Elucidating the role of polygalacturonase genes in strawberry fruit softening.

To disentangle the role of polygalacturonase (PG) genes in strawberry softening, the two PG genes most expressed in ripe receptacles, FaPG1 and FaPG2, were down-regulated. Transgenic ripe fruits were firmer than those of the wild type when PG genes were silenced individually. Simultaneous silencing of both PG genes by transgene stacking did not result in an additional increase in firmness. Cell walls from ripe fruits were characterized by a carbohydrate microarray. Higher signals of homogalacturonan and rhamnogalacturonan I pectin epitopes in polysaccharide fractions tightly bound to the cell wall were observed in the transgenic genotypes, suggesting a lower pectin solubilization. At the transcriptomic level, the suppression of FaPG1 or FaPG2 alone induced few transcriptomic changes in the ripe receptacle, but the amount of differentially expressed genes increased notably when both genes were silenced. Many genes encoding cell wall-modifying enzymes were down-regulated. The expression of a putative high affinity potassium transporter was induced in all transgenic genotypes, indicating that cell wall weakening and loss of cell turgor could be linked. These results suggest that, besides the disassembly of pectins tightly linked to the cell wall, PGs could play other roles in strawberry softening, such as the release of oligogalacturonides exerting a positive feedback in softening.

Metodología para la enseñanza desarrolladora del diagnóstico de anomalías dentomaxilofaciales centrado en el método / Methodology for the teaching of skills required to diagnose dentomaxillofacial anomalies based on the clinical method

Introducción: la utilización del enfoque desarrollador de la didáctica y el método clínico en la enseñanza del diagnóstico de anomalías dentomaxilofaciales es de gran importancia en la asignatura Ortodoncia, para que el estomatólogo general realice la atención integral, según su modelo de formación. Objetivo: valorar la pertinencia de una metodología para la enseñanza del diagnóstico clínico de anomalías dentomaxilofaciales en la asignatura Ortodoncia de la carrera de Estomatología. Métodos: investigación pedagógica. Se emplearon métodos del nivel teórico (histórico-lógico, inductivo-deductivo, analítico-sintético y sistémico) y empírico (criterio de expertos). De 33 posibles expertos, se seleccionó una muestra de 30 según el coeficiente de competencia. Resultados: se elaboró una metodología que se estructura en las etapas de organización, planificación, ejecución y evaluación de la enseñanza del diagnóstico de anomalías dentomaxilofaciales; se distingue porque precisa las relaciones entre las diferentes categorías de la didáctica en el contexto de la atención estomatológica desde un enfoque desarrollador; se centra en el método clínico; y revela la necesidad de partir de los resultados del diagnóstico pedagógico. La metodología fue valorada por expertos, quienes en su totalidad ubicaron a los siete indicadores evaluados en el rango de bastante adecuado. Conclusiones: la metodología propuesta resulta pertinente para su aplicación en la enseñanza del diagnóstico de anomalías dentomaxilofaciales en la asignatura Ortodoncia de la carrera de Estomatología, según la valoración de expertos(AU)

Introduction: use of a skill-developing didactic approach and the clinical method in training for the diagnosis of dentomaxillofacial anomalies is essential in the subject Orthodontics, so that the general dental practitioner can provide comprehensive care following their own training model. Objective: assess the relevance of a methodology for the teaching of skills required to clinically diagnose dentomaxillofacial anomalies in the subject Orthodontics of the dental training curriculum. Methods: a pedagogical study was conducted, based on both theoretical methods (historical-logical, inductive-deductive, analytical-synthetic and systemic) and empirical methods (expert criteria). Of 33 possible experts, a sample of 30 was selected based on their competence coefficient. Results: a methodology was developed which is structured into stages for organization, planning, implementation and evaluation in the teaching of skills required to diagnose dentomaxillofacial anomalies. Distinguishing features of this methodology are that it clearly points out the relationships between the different didactic categories in the context of dental care with a skill-development approach, is centered on the clinical method, and reveals the need to start from the results of the pedagogical diagnosis. The methodology was assessed by experts, all of whom ranked the seven indicators evaluated as quite adequate. Conclusions: the methodology proposed is appropriate for application in the teaching of skills required to diagnose dentomaxillofacial anomalies in the subject Orthodontics of the dental training curriculum, according to the opinion of experts(AU)

Metodología para la enseñanza desarrolladora del diagnóstico de anomalías dentomaxilofaciales centrado en el método / Methodology for the teaching of skills required to diagnose dentomaxillofacial anomalies based on the clinical method

Introducción: la utilización del enfoque desarrollador de la didáctica y el método clínico en la enseñanza del diagnóstico de anomalías dentomaxilofaciales es de gran importancia en la asignatura Ortodoncia, para que el estomatólogo general realice la atención integral, según su modelo de formación. Objetivo: valorar la pertinencia de una metodología para la enseñanza del diagnóstico clínico de anomalías dentomaxilofaciales en la asignatura Ortodoncia de la carrera de Estomatología. Métodos: investigación pedagógica. Se emplearon métodos del nivel teórico (histórico-lógico, inductivo-deductivo, analítico-sintético y sistémico) y empírico (criterio de expertos). De 33 posibles expertos, se seleccionó una muestra de 30 según el coeficiente de competencia. Resultados: se elaboró una metodología que se estructura en las etapas de organización, planificación, ejecución y evaluación de la enseñanza del diagnóstico de anomalías dentomaxilofaciales; se distingue porque precisa las relaciones entre las diferentes categorías de la didáctica en el contexto de la atención estomatológica desde un enfoque desarrollador; se centra en el método clínico; y revela la necesidad de partir de los resultados del diagnóstico pedagógico. La metodología fue valorada por expertos, quienes en su totalidad ubicaron a los siete indicadores evaluados en el rango de bastante adecuado. Conclusiones: la metodología propuesta resulta pertinente para su aplicación en la enseñanza del diagnóstico de anomalías dentomaxilofaciales en la asignatura Ortodoncia de la carrera de Estomatología, según la valoración de expertos(AU)

Introduction: use of a skill-developing didactic approach and the clinical method in training for the diagnosis of dentomaxillofacial anomalies is essential in the subject Orthodontics, so that the general dental practitioner can provide comprehensive care following their own training model. Objective: assess the relevance of a methodology for the teaching of skills required to clinically diagnose dentomaxillofacial anomalies in the subject Orthodontics of the dental training curriculum. Methods: a pedagogical study was conducted, based on both theoretical methods (historical-logical, inductive-deductive, analytical-synthetic and systemic) and empirical methods (expert criteria). Of 33 possible experts, a sample of 30 was selected based on their competence coefficient. Results: a methodology was developed which is structured into stages for organization, planning, implementation and evaluation in the teaching of skills required to diagnose dentomaxillofacial anomalies. Distinguishing features of this methodology are that it clearly points out the relationships between the different didactic categories in the context of dental care with a skill-development approach, is centered on the clinical method, and reveals the need to start from the results of the pedagogical diagnosis. The methodology was assessed by experts, all of whom ranked the seven indicators evaluated as quite adequate. Conclusions: the methodology proposed is appropriate for application in the teaching of skills required to diagnose dentomaxillofacial anomalies in the subject Orthodontics of the dental training curriculum, according to the opinion of experts(AU)

Structural changes in cell wall pectins during strawberry fruit development.

Strawberry (Fragaria × anannasa Duch.) is one of the most important soft fruit. Rapid loss of firmness occurs during the ripening process, resulting in a short shelf life and high economic losses. To get insight into the role of pectin matrix in the softening process, cell walls from strawberry fruit at two developmental stages, unripe-green and ripe-red, were extracted and sequentially fractionated with different solvents to obtain fractions enriched in a specific component. The yield of cell wall material as well as the per fresh weight contents of the different fractions decreased in ripe fruit. The largest reduction was observed in the pectic fractions extracted with a chelating agent (trans-1,2- diaminocyclohexane-N,N,N'N'-tetraacetic acid, CDTA fraction) and those covalently bound to the wall (extracted with Na2CO3). Uronic acid content of these two fractions also decreased significantly during ripening, but the amount of soluble pectins extracted with phenol:acetic acid:water (PAW) and water increased in ripe fruit. Fourier transform infrared spectroscopy of the different fractions showed that the degree of esterification decreased in CDTA pectins but increased in soluble fractions at ripen stage. The chromatographic analysis of pectin fractions by gel filtration revealed that CDTA, water and, mainly PAW polyuronides were depolymerised in ripe fruit. By contrast, the size of Na2CO3 pectins was not modified. The nanostructural characteristics of CDTA and Na2CO3 pectins were analysed by atomic force microscopy (AFM). Isolated pectic chains present in the CDTA fractions were significantly longer and more branched in samples from green fruit than those from red fruit. No differences in contour length were observed in Na2CO3 strands between samples of both stages. However, the percentage of branched chains decreased from 19.7% in unripe samples to 3.4% in ripe fruit. The number of pectin aggregates was higher in green fruit samples of both fractions. These results show that the nanostructural complexity of pectins present in CDTA and Na2CO3 fractions diminishes during fruit development, and this correlates with the solubilisation of pectins and the softening of the fruit.

Unravelling the nanostructure of strawberry fruit pectins by endo-polygalacturonase digestion and atomic force microscopy.

Pectins analysed by AFM are visualized as individual chains, branched or unbranched, and aggregates. To investigate the nature of these structures, sodium carbonate soluble pectins from strawberry fruits were digested with endo-polygalacturonase M2 from Aspergillus aculeatus and visualized by AFM. A gradual decrease in the length of chains was observed as result of the treatment, reaching a minimum LN value of 22nm. The branches were not visible after 2h of enzymatic incubation. The size of complexes also diminished significantly with the enzymatic digestion. A treatment to hydrolyse rhamnogalacturonan II borate diester bonds neither affected chains length or branching nor complex size but reduced the density of aggregates. These results suggest that chains are formed by a mixture of homogalacturonan and more complex molecules composed by a homogalacturonan unit linked to an endo-PG resistant unit. Homogalacturonan is a structural component of the complexes and rhamnogalacturonan II could be involved in their formation.

The nanostructural characterization of strawberry pectins in pectate lyase or polygalacturonase silenced fruits elucidates their role in softening.

To ascertain the role of pectin disassembly in fruit softening, chelated- (CSP) and sodium carbonate-soluble (SSP) pectins from plants with a pectate lyase, FaplC, or a polygalacturonase, FaPG1, downregulated by antisense transformation were characterized at the nanostructural level. Fruits from transgenic plants were firmer than the control, although FaPG1 suppression had a greater effect on firmness. Size exclusion chromatography showed that the average molecular masses of both transgenic pectins were higher than that of the control. Atomic force microscopy analysis of pectins confirmed the higher degree of polymerization as result of pectinase silencing. The mean length values for CSP chains increased from 84 nm in the control to 95.5 and 101 nm, in antisense FaplC and antisense FaPG1 samples, respectively. Similarly, SSP polyuronides were longer in transgenic fruits (61, 67.5 and 71 nm, in the control, antisense FaplC and antisense FaPG1 samples, respectively). Transgenic pectins showed a more complex structure, with a higher percentage of branched chains than the control, especially in the case of FaPG1 silenced fruits. Supramolecular pectin aggregates, supposedly formed by homogalacturonan and rhamnogalacturonan I, were more frequently observed in antisense FaPG1 samples. The larger modifications in the nanostructure of pectins in FaPG1 silenced fruits when compared with antisense pectate lyase plants correlate with the higher impact of polygalacturonase silencing on reducing strawberry fruit softening.

La enseñanza-aprendizaje del diagnóstico de anomalías dentomaxilofaciales sustentado en el método clínico / The teaching and learning of the diagnosis of dentofacial anomalies, based on the clinical method

La enseñanza del método clínico para el diagnóstico constituye una demanda de la sociedad, demanda que se expresa en los lineamientos de la política económica y social del país, aprobados en el VI Congreso del Partido Comunista de Cuba; sin embargo existen insuficiencias en su ejecución en la asignatura Ortodoncia de la carrera de Estomatología. Objetivo: Facilitar a los profesores de ortodoncia elementos del método clínico como sustento para la enseñanza-aprendizaje del diagnóstico de anomalías dentomaxilofaciales en la carrera de Estomatología. Desarrollo: A partir de un marco teórico referencial, se ofrecen consideraciones para la enseñanza-aprendizaje del diagnóstico de anomalías dentomaxilofaciales sustentada en el método clínico, como contenido de la asignatura Ortodoncia. Conclusiones: Las consideraciones realizadas permitieron fundamentar desde la teoría, la utilización del método clínico para el perfeccionamiento de la enseñanza-aprendizaje del diagnóstico de anomalías dentomaxilofaciales.(AU)

The teaching of the clinical method for diagnosis is a demand of the society expressed in the Guidelines of the Economic and Social Policy of the Cuban Party and the Revolution; however, there are weaknesses in its performance in the course in the subject orthodontics of the stomatology career. Objective: To provide orthodontic teachers with elements of the clinical method as support for the teaching and learning of the diagnosis of dentofacial anomalies in the career of stomatology. Development: From identifying the shortcomings of the process and a theoretical framework, considerations for the teaching and learning of the diagnosis of dentofacial anomalies are offered supported by the clinical method in education in the work of the subject of orthodontics. Conclusions: The findings made it possible to base from theory, using the clinical method for improving the teaching and learning of the diagnosis of dentofacial anomalies.(AU)

Fruit softening and pectin disassembly: an overview of nanostructural pectin modifications assessed by atomic force microscopy.

BACKGROUND: One of the main factors that reduce fruit quality and lead to economically important losses is oversoftening. Textural changes during fruit ripening are mainly due to the dissolution of the middle lamella, the reduction of cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Pectins, major components of fruit cell walls, are extensively modified during ripening. These changes include solubilization, depolymerization and the loss of neutral side chains. Recent evidence in strawberry and apple, fruits with a soft or crisp texture at ripening, suggests that pectin disassembly is a key factor in textural changes. In both these fruits, softening was reduced as result of antisense downregulation of polygalacturonase genes. Changes in pectic polymer size, composition and structure have traditionally been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides, and studies focusing on modifications at the nanostructural level are scarce. Atomic force microscopy (AFM) allows the study of individual polymers at high magnification and with minimal sample preparation; however, AFM has rarely been employed to analyse pectin disassembly during fruit ripening. SCOPE: In this review, the main features of the pectin disassembly process during fruit ripening are first discussed, and then the nanostructural characterization of fruit pectins by AFM and its relationship with texture and postharvest fruit shelf life is reviewed. In general, fruit pectins are visualized under AFM as linear chains, a few of which show long branches, and aggregates. Number- and weight-average values obtained from these images are in good agreement with chromatographic analyses. Most AFM studies indicate reductions in the length of individual pectin chains and the frequency of aggregates as the fruits ripen. Pectins extracted with sodium carbonate, supposedly located within the primary cell wall, are the most affected.

Insights into the effects of polygalacturonase FaPG1 gene silencing on pectin matrix disassembly, enhanced tissue integrity, and firmness in ripe strawberry fruits.

Antisense-mediated down-regulation of the fruit-specific polygalacturonase (PG) gene FaPG1 in strawberries (Fragaria×ananassa Duch.) has been previously demonstrated to reduce fruit softening and to extend post-harvest shelf life, despite the low PG activity detected in this fruit. The improved fruit traits were suggested to be attributable to a reduced cell wall disassembly due to FaPG1 silencing. This research provides empirical evidence that supports this assumption at the biochemical, cellular, and tissue levels. Cell wall modifications of two independent transgenic antisense lines that demonstrated a >90% reduction in FaPG1 transcript levels were analysed. Sequential extraction of cell wall fractions from control and ripe fruits exhibited a 42% decrease in pectin solubilization in transgenic fruits. A detailed chromatographic analysis of the gel filtration pectin profiles of the different cell wall fractions revealed a diminished depolymerization of the more tightly bound pectins in transgenic fruits, which were solubilized with both a chelating agent and sodium carbonate. The cell wall extracts from antisense FaPG1 fruits also displayed less severe in vitro swelling. A histological analysis revealed more extended cell-cell adhesion areas and an enhanced tissue integrity in transgenic ripe fruits. An immunohistological analysis of fruit sections using the JIM5 antibody against low methyl-esterified pectins demonstrated a higher labelling in transgenic fruit sections, whereas minor differences were observed with JIM7, an antibody that recognizes highly methyl-esterified pectins. These results support that the increased firmness of transgenic antisense FaPG1 strawberry fruits is predominantly due to a decrease in pectin solubilization and depolymerization that correlates with more tightly attached cell wall-bound pectins. This limited disassembly in the transgenic lines indicates that these pectin fractions could play a key role in tissue integrity maintenance that results in firmer ripe fruit.

Improvement of bone formation biomarkers after 1-year consumption with milk fortified with eicosapentaenoic acid, docosahexaenoic acid, oleic acid, and selected vitamins.

The hypothesis of this study was that the replacement of regular milk with fortified milk in hyperlipidemic adults for 1 year would improve bone biomarkers. The fortified milk contained eicosapentaenoic acid and docosahexaenoic acid from fish oils, oleic acid, vitamins A, B(6), and E, as well as folic acid. We believe that the fortified milk will improve the blood fatty acid profile and vitamin status in subjects to benefit bone health biomarkers. From the 84 patients who accepted to participate, 11 of these were excluded for the presence of metabolic diseases and 1 was excluded for noncompliance with the protocol. Seventy-two hyperlipidemic patients (35-65 years) were randomly divided between 2 study groups. The supplement group (E; n = 39) consumed 0.5 L/d of fortified milk that contained fish oil, oleic acid, and vitamins. The control group (C; n = 33) consumed 0.5 L/d of semiskimmed milk containing the same amount of total fat. Blood samples were taken at T(0), T(3), T(6), and T(12) months to determine plasma fatty acids, vitamins B(6), E, and 25-hydroxyvitamin D and serum folate, calcium, soluble osteoprotegerin (OPG), soluble receptor activator of NF-kappaB ligand (RANKL), osteocalcin, parathormone, type I collagen carboxy-terminal telopeptide, and malondialdehyde. After 1 year, the E group showed a significant increase in plasma eicosapentaenoic acid (42%), docosahexaenoic acid (60%), vitamin B6 (38%), OPG (18%), RANKL (7%), OPG/RANKL (10%), red blood cell folate (21%), serum folate (53%), calcium (4%), vitamin D (11%), and osteocalcin (22%). Dietary supplementation with the fortified milk drink improved nutritional status and bone formation markers in adult hyperlipidemic patients.

Antisense inhibition of a pectate lyase gene supports a role for pectin depolymerization in strawberry fruit softening.

Cell wall disassembly in softening fruits is a complex process involving the cumulative action of many families of wall-modifying proteins on interconnected polysaccharide matrices. One strategy to elucidate the in vivo substrates of specific enzymes and their relative importance and contribution to wall modification is to suppress their expression in transgenic fruit. It has been reported previously that inhibiting the expression of pectate lyase genes by antisense technology in strawberry (Fragaria x ananassa Duch.) fruit resulted in prolonged fruit firmness. This suggested that pectin depolymerization might make a more important contribution to strawberry fruit softening than is often stated. In this present study, three independent transgenic lines were identified exhibiting a greater than 90% reduction in pectate lyase transcript abundance. Analyses of sequential cell wall extracts from the transgenic and control fruit collectively showed clear quantitative and qualitative differences in the extractability and molecular masses of populations of pectin polymers. Wall extracts from transgenic fruits showed a reduction in pectin solubility and decreased depolymerization of more tightly bound polyuronides. Additional patterns of differential extraction of other wall-associated pectin subclasses were apparent, particularly in the sodium carbonate- and chelator-soluble polymers. In addition, microscopic studies revealed that the typical ripening-associated loss of cell-cell adhesion was substantially reduced in the transgenic fruits. These results indicate that pectate lyase plays an important degradative role in the primary wall and middle lamella in ripening strawberry fruit, and should be included in synergistic models of cell wall disassembly.

Partial demethylation of oligogalacturonides by pectin methyl esterase 1 is required for eliciting defence responses in wild strawberry (Fragaria vesca).

In addition to the role of the cell wall as a physical barrier against pathogens, some of its constituents, such as pectin-derived oligogalacturonides (OGA), are essential components for elicitation of defence responses. To investigate how modifications of pectin alter defence responses, we expressed the fruit-specific Fragaria x ananassa pectin methyl esterase FaPE1 in the wild strawberry Fragaria vesca. Pectin from transgenic ripe fruits differed from the wild-type with regard to the degree and pattern of methyl esterification, as well as the average size of pectin polymers. Purified oligogalacturonides from the transgenic fruits showed a reduced degree of esterification compared to oligogalacturonides from wild-type fruits. This reduced esterification is necessary to elicit defence responses in strawberry. The transgenic F. vesca lines had constitutively activated pathogen defence responses, resulting in higher resistance to the necrotropic fungus Botrytis cinerea. Further studies in F. vesca and Nicotiana benthamiana leaves showed that the elicitation capacity of the oligogalacturonides is more specific than previously envisaged.

Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene.

Strawberry (Fragaria x ananassa, Duch., cv Chandler) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, we obtained transgenic plants that incorporate an antisense sequence of a strawberry pectate lyase gene under the control of the 35S promoter. Forty-one independent transgenic lines (Apel lines) were obtained, propagated in the greenhouse for agronomical analysis, and compared with control plants, non-transformed plants, and transgenic lines transformed with the pGUSINT plasmid. Total yield was significantly reduced in 33 of the 41 Apel lines. At the stage of full ripen, no differences in color, size, shape, and weight were observed between Apel and control fruit. However, in most of the Apel lines, ripened fruits were significantly firmer than controls. Six Apel lines were selected for further analysis. In all these lines, the pectate lyase gene expression in ripened fruit was 30% lower than in control, being totally suppressed in three of them. Cell wall material isolated from ripened Apel fruit showed a lower degree of in vitro swelling and a lower amount of ionically bound pectins than control fruit. An analysis of firmness at three different stages of fruit development (green, white, and red) showed that the highest reduction of softening in Apel fruit occurred during the transition from the white to the red stage. The postharvest softening of Apel fruit was also diminished. Our results indicate that pectate lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry.