Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

Abstract Background Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. Results A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way. Conclusions We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data.This work was supported by grants of the Spanish Ministerio de Ciencia e Innovación (BIO2009-09523 to A.B., including European Regional Development Funds), the Spanish Consolider-Ingenio 2010 Program (CSD2007-00036 Centre for Research in Agrigenomics) and the Generalitat de Catalunya (2009SGR0026). DMA was recipient of a predoctoral fellowship from Generalitat de Catalunya (DEBEQ 2004).Peer Reviewe

Similar effect of running on visual and auditory time perception in the ranges of milliseconds and seconds

IntroductionThe ability to accurately encode events’ duration is of critical importance for almost all everyday activities, yet numerous factors have been reported to robustly distort time perception. One of these is physical activity (i.e., running, walking) but, partly due to the variety of methodologies employed, a full comprehension of the role of exercise on the encoding of time has still to be achieved.MethodsHere we tackle the issue with a multifaceted approach by measuring the effect of vigorous running with a time generalization task for visual and auditory stimuli in the range of milliseconds (0.2–0.8 s) as well as seconds (1–4 s). At baseline, participants performed both the encoding and decoding at rest while in the experimental conditions the decoding was performed while running.ResultsOur results indicate that physical activity in both duration ranges (sub-second and seconds) was expanded during running regardless of the sensory modality used to present the stimuli. Despite this generalized effect of running on perceived duration, we found evidence for the existence of independent timing mechanisms: (1) the perceptual biases induced by running in the two temporal regimes were uncorrelated, (2) sensory precision levels (Weber fraction) were higher for stimuli in the seconds range, (3) sensory precision levels were higher for auditory than for visual stimuli, but only within the sub-second range.DiscussionOverall, our results support previous findings suggesting (at least partially) separate timing mechanisms for short/long durations and for visual and auditory stimuli. However, they also indicate that physical activity affects all these temporal modules, suggesting a generalized interaction—via generalized and shared resources—between the motor system and the brain time mechanisms

Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

Background Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. Results A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way. Conclusions We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data

Did Late Cretaceous cooling trigger the Campanian–Maastrichtian Boundary Event?

The Campanian-Maastrichtian (83-66 Ma) was a period of global climate cooling, featuring significant negative carbon-isotope (delta C-13) anomalies, such as the Late Campanian Event (LCE) and the Campanian-Maastrichtian Boundary Event (CMBE). A variety of factors, including changes in temperature, oceanic circulation and gateway opening, have been invoked to explain these delta C-13 perturbations, but no precise mechanism has yet been well constrained. In order to improve our understanding of these events, we measured stable carbon and oxygen isotopes of hemipelagic sediments from the Shuqualak-Evans cored borehole (Mississippi, USA) and compared the data with previously published sea-surface temperature (SST) estimates from the same core. We found that the CMBE can be recognised, unambiguously, in the Shuqualak-Evans core, and that it is associated with an interval of cooler SSTs suggesting a possible mechanistic link between palaeotemperat ure change and this event. Determining the precise position of the LCE in the Shuqualak-Evans core is more problematic, but it may also be associated with cooler SSTs. Our combined records of carbon cycling and SSTs compare well with other studies and provide evidence that cooling during the CMBE (and possibly LCE) was global in nature and affected surface waters, in addition to the deep-ocean. We suggest that short-term cooling drove intensification of high-latitude deep-water formation, which in turn led to changes in the ratio of carbonate to organic carbon burial that led to a negative delta C-13 excursion. Critically, the absence of warming during these intervals implies that the Late Cretaceous events must not have been associated with an appreciable increase in atmospheric pCO(2), and was likely associated with decreased pCO(2)

A Predictive Pedestrian Crash Model Based on Artificial Intelligence Techniques

Every year in Italy, there are about 20,000 road accidents involving pedestrians, with a significant number of injuries and deaths. Out of these, about 30% occur at pedestrian crossings, where pedestrians should be protected the most. Here, we propose a new accident prediction model to improve pedestrian safety assessments that allows us to accurately identify the sites with the largest potential safety improvements and define the best treatments to be applied. The accident prediction model was developed using the ISTAT dataset, including information about the fatal and injurious crashes that occurred in Italy in a 5-year period. The model allowed us to estimate the risk level of a road section through a machine-learning approach. Gradient Boosting seems to be an appropriate tool to fit classification models for its flexibility that allows us to capture non-linear relationships that would be difficult to detect via a classical approach. The results show the ability of the model to perform an accurate analysis of the sites included in the dataset. The locations analyzed have been classified based on the potential risk in the following three classes: High, medium, and low. The proposed model represents a solid and reliable tool for practitioners to perform accident analysis with pedestrian involvement

Visual P2p component responds to perceived numerosity

The folder Data contains two subfolders one for Baseline data and one for Adaptation data. Each subfolder contains Raw EEG traces (.set and .fdt files) and Raw Behavioral data (.mat files) of each participant. Behavioral Data Consist of a table (“Tbase” for Baseline and “Tadapt” for Adaptation) with each column reporting the following information: 1st column: Numerosity of the test stimulus. 2nd column: Side of presentation (in Baseline trials: 1 = Left; 2 = Right; in Adaptation trials: 1 = Adapted condition; 2 = Unadapted condition). 3rd column: Response request (1 meaning “yes” and 0 meaning “no”) 4th column: Response 5th column (only in Adaptation): Adaptors’ arrangement: 1 = High Adaptor on the left and Neutral Adaptor on the right; 2 = High Adaptor on the right and Neutral Adaptor on the left EEG Data All EEG traces contain labeled triggers following this code: Baseline traces: “Side” (Left or Right) – “Numerosity” Adaptation traces: “Condition” (AdaptSX, UnadaptSX, AdaptDX, UnadaptDX) – “Numerosity”. Legend for the adaptation conditions: AdaptSX: test stimulus being presented in the left side when the real adaptor appeared on the left side UnadaptSX: test stimulus being presented in the right side when the real adaptor appeared on the left side AdaptDX: test stimulus being presented in the right side when the real adaptor appeared on the right side UnadaptDX: test stimulus being presented in the left side when the real adaptor appeared on the right side Trigger 1: appearance of the two Adaptor

Top-down determinants of the numerosity–time interaction

Abstract Previous studies have reported that larger visual stimuli are perceived as lasting longer than smaller ones. However, this effect disappears when participants provide a qualitative judgment, by stating whether two stimuli have the “same or different” duration, instead of providing an explicit quantitative judgment (which stimulus lasts longer). Here, we extended these observations to the interaction between the numerosity of visual stimuli, i.e. clouds of dots, and their duration. With “longer vs shorter” responses, participants judged larger numerosities as lasting longer than smaller ones, both when the responses were related to the order (Experiment 1) or color (Experiment 4) of stimuli. In contrast, no similar effect was found with “same vs different” responses (Experiment 2) and in a time motor reproduction task (Experiment 3). The numerosity–time interference in Experiment 1 and Experiment 4 was not due to task difficulty, as sensory precision was equivalent to that of Experiment 2. We conclude that in humans the functional interaction between numerosity and time is not guided, in the main, by a shared bottom-up mechanism of magnitude coding. Rather, high-level and top-down processes involved in decision-making and guided by the use of “magnitude-related” response codes play a crucial role in triggering interference among different magnitude domains

Drivers' performance assessment approaching pedestrian crossings through the analysis of the speed and perceptive data recorded during on-field tests

Pedestrian fatalities in road accidents represent one of the biggest causes of death in the world despite the great efforts that have been made to decrease the involvement of vulnerable road users in road accidents. Literature analysis revealed the presence of several studies aimed at investigating the phenomenon and proposing strategies to improve pedestrian safety, but this is still not enough to considerably reduce the number of pedestrians killed on the road.In this context, with the aim to take a step forward in the topic, this paper describes a naturalistic driving assessment carried out in Firenze aimed at evaluating the effect of different pedestrian crossing configurations on the drivers' behavior, especially concerning the reduction of the speeding phenomenon approaching a pedestrian crossing. The experiment was conducted on a section of an urban collector road within the Firenze suburban area. Crucially, over the past few years, different traffic calming interventions have been implemented along this street. Among the different traffic calming countermeasures, both the presence of a traffic light and trapezoidal deflection have been considered to assess their effect on drivers' behavior, also with reference to specific aspects related to the drivers’ perception. During the experiment, thirty-six users drove their own vehicles along the street, encountering different pedestrian crossing configurations. During the driving speed, deceleration and ocular fixation were recorded.This study shows the difference in drivers' behavior in response to different traffic calming countermeasures. It demonstrates also that the raised pedestrian crossing caused a significant effect on reducing the speed approaching a pedestrian crossing. Moreover, it is observed that, when perceptive countermeasures are present, the drivers’ behavior changes only if the pedestrian crossing configuration is perceived in foveal vision; suggesting that the correct identification of the configuration is crucial to implement a congruent and safe driving behavior

A Predictive Pedestrian Crash Model Based on Artificial Intelligence Techniques

Every year in Italy, there are about 20,000 road accidents involving pedestrians, with a significant number of injuries and deaths. Out of these, about 30% occur at pedestrian crossings, where pedestrians should be protected the most. Here, we propose a new accident prediction model to improve pedestrian safety assessments that allows us to accurately identify the sites with the largest potential safety improvements and define the best treatments to be applied. The accident prediction model was developed using the ISTAT dataset, including information about the fatal and injurious crashes that occurred in Italy in a 5-year period. The model allowed us to estimate the risk level of a road section through a machine-learning approach. Gradient Boosting seems to be an appropriate tool to fit classification models for its flexibility that allows us to capture non-linear relationships that would be difficult to detect via a classical approach. The results show the ability of the model to perform an accurate analysis of the sites included in the dataset. The locations analyzed have been classified based on the potential risk in the following three classes: High, medium, and low. The proposed model represents a solid and reliable tool for practitioners to perform accident analysis with pedestrian involvement

Reshaping the peripersonal space in virtual reality

Abstract Peripersonal space (PPS) is defined as the space that lies within reach. Previous research revealed that PPS can be dynamically reshaped with the use of tools extending the arm’s reach. Here we investigated whether PPS reshaping depends on the kind of selected tool and/or the motor routine associated with its use. Participants carried out a visuo-tactile detection task in an immersive VR environment that allowed to measure the PPS size before and after a short period of tools use. In Experiment 1, participants had to pull or push objects towards or away from themselves using a shovel. In Experiment 2, they were required to either hammer or shoot an avatar placed in the Extrapersonal space. We found, for the first time in a VR environment, that a period of pull training was effective in enlarging the PPS, a result that replicates and expands previous findings carried out in real life conditions. However, no significant change in PPS size was achieved for training with other tools and motor routines. Our results suggest that the reshaping of PPS is a complex phenomenon in which the kind of interaction between the agent, the targets and the exploited motor routines all play a critical role