Search Results (66)

Heat stress is a major constraint on plant growth and productivity, and excessive accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), especially hydrogen peroxide (H₂O₂), is a primary cause of heat-induced cellular damage. Photorespiration becomes accelerated at high temperature and generates the toxic metabolite 2-phosphoglycolate (2PG), whose accumulation is prevented by the first photorespiratory enzyme 2-phosphoglycolate phosphatase (PGLP1). Here, we identify the auxilin-like J-domain protein JAC1 (AT1G75100) as a positive regulator of thermotolerance in Arabidopsis thaliana (A. thaliana). JAC1 transcripts were rapidly induced by heat treatment, and loss-of-function jac1 mutants (jac1-1 and jac1-2) were hypersensitive to long-term heat stress (38 °C, 7 d), whereas three independent JAC1 overexpression lines (#2, #4, #5) exhibited enhanced survival. Consistent with a role in ROS homeostasis, heat-treated jac1 mutants showed stronger DAB staining, consistent with higher apparent H₂O₂ levels, whereas JAC1 overexpression lines showed weaker staining. Although PGLP1 abundance (mRNA and protein) was unchanged among genotypes, heat treatment (38 °C, 12 h) caused a marked reduction in leaf PGLP1 enzymatic activity in jac1 mutants and a significant increase in JAC1 overexpression lines, leading to corresponding changes in 2PG content. PGLP1 sulfenylation, a H₂O₂-dependent post-translational modification that inhibits PGLP1, was detectable only after heat and was enhanced in jac1 mutants but suppressed in JAC1 overexpression lines. Genetic manipulation of PGLP1 supported that PGLP1 functions downstream of JAC1: overexpressing PGLP1 in jac1-1 rescued heat sensitivity, whereas knocking down PGLP1 in the JAC1 overexpression background reversed thermotolerance. Together, our results support a model in which JAC1 maintains heat-induced H₂O₂ homeostasis, thereby limiting PGLP1 sulfenylation, sustaining PGLP1 activity and preventing toxic 2PG accumulation during heat stress. Full article

Small heat shock proteins (HSP20s) are known to function as molecular chaperones that bind to denatured proteins under high-temperature stress and assist in their conformational recovery, thereby contributing to plant thermotolerance. In the present study, three HSP20 genes—PcHSP12.8, PcHSP12.9, and PcHSP13.4—were identified in the transcriptome of Polygonatum cyrtonema Hua. Bioinformatics analysis indicated their phylogenetic relationships, conserved domains, and potential tertiary structures. RT-qPCR analysis revealed up-regulation of all three genes in response to heat stress. Subcellular localization studies further suggested that PcHSP12.8, PcHSP12.9, and PcHSP13.4 are predominantly localized in the nucleus. Heterologous expression of these genes in a heat-sensitive yeast mutant appeared to improve cell survival under heat stress relative to the control strain. In Arabidopsis thaliana overexpressing these genes, moderate improvements in germination rate, root elongation, and stress survival were observed compared to wild-type plants under heat stress. Transgenic lines also showed a tendency toward reduced reactive oxygen species accumulation, as reflected by decreased 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining, together with increased activities of catalase (CAT) and peroxidase (POD), as well as higher chlorophyll retention under thermal stress. Taken together, these findings imply that the three PcHSP20 genes could be involved in thermotolerance in P. cyrtonema. Full article

During tendon healing, collagen III expression precedes that of collagen I. The collagen I-to-III ratio at a certain time point post-laceration serves as an indicator of the healing status. Consequently, it is crucial to understand how different therapeutic approaches to support tendon healing affect the collagen I-to-III ratio in the extracellular matrix of a healing tendon, particularly across distinct anatomical zones. We compared the impact of a platelet-derived growth factor-BB (PDGF-BB) treatment via controlled release from coaxially electrospun DegraPol^® (Ab medica, Cerro Maggiore, Italy) hollow-fiber mesh with a treatment by the vehicle alone (no PDGF-BB) in the rabbit Achilles tendon full transection model and provide data on the collagen I-to-III ratio 3 weeks post-operation. For this purpose, we compared a dual-color Herovici staining to two single IHC labeling, for collagen I and collagen III, respectively. Herovici staining (HV) was expected to offer a more precise approach (pink-to-blue histogram) than the two separately labeled IHC stainings, both with chromogenic DAB labeling (red-to-green histogram), despite an anticipated positive correlation of the data assessed by these methods. Different zones were compared, i.e., native tendon tissue, reactive zone at interface to implant, hot zone within the core of the healing tendon and the zone within the scaffold, meaning the collagen deposited within the fibers of the implanted DegraPol^® tube, respectively. The analysis revealed that the ratios obtained via HV correlated weakly with the ratios obtained by IHC. Based on HV, PDGF-BB therapy led to higher collagen I-to-III ratios in all zones, except for the zone within the scaffold pores, while IHC did not reveal significant differences. Notably, collagen I-to-III ratios were not higher in immediate proximity, but rather distal from the PDGF-BB releasing implant, specifically in the core of the healing tendon tissue. Hence, a PDGF-BB therapy is suggestive of greater collagen maturation in specific zones of the healing tendon. Full article

Maize (Zea mays L.) is a major economic crop highly susceptible to Colletotrichum graminicola, the causal agent of anthracnose leaf blight, which causes substantial annual yield losses. This fungal pathogen employs numerous effectors to manipulate plant immunity, yet the functions of many secreted proteins during biphasic infection remain poorly characterized. In this study, we identified CgMas2, a candidate secreted protein in C. graminicola and a homolog of Magnaporthe oryzae MoMas2. Deletion of CgMAS2 in the wild-type strain CgM2 did not affect fungal vegetative growth or conidial morphology but significantly impaired virulence on maize leaves. Leaf sheath infection assays revealed that CgMas2 is required for biotrophic invasive hyphal growth, as the mutant showed defective spreading of invasive hyphae to adjacent cells. Subcellular localization analysis indicated that CgMas2 localizes to the cytoplasm of conidia and to the primary infection hyphae. Furthermore, DAB staining demonstrated that disrupt of CgMAS2 leads to host reactive oxygen species (ROS) accumulation. Comparative transcriptome analysis of maize infected with ΔCgmas2 versus CgM2 revealed enrichment of GO terms related to peroxisome and defense response, along with up-regulation of benzoxazinoid biosynthesis genes (benzoxazinone biosynthesis 3, 4 and 5) at 60 h post-inoculation (hpi). Conversely, six ethylene-responsive transcription factors (ERF2, ERF3, ERF56, ERF112, ERF115 and ERF118) involved in ethylene signaling pathways were down-regulated at 96 hpi. These expression patterns were validated by RT-qPCR. Collectively, our results demonstrate that CgMas2 not only promotes invasive hyphal growth during the biotrophic stage but may also modulate phytohormone signaling and defense compound biosynthesis during the necrotrophic phase of infection. Full article

Drought and salinity are critical abiotic stresses that constrain plant growth. Although MYB transcription factors mediate plant responses to abiotic stresses, their functions in the monocot I. laevigata remain unexplored. Here, we identified a nuclear-localized gene, IlMYB108, which was rapidly upregulated under NaCl and PEG-6000 treatments. Overexpression of IlMYB108 in tobacco enhanced root growth under salt and drought conditions. At the seedling stage, transgenic lines maintained higher leaf growth rates and plant height with reduced wilting during 14 days of continuous stress. Physiologically, transgenic plants exhibited a higher net photosynthetic rate (Pn), maximum photochemical efficiency of photosystem II (Fv/Fm), and chlorophyll content, alongside lower stomatal conductance (Gs), intercellular CO₂ concentration (Ci), and transpiration rate (Tr). They also accumulated less malondialdehyde (MDA), superoxide anion (O₂⁻), and hydrogen peroxide (H₂O₂), which was attributed to enhanced activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as confirmed by p-Nitro-Blue tetrazolium chloride (NBT) and 3,3′-diaminobenzidine tetrahydrochloride (DAB) staining. Moreover, IlMYB108 up-regulated stress-responsive and antioxidant genes. Collectively, IlMYB108 functions as a key gene that enhances tobacco tolerance to salt and drought stress by coordinating root development, photosynthetic efficiency, water balance and antioxidant defense, thereby providing a valuable genetic resource for breeding stress-resilient plants. Full article

Background/Objectives: The meninges, the protective membranes covering the central nervous system, undergo complex developmental processes that are critical for CNS integrity and function. Connexin 37 (Cx37) and 40 (Cx40), members of the connexin family of gap junction proteins, have been implicated in various physiological and pathological processes. They play a critical role in cell–cell communication. The aim of our study was to investigate the expression of connexins Cx37, Cx40, and Panx1 in the meninges of both human and murine models (yotari and wild type) at the 6th week/E13.5 and 8th week/E15.5 of developmental stages. Methods: Human embryonic tissues (6th–8th week, n = 4 for the 6th week and n = 4 for the 8th week) and mouse embryos (yotari Dab1⁻/⁻ and wild type, E13.5–E15.5) were collected and fixed in 4% paraformaldehyde. Paraffin sections were stained for Cx37, Cx40, and Panx1 using immunofluorescence. Images were analyzed in ImageJ, and statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test (p < 0.05). Results: Cx37 was consistently expressed across all developmental stages, with the highest threshold area percentage observed at E13.5 and E15.5 in murine leptomeninges, demonstrating statistically significant differences compared to controls (p < 0.05) and notably from corresponding human stages (p < 0.001). Strong Cx37 staining intensity at E13.5 was noted in both wild-type and yot mice, while human leptomeninges displayed mild staining at the 6th week of development. In contrast, both human and murine pachymeninges exhibited moderate Cx37 expression. Additionally, the expression of Cx37 in wild-type mice surpassed that of human samples at both E13.5 and E15.5 stages (p < 0.01 and p < 0.001, respectively). For the developing dura mater, Cx37 expression peaked at E15.5 in yot mice, significantly different from both wild-type and human dura mater (p < 0.01 and p < 0.05). Cx40 expression was highest in the leptomeninges at E15.5. Panx1 was similarly expressed across stages, with the highest threshold area percent observed in wild-type leptomeninges and pachymeninges at E15.5, showing significant differences compared to yot mice and human samples (p < 0.05). Both leptomeninges and pachymeninges exhibited mild Panx1 staining at E13.5, while stronger staining was observed at E15.5 in murine samples, contrasting with mild intensity in human counterparts. Conclusions: These findings highlight the implications of Dab1 deficiency for the expression of gap junction proteins during meninges development, implicating their importance in intercellular communication that is essential for normal meningeal and neurodevelopmental processes. Full article

Background: Accumulating evidence regarding the association between tumor-infiltrating lymphocyte (TIL) subtypes and prognosis in colorectal cancer has emerged recently in the literature. Whether the prognostic impact of TIL subsets is different according to tumor location remains unknown, despite genetic, epigenetic and molecular differences between the proximal and distal colon. Our study aimed to investigate the value of CD3⁺ lymphocytes, reflecting overall T-cell infiltration, CD8⁺ cells identifying cytotoxic effector T-cells and CD73⁺ cells acting as a modulator of immunosuppression, stratified by primary tumor location. Methods: The density of CD73⁺, CD3⁺ and CD8⁺ tumor-infiltrating B- and T-cells was determined in colon cancer patients using whole-section tissue sampling, heat-induced epitope retrieval, primary antibodies and DAB visualization. QuPath Cell counter function quantified nucleated cells and immune-positive percentages; ImageJ assessed staining intensity via color deconvolution and optical density. An Immunoreactive Score combined intensity and positivity for immune profiling. The Receiver Operating Characteristic (ROC) curve analysis was used to determine the optimal cut-off values for CD3⁺, CD8⁺ and CD73⁺ lymphocytes. Statistical analysis was performed in order to identify potential associations between TILs expression and pathological characteristics, according to the location of the primary tumor. Survival analysis was carried out using the Kaplan–Meier method. Results: A total of 100 patients were included in the study. CD3⁺ T-cells were the most abundantly expressed and were more predominantly encountered in the right colon. Total CD3⁺ numbers were correlated with T stage and the presence of perineural invasion in left-sided tumors, as well as with tumor grading in the right colon. Correlation analysis based on CD3⁺ threshold values according to tumor location demonstrated a statistically significant association between a higher N stage and low CD3⁺ cell values (p value = 0.0306), and higher perineural invasion and low CD3⁺ TILs values in the left colon (p value = 0.0123). In addition, low CD8⁺ values were associated with a higher T stage in the left colon (p value = 0.0382). Survival analysis did not demonstrate statistically significant differences between the investigated groups. Conclusions: TIL subtypes in colon cancer patients demonstrate significant variability according to the location of the primary tumor and are associated with different clinical and pathological characteristics. This exploratory study requires larger validation before TIL densities can guide therapy. Full article

Background/Objectives: Alfalfa is a widely cultivated high-quality forage crop, and salinity tolerance is one of the most important breeding goals. Glycine max SALT INDUCED NAC 1 (GmSIN1) was found to enhance salinity tolerance in soybean plants. The phylogenetic analysis showed there were two homologs of GmSIN1 in Medicago truncatula, MtSIN1a and MtSIN1b. This raised questions regarding the roles of MtSIN1s in alfalfa under salinity stress. Methods: From a Tnt1 mutant collection, we identified the mutants of MtSIN1a. We recorded the survival rate and plant height of mtsin1a-1 and mtsin1a-2 after 100 mM NaCl treatment. Subsequently, we generated 35S:MtSIN1a-GFP transgenic alfalfa lines via genetic transformation. Two lines with relatively high MtSIN1a expression, 35S:MtSIN1a-GFP#3 and 35S:MtSIN1a-GFP#4, were selected for gradient NaCl treatments. In addition, DAB and NBT staining were performed, and the H₂O₂ content and catalase (CAT) activity were determined. Then, we used RNA-seq analysis and RT-qPCR to study the mechanism of its tolerance. Results: This study found that after salt treatment, the survival rate and plant height of mtsin1a-1 and mtsin1a-2 were significantly lower than those of the WT. The mutants of MtSIN1a were sensitive to salinity stress. The transgenic alfalfa plants exhibited higher plant height, weaker DAB staining, stronger NBT staining, less H₂O₂ content, and enhanced CAT activity. The transgenic alfalfa constructed by transforming MtSIN1a showed enhanced salinity tolerance with elevated ROS scavenging. We identified MsSOD1 showing elevated expression levels in transcriptomic analysis. Conclusions: MtSIN1a is a positive regulator for enhancing salinity tolerance in alfalfa with activated ROS scavenging. Full article

Background: Chronic exercise has been linked to positive effects on cognitive function and brain health. The aim of our study was to investigate how exercise affects cerebral resistance artery morphology, with an underlying focus on potential sex differences. Methods: Wistar rats were divided into male exercising (M.Ex; n = 6), female exercising (F.Ex; n = 5), male sedentary (M.Sed; n = 5), and female sedentary (F.Sed; n = 5) groups. After a 12-week swimming program, histological examinations of the intracerebral and pial arterioles were performed. SMA-DAB (smooth muscle actin) and resorcin-fuchsin (elastica) stained brain coronal sections were used for quantitative colorimetric analysis. Results: Investigating the effect of exercise, we found that in both pial and intracerebral arterioles, the elastic fiber density increased in both female and male exercising animals compared to the sedentary groups (p < 0.05 (M.Sed vs. M.Ex); p < 0.0001 (F.Sed vs. F.Ex)). As sex differences, we found that in female animals’ pial arterioles, the density of elastic fiber was increased compared to the male exercising group (p < 0.001 (M.Ex vs. F.Ex)). In pial arterioles, the smooth muscle density was higher in the male sedentary animals (p < 0.01 (M.Sed vs. F.Sed)); in intracerebral arterioles, the smooth muscle density increased with exercise in the male animals as well (p < 0.0001 (M.Ex vs. F.Ex)). Conclusions: Our results demonstrate that the increase in vascular elasticity is more pronounced overall in female animals. Full article

Sequential drought and heat stress pose a growing threat to forest ecosystems in the context of climate change, yet systematic evaluation methods for woody plants remain limited. This study aimed to develop a comprehensive screening platform for identifying woody plant species tolerant to sequential drought and heat stress among 27 native species growing in Korea. A sequential stress protocol was applied: drought stress for 2 weeks, followed by high-temperature exposure at 45 °C. Physiological indicators, including relative water content (RWC) and electrolyte leakage index (ELI), were used for preliminary screening, supported by phenotypic observations, Evans blue staining for cell death, and DAB staining to assess oxidative stress and recovery ability. The results revealed clear differences among species. Chamaecyparis obtusa, Quercus glauca, and Q. myrsinaefolia exhibited strong tolerance, maintaining high RWC and low ELI values, while Albizia julibrissin was highly susceptible, showing severe membrane damage and low survival. DAB staining successfully distinguished tolerance levels based on oxidative recovery. Additional species such as Camellia sinensis, Q. acuta, Q. phillyraeoides, Q. salicina, and Ternstroemia japonica showed varied responses: Q. phillyraeoides demonstrated high tolerance, T. japonica showed moderate tolerance, and Q. salicina was relatively sensitive. The integrated screening system effectively differentiated tolerant species through multiscale analysis—physiological, cellular, and morphological—demonstrating its robustness and applicability. This study provides a practical and reproducible framework for evaluating sequential drought and heat stress in trees and offers valuable resources for urban forestry, reforestation, and climate-resilient species selection. Full article

Asian chestnut gall wasp (ACGW) (Dryocosmus kuriphilus Yasumatsu), native to China, is an invasive pest that causes significant economic losses in Castanea species. While some cultivars show full resistance by inhibiting insect development in buds, the underlying defense mechanisms remain unclear. In this study, the accumulation and distribution of hydrogen peroxide (H₂O₂) were investigated in dormant buds of chestnut cultivars that are resistant and susceptible to D. kuriphilus by using the 3,3′-diaminobenzidine (DAB) staining method. Buds were examined under a stereomicroscope during key stages of pest development, including oviposition, transition from egg to larva, gall induction, and gall development. Baseline levels of H₂O₂ were detected in all buds; however, these levels varied among cultivars, with resistant cultivars exhibiting lower basal levels. The degree of H₂O₂ accumulation was found to vary depending on plant–insect interaction, physiological processes, and cultivar-specific traits. Histochemical staining revealed that brown spots indicative of H₂O₂ accumulation were concentrated in the vascular bundles of leaf primordia and in the apical regions. In resistant hybrid cultivars, the defense response was activated at an earlier stage, while in resistant Castanea sativa Mill. cultivars, the response was delayed but more robust. Although consistently high levels of H₂O₂ were observed throughout the pest interaction in susceptible cultivars, gall development was not inhibited. During the onset of physiological bud break, increased H₂O₂ accumulation was observed across all cultivars. This increase was associated with endodormancy in susceptible cultivars and with both defense mechanisms and endodormancy processes in resistant cultivars. These findings highlight the significant role of H₂O₂ in plant defense responses, while also supporting its function as a multifunctional signaling molecule involved in gall development and the regulation of physiological processes. Full article

Foxtail millet (Setaria italica L.), a representative C4 species, is recognized for its efficient nutrient utilization and robust abiotic stress responses. However, the molecular mechanisms mediating its tolerance to biotic stresses are poorly understood. In this study, we investigated the root transcriptomic response of foxtail millet to the damage-associated molecular pattern (DAMP), the plant elicitor peptide 1 (Pep1). Transcriptome analysis of Pep1-treated roots identified 401 differentially expressed genes (DEGs), comprising 144 up-regulated and 257 down-regulated genes. Gene Ontology (GO) enrichment analysis revealed a significant enrichment of ‘peroxidase activity’. This finding was corroborated by DAB staining, which confirmed H₂O₂ accumulation, along with elevated malondialdehyde (MDA) levels, collectively indicating oxidative stress. Notably, Pep1 treatment also resulted in a marked up-regulation of the pathogenesis-related protein 1 (PR1) gene in leaves, suggesting the activation of systemic acquired resistance. Together, these results demonstrate that Pep1 triggers substantial transcriptional reprogramming in roots, induces oxidative stress, and activates systemic defense signaling in foxtail millet. Full article

Environmental concerns regarding synthetic herbicides have sparked interest in plant-derived bioactive compounds as eco-friendly alternatives. This study investigated the cellular targets of sweet flag essential oil (Acorus calamus L., SEO at IC₅₀ concentration) in root meristem cells of Fabaceae (Vicia faba, Lupinus luteus) and Brassicaceae (Brassica napus, Arabidopsis thaliana), focusing on reactive oxygen species (ROS) accumulation (DAB, NBT staining), DNA replication dynamics (EdU labeling), and genome integrity (γ-H2AX immunocytochemistry, TUNEL assay, and DNA electrophoresis). SEO induced oxidative stress (200–250% of control depending on the species) and replication stress, causing DNA double-strand breaks in 50% of proliferating cells, confirmed by γ-H2AX/TUNEL. Consequently, cells were prolonged in the G₁ phase, replication activity dropped to 70% of control in Fabaceae and 80% in Brassicaceae, and EdU incorporation intensity decreased to 80% and 70% of control, respectively. An increased proportion of cells replicating heterochromatin indicated slowed S-phase progression. Despite genotoxic effects, SEO did not trigger endoreplication, apoptotic DNA fragmentation, or extensive cell death. All species exhibited a uniform stress response, although sensitivity varied, which previously enabled the establishment of selective SEO doses between Fabaceae and Brassicaceae. These findings suggest that SEO exerts phytotoxicity by disrupting S-phase progression, supporting its potential as a selective bioherbicide. Full article

Drought and soil salinization significantly constrain agricultural productivity, driving the need for molecular breeding strategies to enhance stress resistance. Zinc finger proteins play a critical role in plant response to abiotic stress. In this study, a gene encoding a C2H2-type zinc finger protein (AfZFP5) was cloned from Amorpha fruticosa, a species known for its strong adaptability. qRT-PCR analysis revealed that AfZFP5 expression is regulated by sorbitol, H₂O₂, NaCl, and NaHCO₃. And all four treatments can cause upregulation of AFZFP5 expression in the roots or leaves of Amorpha fruticosa within 48 h. Transgenic tobacco lines overexpressing AfZFP5 demonstrated enhanced tolerance to drought and salt–alkali stress at germination, seedling, and vegetative stages. Compared to wild-type plants, transgenic lines exhibited significantly higher germination rates, root lengths, and fresh weights when treated with sorbitol, NaCl, and NaHCO₃. Under natural drought and salt–alkali stress conditions, transgenic plants showed elevated activities of superoxide dismutase (SOD) and peroxidase (POD), and upregulated expression of oxidative stress-related kinase genes (NtSOD, NtPOD) during the vegetative stage. Additionally, transgenic tobacco displayed lower malondialdehyde (MDA) content and reduced staining levels with 3,3′diaminobenzidine (DAB) and Nitro blue tetrazolium (NBT), indicating enhanced reactive oxygen species (ROS) scavenging capacity by AfZFP5 upon salt–alkali stress. Under simulated drought with PEG6000 and salt–alkali stress, chlorophyll fluorescence intensity and Fv/Fm values in transgenic tobacco were significantly higher than in wild-type plants during the vegetative stage, suggesting that AfZFP5 mitigates stress-induced damage to the photosynthetic system. This study highlights the role of AfZFP5 in conferring drought and salt–alkali stress tolerance, providing genetic resources and a theoretical foundation for breeding stress-resistance crops. Full article

Background/Objectives: The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme, encoded by OsGAPDHC6, plays a crucial role in glycolysis while participating in various physiological and stress response pathways. Methods: In this study, the expression levels of the OsGAPDHC1 and OsGAPDHC6 genes were investigated over time by treating various abiotic stresses (ABA, PEG, NaCl, heat, and cold) in rice seedlings. Results: As a result, the expression levels of both genes in the ABA-treated group increased continuously for 0–6 h and then de-creased sharply from 12 h onwards. The mutational induction of the GAPDHC6 gene by the CRISPR/Cas9 system generated a stop codon through a 1 bp insertion into protein production. The knockout (KO) lines showed differences in seed length, seed width, and seed thickness compared to wild-type (WT) varieties. In addition, KO lines showed a lower germination rate, germination ability, and germination index of seeds under salt treatment compared to WT, and leaf damage due to 3,3′-diaminobenzidine (DAB) staining was very high due to malondialdehyde (MDA) accumulation. The KO line was lower regarding the expression level of stress-related genes compared to WT. Conclusions: Therefore, the OsGAPDHC6 gene is evaluated as a gene that can increase salt resistance in rice as it actively responds to salt stress in the early stages of growth, occurring from seed germination to just before the tilling stage. Full article