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During nutrient scarcity, plants can adapt their developmental strategy to maximize their chance of survival. Such plasticity in development is underpinned by hormonal regulation, which mediates the relationship between environmental cues and developmental outputs. In legumes, endosymbiosis with nitrogen fixing bacteria (rhizobia) is a key adaptation for supplying the plant with nitrogen in the form of ammonium. Rhizobia are housed in lateral root-derived organs termed nodules that maintain an environment conducive to Nitrogenase in these bacteria. Several phytohormones are important for regulating the formation of nodules, with both positive and negative roles proposed for gibberellin (GA). In this study, we determine the cellular location and function of bioactive GA during nodule organogenesis using a genetically-encoded second generation GA biosensor, GIBBERELLIN PERCEPTION SENSOR 2 in Medicago truncatula. We find endogenous bioactive GA accumulates locally at the site of nodule primordia, increasing dramatically in the cortical cell layers, persisting through cell divisions and maintaining accumulation in the mature nodule meristem. We show, through mis-expression of GA catabolic enzymes that suppress GA accumulation, that GA acts as a positive regulator of nodule growth and development. Furthermore, increasing or decreasing GA through perturbation of biosynthesis gene expression can increase or decrease the size of nodules, respectively. This is unique from lateral root formation, a developmental program that shares common organogenesis regulators. We link GA to a wider gene regulatory program by showing that nodule-identity genes induce and sustain GA accumulation necessary for proper nodule formation.
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Arbuscular mycorrhizal (AM) fungi engage with land plants in a widespread, mutualistic endosymbiosis which provides their hosts with increased access to nutrients and enhanced biotic and abiotic stress resistance. The potential for reducing fertiliser use and improving crop resilience has resulted in rapidly increasing scientific interest. Microscopic quantification of the level of AM colonization is of fundamental importance to this research, however the methods for recording and processing these data are time-consuming and tedious. In order to streamline these processes, we have developed AMScorer, an easy-to-use Excel spreadsheet, which enables the user to record data rapidly during from microscopy-based assays, and instantly performs the subsequent data processing steps. In our hands, AMScorer has more than halved the time required for data collection compared to paper-based methods. Subsequently, we developed AMReader, a user-friendly R package, which enables easy visualization and statistical analyses of data from AMScorer. These tools require only limited skills in Excel and R, and can accelerate research into AM symbioses, help researchers with variable resources to conduct research, and facilitate the storage and sharing of data from AM colonization assays. They are available for download at https://github.com/EJarrattBarnham/AMReader, along with an extensive user manual.
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BACKGROUND: Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause a severe neurological disorder characterised by early-onset epileptic seizures, autism and intellectual disability (ID). Impaired hippocampal function has been implicated in other models of monogenic forms of autism spectrum disorders and ID and is often linked to epilepsy and behavioural abnormalities. Many individuals with CDKL5 deficiency disorder (CDD) have null mutations and complete loss of CDKL5 protein, therefore in the current study we used a Cdkl5-/y rat model to elucidate the impact of CDKL5 loss on cellular excitability and synaptic function of CA1 pyramidal cells (PCs). We hypothesised abnormal pre and/or post synaptic function and plasticity would be observed in the hippocampus of Cdkl5-/y rats. METHODS: To allow cross-species comparisons of phenotypes associated with the loss of CDKL5, we generated a loss of function mutation in exon 8 of the rat Cdkl5 gene and assessed the impact of the loss of CDLK5 using a combination of extracellular and whole-cell electrophysiological recordings, biochemistry, and histology. RESULTS: Our results indicate that CA1 hippocampal long-term potentiation (LTP) is enhanced in slices prepared from juvenile, but not adult, Cdkl5-/y rats. Enhanced LTP does not result from changes in NMDA receptor function or subunit expression as these remain unaltered throughout development. Furthermore, Ca2+ permeable AMPA receptor mediated currents are unchanged in Cdkl5-/y rats. We observe reduced mEPSC frequency accompanied by increased spine density in basal dendrites of CA1 PCs, however we find no evidence supporting an increase in silent synapses when assessed using a minimal stimulation protocol in slices. Additionally, we found no change in paired-pulse ratio, consistent with normal release probability at Schaffer collateral to CA1 PC synapses. CONCLUSIONS: Our data indicate a role for CDKL5 in hippocampal synaptic function and raise the possibility that altered intracellular signalling rather than synaptic deficits contribute to the altered plasticity. LIMITATIONS: This study has focussed on the electrophysiological and anatomical properties of hippocampal CA1 PCs across early postnatal development. Studies involving other brain regions, older animals and behavioural phenotypes associated with the loss of CDKL5 are needed to understand the pathophysiology of CDD.
Assuntos
Modelos Animais de Doenças , Potenciação de Longa Duração , Proteínas Serina-Treonina Quinases , Receptores de AMPA , Receptores de N-Metil-D-Aspartato , Espasmos Infantis , Animais , Masculino , Ratos , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Região CA1 Hipocampal/fisiopatologia , Síndromes Epilépticas/genética , Síndromes Epilépticas/metabolismo , Potenciais Pós-Sinápticos Excitadores , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/fisiopatologia , Hipocampo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Células Piramidais/metabolismo , Células Piramidais/patologia , Receptores de AMPA/metabolismo , Receptores de AMPA/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Espasmos Infantis/genética , Espasmos Infantis/metabolismo , Sinapses/metabolismoRESUMO
Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.
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Neurônios , Receptores de N-Metil-D-Aspartato , Transfecção , Neurônios/metabolismo , Transfecção/métodos , Cultura Primária de Células , Receptores de N-Metil-D-Aspartato/genética , DNA Complementar/química , DNA Complementar/genética , Animais , Roedores , LipídeosRESUMO
Chronobiology investigations have revealed much about cellular and physiological clockworks but we are far from having a complete mechanistic understanding of the physiological and ecological implications. Here we present some unresolved questions in circadian biology research as posed by the editorial staff and guest contributors to the Journal of Circadian Rhythms. This collection of ideas is not meant to be comprehensive but does reveal the breadth of our observations on emerging trends in chronobiology and circadian biology. It is amazing what could be achieved with various expected innovations in technologies, techniques, and mathematical tools that are being developed. We fully expect strengthening mechanistic work will be linked to health care and environmental understandings of circadian function. Now that most clock genes are known, linking these to physiological, metabolic, and developmental traits requires investigations from the single molecule to the terrestrial ecological scales. Real answers are expected for these questions over the next decade. Where are the circadian clocks at a cellular level? How are clocks coupled cellularly to generate organism level outcomes? How do communities of circadian organisms rhythmically interact with each other? In what way does the natural genetic variation in populations sculpt community behaviors? How will methods development for circadian research be used in disparate academic and commercial endeavors? These and other questions make it a very exciting time to be working as a chronobiologist.
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Circadian Biology intersects with diverse scientific domains, intricately woven into the fabric of organismal physiology and behavior. The rhythmic orchestration of life by the circadian clock serves as a focal point for researchers across disciplines. This retrospective examination delves into several of the scientific milestones that have fundamentally shaped our contemporary understanding of circadian rhythms. From deciphering the complexities of clock genes at a cellular level to exploring the nuances of coupled oscillators in whole organism responses to stimuli. The field has undergone significant evolution lately guided by genetics approaches. Our exploration here considers key moments in the circadian-research landscape, elucidating the trajectory of this discipline with a keen eye on scientific advancements and paradigm shifts.
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Plants establish symbiotic associations with arbuscular mycorrhizal fungi (AMF) to facilitate nutrient uptake, particularly in nutrient-limited conditions. This partnership is rooted in the plant's ability to recognize fungal signaling molecules, such as chitooligosaccharides (chitin) and lipo-chitooligosaccharides. In the legume Medicago truncatula, chitooligosaccharides trigger both symbiotic and immune responses via the same lysin-motif-receptor-like kinases (LysM-RLKs), notably CERK1 and LYR4. The nature of plant-fungal engagement is opposite according to the outcomes of immunity or symbiosis signaling, and as such, discrimination is necessary, which is challenged by the dual roles of CERK1/LYR4 in both processes. Here, we describe a LysM-RLK, LYK8, that is functionally redundant with CERK1 for mycorrhizal colonization but is not involved in chitooligosaccharides-induced immunity. Genetic mutation of both LYK8 and CERK1 blocks chitooligosaccharides-triggered symbiosis signaling, as well as mycorrhizal colonization, but shows no further impact on immunity signaling triggered by chitooligosaccharides, compared with the mutation of CERK1 alone. LYK8 interacts with CERK1 and forms a receptor complex that appears essential for chitooligosaccharides activation of symbiosis signaling, with the lyk8/cerk1 double mutant recapitulating the impact of mutations in the symbiosis signaling pathway. We conclude that this novel receptor complex allows chitooligosaccharides activation specifically of symbiosis signaling and helps the plant to differentiate between activation of these opposing signaling processes.
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Quitina , Quitosana , Medicago truncatula , Micorrizas , Proteínas de Plantas , Simbiose , Micorrizas/fisiologia , Quitina/metabolismo , Medicago truncatula/microbiologia , Medicago truncatula/metabolismo , Medicago truncatula/imunologia , Medicago truncatula/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Imunidade Vegetal , Oligossacarídeos/metabolismo , Raízes de Plantas/microbiologia , Raízes de Plantas/metabolismoRESUMO
A key step in understanding the results of biological experiments is visualization of the data. Many laboratory experiments contain a range of measurements that exist within a hierarchy of interdependence. An automated and facile way to visualize and interrogate such multilevel data, across many experimental variables, would (i) lead to improved understanding of the results, (ii) help to avoid misleading interpretation of statistics and (iii) easily identify outliers and sources of batch and confounding effects. While many excellent graphing solutions already exist, they are often geared towards the production of publication-ready plots and the analysis of a single variable at a time, require programming expertise or are unnecessarily complex for the task at hand. Here, we present Laboratory Automated Interrogation of Data (LAB-AID), an interactive tool specifically designed to automatically visualize and query hierarchical data resulting from biological experiments.
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The conservation of GOLVEN (GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide encoding genes across plant genomes capable of forming roots or root-like structures underscores their potential significance in the terrestrial adaptation of plants. This study investigates the function and role of GOLVEN peptide-coding genes in Medicago truncatula. Five out of fifteen GLV/RGF genes were notably upregulated during nodule organogenesis and were differentially responsive to nitrogen deficiency and auxin treatment. Specifically, the expression of MtGLV9 and MtGLV10 at nodule initiation sites was contingent upon the NODULE INCEPTION transcription factor. Overexpression of these five nodule-induced GLV genes in hairy roots of M. truncatula and application of their synthetic peptide analogues led to a decrease in nodule count by 25-50%. Uniquely, the GOLVEN10 peptide altered the positioning of the first formed lateral root and nodule on the primary root axis, an observation we term 'noduletaxis'; this decreased the length of the lateral organ formation zone on roots. Histological section of roots treated with synthetic GOLVEN10 peptide revealed an increased cell number within the root cortical cell layers without a corresponding increase in cell length, leading to an elongation of the root likely introducing a spatiotemporal delay in organ formation. At the transcription level, the GOLVEN10 peptide suppressed expression of microtubule-related genes and exerted its effects by changing expression of a large subset of Auxin responsive genes. These findings advance our understanding of the molecular mechanisms by which GOLVEN peptides modulate root morphology, nodule ontogeny, and interactions with key transcriptional pathways.
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Regulação da Expressão Gênica de Plantas , Medicago truncatula , Proteínas de Plantas , Raízes de Plantas , Nódulos Radiculares de Plantas , Medicago truncatula/genética , Medicago truncatula/crescimento & desenvolvimento , Medicago truncatula/metabolismo , Medicago truncatula/efeitos dos fármacos , Medicago truncatula/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/efeitos dos fármacos , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/farmacologia , Nodulação/genética , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/efeitos dos fármacos , Peptídeos/metabolismo , Peptídeos/genéticaRESUMO
Legumes produce specialized root nodules that are distinct from lateral roots in morphology and function, with nodules intracellularly hosting nitrogen-fixing bacteria. We have previously shown that a lateral root program underpins nodule initiation, but there must be additional developmental regulators that confer nodule identity. Here, we show two members of the LIGHT-SENSITIVE SHORT HYPOCOTYL (LSH) transcription factor family, predominantly known to define shoot meristem complexity and organ boundaries, function as regulators of nodule organ identity. In parallel to the root initiation program, LSH1/LSH2 recruit a program into the root cortex that mediates the divergence into nodules, in particular with cell divisions in the mid-cortex. This includes regulation of auxin and cytokinin, promotion of NODULE ROOT1/2 and Nuclear Factor YA1, and suppression of the lateral root program. A principal outcome of LSH1/LSH2 function is the production of cells able to accommodate nitrogen-fixing bacteria, a key feature unique to nodules.
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Medicago truncatula , Medicago truncatula/genética , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/microbiologia , Hipocótilo/genética , Hipocótilo/metabolismo , Citocininas/genética , Meristema/metabolismo , Simbiose/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/metabolismoRESUMO
Mosquitoes express a rich repertoire of daily 24-hour rhythms in biochemistry, physiology, and behavior. The nocturnal Anopheles and Culex and diurnal Aedes mosquitoes are major vectors of human disease, transmitting parasites and arboviruses, such as malaria and dengue. In this review, we explore the role that 24-hour diel and circadian rhythms play in shaping the temporal life of the mosquito. We focus on recent advances in our understanding of behavioral rhythms, focusing on locomotor/flight activity, host-seeking, biting/blood feeding, and mating. We examine the molecular circadian clock, photocycle, and light signals, which in combination shape the mosquito 24-hour temporal program. We address species- and sex-specific differences and highlight important selective pressures from dynamically changing environments. This work also provides new insights into disease transmission, insect control, and future experimental design.
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Ritmo Circadiano , Mosquitos Vetores , Animais , Mosquitos Vetores/fisiologia , Culicidae/fisiologia , Comportamento AlimentarRESUMO
White matter abnormalities, related to poor cerebral perfusion, are a core feature of small vessel cerebrovascular disease, and critical determinants of vascular cognitive impairment and dementia. Despite this importance there is a lack of treatment options. Proliferation of microglia producing an expanded, reactive population and associated neuroinflammatory alterations have been implicated in the onset and progression of cerebrovascular white matter disease, in patients and in animal models, suggesting that targeting microglial proliferation may exert protection. Colony-stimulating factor-1 receptor (CSF1R) is a key regulator of microglial proliferation. We found that the expression of CSF1R/Csf1r and other markers indicative of increased microglial abundance are significantly elevated in damaged white matter in human cerebrovascular disease and in a clinically relevant mouse model of chronic cerebral hypoperfusion and vascular cognitive impairment. Using the mouse model, we investigated long-term pharmacological CSF1R inhibition, via GW2580, and demonstrated that the expansion of microglial numbers in chronic hypoperfused white matter is prevented. Transcriptomic analysis of hypoperfused white matter tissue showed enrichment of microglial and inflammatory gene sets, including phagocytic genes that were the predominant expression modules modified by CSF1R inhibition. Further, CSF1R inhibition attenuated hypoperfusion-induced white matter pathology and rescued spatial learning impairments and to a lesser extent cognitive flexibility. Overall, this work suggests that inhibition of CSF1R and microglial proliferation mediates protection against chronic cerebrovascular white matter pathology and cognitive deficits. Our study nominates CSF1R as a target for the treatment of vascular cognitive disorders with broader implications for treatment of other chronic white matter diseases.
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Transtornos Cerebrovasculares , Transtornos Cognitivos , Disfunção Cognitiva , Leucoencefalopatias , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos , Substância Branca , Animais , Camundongos , Transtornos Cerebrovasculares/metabolismo , Transtornos Cerebrovasculares/patologia , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/patologia , Disfunção Cognitiva/metabolismo , Modelos Animais de Doenças , Leucoencefalopatias/genética , Leucoencefalopatias/metabolismo , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Receptores de Fator Estimulador de Colônias/metabolismo , Substância Branca/patologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismoRESUMO
Los cuadros delirantes que se presentan en el curso de los trastornos orgánico-cerebrales constituyen un tema de interés psiquiátrico tanto por la frecuencia con que se presentan en la clínica, sobre todo gerontopsiquiátrica, como por la dificultad nosológica que entrañan. Desde un punto de vista clásico la proliferación de términos que han ido surgiendo para denominar estos cuadros psicóticos ha sido en no pocos casos motivo de confusión. Desde una perspectiva más actual las denominaciones clásicas (psicosis exógena, psicosis de fundamento corporal conocido, psicosis somatógena o psicosis orgánica/sintomática) engloban el mismo concepto, a saber: aquel grupo de psicosis condicionadas por una enfermedad somática demostrable en la que además de los factores biológicos - que ocupan un primer y preponderante lugar - intervienen también, desde el punto de vista etiopatogénico, los factores psicológicos y ambientales. Uno de los aspectos nucleares de estas psicosis es su planteamiento como entidades nosográficas. La mayoría de los autores actuales están de acuerdo en admitir dos grandes bloques: psicosis agudas y psicosis crónicas, al margen de todo compromiso etiopatogénico. Ocurre igual con los criterios diagnósticos internacionales (DSM-IV y CIE-10) que abordan a estas psicosis desde un planteamiento sindrómico (AU)