Targeting Induced Local Lesions in the Wheat DEMETER and DRE2 Genes, Responsible for Transcriptional Derepression of Wheat Gluten Proteins in the Developing Endosperm.

Wheat is a major source of energy and nutrition worldwide, but it is also a primary cause of frequent diet-induced health issues, specifically celiac disease, for which the only effective therapy so far is strict dietary abstinence from gluten-containing grains. Wheat gluten proteins are grouped into two major categories: high-molecular-weight glutenin subunits (HMWgs), vital for mixing and baking properties, and gliadins plus low-molecular-weight glutenin subunits (LMWgs) that contain the overwhelming majority of celiac-causing epitopes. We put forth a hypothesis that eliminating gliadins and LMWgs while retaining HMWgs might allow the development of reduced-immunogenicity wheat genotypes relevant to most gluten-sensitive individuals. This hypothesis stems from the knowledge that the molecular structures and regulatory mechanisms of the genes encoding the two groups of gluten proteins are quite different, and blocking one group's transcription, without affecting the other's, is possible. The genes for gliadins and LMWgs have to be de-methylated by 5-methylcytosine DNA glycosylase/lyase (DEMETER) and an iron-sulfur (Fe-S) cluster biogenesis enzyme (DRE2) early during endosperm development to permit their transcription. In this study, a TILLING (Targeting Induced Local Lesions IN Genomes) approach was undertaken to identify mutations in the homoeologous DEMETER (DME) and DRE2 genes in common and durum wheat. Lines with mutations in these genes were obtained that displayed reduced content of immunogenic gluten proteins while retaining essential baking properties. Although our data at first glance suggest new possibilities for treating celiac disease and are therefore of medical and agronomical interest, it also shows that inducing mutations in the DME and DRE2 genes analyzed here affected pollen viability and germination. Hence there is a need to develop other approaches in the future to overcome this undesired effect.

Effects of three biochars on copper immobilization and soil microbial communities in a metal-contaminated soil using a metallophyte and two agricultural plants.

Biochar (BC) is a porous, carbonaceous material produced by slow pyrolysis of biomass under oxygen-limited conditions. BC production has been attracting research interest because it modifies soil physicochemical characteristics and improves the growth of plants in problem soils. These benefits may be best actualized for soils contaminated by metals, where remediation is hampered by metal toxicity to both plants and soil microbial communities. The objectives of this study were to evaluate the impact of the addition of chicken manure biochar (CMB), oat hull biochar (OHB), or pine bark biochar (PBB) on copper (Cu) bioavailability in a Cu-contaminated soil, the effectiveness of these BCs promoting plant growth, and its effects on soil microbial communities supporting these plants. A sandy soil (338 mg Cu kg-1) was amended with CMB, OHB, and PBB, and the metallophyte Oenothera picensis or the agricultural species Solanum lycopersicum and Lolium perenne were grown for 3 months. The BCs produced an increase in soil pH, reduced the exchangeable Cu, and increased Cu bound to organic matter and residual fractions. All BCs enhanced the quality of contaminated soil and increased the plant biomass production, notably for S. lycopersicum, which grew until 12 times more than plants in non-amended soil. While BC addition reduced the concentration of Cu in soil pore water, the amendment did not reduce the concentrations of Cu in shoot tissues. BC additions also stimulated soil microorganisms, increasing basal respiration and DHA activity and modifying microbial communities, especially in soils supporting L. perenne. These results indicate that BCs represent an effective tool to remediate Cu-contaminated sandy soils.

Synthesis of calcium borate nanoparticles and its use as a potential foliar fertilizer in lettuce (Lactuca sativa) and zucchini (Cucurbita pepo).

This study was carried out to evaluate the effects of foliar sprays containing boron (B) nano-fertilizer (NF) on the growth and physiology of lettuce (Lactuca sativa) and zucchini (Cucurbita pepo). Plants were grown under greenhouse conditions for 60 days on a modified Hoagland solution with the presence and absence of boron (+B or -B). A synthesized B-NF foliar spray and a commercial B foliar fertilizer (Bortrac™ 150, BT) was applied at a concentration of 30 mg B L-1 at 10-d intervals throughout the experiment. The B-NF treatment increased the growth of lettuce 2.7- and 1.9-fold for shoots and roots, respectively, with an average production of lettuce biomass by ~58%. Similarly, the NF increased the growth of zucchini by 18 and 66% compared with Control-B (the absence of B), and 13 and 36% compared with BT, both for shoots and roots, respectively. Nevertheless, NF + B mostly decreased lettuce growth with symptoms of B toxicity in leaves. In lettuce, addition of B did not affect concentrations of phenols; however, in zucchini, Control-B induced a higher production of phenolic compounds possibly related to B deficiency. The B addition in lettuce reduced the DPPH activity by 32 and 21% in NF and BT, respectively, compared to Control-B. These responses were similar in zucchini; however, the effect of B was product of its presence in mineral solution rather than due the foliar product applied. This suggests that a NF-based delivery system for B may be highly effective at boosting plant productivity on B-limited soils.

Directed-Mutagenesis of Flavobacterium meningosepticum Prolyl-Oligopeptidase and a Glutamine-Specific Endopeptidase From Barley.

Wheat gluten proteins are the known cause of celiac disease. The repetitive tracts of proline and glutamine residues in these proteins make them exceptionally resilient to digestion in the gastrointestinal tract. These indigested peptides trigger immune reactions in susceptible individuals, which could be either an allergic reaction or celiac disease. Gluten exclusion diet is the only approved remedy for such disorders. Recently, a combination of a glutamine specific endoprotease from barley (EP-B2), and a prolyl endopeptidase from Flavobacterium meningosepticum (Fm-PEP), when expressed in the wheat endosperm, were shown to reasonably detoxify immunogenic gluten peptides under simulated gastrointestinal conditions. However useful, these "glutenases" are limited in application due to their denaturation at high temperatures, which most of the food processes require. Variants of these enzymes from thermophilic organisms exist, but cannot be applied directly due to their optimum activity at temperatures higher than 37°C. Though, these enzymes can serve as a reference to guide the evolution of peptidases of mesophilic origin toward thermostability. Therefore, a sequence guided site-saturation mutagenesis approach was used here to introduce mutations in the genes encoding Fm-PEP and EP-B2. A thermostable variant of Fm-PEP capable of surviving temperatures up to 90°C and EP-B2 variant with a thermostability of up 60°C were identified using this approach. However, the level of thermostability achieved is not sufficient; the present study has provided evidence that the thermostability of glutenases can be improved. And this pilot study has paved the way for more detailed structural studies in the future to obtain variants of Fm-PEP and EP-B2 that can survive temperatures ~100°C to allow their packing in grains and use of such grains in the food industry.

Gluten Detection Methods and Their Critical Role in Assuring Safe Diets for Celiac Patients.

Celiac disease, wheat sensitivity, and allergy represent three different reactions, which may occur in genetically predisposed individuals on the ingestion of wheat and derived products with various manifestations. Improvements in the disease diagnostics and understanding of disease etiology unveiled that these disorders are widespread around the globe affecting about 7% of the population. The only known treatment so far is a life-long gluten-free diet, which is almost impossible to follow because of the contamination of allegedly "gluten-free" products. Accidental contamination of inherently gluten-free products could take place at any level from field to shelf because of the ubiquity of these proteins/grains. Gluten contamination of allegedly "gluten-free" products is a constant threat to celiac patients and a major health concern. Several detection procedures have been proposed to determine the level of contamination in products for celiac patients. The present article aims to review the advantages and disadvantages of different gluten detection methods, with emphasis on the recent technology that allows identification of the immunogenic-gluten peptides without the use of antibodies. The possibility to detect gluten contamination by different approaches with similar or better detection efficiency in different raw and processed foods will guarantee the safety of the foods for celiac patients.

Development of wheat genotypes expressing a glutamine-specific endoprotease from barley and a prolyl endopeptidase from Flavobacterium meningosepticum or Pyrococcus furiosus as a potential remedy to celiac disease.

Ubiquitous nature of prolamin proteins dubbed gluten from wheat and allied cereals imposes a major challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of hydrolytic glutenases as food supplement is an alternative to deliver the therapeutic agents directly to the small intestine, where sensitization of immune system and downstream reactions take place. The aim of the present research was to evaluate the capacity of wheat grain to express and store hydrolytic enzymes capable of gluten detoxification. For this purpose, wheat scutellar calli were biolistically transformed to generate plants expressing a combination of glutenase genes for prolamin detoxification. Digestion of prolamins with barley endoprotease B2 (EP-HvB2) combined with Flavobacterium meningosepticum prolyl endopeptidase (PE-FmPep) or Pyrococcus furiosus prolyl endopeptidase (PE-PfuPep) significantly reduced (up to 67%) the amount of the indigestible gluten peptides of all prolamin families tested. Seven of the 168 generated lines showed inheritance of transgene to the T2 generation. Reversed phase high-performance liquid chromatography of gluten extracts under simulated gastrointestinal conditions allowed the identification of five T2 lines that contained significantly reduced amounts of immunogenic, celiac disease-provoking gliadin peptides. These findings were complemented by the R5 ELISA test results where up to 72% reduction was observed in the content of immunogenic peptides. The developed wheat genotypes open new horizons for treating celiac disease by an intraluminal enzyme therapy without compromising their agronomical performance.

Copper immobilization by biochar and microbial community abundance in metal-contaminated soils.

Biochar (BC) is gaining attention as a soil amendment that can remediate metal polluted soils. The simultaneous effects of BC on copper (Cu) mobility, microbial activities in soil using metallophytes have scarcely been addressed. The objective of this study was to evaluate the effects of biochar BCs on Cu immobilization and over soil microbial communities in a Cu-contaminated soil evaluated over a two-year trial. A Cu-contaminated soil (338mgkg-1) was incubated with chicken manure biochar (CMB) or oat hull biochar (OHB) at rates of 1 and 5% w/w. Metallophyte Oenothera picensis was grown over one season (six months). The above process was repeated for 3 more consecutive seasons using the same soils. The BCs increased the soil pH and decreased the Cu exchangeable fraction Cu by 5 and 10 times (for OHB and CMB, respectively) by increasing the Cu bound in organic matter and residual fractions, and its effects were consistent across all seasons evaluated. BCs provided favorable habitat for microorganisms that was evident in increased microbial activity. The DHA activity was increased in all BC treatments, reaching a maximum of 7 and 6 times higher than control soils in CMB and OHB. Similar results were observed in microbial respiration, which increased 53% in OHB and 61% in CMB with respect to control. The BCs produced changes in microbial communities in all seasons evaluated. The fungal and bacterial richness were increased by CMB and OHB treatments; however, no clear effects were observed in the microbial diversity estimators. The physiochemical and microbiological effects produced by BC result in an increase of plant biomass production, which was on average 3 times higher than control treatments. However, despite being a metallophyte, O. picensis did not uptake Cu efficiently. Root and shoot Cu concentrations decreased or changed insignificantly in most BC treatments.

Assessment of genetic diversity among barley cultivars and breeding lines adapted to the US Pacific Northwest, and its implications in breeding barley for imidazolinone-resistance.

Extensive application of imidazolinone (IMI) herbicides had a significant impact on barley productivity contributing to a continuous decline in its acreage over the last two decades. A possible solution to this problem is to transfer IMI-resistance from a recently characterized mutation in the 'Bob' barley AHAS (acetohydroxy acid synthase) gene to other food, feed and malting barley cultivars. We focused our efforts on transferring IMI-resistance to barley varieties adapted to the US Pacific Northwest (PNW), since it comprises ∼23% (335,000 ha) of the US agricultural land under barley production. To effectively breed for IMI-resistance, we studied the genetic diversity among 13 two-rowed spring barley cultivars/breeding-lines from the PNW using 61 microsatellite markers, and selected six barley genotypes that showed medium to high genetic dissimilarity with the 'Bob' AHAS mutant. The six selected genotypes were used to make 29-53 crosses with the AHAS mutant and a range of 358-471 F1 seeds were obtained. To make informed selection for the recovery of the recipient parent genome, the genetic location of the AHAS gene was determined and its genetic nature assessed. Large F2 populations ranging in size from 2158-2846 individuals were evaluated for herbicide resistance and seedling vigor. Based on the results, F3 lines from the six most vigorous F2 genotypes per cross combination were evaluated for their genetic background. A range of 20%-90% recovery of the recipient parent genome for the carrier chromosome was observed. An effort was made to determine the critical dose of herbicide to distinguish between heterozygotes and homozygotes for the mutant allele. Results suggested that the mutant can survive up to the 10× field recommended dose of herbicide, and the 8× and 10× herbicide doses can distinguish between the two AHAS mutant genotypes. Finally, implications of this research in sustaining barley productivity in the PNW are discussed.

Analysis of wheat prolamins, the causative agents of celiac sprue, using reversed phase high performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).

Wheat prolamins, commonly known as "gluten", are a complex mixture of 71-78 proteins, which constitute ~80% of the proteins in the wheat grains and supply 50% of the global dietary protein demand. Prolamins are also responsible for numerous gluten-induced disorders and determine the unique visco-elastic properties of the wheat dough. These properties necessitate the reliable determination of the prolamin composition in wheat grains and their derived products. Therefore, this study examined the impact of HPLC conditions, including column type, column temperature, flow rate, and the gradient of polar and non-polar solvents in the mobile phase, to improve the analytical resolution of prolamins. The following conditions were found optimal for analyses: column temperature 60 °C, flow rate 1.0 mL/min and an elution gradient of 20%-60% of 0.1% trifluoroacetic acid + acetonitrile in 60 min. For further improvement of resolution, gliadin and glutenin extracts were analyzed using MALDI-TOF-MS in combination with HPLC fractionation. Two semi-quantitative methods, densitometry of stained polyacrylamide gels and HPLC, were used to determine relative prolamin quantities and the correspondence between the methods was established. The combinatorial gluten analyses approach developed during the present study was used to analyze prolamin profiles of wheat transformants expressing DEMETER silencing artificial microRNA, and the results are discussed.

Structural genes of wheat and barley 5-methylcytosine DNA glycosylases and their potential applications for human health.

Wheat supplies about 20% of the total food calories consumed worldwide and is a national staple in many countries. Besides being a key source of plant proteins, it is also a major cause of many diet-induced health issues, especially celiac disease. The only effective treatment for this disease is a total gluten-free diet. The present report describes an effort to develop a natural dietary therapy for this disorder by transcriptional suppression of wheat DEMETER (DME) homeologs using RNA interference. DME encodes a 5-methylcytosine DNA glycosylase responsible for transcriptional derepression of gliadins and low-molecular-weight glutenins (LMWgs) by active demethylation of their promoters in the wheat endosperm. Previous research has demonstrated these proteins to be the major source of immunogenic epitopes. In this research, barley and wheat DME genes were cloned and localized on the syntenous chromosomes. Nucleotide diversity among DME homeologs was studied and used for their virtual transcript profiling. Functional conservation of DME enzyme was confirmed by comparing the motif and domain structure within and across the plant kingdom. Presence and absence of CpG islands in prolamin gene sequences was studied as a hallmark of hypo- and hypermethylation, respectively. Finally the epigenetic influence of DME silencing on accumulation of LMWgs and gliadins was studied using 20 transformants expressing hairpin RNA in their endosperm. These transformants showed up to 85.6% suppression in DME transcript abundance and up to 76.4% reduction in the amount of immunogenic prolamins, demonstrating the possibility of developing wheat varieties compatible for the celiac patients.

Suplemento de calcio en gestantes multiparas: con antecedentes de trastornos hipertensivos como factor predictivo para su aparición / Supplement of calcium in multiparous gestating with antecedents of hypertensive disorder as predictive factor for the its apparition

El propósito de la presente investigación es conocer la importancia que tiene el calcio como factor preventivo de la hipertensión arterial gestacional en embarazadas multíparas. Determinar la influencia del calcio en la prevención de la hipertensión arterial gestacional en embarazadas multíparas. Establecer los posibles mecanismos por los que el calcio actúa en la prevención de la hipertensión arterial gestacional. Informar a médicos y estudiantes de Medicina acerca de los medios para la prevención de la HTA gestacional utilizando un suplemento de calcio. Señalar las consecuencias de una ingesta inadecuada de calcio en la dieta de la mujer embarazada

Prevención médica a los empleados de galvanoplastia: estudio y enfoque actual / Medical prevention to the galvanoplasty employer: study and actual focussing

Experimentar en el medio físico del trabajo, ya que estamos acostumbrados a recibir información sin estar en contacto directo con un trabajador que por su necesidad de subsistir se expone a una serie de riesgos que comprometen su salud física y mental. Para constatar que las empresas ven al individuo sólo como recurso humano, para acrecentar sus riquezas sin considerar con medidas preventivas y educación el bienestar y la adaptación del trabajo al hombre. En menos escala, consideramos que aun el mismo trabajador no coopera con las medidas mínimas por exceptisismo. El poder a través de este trabajo apoyar con una extensa bibliografía; los riesgos de enfermedades ocupacionales y de accidentes en el área laboral de la industria del Cromo, de puntos de vista distintos, como lo es la Medicina, de Ingenieria, las Ciencias Sociales, las Estadísticas, Psicología, Toxicología, etc., a los cuales está sometido el hombre. Permitir intercambiar ideas entre los estudiantes, trabajar en grupo y en particular nosotros que fue a las afuera de la UCV, cambiando un poco los esquemas

Diabetes mellitus en la infancia: repercusión con el entorno familiar / Diabetes mellitus in the infance: repercussion with family environment

Debido a que la DMID es una enfermedad crónica, que al afectar tempranamente a los niños cambia radicalmente sus vidas (2), se pretende relacionar el entorno familiar como un posible factor de riesgo para la aceptación de su enfermedad (1) lo cual se traduciría en un mejor o peor control metabólico de la Diabetes (1,2,3). Con este estudio se pretende probar si un niño sometido a situaciones de stress familiar tiende a presentar mayor descontrol en el metabolismo de los carbohidratos, con lo cual aumentan sus posibilidades de complicaciones (4). Se escogieron las edades comprendidas entre los seis y doce años debido a que en edades inferiores hay pocos casos (7), y a partir de los doce años hay cambios naturales, debido a que la hormona de crecimiento, hace que aumenten los niveles de glucosa en sangre por lo que se puede obtener el porcentaje de hemoglobina glicosilada en sangre elevada (4,5). Consideramos importante determinar la influencia de la situación familiar para poder catalogar a esos niños como de alto riesgo para un descontrol metabólico y de alguna manera seguirlos más de cerca para prevenir complicaciones

Adolescencia: un problema actual: una aproximación a la realidad del adolescente venezolano / Adolescence: an actual problem: an approximation to the reality of venezuelan adolescent

El período de la adolescencia tiene una morbilidad propia, diferente a la del niño y a la del adulto, y es por eso que decidimos realizar un estudio retrospectivo de las 5 primeras causas de consulta en los adolescentes. La adolescencia, ese período de transición entre la infancia y la madurez, constituye al parecer uno de los puntos ciegos de la visión médica del hombre, en la que aparentemente el niño se hace adulto sin que medie para ello ningún tipo de cambio progresivo. ¿Acaso las enfermedades tienen su mayor incidencia en este período de transformación física y mental son diferentes a las del niño y a las del adulto?; y de ser así, ¿estaremos listos para atender y enfrentarlas de la mejor forma posible?. Con la mirada fija en este objetivo, iniciamos una evaluación estadística. Sin embargo, tan noble propósito no soportó el embate inicial de la realidad, en la que un sistema de archivo y registro anacrónico e ineficaz constituyeron el obstáculo insalvable. De este modo, tuvimos que cambiar nuestro objetivo principal. Así, prescindiendo de los datos estadísticos previos, diseñamos una encuesta para adolescentes con edades comprendidas entre los 12 y 17 años inclusive, que aborda de una manera general, temas de interes como: sexo y embarazo, drogadicción, relación con los padres, depresión y suicidio, actividades personales y accidentes entre otros; apoyándonos en un marco teórico que nos nutre e informa, tratando de esta manera de plantear una aproximación a la problemática del adolescente. No obstante, si con este trabajo logramos despertar un mínimo de interés por pequeño que este sea respecto al adolescente, consideraremos cubierta con creces, la esencia de nuestro objetivo principal