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Achieving global restoration targets poses challenges including the need for long-term research and effective monitoring of success, fostering collaborations across diverse fields and actors, ensuring the availability of suitable reference ecosystems, and securing sustained funding. Yet, these conditions are often lacking, limiting the effectiveness of restoration. We provide an overview of ecological restoration practices in the pan-European region of the Long-term Ecological Research Network (eLTER) and demonstrate the importance of eLTER and its potential contributions to support the implementation of the EU Nature Restoration Law. We developed an online questionnaire to collect information on eLTER restoration experts and restoration projects details including the use of eLTER contributions (e.g. infrastructure, data and knowledge), between November 2021 and March 2022. We identified 62 restoration experts and 42 restoration projects from 18 countries. Our results show that eLTER restoration expertise covers most of the European habitats, diverse degradation states and restoration techniques. Most restoration projects (78%) involved long-term monitoring exceeding the average project lifespan, which has proven necessary to achieve restoration success. No common protocol was used for monitoring and evaluation or cost-benefit estimates, but respondents reported effective projects, mostly financed from national funds, and benefits in five ecosystem services on average covered per project. Key eLTER contributions included providing reference ecosystems, biotic and abiotic background data, and interdisciplinary discussion or stakeholder management. Ecological restoration is time intensive and requires long-term research and monitoring standardization to fully understand the restoration process and to ensure comparability across ecosystems. The eLTER network can help address these challenges providing added-value contributions through its infrastructure, long-term datasets, diversity of expertise and strategies that can help identify best restoration practices and support the EU Nature Restoration Law. Finally, additional and long-term funding from the EU and the private sector is needed to achieve global larger-scale restoration targets.
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Conservação dos Recursos Naturais , Ecologia , Ecossistema , Europa (Continente) , Conservação dos Recursos Naturais/métodos , Inquéritos e Questionários , Recuperação e Remediação AmbientalRESUMO
Ca2+/calmodulin (Ca2+/CaM) interaction with connexins (Cx) is well-established; however, the mechanistic basis of regulation of gap junction function by Ca2+/CaM is not fully understood. Ca2+/CaM is predicted to bind to a domain in the C-terminal portion of the intracellular loop (CL2) in the vast majority of Cx isoforms and for a number of Cx-s this prediction has proved correct. In this study, we investigate and characterise both Ca2+/CaM and apo-CaM binding to selected representatives of each of the α, ß and γ connexin family to develop a better mechanistic understanding of CaM effects on gap junction function. The affinity and kinetics Ca2+/CaM and apo-CaM interactions of CL2 peptides of ß-Cx32, γ-Cx35, α-Cx43, α-Cx45 and α-Cx57 were investigated. All five Cx CL2 peptides were found to have high affinity for Ca2+/CaM with dissociation constants (Kd(+Ca)) from 20 to 150 nM. The limiting rate of binding and the rates of dissociation covered a broad range. In addition, we obtained evidence for high affinity Ca2+-independent interaction of all five peptides with CaM, consistent with CaM remaining anchored to gap junctions in resting cells. However, for the α-Cx45 and α-Cx57 CL2 peptides, Ca2+-dependent association at resting [Ca2+] of 50-100 nM is indicated in these complexes as one of the CaM Ca2+ binding sites displays high affinity with Kd of 70 and 30 nM for Ca2+, respectively. Furthermore, complex conformational changes were observed in peptide-apo-CaM complexes with the structure of CaM compacted or stretched by the peptide in a concentration dependent manner suggesting that the CL2 domain may undergo helix-to-coil transition and/or forms bundles, which may be relevant in the hexameric gap junction. We demonstrate inhibition of gap junction permeability by Ca2+/CaM in a dose dependent manner, further cementing Ca2+/CaM as a regulator of gap junction function. The motion of a stretched CaM-CL2 complex compacting upon Ca2+ binding may bring about the Ca2+/CaM block of the gap junction pore by a push and pull action on the CL2 C-terminal hydrophobic residues of transmembrane domain 3 (TM3) in and out of the membrane.
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Calmodulina , Conexinas , Conexinas/metabolismo , Calmodulina/metabolismo , Junções Comunicantes/metabolismo , Ligação Proteica , Sinalização do Cálcio , Sítios de Ligação , Cálcio/metabolismoRESUMO
BACKGROUND: Office workers near retirement tend to be sedentary and can be prone to mobility limitations and diseases. We examined the dose effects of exergaming volume and duration of detraining on motor and cognitive function in office workers at late midlife to reduce sedentariness and mobility limitations. METHODS: In an assessor-blinded randomized trial, 160 workers aged 55-65 years performed physically active video games in a nonimmersive form of virtual reality (exergaming) in small, supervised groups for 1 h, 1×, 2×, or 3×/week for 8 weeks followed by detraining for 8 and 16 weeks. Exergaming comprises high-intensity, full-body sensorimotor coordination, balance, endurance, and strengthening exercises. The primary outcome was the 6-minute walk test (6MWT), and secondary outcomes were body mass, self-reported physical activity, sleep quality, Berg Balance Scale, Short Physical Performance Battery, fast gait speed, dynamic balance, heart rate recovery after step test, and 6 cognitive tests. RESULTS: The 3 groups were not different in any of the outcomes at baseline (all p > 0.05). The outcomes were stable and had acceptable reliability (intraclass correlation coefficients ≥0.334) over an 8-week control period. Training produced an inverted U-shaped dose response of no (1×), most (2×), and medium (3×/week) effects of exergaming volume in most motor and selected cognitive outcomes. The distance walked in the 6MWT (primary outcome) increased most (94 m, 19%, p < 0.05), medium (57 m, 12%, p < 0.05), and least (4 m, 1%) after exergaming 2×, 3×, or 0× (control) (all different p < 0.05). The highest responders tended to retain the exercise effects over 8 weeks of detraining, independent of training volume. This maintenance effect was less consistent after 16 weeks of detraining. CONCLUSION: Less was more during training and lasted longer after detraining. A medium dose volume of exergaming produced the largest clinically meaningful improvements in mobility and selected cognitive tests in 60-year-old office workers with mild mobility limitations and intact cognition.
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Exercício Físico , Equilíbrio Postural , Terapia por Exercício , Humanos , Reprodutibilidade dos Testes , CaminhadaRESUMO
OBJECTIVES: To determine the effects of exergaming on quality of life (QoL), motor, and clinical symptoms in subacute stroke patients. DESIGN: A pseudorandomized controlled trial, using a before-after test design. SETTING: University hospital. PARTICIPANTS: Subacute, ischemic stroke outpatients (N=3857), 680 of whom were randomized and 641 completed the study. INTERVENTIONS: We determined the effects of 5 times a week twice daily (EX2; 50 sessions; n=286) and once daily (EX1; 25 sessions; n=272) exergaming and low-intensity standard care (control [CON]; 25 sessions; n=83) on clinical, mobility, blood pressure (BP), and QoL outcomes. MAIN OUTCOME MEASURES: The primary outcome was Modified Rankin Scale. Secondary outcomes were activities of daily living, 5 aspects of health-related QoL, Beck Depression Inventory, 6-minute walk test (6MWT), Berg Balance Scale (BBS), and static balance (center of pressure). RESULTS: During exercise, the peak heart rate was 134, 134, and 126 beats per minute in the EX2, EX1, and CON groups, respectively. mRS improved similarly in the EX2 (-1.8; effect size, d=-4.0) and EX1 (-1.4; d=-2.6) groups, but more than in the CON group (-0.7; d=-0.6). QoL, Barthel Index, BBS, 6MWT, and standing posturography improved more in the EX2 group and the same in the EX1 and CON groups. Systolic and diastolic resting BP decreased more in the EX2 and EX1 groups than in the CON group. The intervention effects did not differ between men (n=349) and women (n=292). CONCLUSIONS: Twice daily compared with once daily high-intensity exergaming or once daily lower intensity standard care produced superior effects on clinical and motor symptoms, BP, and QoL in male and female subacute ischemic stroke participants.
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Terapia por Exercício/métodos , AVC Isquêmico/reabilitação , Reabilitação do Acidente Vascular Cerebral/métodos , Jogos de Vídeo , Atividades Cotidianas , Idoso , Pressão Sanguínea , Comorbidade , Feminino , Marcha/fisiologia , Frequência Cardíaca/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Limitação da Mobilidade , Equilíbrio Postural/fisiologia , Qualidade de Vida , Método Simples-CegoRESUMO
Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to â¼10 Hz. However, to resolve glutamate dynamics during high-frequency bursts, faster indicators are required. Here, we report the development of fast (iGlu f ) and ultrafast (iGlu u ) variants with comparable brightness but increased Kd for glutamate (137 µM and 600 µM, respectively). Compared with iGluSnFR, iGlu u has a sixfold faster dissociation rate in vitro and fivefold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGlu u is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGlu u responses during 100-Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high-frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons.
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Ácido Glutâmico/metabolismo , Hipocampo/fisiologia , Terminações Pré-Sinápticas/fisiologia , Células Piramidais/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Masculino , Plasticidade Neuronal , Técnicas de Patch-Clamp , Ratos , Ratos WistarRESUMO
We have developed probes based on the bacterial periplasmic glutamate/aspartate binding protein with either an endogenously fluorescent protein or a synthetic fluorophore as the indicator of glutamate binding for studying the kinetic mechanism of glutamate binding. iGluSnFR variants termed iGluh, iGlum, and iGlul cover a broad range of Kd-s (5.8 µM and 2.1 and 50 mM, respectively), and a novel fluorescently labeled indicator, Fl-GluBP, has a Kd of 9.7 µM. The fluorescence response kinetics of all the probes are consistent with a two-step mechanism involving ligand binding and isomerization either of the apo or the ligand-bound binding protein. Although the previously characterized ultrafast indicators iGluu and iGluf had monophasic fluorescence enhancement that occurred in the rate limiting isomerization step, the sensors described here all have biphasic binding kinetics with fluorescence increases occurring both in the glutamate binding and the isomerization steps. For iGlum and iGlul, the data indicate prebinding conformational change followed by ligand binding. In contrast, for iGluh and Fl-GluBP, glutamate binding is followed by isomerization. Thus, the effects of structural heterogeneity introduced by single amino acid changes around the binding site on the kinetic path of interactions with glutamate are revealed. Remarkably, glutamate binding with a diffusion-limited rate constant to iGluh and Fl-GluBP is detected for the first time, hinting at the underlying mechanism of the supremely rapid activation of the highly homologous α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor by glutamate binding.
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Técnicas Biossensoriais/métodos , Ácido Glutâmico/análise , Proteínas Luminescentes/química , Ácido Glutâmico/química , Cinética , Ligantes , Modelos Moleculares , Conformação ProteicaRESUMO
Changes in the balance between glutamate (Glu) release and uptake may stimulate synaptic reorganization and even synapse loss. In the case of neurodegeneration, a mismatch between astroglial Glu uptake and presynaptic Glu release could be detected if both parameters were assessed independently and at a single-synapse level. This has now become possible due to a new imaging assay with the genetically encoded ultrafast Glu sensor iGlu u We report findings from individual corticostriatal synapses in acute slices prepared from mice of either sex that were >1 year of age. Contrasting patterns of short-term plasticity and a size criterion identified two classes of terminals, presumably corresponding to the previously defined IT (intratelencephalic) and PT (pyramidal tract) synapses. The latter exhibited a higher degree of frequency potentiation/residual Glu accumulation and were selected for our first iGlu u single-synapse study in Q175 mice, a model of Huntington's disease (HD). In HD mice, the decay time constant of the perisynaptic Glu concentration (TauD), as an indicator of uptake, and the peak iGlu u amplitude, as an indicator of release, were prolonged and reduced, respectively. Treatment of WT preparations with the astrocytic Glu uptake blocker TFB-TBOA (100 nm) mimicked the TauD changes in homozygotes. Considering the largest TauD values encountered in WT, â¼40% of PT synapses tested in Q175 heterozygotes can be classified as dysfunctional. Moreover, HD but not WT synapses exhibited a positive correlation between TauD and the peak amplitude of iGlu u Finally, EAAT2 (excitatory amino acid transport protein 2) immunoreactivity was reduced next to corticostriatal terminals. Thus, astrocytic Glu transport remains a promising target for therapeutic intervention.SIGNIFICANCE STATEMENT Alterations in astrocytic Glu uptake can play a role in synaptic plasticity and neurodegeneration. Until now, the sensitivity of synaptic responses to pharmacological transport block and the resulting activation of NMDA receptors were regarded as reliable evidence for a mismatch between synaptic uptake and release. But the latter parameters are interdependent. Using a new genetically encoded sensor to monitor extracellular glutamate concentration ([Glu]) at individual corticostriatal synapses, we can now quantify the time constant of perisynaptic [Glu] decay (as an indicator of uptake) and the maximal [Glu] elevation next to the active zone (as an indicator of Glu release). The results provide a positive answer to the hitherto unresolved question of whether neurodegeneration (e.g., Huntington's disease) associates with a glutamate uptake deficit at tripartite excitatory synapses.
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Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Ácido Glutâmico/metabolismo , Doença de Huntington/metabolismo , Sinapses/metabolismo , Vias Aferentes/metabolismo , Animais , Técnicas Biossensoriais/métodos , Modelos Animais de Doenças , Feminino , Masculino , Potenciais da Membrana , Camundongos Transgênicos , Plasticidade NeuronalRESUMO
Genetically encoded calcium indicators (GECIs) are useful reporters of cell-signaling, neuronal, and network activities. We have generated novel fast variants and investigated the kinetic mechanisms of two recently developed red-fluorescent GECIs (RGECIs), mApple-based jRGECO1a and mRuby-based jRCaMP1a. In the formation of fluorescent jRGECO1a and jRCaMP1a complexes, calcium binding is followed by rate-limiting isomerization. However, fluorescence decay of calcium-bound jRGECO1a follows a different pathway from its formation: dissociation of calcium occurs first, followed by the peptide, similarly to GCaMP-s. In contrast, fluorescence decay of calcium-bound jRCaMP1a occurs by the reversal of the on-pathway: peptide dissociation is followed by calcium. The mechanistic differences explain the generally slower off-kinetics of jRCaMP1a-type indicators compared with GCaMP-s and jRGECO1a-type GECI: the fluorescence decay rate of f-RCaMP1 was 21 s-1, compared with 109 s-1 for f-RGECO1 and f-RGECO2 (37 °C). Thus, the CaM-peptide interface is an important determinant of the kinetic responses of GECIs; however, the topology of the structural link to the fluorescent protein demonstrably affects the internal dynamics of the CaM-peptide complex. In the dendrites of hippocampal CA3 neurons, f-RGECO1 indicates calcium elevation in response to a 100 action potential train in a linear fashion, making the probe particularly useful for monitoring large-amplitude, fast signals, e.g. those in dendrites, muscle cells, and immune cells.
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Antraquinonas/metabolismo , Sinalização do Cálcio , Cálcio/análise , Cálcio/metabolismo , Antraquinonas/química , Células HEK293 , Humanos , Cinética , Modelos MolecularesRESUMO
Improving efficiency of oligonucleotide-directed mutagenesis (ODM) is a prerequisite for wide application of this gene-editing approach in plant science and breeding. Here we have tested histone deacetylase inhibitor treatments for induction of relaxed chromatin and for increasing the efficiency of ODM in cultured maize cells. For phenotypic assay we produced transgenic maize cell lines expressing the non-functional Green Fluorescent Protein (mGFP) gene carrying a TAG stop codon. These transgenic cells were bombarded with corrective oligonucleotide as editing reagent to recover GFP expression. Repair of green fluorescent protein function was monitored by confocal fluorescence microscopy and flow cytometry was used for quantification of correction events. Sequencing PCR fragments of the GFP gene from corrected cells indicated a nucleotide exchange in the stop codon (TAG) from T to G nucleotide that resulted in the restoration of GFP function. We show that pretreatment of maize cells with sodium butyrate (5-10 mM) and nicotinamide (1-5 mM) as known inhibitors of histone deacetylases can cause elevated chromatin sensitivity to DNase I that was visualized in agarose gels and confirmed by the reduced presence of intact PCR template for the inserted exogenous mGFP gene. Maize cells with more relaxed chromatin could serve as an improved recipient for targeted nucleotide exchange as indicated by an average of 2.67- to 3.62-fold increase in GFP-positive cells. Our results stimulate further studies on the role of the condition of the recipient cells in ODM and testing the application of chromatin modifying agents in other, programmable nuclease-based genome-editing techniques in higher plants.
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Cromatina/genética , Edição de Genes , Inibidores de Histona Desacetilases/metabolismo , Oligonucleotídeos/metabolismo , Proteínas de Plantas/genética , Zea mays/genética , Cromatina/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Zea mays/metabolismoRESUMO
Amyloid ß-peptides (Aß) are a major hallmark of Alzheimer's disease (AD) and their neurotoxicity develop with cytosolic calcium dysregulation. On the other hand, calmodulin (CaM), a protein which plays a major multifunctional role in neuronal calcium signaling, has been shown to be involved in the regulation of non-amyloidogenic processing of amyloid ß precursor protein (APP). Using fluorescent 6-bromoacetyl-2-dimethylaminonaphthalene derivatives of CaM, Badan-CaM, and human amyloid ß(1-42) HiLyte™-Fluor555, we show in this work that Aß binds with high affinity to CaM through the neurotoxic Aß25-35 domain. In addition, the affinity of Aß for calcium-saturated CaM conformation is approximately 20-fold higher than for CaM conformation in the absence of calcium (apo-CaM). Moreover, the value of Kd of 0.98 ± 0.11 nM obtained for Aß1-42 dissociation from CaM saturated by calcium points out that CaM is one of the cellular targets with highest affinity for neurotoxic Aß peptides. A major functional consequence of Aß-CaM interaction is that it slowdowns Aß fibrillation. The novel and high affinity interaction between calmodulin and Aß shown in this work opens a yet-unexplored gateway to further understand the neurotoxic effect of Aß in different neural cells and also to address the potential of calmodulin and calmodulin-derived peptides as therapeutic agents in AD.
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Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/ultraestrutura , Cálcio/química , Calmodulina/química , Calmodulina/ultraestrutura , Simulação de Acoplamento Molecular/métodos , Sítios de Ligação , Modelos Químicos , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
The biomass productivity of the energy willow Salix viminalis as a short-rotation woody crop depends on organ structure and functions that are under the control of genome size. Colchicine treatment of axillary buds resulted in a set of autotetraploid S. viminalis var. Energo genotypes (polyploid Energo [PP-E]; 2n = 4x = 76) with variation in the green pixel-based shoot surface area. In cases where increased shoot biomass was observed, it was primarily derived from larger leaf size and wider stem diameter. Autotetraploidy slowed primary growth and increased shoot diameter (a parameter of secondary growth). The duplicated genome size enlarged bark and wood layers in twigs sampled in the field. The PP-E plants developed wider leaves with thicker midrib and enlarged palisade parenchyma cells. Autotetraploid leaves contained significantly increased amounts of active gibberellins, cytokinins, salicylic acid, and jasmonate compared with diploid individuals. Greater net photosynthetic CO2 uptake was detected in leaves of PP-E plants with increased chlorophyll and carotenoid contents. Improved photosynthetic functions in tetraploids were also shown by more efficient electron transport rates of photosystems I and II. Autotetraploidization increased the biomass of the root system of PP-E plants relative to diploids. Sections of tetraploid roots showed thickening with enlarged cortex cells. Elevated amounts of indole acetic acid, active cytokinins, active gibberellin, and salicylic acid were detected in the root tips of these plants. The presented variation in traits of tetraploid willow genotypes provides a basis to use autopolyploidization as a chromosome engineering technique to alter the organ development of energy plants in order to improve biomass productivity.
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Folhas de Planta/genética , Raízes de Plantas/genética , Caules de Planta/genética , Salix/genética , Tetraploidia , Biomassa , Carotenoides/metabolismo , Clorofila/metabolismo , Duplicação Cromossômica , Cromossomos de Plantas/genética , Diploide , Genoma de Planta/genética , Genótipo , Microscopia Confocal , Fenótipo , Fotossíntese/genética , Fotossíntese/fisiologia , Casca de Planta/genética , Casca de Planta/fisiologia , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/fisiologia , Raízes de Plantas/fisiologia , Caules de Planta/fisiologia , Salix/fisiologia , Madeira/genética , Madeira/fisiologiaRESUMO
PURPOSE: In-stent restenosis (ISR) following internal carotid artery (ICA) stenting is relatively common with an estimated incidence of 5%. Treatment options include repeat angioplasty with conventional or drug-eluting balloons (DEB), repeat stent angioplasty and surgical intervention. Application of DEB in ISR of the coronary and peripheral arteries is an established method; however, data on DEB treatment of ICA ISR are sparse. In this work, results from a retrospective cohort of 45 patients harboring 46 ICA ISR lesions treated with DEB angioplasty are presented. METHODS: Clinical, procedural and imaging data from DEB angioplasty treatment of 46 high-grade ICA ISR lesions in 45 patients, performed between 2013 and 2021 were collected. A single type of DEB (Elutax, Aachen Resonance, Aachen, Germany) was used in all procedures. Imaging follow-up was performed by regular Doppler ultrasound (DUS), verified by computed tomography angiography (CTA) in cases suspicious for a recurrent ISR. RESULTS: Technical success was 100%. Intraprocedural and postprocedural complications were not encountered. Clinical follow-up was obtained in all patients. Recurrent stroke in the affected territory was not encountered. A recurrent ISR following DEB treatment was confirmed by DUS and CTA in 4/46 (8.7%) of the lesions and were retreated with DEB. A third recurrent ISR occurred in a single case (2%) and following a second DEB retreatment there were no signs of a fourth recurrence after 36 months follow-up. CONCLUSION: The use of DEB angioplasty is a safe and effective treatment of ICA ISR lesions, yielding significantly better results compared to other modalities. Randomized multicenter studies are warranted.
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Reestenose Coronária , Stents Farmacológicos , Humanos , Artéria Carótida Interna/diagnóstico por imagem , Estudos Retrospectivos , Reestenose Coronária/etiologia , Reestenose Coronária/terapia , Stents Farmacológicos/efeitos adversos , Stents/efeitos adversos , Constrição Patológica , Resultado do TratamentoRESUMO
In this study, we examined over 200-year-old Ginkgo biloba L. specimens under different environmental conditions. The overall aim was to explore which factors influence their vitality and general fitness in urban environments and thus their ability to tolerate stressful habitats. In order to determine this, we used a number of different methods, including histological examinations (stomatal density and size) and physiological measurements (peroxidase enzyme activity), as well as assessing the air pollution tolerance index (APTI). The investigation of the genetic relationships between individuals was performed using flow cytometry and miRNA marker methods. The genetic tests revealed that all individuals are diploid, whereas the lus-miR168 and lus-miR408 markers indicated a kinship relation between them. These results show that the effect of different habitat characteristics can be detected through morphological and physiological responses, thus indicating relatively higher stress values for all studied individuals. A significant correlation can be found between the level of adaptability and the relatedness of the examined individuals. These results suggest that Ginkgo biloba L. is well adapted to an environment with increased stress factors and therefore suitable for use in urban areas.
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Introduction: Genetically encoded biosensors for monitoring intracellular calcium changes have advanced our understanding of cell signaling and neuronal activity patterns in health and disease. Successful application of GCaMP biosensors to a wide range of biological questions requires that sensor properties such as brightness and dynamic range, ligand affinity and response kinetics be tuned to the specific conditions or phenomena to be investigated. Random as well as rational targeted mutations of such sensor molecules have led to a number of important breakthroughs in this field, including the calcium sensors GCaMP6f and GCaMP6fu. jGCaMP8f of the most recently developed generation is promising a step-change in in vivo imaging with further increased fluorescence dynamic range. Here, we critically examine the biophysical properties of jGCaMP8f and report development by rational design of two novel variants of jGCaMP8f. Methods: We determined the in vitro biophysical properties of jGCaMP8f and selected variants by fluorescence spectroscopies and compared their performance monitoring intracellular Ca2+ transients with previously developed fast and bright GCaMP sensors by live cell imaging. Results: We demonstrate that the physiologically highly relevant Mg2+ not only majorly affects the kinetic responses of GCaMPs but also their brightness and fluorescence dynamic range. We developed novel variants jGCaMP8f L27A which has threefold faster off-kinetics and jGCaMP8f F366H which shows a â¼3-fold greater dynamic range than jGCaMP8f, in vitro as well as in HEK293T cells and endothelial cell line HUVEC in response to ATP stimulation. Discussion: We discuss the importance of optimization of biosensors for studying neurobiology in the context of the novel variants of jGCaMP8f. The jGCaMP8f F366H variant with a large dynamic range has the potential to improve in vivo imaging outcomes with increased signal-to-noise ratio. The L27A variant with faster kinetics than jGCaMP8f has larger cellular responses than previous fast GCaMP variants. The jGCaMP8f generation and novel improved variants presented here will further increase the application potential of GECIs in health and disease.
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The restoration of invasion-resistant plant communities is an important strategy to combat the negative impacts of alien invasions. Based on a systematic review and meta-analysis of seed-based ecological restoration experiments, here we demonstrate the potential of functional similarity, seeding density and priority effect in increasing invasion resistance. Our results indicate that native priority is the most promising mechanism to control invasion that can reduce the performance of invasive alien species by more than 50%. High-density seeding is effective in controlling invasive species, but threshold seeding rates may exist. Overall seeding functionally similar species do not have a significant effect. Generally, the impacts are more pronounced on perennial and grassy invaders and on the short-term. Our results suggest that biotic resistance can be best enhanced by the early introduction of native plant species during restoration. Seeding of a single species with high functional similarity to invasive alien species is unpromising, and instead, preference should be given to high-density multifunctional seed mixtures, possibly including native species favored by the priority effect. We highlight the need to integrate research across geographical regions, global invasive species and potential resistance mechanisms.
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Espécies Introduzidas , Plantas , SementesRESUMO
Successful use of woody species in reducing climatic and environmental risks of energy shortage and spreading pollution requires deeper understanding of the physiological functions controlling biomass productivity and phytoremediation efficiency. Targets in the breeding of energy willow include the size and the functionality of the root system. For the combination of polyploidy and heterosis, we have generated triploid hybrids (THs) of energy willow by crossing autotetraploid willow plants with leading cultivars (Tordis and Inger). These novel Salix genotypes (TH3/12, TH17/17, TH21/2) have provided a unique experimental material for characterization of Mid-Parent Heterosis (MPH) in various root traits. Using a root phenotyping platform, we detected heterosis (TH3/12: MPH 43.99%; TH21/2: MPH 26.93%) in the size of the root system in soil. Triploid heterosis was also recorded in the fresh root weights, but it was less pronounced (MPH%: 9.63-19.31). In agreement with root growth characteristics in soil, the TH3/12 hybrids showed considerable heterosis (MPH: 70.08%) under in vitro conditions. Confocal microscopy-based imaging and quantitative analysis of root parenchyma cells at the division-elongation transition zone showed increased average cell diameter as a sign of cellular heterosis in plants from TH17/17 and TH21/2 triploid lines. Analysis of the hormonal background revealed that the auxin level was seven times higher than the total cytokinin contents in root tips of parental Tordis plants. In triploid hybrids, the auxin-cytokinin ratios were considerably reduced in TH3/12 and TH17/17 roots. In particular, the contents of cytokinin precursor, such as isopentenyl adenosine monophosphate, were elevated in all three triploid hybrids. Heterosis was also recorded in the amounts of active gibberellin precursor, GA19, in roots of TH3/12 plants. The presented experimental findings highlight the physiological basics of triploid heterosis in energy willow roots.
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Vigor Híbrido , Salix , Vigor Híbrido/genética , Triploidia , Diploide , Salix/genética , Melhoramento Vegetal , Citocininas , Solo , Ácidos IndolacéticosRESUMO
Mechanosensing is a ubiquitous process to translate external mechanical stimuli into biological responses. Piezo1 ion channels are directly gated by mechanical forces and play an essential role in cellular mechanotransduction. However, readouts of Piezo1 activity are mainly examined by invasive or indirect techniques, such as electrophysiological analyses and cytosolic calcium imaging. Here, we introduce GenEPi, a genetically-encoded fluorescent reporter for non-invasive optical monitoring of Piezo1-dependent activity. We demonstrate that GenEPi has high spatiotemporal resolution for Piezo1-dependent stimuli from the single-cell level to that of the entire organism. GenEPi reveals transient, local mechanical stimuli in the plasma membrane of single cells, resolves repetitive contraction-triggered stimulation of beating cardiomyocytes within microtissues, and allows for robust and reliable monitoring of Piezo1-dependent activity in vivo. GenEPi will enable non-invasive optical monitoring of Piezo1 activity in mechanochemical feedback loops during development, homeostatic regulation, and disease.
Assuntos
Canais Iônicos , Mecanotransdução Celular , Mecanotransdução Celular/fisiologia , Canais Iônicos/metabolismo , Membrana Celular/metabolismo , Fenômenos MecânicosRESUMO
N-methyl-D-aspartic acid receptor-dependent long term potentiation (LTP), a model of memory formation, requires Ca2+·calmodulin-dependent protein kinase II (αCaMKII) activity and Thr286 autophosphorylation via both global and local Ca2+ signaling, but the mechanisms of signal transduction are not understood. We tested the hypothesis that the Ca2+-binding activator protein calmodulin (CaM) is the primary decoder of Ca2+ signals, thereby determining the output, e.g. LTP. Thus, we investigated the function of CaM mutants, deficient in Ca2+ binding at sites 1 and 2 of the N-terminal lobe or sites 3 and 4 of the C-terminal CaM lobe, in the activation of αCaMKII. Occupancy of CaM Ca2+ binding sites 1, 3, and 4 is necessary and sufficient for full activation. Moreover, the N- and C-terminal CaM lobes have distinct functions. Ca2+ binding to N lobe Ca2+ binding site 1 increases the turnover rate of the enzyme 5-fold, whereas the C lobe plays a dual role; it is required for full activity, but in addition, via Ca2+ binding site 3, it stabilizes ATP binding to αCaMKII 4-fold. Thr286 autophosphorylation is also dependent on Ca2+ binding sites on both the N and the C lobes of CaM. As the CaM C lobe sites are populated by low amplitude/low frequency (global) Ca2+ signals, but occupancy of N lobe site 1 and thus activation of αCaMKII requires high amplitude/high frequency (local) Ca2+ signals, lobe-specific sensing of Ca2+-signaling patterns by CaM is proposed to explain the requirement for both global and local Ca2+ signaling in the induction of LTP via αCaMKII.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Animais , Linhagem Celular , Humanos , Potenciação de Longa Duração/fisiologia , Fosforilação , SpodopteraRESUMO
OBJECTIVE: To compare the outcomes of a 6-month-long accelerated rehabilitation with a 12-month-long rehabilitation. There is no consensus on the optimal duration of rehabilitation after anterior cruciate ligament reconstruction (ACLR). Trends in the past decades have shifted towards accelerated programs, often resulting in a return to play (RTP) at 4-6 months, postoperatively. However, longer rehabilitation cycles have recently experienced renaissance due to a greater understanding of graft remodeling. METHODS: Adult athletes who underwent ACLR between 2015 and 2018 by the same surgeon were included and followed-up prospectively for 24 months. Participants were allocated into two groups based on their RTP (6 months vs. 12 months) and compared with graft elongation, reoperation rate, and sports career (quit or continue) outcomes. RESULTS: Fifty-four patients underwent accelerated rehabilitation and 92 completed conventional rehabilitation. The accelerated rehabilitation was significantly associated with graft elongation-the accelerated rehabilitation group (n=9) and the conventional rehabilitation group (n=0), p<0.001-and need for reoperation-the accelerated rehabilitation group (n=5) and the conventional rehabilitation group (n=1), p=0.026. Although the relationship between rehabilitation time and quitting competitive sports did not reach significance at 0.05 level (p=0.063), it was significant when p<0.1, thereby showing a clear trend. CONCLUSION: Accelerated rehabilitation increased graft elongation risk. Knee laxity ≥3 mm measured at 6 months after ACLR should be accompanied by RTP time frame re-evaluation. Arthrometry checkups or routine magnetic resonance imaging shortly after RTP may be considered in cases of accelerated rehabilitation.
RESUMO
Information processing in the brain is controlled by quantal release of neurotransmitters, a tightly regulated process. From ultrastructural analysis, it is known that presynaptic boutons along single axons differ in the number of vesicles docked at the active zone. It is not clear whether the probability of these vesicles to get released (pves) is homogenous or also varies between individual boutons. Here, we optically measure evoked transmitter release at individual Schaffer collateral synapses at different calcium concentrations, using the genetically encoded glutamate sensor iGluSnFR. Fitting a binomial model to measured response amplitude distributions allowed us to extract the quantal parameters N, pves, and q. We find that Schaffer collateral boutons typically release single vesicles under low pves conditions and switch to multivesicular release in high calcium saline. The potency of individual boutons is highly correlated with their vesicular release probability while the number of releasable vesicles affects synaptic output only under high pves conditions.