Profiling of 1-aminocyclopropane-1-carboxylic acid and selected phytohormones in Arabidopsis using liquid chromatography-tandem mass spectrometry.

BACKGROUND: Gaseous phytohormone ethylene levels are directly influenced by the production of its immediate non-volatile precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Owing to the strongly acidic character of the ACC molecule, its quantification has been difficult to perform. Here, we present a simple and straightforward validated method for accurate quantification of not only ACC levels, but also major members of other important phytohormonal classes - auxins, cytokinins, jasmonic acid, abscisic acid and salicylic acid from the same biological sample. RESULTS: The presented technique facilitates the analysis of 15 compounds by liquid chromatography coupled with tandem mass spectrometry. It was optimized and validated for 10 mg of fresh weight plant material. The extraction procedure is composed of a minimal amount of necessary steps. Accuracy and precision were the basis for evaluating the method, together with process efficiency, recovery and matrix effects as validation parameters. The examined compounds comprise important groups of phytohormones, their active forms and some of their metabolites, including six cytokinins, four auxins, two jasmonates, abscisic acid, salicylic acid and 1-aminocyclopropane-1-carboxylic acid. The resulting method was used to examine their contents in selected Arabidopsis thaliana mutant lines. CONCLUSION: This profiling method enables a very straightforward approach for indirect ethylene study and explores how it interacts, based on content levels, with other phytohormonal groups in plants.

Training Peer Support Workers in Mental Health Care: A Mixed Methods Study in Central Catalonia.

Introduction: A mental health peer support program was implemented at two reference institutions in Central Catalonia. The program culturally and contextually adapted successful international projects by training people with experience of mental health problems and ensuring their employment in multidisciplinary health care teams. This study explores the influence of peer interventions in mental health on the three groups of participants: peer support workers, service users, and mental health professionals. Methods: A mixed observational method design included pre-, inter-, and post-experimental components and a qualitative description of the impact. The triangulation of the qualitative and quantitative findings showed its coherence and facilitated the understanding of the results. Outcomes and measures were as follows: self-stigma (Self-Stigma Questionnaire); life satisfaction (Scale of Satisfaction with Life); participation in relevant activities (Engagement in Meaningful Activities Survey); personal recovery (Scale-revised Recovery Assessment); occupational performance (Canadian Occupational Performance Measure); and attitudes toward mental illness (Community Attitudes toward Mental Illness). Results: The program showed beneficial effects on peer support workers' (PSW) perceptions of occupational performance, specifically on the ability to find work (p = 0.038), work as a peer support worker (p = 0.016), give to the community (p = 0.011), and satisfaction in the ability to find work (p = 0.031). The assessment made by the three groups of participants was very positive: the PSWs showed an increase in self-esteem and a feeling of usefulness; users of the service described the experience as a source of hope and optimism in their recovery process; and professionals described the program as a positive step in their professional growth. Discussion: The peer-to-peer strategy is a source of hope in the personal recovery process, providing meaning to life for the PSWs while providing an extra source of support to service users in their process of personal recovery. The results offer us lines of improvement for future implementations. PSW's final emphasis has us reflecting on improvements to enhance their own wellness in mental health care services. The findings show the importance of working on life projects and their impact on the recovery process.

Metabolic impact of a nutrition education program for the promotion of fruit and vegetable consumption with people with severe mental disorders (DIETMENT).

OBJECTIVES: The aim of this study is to determine the metabolic impact of a nutrition education program on metabolic parameters and the presence of metabolic syndrome (MetS). RESULTS: Seventy-four patients were included (mean age, 48.7 years [Standard deviation, SD: 10.8], 55.4% men). The diagnoses of SMD were 37.8% schizophrenia and related disorders; 29.7% bipolar disorder; 25.7% depressive disorder; 4.1% personality disorders; and 2.7% obsessive compulsive disorders. Thirty-seven individuals were distributed in both the intervention group (IG) and the control group (CG). In the IG the presence of MetS was 56.3% and in the CG 46.7%, with no statistically significant difference (p = 0.309). At the end of the study, glomerular filtrate decreased in the IG, body mass index and abdominal perimeter increased in both groups, and there were no changes in metabolic parameters between the groups. Between the baseline and the end of the study, there was no increase in the number of patients diagnosed with MetS (14 at both points); and in the CG the increase was from 8 to 12 (p = 0.005). An intervention based on fruit and vegetable intake could prevent progression to MetS in individuals with SMD, decreasing the likelihood of cardiovascular disease. Trial registration The trial was retrospectively registered on International Standard Randomised Controlled Trial Number (ISRCTN) Register on 11 March 2022 (ISRCTN12024347).

Inactivation of the entire Arabidopsis group II GH3s confers tolerance to salinity and water deficit.

Indole-3-acetic acid (IAA) controls a plethora of developmental processes. Thus, regulation of its concentration is of great relevance for plant performance. Cellular IAA concentration depends on its transport, biosynthesis and the various pathways for IAA inactivation, including oxidation and conjugation. Group II members of the GRETCHEN HAGEN 3 (GH3) gene family code for acyl acid amido synthetases catalysing the conjugation of IAA to amino acids. However, the high degree of functional redundancy among them has hampered thorough analysis of their roles in plant development. In this work, we generated an Arabidopsis gh3.1,2,3,4,5,6,9,17 (gh3oct) mutant to knock out the group II GH3 pathway. The gh3oct plants had an elaborated root architecture, showed an increased tolerance to different osmotic stresses, including an IAA-dependent tolerance to salinity, and were more tolerant to water deficit. Indole-3-acetic acid metabolite quantification in gh3oct plants suggested the existence of additional GH3-like enzymes in IAA metabolism. Moreover, our data suggested that 2-oxindole-3-acetic acid production depends, at least in part, on the GH3 pathway. Targeted stress-hormone analysis further suggested involvement of abscisic acid in the differential response to salinity of gh3oct plants. Taken together, our data provide new insights into the roles of group II GH3s in IAA metabolism and hormone-regulated plant development.

Applying the Stages of Change Model in a Nutrition Education Programme for the Promotion of Fruit and Vegetable Consumption among People with Severe Mental Disorders (DIETMENT).

Despite growing evidence of the benefits of adequate intake of fruit and vegetables (F&V) and the recommendation to consume five servings daily, the adoption of these habits is poor among people with severe mental disorder (SMD). The main aim of the present study is to determine changes in the intake of F&V and motivation to do so among people with SMDs after participating in a food education programme. A community-based randomized controlled trial was conducted in Spain, with the intervention group (IG) participating in a food education programme based on the stages of change model to promote consumption of F&V and the control group (CG) receiving three informative sessions on basic healthy eating. The main outcomes were related to the intake of F&V and stages of change. Data collection was performed at baseline, post intervention, and 12-month follow-up. Seventy-four participants enrolled in the study and sixty completed the 12-month follow-up. An increase in motivation towards the intake of F&V was observed in the IG but not in the CG (McNemar's test p = 0.016, p = 0.625). No significant difference was observed for the intake of fruit, vegetables, or F&V. Basing food education strategies on the stages of change model shows positive results, increasing the awareness and disposition of people with SMD towards the intake of F&V. More research is needed to identify the most appropriate eating intervention to increase the intake of F&V.

Broadening the roles of UDP-glycosyltransferases in auxin homeostasis and plant development.

The levels of the important plant growth regulator indole-3-acetic acid (IAA) are tightly controlled within plant tissues to spatiotemporally orchestrate concentration gradients that drive plant growth and development. Metabolic inactivation of bioactive IAA is known to participate in the modulation of IAA maxima and minima. IAA can be irreversibly inactivated by oxidation and conjugation to aspartate and glutamate. Usually overlooked because of its reversible nature, the most abundant inactive IAA form is the IAA-glucose (IAA-glc) conjugate. Glycosylation of IAA in Arabidopsis thaliana is reported to be carried out by UDP-glycosyltransferase 84B1 (UGT84B1), while UGT74D1 has been implicated in the glycosylation of the irreversibly formed IAA catabolite oxIAA. Here we demonstrated that both UGT84B1 and UGT74D1 modulate IAA levels throughout plant development by dual IAA and oxIAA glycosylation. Moreover, we identified a novel UGT subfamily whose members redundantly mediate the glycosylation of oxIAA and modulate skotomorphogenic growth.

Dynamics of Auxin and Cytokinin Metabolism during Early Root and Hypocotyl Growth in Theobroma cacao.

The spatial location and timing of plant developmental events are largely regulated by the well balanced effects of auxin and cytokinin phytohormone interplay. Together with transport, localized metabolism regulates the concentration gradients of their bioactive forms, ultimately eliciting growth responses. In order to explore the dynamics of auxin and cytokinin metabolism during early seedling growth in Theobroma cacao (cacao), we have performed auxin and cytokinin metabolite profiling in hypocotyls and root developmental sections at different times by using ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS). Our work provides quantitative characterization of auxin and cytokinin metabolites throughout early root and hypocotyl development and identifies common and distinctive features of auxin and cytokinin metabolism during cacao seedling development.

Auxin Metabolism in Plants.

The major natural auxin in plants, indole-3-acetic acid (IAA), orchestrates a plethora of developmental responses that largely depend on the formation of auxin concentration gradients within plant tissues. Together with inter- and intracellular transport, IAA metabolism-which comprises biosynthesis, conjugation, and degradation-modulates auxin gradients and is therefore critical for plant growth. It is now very well established that IAA is mainly produced from Trp and that the IPyA pathway is a major and universally conserved biosynthetic route in plants, while other redundant pathways operate in parallel. Recent findings have shown that metabolic inactivation of IAA is also redundantly performed by oxidation and conjugation processes. An exquisite spatiotemporal expression of the genes for auxin synthesis and inactivation have been shown to drive several plant developmental processes. Moreover, a group of transcription factors and epigenetic regulators controlling the expression of auxin metabolic genes have been identified in past years, which are illuminating the road to understanding the molecular mechanisms behind the coordinated responses of local auxin metabolism to specific cues. Besides transcriptional regulation, subcellular compartmentalization of the IAA metabolism and posttranslational modifications of the metabolic enzymes are emerging as important contributors to IAA homeostasis. In this review, we summarize the current knowledge on (1) the pathways for IAA biosynthesis and inactivation in plants, (2) the influence of spatiotemporally regulated IAA metabolism on auxin-mediated responses, and (3) the regulatory mechanisms that modulate IAA levels in response to external and internal cues during plant development.

Conifers exhibit a characteristic inactivation of auxin to maintain tissue homeostasis.

Dynamic regulation of the concentration of the natural auxin (IAA) is essential to coordinate most of the physiological and developmental processes and responses to environmental changes. Oxidation of IAA is a major pathway to control auxin concentrations in angiosperms and, along with IAA conjugation, to respond to perturbation of IAA homeostasis. However, these regulatory mechanisms remain poorly investigated in conifers. To reduce this knowledge gap, we investigated the different contributions of the IAA inactivation pathways in conifers. MS-based quantification of IAA metabolites under steady-state conditions and after perturbation was investigated to evaluate IAA homeostasis in conifers. Putative Picea abies GH3 genes (PaGH3) were identified based on a comprehensive phylogenetic analysis including angiosperms and basal land plants. Auxin-inducible PaGH3 genes were identified by expression analysis and their IAA-conjugating activity was explored. Compared to Arabidopsis, oxidative and conjugative pathways differentially contribute to reduce IAA concentrations in conifers. We demonstrated that the oxidation pathway plays a marginal role in controlling IAA homeostasis in spruce. By contrast, an excess of IAA rapidly activates GH3-mediated irreversible conjugation pathways. Taken together, these data indicate that a diversification of IAA inactivation mechanisms evolved specifically in conifers.

Epigenetic Regulation of Auxin Homeostasis.

Epigenetic regulation involves a myriad of mechanisms that regulate the expression of loci without altering the DNA sequence. These different mechanisms primarily result in modifications of the chromatin topology or DNA chemical structure that can be heritable or transient as a dynamic response to environmental cues. The phytohormone auxin plays an important role in almost every aspect of plant life via gradient formation. Auxin maxima/minima result from a complex balance of metabolism, transport, and signaling. Although epigenetic regulation of gene expression during development has been known for decades, the specific mechanisms behind the spatiotemporal dynamics of auxin levels in plants are only just being elucidated. In this review, we gather current knowledge on the epigenetic mechanisms regulating the expression of genes for indole-3-acetic acid (IAA) metabolism and transport in Arabidopsis and discuss future perspectives of this emerging field.

Auxin Metabolism Controls Developmental Decisions in Land Plants.

Unlike animals, whose body plans are set during embryo development, plants maintain the ability to initiate new organs throughout their life cycle. Auxin is a key regulator of almost all aspects of plant development, including morphogenesis and adaptive responses. Cellular auxin concentrations influence whether a cell will divide, grow, or differentiate, thereby contributing to organ formation, growth, and ultimately plant shape. Auxin gradients are established and maintained by a tightly regulated interplay between metabolism, signalling, and transport. Auxin is synthesised, stored, and inactivated by a multitude of parallel pathways that are all tightly regulated. Here we summarise the remarkable progress that has been achieved in identifying some key components of these pathways and the genetic complexity underlying their precise regulation.

Controlled living anionic polymerization of cyanoacrylates by frustrated Lewis pair based initiators.

A hydrogenated frustrated Lewis pair ([TMPH+][HB(C6F5)3 -]) promotes controlled living ionic polymerization of cyanoacrylates. Controlled growth of various homopolymeric CAs through sequential monomer addition has been achieved, in addition to CA block copolymers with controlled block sequences for the first time in the long history of these materials.

Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis.

Auxin (indole-3-acetic acid, IAA) plays fundamental roles as a signalling molecule during numerous plant growth and development processes. The formation of local auxin gradients and auxin maxima/minima, which is very important for these processes, is regulated by auxin metabolism (biosynthesis, degradation, and conjugation) as well as transport. When studying auxin metabolism pathways it is crucial to combine data obtained from genetic investigations with the identification and quantification of individual metabolites. Thus, to facilitate efforts to elucidate auxin metabolism and its roles in plants, we have developed a high-throughput method for simultaneously quantifying IAA and its key metabolites in minute samples (<10 mg FW) of Arabidopsis thaliana tissues by in-tip micro solid-phase extraction and fast LC-tandem MS. As a proof of concept, we applied the method to a collection of Arabidopsis mutant lines and identified lines with altered IAA metabolite profiles using multivariate data analysis. Finally, we explored the correlation between IAA metabolite profiles and IAA-related phenotypes. The developed rapid analysis of large numbers of samples (>100 samples d-1) is a valuable tool to screen for novel regulators of auxin metabolism and homeostasis among large collections of genotypes.

Dioxygenase-encoding AtDAO1 gene controls IAA oxidation and homeostasis in Arabidopsis.

Auxin represents a key signal in plants, regulating almost every aspect of their growth and development. Major breakthroughs have been made dissecting the molecular basis of auxin transport, perception, and response. In contrast, how plants control the metabolism and homeostasis of the major form of auxin in plants, indole-3-acetic acid (IAA), remains unclear. In this paper, we initially describe the function of the Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1 (AtDAO1). Transcriptional and translational reporter lines revealed that AtDAO1 encodes a highly root-expressed, cytoplasmically localized IAA oxidase. Stable isotope-labeled IAA feeding studies of loss and gain of function AtDAO1 lines showed that this oxidase represents the major regulator of auxin degradation to 2-oxoindole-3-acetic acid (oxIAA) in Arabidopsis Surprisingly, AtDAO1 loss and gain of function lines exhibited relatively subtle auxin-related phenotypes, such as altered root hair length. Metabolite profiling of mutant lines revealed that disrupting AtDAO1 regulation resulted in major changes in steady-state levels of oxIAA and IAA conjugates but not IAA. Hence, IAA conjugation and catabolism seem to regulate auxin levels in Arabidopsis in a highly redundant manner. We observed that transcripts of AtDOA1 IAA oxidase and GH3 IAA-conjugating enzymes are auxin-inducible, providing a molecular basis for their observed functional redundancy. We conclude that the AtDAO1 gene plays a key role regulating auxin homeostasis in Arabidopsis, acting in concert with GH3 genes, to maintain auxin concentration at optimal levels for plant growth and development.

Plastid control of abaxial-adaxial patterning.

Translational regulation, exerted by the cytosolic ribosome, has been shown to participate in the establishment of abaxial-adaxial polarity in Arabidopsis thaliana: many hypomorphic and null alleles of genes encoding proteins of the cytosolic ribosome enhance the leaf polarity defects of asymmetric leaves1 (as1) and as2 mutants. Here, we report the identification of the SCABRA1 (SCA1) nuclear gene, whose loss-of-function mutations also enhance the polarity defects of the as2 mutants. In striking contrast to other previously known enhancers of the phenotypes caused by the as1 and as2 mutations, we found that SCA1 encodes a plastid-type ribosomal protein that functions as a structural component of the 70S plastid ribosome and, therefore, its role in abaxial-adaxial patterning was not expected.

Chemoradiotherapeutic Magnetic Nanoparticles for Targeted Treatment of Nonsmall Cell Lung Cancer.

Lung cancer is the leading cause of cancer-related death in the United States and approximately 85% of all lung cancers are classified as nonsmall cell (NSCLC). We here use an innovative approach that may ultimately allow for the clinician to target tumors and aggressively reduce tumor burden in patients with NSCLC. In this study, a platinum (Pt)-based chemotherapeutic (cisplatin, carboplatin, or oxaliplatin) and holmium-165 (Ho), which can be neutron-activated to produce the holmium-166 radionuclide, have been incorporated together in a garnet magnetic nanoparticle (HoIG-Pt) for selective delivery to tumors using an external magnet. The synthesized magnetic HoIG nanoparticles were characterized using PXRD, TEM, ICP-MS, and neutron-activation. Platinum(II) drugs were incorporated onto HoIG, and these were characterized using FTIR, EDX, ICP-MS, and zeta potential measurements, and in vitro and in vivo studies were performed using a HoIG-platinum system. Results indicate that neutron-activated (166)HoIG-cisplatin is more toxic toward NSCLC A549 cells than is blank (166)HoIG and free cisplatin, and that when an external magnetic field is applied in vivo, higher tumor to liver ratios of Ho are observed than when no magnet is applied, suggesting that magnetic targeting is achieved using this system. Furthermore, an efficacy study demonstrated the inhibition of tumor growth by chemoradiotherapeutic magnetic nanoparticles, compared to no treatment controls.

Getting started in mapping-by-sequencing.

Next-generation sequencing (NGS) technologies allow the cost-effective sequencing of whole genomes and have expanded the scope of genomics to novel applications, such as the genome-wide characterization of intraspecific polymorphisms and the rapid mapping and identification of point mutations. Next-generation sequencing platforms, such as the Illumina HiSeq2000 platform, are now commercially available at affordable prices and routinely produce an enormous amount of sequence data, but their wide use is often hindered by a lack of knowledge on how to manipulate and process the information produced. In this review, we focus on the strategies that are available to geneticists who wish to incorporate these novel approaches into their research but who are not familiar with the necessary bioinformatic concepts and computational tools. In particular, we comprehensively summarize case studies where the use of NGS technologies has led to the identification of point mutations, a strategy that has been dubbed "mapping-by-sequencing", and review examples from plants and other model species such as Caenorhabditis elegans, Saccharomyces cerevisiae, and Drosophila melanogaster. As these technologies are becoming cheaper and more powerful, their use is also expanding to allow mutation identification in species with larger genomes, such as many crop plants.

Rapid identification of angulata leaf mutations using next-generation sequencing.

Map-based (positional) cloning has traditionally been the preferred strategy for identifying the causal genes underlying the phenotypes of mutants isolated in forward genetic screens. Massively parallel sequencing technologies are enabling the rapid cloning of genes identified in such screens. We have used a combination of linkage mapping and whole-genome re-sequencing to identify the causal mutations in four loss-of-function angulata (anu) mutants. These mutants were isolated in a screen for mutants with defects in leaf shape and leaf pigmentation. Our results show that the anu1-1, anu4-1, anu9-1 and anu12-1 mutants carry new alleles of the previously characterized SECA2, TRANSLOCON AT THE OUTER MEMBRANE OF CHLOROPLASTS 33 (TOC33), NON-INTRINSIC ABC PROTEIN 14 (NAP14) and CLP PROTEASE PROTEOLYTIC SUBUNIT 1 (CLPR1) genes. Re-sequencing the genomes of fine mapped mutants is a feasible approach that has allowed us to identify a moderate number of candidate mutations, including the one that causes the mutant phenotype, in a nonstandard genetic background. Our results indicate that anu mutations specifically affect plastid-localized proteins involved in diverse processes, such as the movement of peptides through chloroplast membranes (ANU1 and ANU4), metal homeostasis (ANU9) and protein degradation (ANU12).

Influence of PPh3 moiety in the anticancer activity of new organometallic ruthenium complexes.

The effect of the PPh3 group in the antitumor activity of some new organometallic ruthenium(II) complexes has been investigated. Several complexes of the type [Ru((II))(Cl)(PPh3)(Lig-N)], [Ru((II))(Cl)2(Lig-N)] (where Lig-N=pyridine derivate) and [Ru((II))(Cl)(PPh3)2], have been synthesized and characterized. A noticeable increment of the antitumor activity and cytotoxicity of the complexes due to the presence of PPh3 moiety has also been demonstrated, affording IC50 values of 5.2 µM in HL-60 tumor cell lines. Atomic force microscopy, circular dichroism and electrophoresis experiments have proved that these complexes can bind DNA resulting in a distortion of both secondary and tertiary structures. Ethidium bromide displacement fluorescence spectroscopy studies and viscosity measurements support that the presence of PPh3 group induces intercalation interactions with DNA. Indeed, crystallographic analysis, suggest that intra-molecular π-π interactions could be involved in the intercalation within DNA base pairs. Furthermore, high performance liquid chromatography mass spectrometry (HPLC-MS) studies have confirmed a strong interaction between ruthenium complexes and proteins (ubiquitin and potato carboxypeptidase inhibitor - PCI) including slower kinetics due to the presence of PPh3 moiety, which could have an important role in detoxification mechanism and others. Finally, ion mobility mass spectrometry (IMMS) experiments have proved that there is no significant change in the gas phase structural conformation of the proteins owing to their bonding to ruthenium complexes.

Arabidopsis ANGULATA10 is required for thylakoid biogenesis and mesophyll development.

The chloroplasts of land plants contain internal membrane systems, the thylakoids, which are arranged in stacks called grana. Because grana have not been found in Cyanobacteria, the evolutionary origin of genes controlling the structural and functional diversification of thylakoidal membranes in land plants remains unclear. The angulata10-1 (anu10-1) mutant, which exhibits pale-green rosettes, reduced growth, and deficient leaf lateral expansion, resulting in the presence of prominent marginal teeth, was isolated. Palisade cells in anu10-1 are larger and less packed than in the wild type, giving rise to large intercellular spaces. The ANU10 gene encodes a protein of unknown function that localizes to both chloroplasts and amyloplasts. In chloroplasts, ANU10 associates with thylakoidal membranes. Mutant anu10-1 chloroplasts accumulate H2O2, and have reduced levels of chlorophyll and carotenoids. Moreover, these chloroplasts are small and abnormally shaped, thylakoidal membranes are less abundant, and their grana are absent due to impaired thylakoid stacking in the anu10-1 mutant. Because the trimeric light-harvesting complex II (LHCII) has been reported to be required for thylakoid stacking, its levels were determined in anu10-1 thylakoids and they were found to be reduced. Together, the data point to a requirement for ANU10 for chloroplast and mesophyll development.