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Small GTPases switch between GDP- and GTP-bound states during cell signaling. The ADP-ribosylation factor (ARF) family of small GTPases is involved in vesicle trafficking. Although evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. We characterized biochemical properties and cellular localization of the essential small ARF-like GTPase TITAN 5 (TTN5; also known as HALLIMASCH, ARL2 and ARLC1) from Arabidopsis thaliana, and two TTN5 proteins with point mutants in conserved residues, TTN5T30N and TTN5Q70L, that were expected to be unable to perform nucleotide exchange and GTP hydrolysis, respectively. TTN5 exhibited very rapid intrinsic nucleotide exchange and remarkably low GTP hydrolysis activity, functioning as a non-classical small GTPase being likely present in a GTP-loaded active form. We analyzed signals from YFP-TTN5 and HA3-TTN5 by in situ immunolocalization in Arabidopsis seedlings and through use of a transient expression system. Colocalization with endomembrane markers and pharmacological treatments suggests that TTN5 can be present at the plasma membrane and that it dynamically associates with membranes of vesicles, Golgi stacks and multivesicular bodies. Although TTN5Q70L mirrored wild-type TTN5 behavior, the TTN5T30N mutant differed in some aspects. Hence, the unusual rapid nucleotide exchange activity of TTN5 is linked with its membrane dynamics, and TTN5 likely has a role in vesicle transport within the endomembrane system.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Guanosina Trifosfato/metabolismo , Membrana Celular/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Hidrólise , Complexo de Golgi/metabolismoRESUMO
The functional importance of nuclear protein condensation remains often unclear. The bHLH FER-like iron deficiency-induced transcription factor (FIT) controls iron acquisition and growth in plants. Previously described C-terminal serine residues allow FIT to interact and form active transcription factor complexes with subgroup Ib bHLH factors such as bHLH039. FIT has lower nuclear mobility than mutant FITmSS271AA. Here, we show that FIT undergoes a light-inducible subnuclear partitioning into FIT nuclear bodies (NBs). Using quantitative and qualitative microscopy-based approaches, we characterized FIT NBs as condensates that were reversible and likely formed by liquid-liquid phase separation. FIT accumulated preferentially in NBs versus nucleoplasm when engaged in protein complexes with itself and with bHLH039. FITmSS271AA, instead, localized to NBs with different dynamics. FIT colocalized with splicing and light signaling NB markers. The NB-inducing light conditions were linked with active FIT and elevated FIT target gene expression in roots. FIT condensation may affect nuclear mobility and be relevant for integrating environmental and Fe nutrition signals.
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Proteínas de Arabidopsis , Arabidopsis , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Ferro , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica de Plantas , Ferro/metabolismo , Corpos Nucleares/genética , Corpos Nucleares/metabolismoRESUMO
Small GTPases comprise key proteins in signal transduction that function by conformational switching ability between GDP- and GTP-bound states. The ADP-ribosylation factor (ARF) family is involved in vesicle trafficking and cellular functions. Though evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. Here, we characterized functional properties and cellular localization of the essential small ARF-like GTPase TITAN5/HALLIMASCH/ARL2/ARLC1 (hereafter termed TTN5) from Arabidopsis thaliana. TTN5 showed rapid guanine nucleotide exchange capacity comparable to that of human counterparts, but a remarkably low GTP hydrolysis reaction. A TTN5Q70L mutant had enhanced nucleotide exchange activity, indicative of intracellular activation, while TTN5T30N with fast nucleotide dissociation can be considered a dominant-negative form. This suggests that TTN5 is present in GTP-loaded active form in the cells. YFP-tagged TTN5 and the two derived mutant variants were located at multiple sites of the endomembrane system in the epidermis of Arabidopsis seedlings and Nicotiana benthamiana leaves. While YFP-TTN5 and YFP-TTN5Q70L were highly mobile in the cells, mobility was reduced for TTN5T30N. Colocalization with endomembrane markers in combination with pharmacological treatments resolved localization at membrane sites and showed that YFP-TTN5 and YFP-TTN5Q70L were located in Golgi stacks, multivesicular bodies, while this was less the case for YFP-TTN5T30N. On the other hand, all three TTN5 forms were located at the plasma membrane. Hence, the unusual capacity of rapid nucleotide exchange activity of the small ARF-like GTPase TTN5 is linked with cell membrane dynamics, likely associated with vesicle transport pathways in the endomembrane system.
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Membrane identity and dynamic processes, that act at membrane sites, provide important cues for regulating transport, signal transduction and communication across membranes. There are still numerous open questions as to how membrane identity changes and the dynamic processes acting at the surface of membranes are regulated in diverse eukaryotes in particular plants and which roles are being played by protein interaction complexes composed of peripheral and integral membrane proteins. One class of peripheral membrane proteins conserved across eukaryotes comprises the SEC14-like phosphatidylinositol transfer proteins (SEC14L-PITPs). These proteins share a SEC14 domain that contributes to membrane identity and fulfills regulatory functions in membrane trafficking by its ability to sense, bind, transport and exchange lipophilic substances between membranes, such as phosphoinositides and diverse other lipophilic substances. SEC14L-PITPs can occur as single-domain SEC14-only proteins in all investigated organisms or with a modular domain structure as multi-domain proteins in animals and streptophytes (comprising charales and land plants). Here, we present an overview on the functional roles of SEC14L-PITPs, with a special focus on the multi-domain SEC14L-PITPs of the SEC14-nodulin and SEC14-GOLD group (PATELLINs, PATLs in plants). This indicates that SEC14L-PITPs play diverse roles from membrane trafficking to organism fitness in plants. We concentrate on the structure of SEC14L-PITPs, their ability to not only bind phospholipids but also other lipophilic ligands, and their ability to regulate complex cellular responses through interacting with proteins at membrane sites.
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Organisms require micronutrients, and Arabidopsis (Arabidopsis thaliana) IRON-REGULATED TRANSPORTER1 (IRT1) is essential for iron (Fe2+) acquisition into root cells. Uptake of reactive Fe2+ exposes cells to the risk of membrane lipid peroxidation. Surprisingly little is known about how this is avoided. IRT1 activity is controlled by an intracellular variable region (IRT1vr) that acts as a regulatory protein interaction platform. Here, we describe that IRT1vr interacted with peripheral plasma membrane SEC14-Golgi dynamics (SEC14-GOLD) protein PATELLIN2 (PATL2). SEC14 proteins bind lipophilic substrates and transport or present them at the membrane. To date, no direct roles have been attributed to SEC14 proteins in Fe import. PATL2 affected root Fe acquisition responses, interacted with ROS response proteins in roots, and alleviated root lipid peroxidation. PATL2 had high affinity in vitro for the major lipophilic antioxidant vitamin E compound α-tocopherol. Molecular dynamics simulations provided insight into energetic constraints and the orientation and stability of the PATL2-ligand interaction in atomic detail. Hence, this work highlights a compelling mechanism connecting vitamin E with root metal ion transport at the plasma membrane with the participation of an IRT1-interacting and α-tocopherol-binding SEC14 protein.
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Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Vitamina E/metabolismo , alfa-Tocoferol , Transporte Biológico , Arabidopsis/genética , Arabidopsis/metabolismo , Raízes de Plantas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
BACKGROUND AND OBJECTIVES: People with multiple sclerosis (MS) are a vulnerable group for severe coronavirus disease 2019 (COVID-19), particularly those taking immunosuppressive disease-modifying therapies (DMTs). We examined the characteristics of COVID-19 severity in an international sample of people with MS. METHODS: Data from 12 data sources in 28 countries were aggregated (sources could include patients from 1-12 countries). Demographic (age, sex), clinical (MS phenotype, disability), and DMT (untreated, alemtuzumab, cladribine, dimethyl fumarate, glatiramer acetate, interferon, natalizumab, ocrelizumab, rituximab, siponimod, other DMTs) covariates were queried, along with COVID-19 severity outcomes, hospitalization, intensive care unit (ICU) admission, need for artificial ventilation, and death. Characteristics of outcomes were assessed in patients with suspected/confirmed COVID-19 using multilevel mixed-effects logistic regression adjusted for age, sex, MS phenotype, and Expanded Disability Status Scale (EDSS) score. RESULTS: Six hundred fifty-seven (28.1%) with suspected and 1,683 (61.9%) with confirmed COVID-19 were analyzed. Among suspected plus confirmed and confirmed-only COVID-19, 20.9% and 26.9% were hospitalized, 5.4% and 7.2% were admitted to ICU, 4.1% and 5.4% required artificial ventilation, and 3.2% and 3.9% died. Older age, progressive MS phenotype, and higher disability were associated with worse COVID-19 outcomes. Compared to dimethyl fumarate, ocrelizumab and rituximab were associated with hospitalization (adjusted odds ratio [aOR] 1.56, 95% confidence interval [CI] 1.01-2.41; aOR 2.43, 95% CI 1.48-4.02) and ICU admission (aOR 2.30, 95% CI 0.98-5.39; aOR 3.93, 95% CI 1.56-9.89), although only rituximab was associated with higher risk of artificial ventilation (aOR 4.00, 95% CI 1.54-10.39). Compared to pooled other DMTs, ocrelizumab and rituximab were associated with hospitalization (aOR 1.75, 95% CI 1.29-2.38; aOR 2.76, 95% CI 1.87-4.07) and ICU admission (aOR 2.55, 95% CI 1.49-4.36; aOR 4.32, 95% CI 2.27-8.23), but only rituximab was associated with artificial ventilation (aOR 6.15, 95% CI 3.09-12.27). Compared to natalizumab, ocrelizumab and rituximab were associated with hospitalization (aOR 1.86, 95% CI 1.13-3.07; aOR 2.88, 95% CI 1.68-4.92) and ICU admission (aOR 2.13, 95% CI 0.85-5.35; aOR 3.23, 95% CI 1.17-8.91), but only rituximab was associated with ventilation (aOR 5.52, 95% CI 1.71-17.84). Associations persisted on restriction to confirmed COVID-19 cases. No associations were observed between DMTs and death. Stratification by age, MS phenotype, and EDSS score found no indications that DMT associations with COVID-19 severity reflected differential DMT allocation by underlying COVID-19 severity. DISCUSSION: Using the largest cohort of people with MS and COVID-19 available, we demonstrated consistent associations of rituximab with increased risk of hospitalization, ICU admission, and need for artificial ventilation and of ocrelizumab with hospitalization and ICU admission. Despite the cross-sectional design of the study, the internal and external consistency of these results with prior studies suggests that rituximab/ocrelizumab use may be a risk factor for more severe COVID-19.
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COVID-19/complicações , Hospitalização/estatística & dados numéricos , Esclerose Múltipla/complicações , Esclerose Múltipla/tratamento farmacológico , Adolescente , Adulto , Idoso , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/uso terapêutico , COVID-19/patologia , COVID-19/fisiopatologia , Estudos Transversais , Fumarato de Dimetilo/efeitos adversos , Fumarato de Dimetilo/uso terapêutico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Natalizumab/efeitos adversos , Natalizumab/uso terapêutico , Respiração Artificial/estatística & dados numéricos , Rituximab/efeitos adversos , Rituximab/uso terapêutico , SARS-CoV-2 , Adulto JovemRESUMO
Photoacoustic spectroscopy is applied to evaluate the impact of Moringa at different concentrations (0, 1.25, 2.5, 5 and 10%) on the elaboration, sanity, texture, and color of wheat bread. It was found that: i) Photoacoustic signal amplitude values of bread significantly increase from 37 to 90% when moringa powder concentration raises from 1.25% to 10%, at 300 nm wavelength. ii) Comparing the photoacoustic signal values at 300, 330, and 370 nm wavelengths, produced by the different bread types, there were statistically significant differences. iii) The sanitary quality of bread mixed with a 2.5% of moringa is relatively higher than the ones obtained for other concentrations, such that the number of fungal colonies were reduced by 99% in comparison with the control bread without moringa, after six days of storage. Moringa at 2.5% of concentration could thus improve the sanitary quality of wheat bread. iv) The addition of moringa for making bread slows down its textural changes (hardness, elasticity, cohesiveness, resilience, and chewiness) during storage. v) Finally, the highest correlation between the photoacoustic amplitude and the moringa concentration occurs at the wavelengths of 300 and 330 nm, which could be related to significant changes in the content of flavonoids and phenolic acids.
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Due to its redox properties, iron is both essential and toxic. Therefore, soil iron availability variations pose a significant problem for plants. Recent evidence suggests that calcium and reactive oxygen species coordinate signaling events related to soil iron acquisition. Calcium was found to affect directly IRT1-mediated iron import through the lipid-binding protein EHB1 and to trigger a CBL-CIPK-mediated signaling influencing the activity of the key iron-acquisition transcription factor FIT. In parallel, under prolonged iron deficiency, reactive oxygen species both inhibit FIT function and depend on FIT through the function of the catalase CAT2. We discuss the role of calcium and reactive oxygen species signaling in iron acquisition, with post-translational mechanisms influencing the localization and activity of iron-acquisition regulators and effectors.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cálcio , Regulação da Expressão Gênica de Plantas , Espécies Reativas de Oxigênio/metabolismoRESUMO
The contribution of amino acids (AAs) to soil nitrogen (N) fluxes is higher than previously thought. The fact that AA uptake is pivotal for N nutrition in boreal ecosystems highlights plant AA transporters as key components of the N cycle. At the same time, very little is known about AA transport and respective transporters in trees. Tree genomes may contain 13 or more genes encoding the lysine histidine transporter (LHT) family proteins, and this complicates the study of their significance for tree N-use efficiency. With the strategy of obtaining a tool to study N-use efficiency, our aim was to identify and characterize a relevant AA transporter in hybrid aspen (Populus tremula L. x tremuloides Michx.). We identified PtrLHT1.2, the closest homolog of Arabidopsis thaliana (L.) Heynh AtLHT1, which is expressed in leaves, stems and roots. Complementation of a yeast AA uptake mutant verified the function of PtrLHT1.2 as an AA transporter. Furthermore, PtrLHT1.2 was able to fully complement the phenotypes of the Arabidopsis AA uptake mutant lht1 aap5, including early leaf senescence-like phenotype, reduced growth, decreased plant N levels and reduced root AA uptake. Amino acid uptake studies finally showed that PtrLHT1.2 is a high affinity transporter for neutral and acidic AAs. Thus, we identified a functional AtLHT1 homolog in hybrid aspen, which harbors the potential to enhance overall plant N levels and hence increase biomass production. This finding provides a valuable tool for N nutrition studies in trees and opens new avenues to optimizing tree N-use efficiency.
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Arabidopsis , Populus , Sistemas de Transporte de Aminoácidos/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Ecossistema , Nitrogênio/metabolismo , Populus/genética , Populus/metabolismoRESUMO
BACKGROUND: One of the major objectives of the Multiple Sclerosis Data Alliance (MSDA) is to enable better discovery of multiple sclerosis (MS) real-world data (RWD). METHODS: We implemented the MSDA Catalogue, which is available worldwide. The current version of the MSDA Catalogue collects descriptive information on governance, purpose, inclusion criteria, procedures for data quality control, and how and which data are collected, including the use of e-health technologies and data on collection of COVID-19 variables. The current cataloguing procedure is performed in several manual steps, securing an effective catalogue. RESULTS: Herein we summarize the status of the MSDA Catalogue as of January 6, 2021. To date, 38 data sources across five continents are included in the MSDA Catalogue. These data sources differ in purpose, maturity, and variables collected, but this landscaping effort shows that there is substantial alignment on some domains. The MSDA Catalogue shows that personal data and basic disease data are the most collected categories of variables, whereas data on fatigue measurements and cognition scales are the least collected in MS registries/cohorts. CONCLUSIONS: The Web-based MSDA Catalogue provides strategic overview and allows authorized end users to browse metadata profiles of data cohorts and data sources. There are many existing and arising RWD sources in MS. Detailed cataloguing of MS RWD is a first and useful step toward reducing the time needed to discover MS RWD sets and promoting collaboration.
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The removal of transmembrane proteins from the plasma membrane via endocytosis has emerged as powerful strategy in the regulation of receptor signalling and molecule transport. In the last decade, IRON-REGULATED TRANSPORTER1 (IRT1) has been established as one of the key plant model proteins for studying endomembrane trafficking. The use of IRT1 and additional other metal transporters has uncovered novel factors involved in plant endocytosis and facilitated a better understanding of the role of endocytosis in the fine balancing of plant metal homoeostasis. In this review, we outline the specifics of plant endocytosis compared to what is known in yeast and mammals, and based on several examples, we demonstrate how studying metal transport has contributed to extending our knowledge of endocytic trafficking by shedding light on novel regulatory mechanisms and factors.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Endocitose , Animais , Transporte Biológico , Ferro/metabolismo , Mamíferos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Reactive oxygen species play a central role in the regulation of plant responses to environmental stress. Under prolonged iron (Fe) deficiency, increased levels of hydrogen peroxide (H2O2) initiate signaling events, resulting in the attenuation of Fe acquisition through the inhibition of FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT). As this H2O2 increase occurs in a FIT-dependent manner, our aim was to understand the processes involved in maintaining H2O2 levels under prolonged Fe deficiency and the role of FIT. We identified the CAT2 gene, encoding one of the three Arabidopsis catalase isoforms, as regulated by FIT. CAT2 loss-of-function plants displayed severe susceptibility to Fe deficiency and greatly increased H2O2 levels in roots. Analysis of the Fe homeostasis transcription cascade revealed that H2O2 influences the gene expression of downstream regulators FIT, BHLH genes of group Ib, and POPEYE (PYE); however, H2O2 did not affect their upstream regulators, such as BHLH104 and ILR3. Our data shows that FIT and CAT2 participate in a regulatory loop between H2O2 and prolonged Fe deficiency.
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Proteínas de Arabidopsis , Arabidopsis , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Ferro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio , Raízes de Plantas/metabolismoRESUMO
KEY MESSAGE: SEC14L-PITPs guide membrane recognition and signaling. An increasingly complex modular structure of SEC14L-PITPs evolved in land plants compared to green algae. SEC14/CRAL-TRIO and GOLD domains govern membrane binding specificity. SEC14-like phosphatidylinositol transfer proteins (SEC14L-PITPs) provide cues for membrane identity by exchanging lipophilic substrates, ultimately governing membrane signaling. Flowering plant SEC14L-PITPs often have modular structure and are associated with cell division, development, and stress responses. Yet, structure-function relationships for biochemical-cellular interactions of SEC14L-PITPs are rather enigmatic. Here, we evaluate the phylogenetic relationships of the SEC14L-PITP superfamily in the green lineage. Compared to green algae, land plants have an extended set of SEC14L-PITPs with increasingly complex modular structure. SEC14-GOLD PITPs, present in land plants but not Chara, diverged to three functional subgroups, represented by the six PATELLIN (PATL) proteins in Arabidopsis. Based on the example of Arabidopsis PATL2, we dissect the functional domains for in vitro binding to phosphoinositides and liposomes and for plant cell membrane association. While the SEC14 domain and its CRAL-TRIO-N-terminal extension serve general membrane attachment of the protein, the C-terminal GOLD domain directs it to the plasma membrane by recognizing specific phosphoinositides. We discuss that the different domains of SEC14L-PITPs integrate developmental and environmental signals to control SEC14L-PITP-mediated membrane identity, important to initiate dynamic membrane events.
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Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Arabidopsis/química , Evolução Biológica , Membrana Celular/química , Perfilação da Expressão Gênica , Fosfatidilinositóis/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Filogenia , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
Eukaryotic organisms share many common features in terms of endomembrane trafficking. This fact has helped plant scientists to propose testable hypotheses on how plant intracellular membrane trafficking is achieved and regulated based on knowledge from yeast and mammals. However, when a new compartment has been identified in a plant cell that has a vesicle tethering complex located at a position which is completely different to its counterpart in yeast and mammalian cells, caution is demanded when interpreting possible interactions with other trafficking elements. This is exemplified by the recently discovered EMAC (ER and microtubule-associated compartment). It has been postulated that this compartment is the recipient of vacuolar sorting receptors (VSRs) transported retrogradely via "retromer vesicles" from a post-Golgi location. Unfortunately, this suggestion was based entirely on our knowledge of retromer from yeast and mammalian cells, and did not take into account the available literature on the composition, localization, and function of the plant retromer. It also lacked reference to recent contradictory findings on VSR trafficking. In this short article, we have tried to rectify this situation, pointing out that plant retromer may not function as a pentameric complex of two subunits: the retromer core and the sorting nexins.
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Transporte Biológico/fisiologia , Complexo de Golgi/química , Nexinas de Classificação/química , Vacúolos/químicaRESUMO
BACKGROUND: We need high-quality data to assess the determinants for COVID-19 severity in people with MS (PwMS). Several studies have recently emerged but there is great benefit in aligning data collection efforts at a global scale. OBJECTIVES: Our mission is to scale-up COVID-19 data collection efforts and provide the MS community with data-driven insights as soon as possible. METHODS: Numerous stakeholders were brought together. Small dedicated interdisciplinary task forces were created to speed-up the formulation of the study design and work plan. First step was to agree upon a COVID-19 MS core data set. Second, we worked on providing a user-friendly and rapid pipeline to share COVID-19 data at a global scale. RESULTS: The COVID-19 MS core data set was agreed within 48 hours. To date, 23 data collection partners are involved and the first data imports have been performed successfully. Data processing and analysis is an on-going process. CONCLUSIONS: We reached a consensus on a core data set and established data sharing processes with multiple partners to address an urgent need for information to guide clinical practice. First results show that partners are motivated to share data to attain the ultimate joint goal: better understand the effect of COVID-19 in PwMS.
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Infecções por Coronavirus/fisiopatologia , Esclerose Múltipla/terapia , Pneumonia Viral/fisiopatologia , Sistema de Registros , Betacoronavirus , COVID-19 , Infecções por Coronavirus/complicações , Infecções por Coronavirus/terapia , Coleta de Dados , Humanos , Disseminação de Informação , Cooperação Internacional , Esclerose Múltipla/complicações , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/terapia , Fatores de Risco , SARS-CoV-2 , Resultado do TratamentoRESUMO
The key basic helix-loop-helix (bHLH) transcription factor in iron (Fe) uptake, FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), is controlled by multiple signaling pathways, important to adjust Fe acquisition to growth and environmental constraints. FIT protein exists in active and inactive protein pools, and phosphorylation of serine Ser272 in the C-terminus, a regulatory domain of FIT, provides a trigger for FIT activation. Here, we use phospho-mutant activity assays and study phospho-mimicking and phospho-dead mutations of three additional predicted phosphorylation sites, namely at Ser221 and at tyrosines Tyr238 and Tyr278, besides Ser 272. Phospho-mutations at these sites affect FIT activities in yeast, plant, and mammalian cells. The diverse array of cellular phenotypes is seen at the level of cellular localization, nuclear mobility, homodimerization, and dimerization with the FIT-activating partner bHLH039, promoter transactivation, and protein stability. Phospho-mimicking Tyr mutations of FIT disturb fit mutant plant complementation. Taken together, we provide evidence that FIT is activated through Ser and deactivated through Tyr site phosphorylation. We therefore propose that FIT activity is regulated by alternative phosphorylation pathways.
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Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bioensaio/métodos , Mutação/genética , Sequência de Aminoácidos , Animais , Proteínas de Arabidopsis/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Células CHO , Cricetinae , Cricetulus , Modelos Biológicos , Fosforilação , Fosfotirosina/metabolismo , Multimerização Proteica , Estabilidade Proteica , Ativação Transcricional/genéticaRESUMO
Regulation of iron (Fe) acquisition and homeostasis is critical for plant survival. In Arabidopsis, Fe deficiency-induced bHLH039 forms a complex with the master regulator FIT and activates it to upregulate Fe acquisition genes. FIT is partitioned between cytoplasm and nucleus, whereby active FIT accumulates more in the nucleus than inactive FIT. At the same time, there is so far no information on the subcellular localization of bHLH039 protein and how it is controlled. We report here that the bHLH039 localization pattern changes depending on the presence of FIT in the cell. When expressed in cells lacking FIT, bHLH039 localizes predominantly in the cytoplasm, including cytoplasmic foci in close proximity to the plasma membrane. The presence of FIT enhances the mobility of bHLH039 and redirects the protein toward primarily nuclear localization, abolishing its accumulation in cytoplasmic foci. This FIT-dependent change in localization of bHLH039 found in transient fluorescent protein expression experiments was confirmed in both leaves and roots of Arabidopsis transgenic plants, stably expressing hemagglutinin-tagged bHLH039 in wild-type or fit mutant background. This posttranslational mechanism for intracellular partitioning of Fe-responsive transcription factors suggests a signaling cascade that translates Fe sensing at the plasma membrane to nuclear accumulation of the transcriptional regulators.
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Plants respond actively to changes in their environment. Variations in nutrient availability elicit substantial transcriptional reprogramming, and we aimed to systematically describe these adjustments and identify the regulators responsible. Using gene coexpression analysis based on 13 different nutrient availability anomalies, we defined and analyzed nutrient stress response signatures. We identified known regulators and could predict functions in nutrient responses for transcriptional regulators previously associated with other processes, thus linking development and environmental interaction. Three of the identified transcriptional regulators, PIF4, HY5, and NF-Y, known from their role in light signaling, targeted a substantial part of the network and may participate in remodeling the global Arabidopsis transcriptome in response to variations of nutrient availability. We present gene coexpression and transcriptional regulation networks, which can be used as tools to further explore regulatory events and dependencies even by users with basic informatics skills.
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In her 1929 essay A Room of One's Own, Virginia Wolf famously wrote, "One cannot think well, love well, sleep well, if one has not dined well." While this popular quote is perhaps not the most inspiring, it is an elegant reminder that food and the cultural practices surrounding food are paramount for our wellbeing. However, in our quest to feed a growing global population, we have become focused on increasing the production of a few staple crops and overlooked hundreds or thousands of locally and regionally important crops that may represent the future of agriculture. The growing interest in identifying and developing promising new crops and novel food sources prompted the 1st Cologne Conference on Food for Future, which took place between the 5 and 7th of September 2018 at the Rautenstrauch-Joest museum in Cologne, Germany. It offered a unique platform for researchers, journalists, politicians, and entrepreneurs to present and discuss their views, visions, and concerns on the topics of Food Security. This interdisciplinary meeting acted as a stage to cover diverse aspects of crop science, food research, and food production in the context of global food and nutrition security. Three sessions accommodated scientific contributions on the topics of "Orphan Crops", "Functional food", and "Innovative food sources and production systems", and two public events (a public lecture and a plenary discussion) engaged the citizens with informative discussions on relevant and mediatic topics. With delegates from Africa, Europe, and the United States of America, the conference aimed at building bridges between different communities through scientific exchange.