchangers that are present also on this site, or to an impairment of the action of other H⁺ transporters such as H⁺-ATPases, including the provision of energy for them.This paper is dedicated to Prof. Carlos Chagas Filho, founder of the Institute of Biophysics, on the occasion of its 50th anniversary.     

Isolation and characterization of abscisic acid-deficient Arabidopsis mutants at two new loci

Novel Arabidopsis mutants with lowered levels of endogenous abscisic acid (ABA) were isolated. These were selected in a screen for germination in the presence of the gibberellin biosynthesis inhibitor paclobutrazol. Another mutant was isolated in a screen for NaCl tolerance. The ABA-deficiency was caused by two monogenic, recessive mutations, aba2 and aba3 , that were both located on chromosome 1. The mutants showed a phenotype that is known to be characteristic for ABA-deficiency: a reduced seed dormancy and excessive water loss, leading to a wilty phenotype. Double mutant analysis, combining different aba mutations, indicated the leaky nature of the mutations.