This work describes the development of an ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for the determination of carotenoids (β-carotene, lutein, ...β-criptoxanthin, neoxanthin, violaxanthin) and chlorophylls, as well as their related compounds (chlorophyll A and B, pheophytin A and B and the banned dyes Cu–pyropheophytin A, Cu–pheophytin A and B) in olive oils. For this purpose, the feasibility of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) for the ionization of these compounds was evaluated and compared. Tandem mass spectrometry (MS/MS) fragmentation was discussed for each family of compounds, and the most characteristic and abundant product ions were selected to propose a selective and sensitive UHPLC–MS/MS method. The best results were obtained using APCI and APPI, while ESI provided the worst signal-to-noise ratio (
S
/
N
) for all compounds. For the analysis of olive oils, a simple solid-phase extraction (SPE) with silica cartridges was applied before the determination by UHPLC–MS/MS (APCI and APPI) in multiple reaction monitoring (MRM) mode. Method quality parameters were stablished, and the results demonstrate the good performance of the new methods, providing low limits of detection (0.004–0.9 mg L
−1
), high extraction efficiencies (62–95%) and low matrix effects (< 25%). The developed UHPLC–API–MS/MS (APCI and APPI) methods were applied to the analysis of olive oil samples, and β-carotene, pheophytin A, pheophytin B and lutein were detected and quantified in all of them at concentrations ranging from 0.1 to 9.5 mg L
−1
.
Graphical abstract
We recorded the simple-spike (SS) firing of Purkinje cells (PCs) in the floccular complex both during normal pursuit caused by step-ramp target motions and after learning induced by a consistently ...timed change in the direction of target motion. The encoding of eye movement by the SS firing rate of individual PCs was described by a linear regression model, in which the firing rate is a sum of weighted components related to eye acceleration, velocity, and position. Although the model fit the data well for individual conditions, the regression coefficients for the learned component of firing often differed substantially from those for normal pursuit of step-ramp target motion. We suggest that the different encoding of learned versus normal pursuit responses in individual PCs reflects different amounts of learning in their inputs. The decoded output from the floccular complex, estimated by averaging responses across the population of PCs, also was fitted by the regression model. Regression coefficients were equal for the two conditions for on-direction pursuit, but differed for off-direction target motion. We conclude that the average output from the population of floccular PCs provides some, but not all, of the neural signals that drive the learned component of pursuit and that plasticity outside of the flocculus makes an important contribution.
Plants detect competitors in shaded environments by perceiving a reduction in photosynthetically active radiation (PAR) and the reduction between the red and far‐red light (R:FR) ratio and blue ...photons. These light signals are detected by phytochromes and cryptochromes, which trigger shade avoidance responses such as shoot and petiole elongation and lead to increased susceptibility to pathogen attack. We studied morphological, anatomical, and photosynthesis differences in potato plants (Solanum tuberosum var. Spunta) exposed to sunlight or simulated shade in a greenhouse. We found that simulated shade strongly induced stem and internode elongation with a higher production of free auxin in stems and a lower production of tubers. The mesophyll thickness of the upper leaves of plants grown in simulated shade was lower, but the epidermis was wider compared with the leaves of plants cultivated in sunlight. In addition, the photosynthesis rate was lower in the upper leaves exposed to nonsaturated irradiances and higher in the basal leaves at saturated irradiances compared with control plants. RNA‐seq analysis showed that 146 and 155 genes were up‐ and downregulated by shade, respectively. By quantitative reverse transcription polymerase chain reaction, we confirmed that FLOWERING LOCUS T (FT), WRKY‐like, and PAR1b were induced, while FLAVONOL 4‐SULFOTRANSFERASE was repressed under shade. In shaded plants, leaves and tubers were more susceptible to the necrotrophic fungus Botrytis cinerea attack. Overall, our work demonstrates configurational changes between growth and defense decisions in potato plants cultivated in simulated shade.
We performed a series of experiments to study the interaction between phototropism and gravitropism in Arabidopsis thaliana as part of the Seedling Growth Project on the International Space Station. ...Red-light-based and blue-light-based phototropism were examined in microgravity and at 1g, a control that was produced by an on-board centrifuge. At the end of the experiments, seedlings were frozen and brought back to Earth for gene profiling studies via RNASeq methods. In this paper, we focus on five genes identified in these space studies by their differential expression in space: one involved in auxin transport and four others encoding genes for: a methyltransferase subunit, a transmembrane protein, a transcription factor for endodermis formation, and a cytoskeletal element (an intermediate filament protein). Time course studies using mutant strains of these five genes were performed for blue-light and red-light phototropism studies as well as for gravitropism assays on ground. Interestingly, all five of the genes had some effects on all the tropisms under the conditions studied. In addition, RT-PCR analyses examined expression of the five genes in wild-type seedlings during blue-light-based phototropism. Previous studies have supported a role of both microfilaments and microtubules in tropism pathways. However, the most interesting finding of the present space studies is that NFL, a gene encoding an intermediate filament protein, plays a role in phototropism and gravitropism, which opens the possibility that this cytoskeletal element modulates signal transduction in plants.
•Seedlings grown on the ISS were frozen and returned for analysis via RNASeq.•Five genes with large differences in expression, pertaining to light and/or gravity perception, were selected.•Mutants of these five genes were studied with tropism assays on the ground.•One of these genes encoded an intermediate filament (neurofilament light protein).•NFL appears to play a role in phototropism and gravitropism pathways.
The climbing fiber input to Purkinje cells acts as a teaching signal by triggering a massive influx of dendritic calcium that marks the occurrence of instructive stimuli during cerebellar learning. ...Here, we challenge the view that these calcium spikes are all-or-none and only signal whether the instructive stimulus has occurred, without providing parametric information about its features. We imaged ensembles of Purkinje cell dendrites in awake mice and measured their calcium responses to periocular airpuffs that serve as instructive stimuli during cerebellar-dependent eyeblink conditioning. Information about airpuff duration and pressure was encoded probabilistically across repeated trials, and in two additional signals in single trials: the synchrony of calcium spikes in the Purkinje cell population, and the amplitude of the calcium spikes, which was modulated by a non-climbing fiber pathway. These results indicate that calcium-based teaching signals in Purkinje cells contain analog information that encodes the strength of instructive stimuli trial-by-trial.
Cerebellar inhibitory interneurons are important regulators of neural circuit activity for diverse motor and nonmotor functions. The molecular layer interneurons (MLIs), consisting of basket cells ...(BCs) and stellate cells (SCs), provide dendritic and somatic inhibitory synapses onto Purkinje cells, respectively. They are sequentially generated in an inside-out pattern from Pax2
immature interneurons, which migrate from the prospective white matter to the ML of the cortex. However, little is known about how MLI subtype identities and pool sizes are determined, nor are their contributions to motor learning well understood. Here, we show that GABAergic progenitors fated to generate both BCs and SCs respond to the Sonic hedgehog (Shh) signal. Conditional abrogation of Shh signaling of either sex inhibited proliferation of GABAergic progenitors and reduced the number of Pax2
cells, whereas persistent Shh pathway activation increased their numbers. These changes, however, did not affect early born BC numbers but selectively altered the SC pool size. Moreover, genetic depletion of GABAergic progenitors when BCs are actively generated also resulted in a specific reduction of SCs, suggesting that the specification of MLI subtypes is independent of Shh signaling and their birth order and likely occurs after Pax2
cells settle into their laminar positions in an inside-out sequence. Mutant mice with reduced SC numbers displayed decreased dendritic inhibitory synapses and neurotransmission onto Purkinje cells, resulting in an impaired acquisition of eyeblink conditioning. These findings also reveal an essential role of Shh signaling-dependent SCs in regulating inhibitory dendritic synapses and motor learning.
The cerebellar circuit that enables fine motor learning involves MLIs of BCs and SCs, which provide dendritic and somatic inhibitory synapses onto Purkinje cells. Little is known about how their identities and numbers are determined, nor are their specific contributions to motor learning well understood. We show that MLI subtypes are specified independent of Shh signaling and their birth orders but appear to occur in their terminal laminar positions according to the inside-out sequence. This finding challenges the current view that MLI subtypes are specified sequentially at the progenitor level. We also demonstrate that dendritic inhibition by Shh signaling-dependent SC pool is necessary for motor learning.
Highlights • Errors in sensory estimates are a major source of motor variation. • Correlated sensory noise causes shared variation in neural population responses. • Shared variation propagates ...through the sensory-motor circuit. • Shared variation causes neuron–neuron and neuron–behavior correlations. • Averaging eliminates independent variation in neural firing.
Rett syndrome is a devastating childhood neurological disorder caused by mutations in
. Of the many symptoms, motor deterioration is a significant problem for patients. In mice, deleting
from the ...cortex or basal ganglia causes motor dysfunction, hypoactivity, and tremor, which are abnormalities observed in patients. Little is known about the function of
in the cerebellum, a brain region critical for motor function. Here we show that deleting
from the cerebellum, but not from its neuronal subtypes, causes a delay in motor learning that is overcome by additional training. We observed irregular firing rates of Purkinje cells and altered heterochromatin architecture within the cerebellum of knockout mice. These findings demonstrate that the motor deficits present in Rett syndrome arise, in part, from cerebellar dysfunction. For Rett syndrome and other neurodevelopmental disorders, our results highlight the importance of understanding which brain regions contribute to disease phenotypes.
Glycogen synthase kinase 3β (GSK-3β) is widely recognised as a relevant player in the pathogenesis of several highly prevalent disorders such as Alzheimer's disease, mood disorders, diabetes and ...cancer. Therefore, this enzyme constitutes a highly attractive therapeutic target for the development of selective inhibitors as new promising drugs for the treatment of these pathologies. We describe here the isolation and biochemical characterization of the marine natural sesquiterpene palinurin as a GSK-3β inhibitor. Experimental studies performed for characterizing the inhibitory mechanism indicate that GSK-3β inhibition by palinurin cannot be competed out by ATP nor peptide substrate. Molecular modelling techniques have enabled us to propose an unconventional binding mode to GSK-3β. Moreover, molecular dynamics simulations have identified an allosteric mechanism by which binding of palinurin leads to GSK-3β inhibition. The inhibitory activities determined for a series of structurally related analogues support the proposed binding mode of palinurin, which is the first compound described to target this allosteric site. The results offer new opportunities for designing and developing selective inhibitors with novel mechanisms of action.
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▶ The biochemical characterization of palinurin as a GSK-3β inhibitor is reported. ▶ A non-ATP/substrate-competitive mechanism is found for the inhibition of GSK-3β. ▶ The enzymatic mode of action is justified by the binding to an unconventional site. ▶ Binding to this site provides a basis to explain the selective inhibition of GSK-3.
ABSTRACTAccurate anticipation of the maize harvest date is important in the agricultural market, as it ensures the sustainability of food production in response to the increasing global demand for ...food. This paper proposes a predictive model to determine the optimal harvest time in maize plots using the Normalised Difference Vegetation Index (NDVI) and climatological data. These variables were oversampled and used to train various models, including Random Forest (RF), Gradient Boosting Machine (GBM), Light Gradient Boosting Machine (LGBM), Extreme Gradient Boosting Machine (XGBoost), CatBoost and Support Vector Machine (SVM). Bayesian optimisation has been used to find the best hyperparameters and Shapley values to identify the variables that exert the most significant influence on the prediction in each model instance. As a result of this approach, a model with an accuracy of 92.1% and an Area Under the Curve (AUC) of 0.935 was developed. The variables that determined these results were atmospheric pressure, mean temperature, precipitation, NDVI, and precipitation.
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