Transcriptome and Gene Regulatory Network Analyses Reveal New Transcription Factors in Mature Fruit Associated with Harvest Date in Prunus persica.

Harvest date is a critical parameter for producers and consumers regarding agro-industrial performance. It involves a pleiotropic effect controlling the development of other fruit quality traits through finely controlling regulatory mechanisms. Fruit ripening is a process in which various signals and biological events co-occur and are regulated by hormone signaling that produces the accumulation/degradation of multiple compounds. However, the regulatory mechanisms that control the hormone signaling involved in fruit development and ripening are still unclear. To investigate the issue, we used individuals with early, middle and late harvest dates from a peach segregating population to identify regulatory candidate genes controlling fruit quality traits at the harvest stage and validate them in contrasting peach varieties for this trait. We identified 467 and 654 differentially expressed genes for early and late harvest through a transcriptomic approach. In addition, using the Arabidopsis DAP-seq database and network analysis, six transcription factors were selected. Our results suggest significant hormonal balance and cell wall composition/structure differences between early and late harvest samples. Thus, we propose that higher expression levels of the transcription factors HB7, ERF017 and WRKY70 in early harvest individuals would induce the expression of genes associated with the jasmonic acid pathway, photosynthesis and gibberellins inhibition. While on the other hand, the high expression levels of LHY, CDF3 and NAC083 in late harvest individuals would promote the induction of genes associated with abscisic acid biosynthesis, auxins and cell wall remodeling.

Identification of DNA Methylation and Transcriptomic Profiles Associated With Fruit Mealiness in Prunus persica (L.) Batsch.

Peach (Prunus persica) fruits have a fast ripening process and a shelf-life of days, presenting a challenge for long-distance consuming markets. To prolong shelf-life, peach fruits are stored at low temperatures (0 to 7 °C) for at least two weeks, which can lead to the development of mealiness, a physiological disorder that reduces fruit quality and decreases consumer acceptance. Several studies have been made to understand this disorder, however, the molecular mechanisms underlying mealiness are not fully understood. Epigenetic factors, such as DNA methylation, modulate gene expression according to the genetic background and environmental conditions. In this sense, the aim of this work was to identify differentially methylated regions (DMRs) that could affect gene expression in contrasting individuals for mealiness. Peach flesh was studied at harvest time (E1 stage) and after cold storage (E3 stage) for 30 days. The distribution of DNA methylations within the eight chromosomes of P. persica showed higher methylation levels in pericentromeric regions and most differences between mealy and normal fruits were at Chr1, Chr4, and Chr8. Notably, differences in Chr4 co-localized with previous QTLs associated with mealiness. Additionally, the number of DMRs was higher in CHH cytosines of normal and mealy fruits at E3; however, most DMRs were attributed to mealy fruits from E1, increasing at E3. From RNA-Seq data, we observed that differentially expressed genes (DEGs) between normal and mealy fruits were associated with ethylene signaling, cell wall modification, lipid metabolism, oxidative stress and iron homeostasis. When integrating the annotation of DMRs and DEGs, we identified a CYP450 82A and an UDP-ARABINOSE 4 EPIMERASE 1 gene that were downregulated and hypermethylated in mealy fruits, coinciding with the co-localization of a transposable element (TE). Altogether, this study indicates that genetic differences between tolerant and susceptible individuals is predominantly affecting epigenetic regulation over gene expression, which could contribute to a metabolic alteration from earlier stages of development, resulting in mealiness at later stages. Finally, this epigenetic mark should be further studied for the development of new molecular tools in support of breeding programs.

Dormant but Active: Chilling Accumulation Modulates the Epigenome and Transcriptome of Prunus avium During Bud Dormancy.

Temperate deciduous fruit tree species like sweet cherry (Prunus avium) require long periods of low temperatures to trigger dormancy release and flowering. In addition to sequence-based genetic diversity, epigenetic variation may contribute to different chilling requirements among varieties. For the low chill variety 'Royal Dawn' and high chill variety 'Kordia', we studied the methylome of floral buds during chilling accumulation using MethylC-seq to identify differentially methylated regions (DMRs) during chilling hours (CH) accumulation, followed by transcriptome analysis to correlate changes in gene expression with DNA methylation. We found that during chilling accumulation, DNA methylation increased from 173 CH in 'Royal Dawn' and 443 CH in 'Kordia' and was mostly associated with the CHH context. In addition, transcriptional changes were observed from 443 CH in 'Kordia' with 1,210 differentially expressed genes, increasing to 4,292 genes at 1,295 CH. While 'Royal Dawn' showed approximately 5,000 genes differentially expressed at 348 CH and 516 CH, showing a reprogramming that was specific for each genotype. From conserved upregulated genes that overlapped with hypomethylated regions and downregulated genes that overlapped with hypermethylated regions in both varieties, we identified genes related to cold-sensing, cold-signaling, oxidation-reduction process, metabolism of phenylpropanoids and lipids, and a MADS-box SVP-like gene. As a complementary analysis, we used conserved and non-conserved DEGs that presented a negative correlation between DNA methylations and mRNA levels across all chilling conditions, obtaining Gene Ontology (GO) categories related to abiotic stress, metabolism, and oxidative stress. Altogether, this data indicates that changes in DNA methylation precedes transcript changes and may occur as an early response to low temperatures to increase the cold tolerance in the endodormancy period, contributing with the first methylome information about the effect of environmental cues over two different genotypes of sweet cherry.

Identification of Metabolite and Lipid Profiles in a Segregating Peach Population Associated with Mealiness in Prunus persica (L.) Batsch.

The peach is the third most important temperate fruit crop considering fruit production and harvested area in the world. Exporting peaches represents a challenge due to the long-distance nature of export markets. This requires fruit to be placed in cold storage for a long time, which can induce a physiological disorder known as chilling injury (CI). The main symptom of CI is mealiness, which is perceived as non-juicy fruit by consumers. The purpose of this work was to identify and compare the metabolite and lipid profiles between two siblings from contrasting populations for juice content, at harvest and after 30 days at 0 °C. A total of 119 metabolites and 189 lipids were identified, which showed significant differences in abundance, mainly in amino acids, sugars and lipids. Metabolites displaying significant changes from the E1 to E3 stages corresponded to lipids such as phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG) and lysophosphatidylcholines (LPC), and sugars such as fructose 1 and 1-fructose-6 phosphate. These metabolites might be used as early stage biomarkers associated with mealiness at harvest and after cold storage.

Metabolite Fruit Profile Is Altered in Response to Source-Sink Imbalance and Can Be Used as an Early Predictor of Fruit Quality in Nectarine.

Peaches and nectarines [Prunus persica (L.) Batsch] are among the most exported fresh fruit from Chile to the Northern Hemisphere. Fruit acceptance by final consumers is defined by quality parameters such as the size, weight, taste, aroma, color, and juiciness of the fruit. In peaches and nectarines, the balance between soluble sugars present in the mesocarp and the predominant organic acids determines the taste. Biomass production and metabolite accumulation by fruits occur during the different developmental stages and depend on photosynthesis and carbon export by source leaves. Carbon supply to fruit can be potentiated through the field practice of thinning (removal of flowers and young fruit), leading to a change in the source-sink balance favoring fruit development. Thinning leads to fruit with increased size, but it is not known how this practice could influence fruit quality in terms of individual metabolite composition. In this work, we analyzed soluble metabolite profiles of nectarine fruit cv "Magique" at different developmental stages and from trees subjected to different thinning treatments. Mesocarp metabolites were analyzed throughout fruit development until harvest during two consecutive harvest seasons. Major polar compounds such as soluble sugars, amino acids, organic acids, and some secondary metabolites were measured by quantitative 1H-NMR profiling in the first season and GC-MS profiling in the second season. In addition, harvest and ripening quality parameters such as fruit weight, firmness, and acidity were determined. Our results indicated that thinning (i.e., source-sink imbalance) mainly affects fruit metabolic composition at early developmental stages. Metabolomic data revealed that sugar, organic acid, and phenylpropanoid pathway intermediates at early stages of development can be used to segregate fruits impacted by the change in source-sink balance. In conclusion, we suggest that the metabolite profile at early stages of development could be a metabolic predictor of final fruit quality in nectarines.

Los siete secretos mágicos de la efectividad terapéutica / The seven magic secrets of therapeutic effectiveness

Parecería que el avance de la Psicología nos debería aumentar la confianza respecto a nuestras prácticas, sin embargo la proliferación de técnicas, protocolos, enfoques, ... nos genera, más bien, una sensación de confusión; la duda sobre si quizás estamos en la opción correcta o nos estamos equivocando de paradigma terapéutico. Los metanálisis y revisiones sistemáticas procuran poner orden intentando averiguar qué terapias psicológicas son más efectivas. Sin embargo, los resultados no son concluyentes y más bien parecen indicar que ningún enfoque psicológico predomina sobre otro. Ante esta situación la pregunta que emerge es: ¿qué factores comparten las distintas técnicas que las convierte en eficaces? Son muchas las categorizaciones sobre factores comunes. En este artículo desvelaremos los 7 secretos mágicos (factores comunes) que consideramos clave para explicar la efectividad terapéutica. Esta clasificación no tiene ninguna pretensión más que provocar la reflexión

It would seem that the advancement of psychology should increase our confidence in our practices, however the proliferation of techniques, protocols, approaches, etc. gives us instead a sense of confusion. We have doubts as to whether we are in the correct therapeutic paradigm or not. Meta-analysis and systematic reviews attempt to create order, trying to determine which psychological therapies are most effective. Unfortunately, the results are inconclusive and seem to indicate that no one psychological approach prevails over the others. The question that emerges is: what factors do the different techniques share that make them effective? There are many categorizations concerning common factors. In this article we unveil the seven magic secrets (common factors) that we consider key in explaining therapeutic effectiveness. This classification has no claim other than to provoke reflection

Degree of conversion of a self-adhesive endodontic sealer when used as bulk material.

This study tested the null hypothesis that the opacity of RealSeal SE (RSSE) sealer makes light-curing inefficient, while the degree of conversion (DC) is similar regardless of curing method. Fourteen uniradicular teeth were sectioned at 15 mm from the apex. Root canals were instrumented using the Reciproc file system, bulk-filled with RSSE, and divided randomly into two groups (dual-cure or self-cure). DC was determined by micro-Raman spectroscopy at 24 h, 48 h, and 1 week after filling, at 6, 9, and 12 mm from the coronal end. Contrast ratio (Yb/Yw) was used to determine the opacity of the material. Descriptive statistics and nonparametric tests were used, and significance was defined as a P value of less than 0.05. Opacity was almost total by the first section, at 6 mm. In dual-cure mode, DC values at 24 h were lower in the apical section (63.8%) than in the more coronal sections and were lower than in self-cured specimens (87.4%). Light-curing of the coronal end did not improve DC. These differences remained at 48 h and 1 week. Only a small (2%) but significant increase in DC was observed in evaluations at 24 h and 1 week. (J Oral Sci 58, 333-338, 2016).

Effect of 30 % hydrogen peroxide on mineral chemical composition and surface morphology of bovine enamel.

A combination of atomic absorption spectroscopy (AAS), Fourier transform infrared spectroscopy (FTIR), scanning electronic microscopy (SEM), and gas adsorption techniques was used to characterize the effect of 30 % hydrogen peroxide (HP) on enamel surface. To perform the analyses of AAS, 1 ml of 30 % HP was added to 30 mg of a bovine enamel powder sample (150-200 µm fractions) for times of 5, 20, 60, 90, and 120 min; then 5 ml of the solution was withdrawn after each time period to measure [Ca(2+)] ions. The remaining powder was recovered and analyzed by FTIR. For SEM and gas adsorption tests, 4 × 4 mm(2) enamel sectioned samples were polished and 30 % HP was applied on the surface for the same time periods. AAS data show that 30 % HP treatment mobilized calcium from the enamel at all times studied. FTIR spectra showed that the total amount of phosphate and carbonate mineral contents such as amide I decreased significantly. SEM revealed that randomly distributed areas throughout the smooth enamel surface treatment became rougher and more irregular. These alterations indicate that surface damage increases with increasing durations of HP treatment. Gas adsorption analysis proved that bleached enamel is a typically non-porous material with a small specific surface area which decreases slightly with the 30 % HP treatment. In sum, 30 % HP induced a significant alteration of the organic and mineral part of the enamel, leading to the release of calcium and a rougher, more irregular enamel surface on randomly distributed areas.

Influence of the bracket on bonding and physical behavior of orthodontic resin cements.

The aim of the study is to determine the influence of the type of bracket, on bond strength, microhardness and conversion degree (CD) of four resin orthodontic cements. Micro-tensile bond strength (µTBS) test between the bracket base and the cement was carried out on glass-hour-shaped specimens (n=20). Vickers Hardness Number (VHN) and micro-Raman spectra were recorded in situ under the bracket base. Weibull distribution, ANOVA and non-parametric test were applied for data analysis (p<0.05). The highest values of ή as well as the ß Weibull parameter were obtained for metallic brackets with Transbond™ plastic brackets with the self-curing cement showing the worst performance. The CD was from 80% to 62.5%.

Bond strength to root dentin and fluid filtration test of AH Plus/gutta-percha, EndoREZ and RealSeal systems.

OBJECTIVES: To investigate the bond strength and seal ability produced by AH Plus/gutta-percha, EndoREZ and RealSeal systems to root canal dentin. MATERIAL AND METHODS: Sixty extracted single-root human teeth, instrumented manually to size 40, were divided into three groups (n=20) according to the sealer used; G1: AH Plus, G2: EndoREZ, and G3: RealSeal sealers. After filling using the lateral condensation technique, each sealer group was randomly divided into two subgroups according to the tests applied (n=10 for µPush-out test and n=10 for fluid filtration test). A fluid filtration method was used for quantitative evaluation of apical leakage. Four 1-mm-thick slices (cervical and medium level) were obtained from each root sample and a µPush-out test was performed. Failure modes were examined under microscopy at 40x, and a one-way ANOVA was applied to analyze the permeability. Non-parametrical statistics for related (Friedman's and Wilcoxon's rank tests) or unrelated samples (Kruskal-Wallis' and Mann-Whitney's tests) allowed for comparisons of µPush-out strength values among materials at the different levels. Statistical significance was accepted for p values <.05. RESULTS: There are no significant differences among fluid filtration of the three sealers. The sealer/core material does not significantly influence the µPush-out bond strength values (F=2.49; p=0.10), although statistically significant differences were detected with regard to root level (Chi2=23.93; p<0.001). AH Plus and RealSeal obtained higher bond strength to intraradicular dentin in the medium root slices. CONCLUSIONS: There are no significant differences between the permeability and global µPush-out bond strength to root canal dentin achieved by AH Plus/gutta-percha, EndoREZ and RealSeal systems.

Bond strength to root dentin and fluid filtration test of AH Plus/gutta-percha, EndoREZ and RealSeal systems

OBJECTIVES: To investigate the bond strength and seal ability produced by AH Plus/gutta-percha, EndoREZ and RealSeal systems to root canal dentin. MATERIAL AND METHODS: Sixty extracted single-root human teeth, instrumented manually to size 40, were divided into three groups (n=20) according to the sealer used; G1: AH Plus, G2: EndoREZ, and G3: RealSeal sealers. After filling using the lateral condensation technique, each sealer group was randomly divided into two subgroups according to the tests applied (n=10 for µPush-out test and n=10 for fluid filtration test). A fluid filtration method was used for quantitative evaluation of apical leakage. Four 1-mm-thick slices (cervical and medium level) were obtained from each root sample and a µPush-out test was performed. Failure modes were examined under microscopy at 40x, and a one-way ANOVA was applied to analyze the permeability. Non-parametrical statistics for related (Friedman's and Wilcoxon's rank tests) or unrelated samples (Kruskal-Wallis' and Mann-Whitney's tests) allowed for comparisons of µPush-out strength values among materials at the different levels. Statistical significance was accepted for p values <.05. RESULTS: There are no significant differences among fluid filtration of the three sealers. The sealer/core material does not significantly influence the µPush-out bond strength values (F=2.49; p=0.10), although statistically significant differences were detected with regard to root level (Chi2=23.93; p<0.001). AH Plus and RealSeal obtained higher bond strength to intraradicular dentin in the ...

Percentage exposure of root dentin collagen after application of two irrigation protocols with manual or rotary instrumentation and two methacrylate resin-based sealers.

PURPOSE: To compare the percentage of collagen exposed in dentin root thirds after two irrigation protocols with manual or rotary instrumentation using two methacrylate resin-based sealers. MATERIALS AND METHODS: Forty-eight single-root human teeth were prepared with manual (n = 24) or nickeltitanium ProFile rotary (n = 24) instrumentation, using 5% NaOCl between instruments and 5 ml 17% EDTA as final irrigant or 20% citric acid + 2% chlorhexidine (CHX) between instruments and as the final irrigant. RealSeal or EndoREZ were used as filling materials. One 1-mm slice per third was abraded and stained with Masson's trichrome method. Mean exposed collagen values were obtained in four areas from each section (at 60X magnification) and a complete factorial ANOVA was used to analyze the influence of the study variables. Non-parametric Mann-Whitney's test was used to compare groups. Differences with p < 0.05 were considered significant. RESULTS: A significantly higher percentage of collagen was exposed in all thirds with the use of the 20% citric acid + 2% CHX protocol with rotary vs manual instrumentation, but percent collagen exposed did not differ as a function of the filling material. After the 5% NaOCl + 17% EDTA protocol, the percentage of collagen exposed did not differ between rotary and manual instrumentation but was higher with the use of RealSeal. CONCLUSION: The highest percentage exposure of collagen was with 20% citric acid + 2% CHX using rotary instrumentation, regardless of the filling material.

Regional bond strength to lateral walls in class I and II ceramic inlays luted with four resin cements and glass-ionomer luting agent.

PURPOSE: To investigate regional shear bond strength to lateral walls of ceramic inlays in occlusal and occlusoproximal cavities using etch-and-rinse and self-adhesive resin cements and a glass-ionomer luting agent. MATERIALS AND METHODS: IPS e.max Press ceramic inlays were made in 50 Class I and 50 Class II standardized cavities in intact extracted human molars and divided into 5 luting agent subgroups (n = 10): Variolink II (VL); Multilink Sprint (MLS); Multilink Automix (MLA); RelyX Unicem (RLX), and Ketac Cem Aplicap (KC). Inlays were pre-etched with IPS Ceramic etching gel for 60s. After 48 h, two disks of ca 1.0 mm thickness, one of superficial and the other of deep dentin, were push-out tested in a universal testing machine at a crosshead speed of 1.0 mm/min. The mode of failure was determined under a stereomicroscope at 20X. Data were analyzed with one way ANOVA, and Scheffé's test was used for post-hoc comparisons (α = 0.05). RESULTS: There were no significant differences in shear bond strength between Class I and Class II cavities for the dual-curing system in light-curing mode (VL=MLS=RLX), except that RLX demonstrated greater bond strength to deep dentin in Class II cavities. Bond strength values were significantly higher on deep than on superficial dentin. KC showed the worst result. Failures were mixed (adhesive/cohesive) for the resin luting cements and solely adhesive (cement/ceramic) for the glass-ionomer luting agent. CONCLUSION: Dual-curing etch-and-rinse or self-etching self-adhesive resin luting cements achieved greater bond strength when light curing was applied, with no differences between Class I and Class II cavities but higher values for deep vs superficial dentin. The weakest adhesion was obtained with glass-ionomer luting agent in both cavity types.

Demineralization effects of phosphoric acid on surface and subsurface bovine enamel bleached with in-office hydrogen peroxide.

PURPOSE: To measure the demineralization capacity of 37% phosphoric acid on surface and subsurface bovine enamel after bleaching with hydrogen peroxide (H2O2). MATERIALS AND METHODS: Three equally-sized sections with 16 mm2 of exposed enamel surface were obtained from the enamel of 10 bovine incisors. One specimen sample from each crown was assigned to one of three groups (n = 10): group I, no bleaching agent; group II, bleached with 38% H202 for 20 min; or group III, 30% H202 for 60 min. After 24 h, the thickness of specimens was measured and they were immersed in 37% phosphoric acid solution, of which 5-ml aliquots were collected at 30 s and 60 s. Specimens were then ground to a depth of 25 µm and again immersed in 37% phosphoric acid solution. This procedure was repeated for enamel depths of 50 and 100 µm. Ca2+ concentrations in the phosphoric acid aliquots were measured by atomic absorption spectroscopy. RESULTS: No significant differences were found in the total amounts of extracted Ca2+ between bleached and unbleached specimens (F = 0.142; p = 0.869). The amount of Ca2+ extracted was similar among the four depth levels in the unbleached and in the 30% H202 bleached specimens. A significantly larger amount of Ca2+ was obtained at 25 µm depth (subsurface) from specimens treated with 38% H202. CONCLUSIONS: Pre-bleaching with 38% H202 significantly increased the decalcifying effect of phosphoric acid on subsurface enamel at a depth of 25 µm compared to 100 µm, whereas pre-bleaching with 30% H202 did not modify this effect at any level.

Interfacial morphology and bond strength of self-etching adhesives to primary dentin with or without acid etching.

The aim of the study was to determine the interfacial morphology and bond strength of three current self-etching adhesives (SEAs) to primary dentin and to evaluate the effect of introducing an additional step of phosphoric acid etching. Three human primary molars were assigned to each adhesive group for testing microtensile bond strength (microTBS) and three for studying interface morphology. Groups were: group 1, Excite, a total-etch adhesive (control); group 2, Adhese (ASE); group 3, Adper-Prompt-L-Pop (APLP), and group 4: Xeno III (XE) SEAs; groups 5-7 received application of 37% phosphoric acid for 15 s before applying ASE, APLP, and XE, respectively. A class I cavity was performed in each molar to study the interface morphology. Two halves of each tooth were used for examination either by optical microscopy, using Masson's trichromic dye technique, or by scanning electron microscopy. For microTBS determination, composite/dentin bars (1 mm(2) section) were obtained from each tooth, and tested in tension until debonding. The microTBS was significantly lower in the APLP than in the rest of the groups. The performance of SEAs on primary dentin depends on the product. Inclusion of dentin pre-etching step did not significantly modify microTBS results. All SAEs achieved greater decalcification depth on etched versus nonetched dentin.

Effects of etching time of primary dentin on interface morphology and microtensile bond strength.

OBJECTIVES: To determine the influence of different etching times (5, 15 or 30 s) on the morphology and micro-tensile bond strength (muTBS) of primary dentin. METHODS: For muTBS study, nine primary molars were randomly distributed in three experimental groups. Three Class I cavities per tooth were drilled and etched (37% orthophosphoric acid gel for 5, 15 or 30 s). Excite adhesive was applied and cavities restored with a resin composite (Tetric Ceram). Composite/dentin bars (ca. 1 mm2 section) were obtained from teeth and tested in tension until debonding. Means of muTBS results were compared with ANOVA and Student-Neuman-Keuls post hoc tests. Morphology: Three occlusal cavities were prepared in five primary molars. Each cavity was etched and restored as described for previous groups and teeth were sectioned mesio-distally. One half of each tooth was prepared for using under optical microscopy using Masson's trichromic dye technique and the other half was examined by SEM. RESULTS: muTBS mean (S.D.) results (in MPa) were 5 s etch: 6.20 (2.81), 15 s: 13.43 (5.91), 30 s: 13.04 (5.67). muTBS groups were Excite 5 s < Excite 15 s = Excite 30 s. Masson's trichromic technique stained the demineralized dentin layer red in all specimens. The mean (S.D.) thickness of the demineralized layers (in mu) were 5 s: 3.28 (1.23), 15 s: 3.83 (1.26), 30 s: 4.44 (1.70). There is a statistically significant linear relationship between time of application of etching and mean depth of demineralized layer. This relationship was established as depth (in mu) = 3.08 + 0.05 time (in s). SIGNIFICANCE: The minimum adequate etching time for primary dentin is 15s.

Long-term evolution of a case of direct pulp capping by adhesion to dentin.

This article presents the long-term follow-up of a female patient who suffered pulp exposure during removal of a large caries lesion from the mandibular right first molar. The clinical decision to perform direct pulp capping was made. The tooth was treated with 5% chlorhexidine, etched with 37% phosphoric acid, and then restored with Heliomolar composite after application of Gluma dentin adhesive. The patient was followed for more than 8 years.

Variations in health services utilization by primary care patients.

This study analyses how both ambulatory care groups (ACGs) and physician characteristics explain the variability in health-service use among primary care patients in Spain. During the period 1996-1997, data derived from 52,152 patients and their 38 respective primary care physicians were collected. The response variables were as follows: number of visits; diagnostic tests requested; and referrals to a specialist. ACGs are an important variable that should be taken into account in order to explain health-service utilization. As for professionals, age and the post they hold are essential factors. Most of the unexplained variability is caused by patient characteristics.