Relación estructura-función de la enzima uroporfirinógeno descarboxilasa: implicancias para la comprensión y el tratamiento de la porfiria cutánea tarda / Structure-function relationship of the enzyme uroporphyrinogen decarboxylase: implications for the understanding and treatment of porphyria cutanea tarda / Relação estrutura-função da enzima uroporfirinogênio descarbioxilase: implicações para a compreensão e tratamento da porfiria cutânea tardia / Reconocimiento a la trayectoria del Prof. Dr. Juan Miguel Castagnino†

Resumen La uroporfirinógeno descarboxilasa humana (UROD-h) es la quinta enzima del camino biosintético del hemo y su actividad deficiente, relacionada a mutaciones en su gen, se encuentra asociada a un subgrupo de porfirias. El objetivo de este trabajo fue estudiar la relación entre la dimerización de la enzima y su actividad enzimática y comprobar si la dimerización de UROD-h es imprescindible tanto para la primera etapa de la reacción (urogen→heptagen), como para la segunda etapa (heptagen→coprogen). Con ese objetivo, se expresó y purificó la UROD-h hasta homogeneidad, se analizó el comportamiento dímero-monómero bajo distintas condiciones que pudieran desplazar el equilibrio de dimerización y se evaluó la actividad enzimática en dichas condiciones. Los resultados obtenidos sugieren que la especie activa para la primera etapa de la reacción es el homodímero y que tanto el dímero como el monómero se comportan como especies activas para la segunda etapa de la reacción. Se propone que mutaciones clínicas como la Y311C, existentes en pacientes con porfiria cutánea tarda, podrían afectar la estabilidad del dímero y podrían ser el blanco para futuras terapias génicas.

Abstract Human uroporphyrinogen decarboxylase (UROD-h) is the fifth enzyme in the heme biosynthetic pathway and its deficient activity, related to mutations in its gene, is associated with a subset of porphyrias. The objective of this work was to study the relationship between the dimerisation of the enzyme and its enzymatic activity and to verify if the dimerisation of UROD-h is essential both for the first stage of the reaction (urogen→heptagen), and for the second stage (heptagen→ coprogen). With this objective, the UROD-h was expressed and purified to homogeneity, the dimer- monomer behaviour was analysed under different conditions, which could shift the dimerisation equilibrium, and the enzymatic activity was evaluated under these conditions. The results obtained suggest that the active species for the first stage of the reaction is the homodimer, and both the dimer and the monomer behaved as active species for the second stage of the reaction. It is proposed that clinical mutations such as Y311C, existing in porphyria cutanea tarda patients, could affect dimer stability and could be the target of future gene therapies.

Resumo A enzima uroporfirinogênio descarboxilase humana (UROD-h) é a quinta enzima da via biossintética do heme e sua atividade deficiente, relacionada com mutações em seu gene, está associada a um subgrupo de porfirias. O objetivo deste trabalho foi estudar a relação entre a dimerização da enzima e sua atividade enzimática e comprovar se a dimerização da UROD-h é imprescindível tanto para a primeira etapa da reação (urogênio→heptagênio), quanto para a segunda etapa (heptagênio→coprogênio). Com esse objetivo, a UROD-h foi expressa e purificada até a homogeneidade, o comportamento de dímero-monômero foi analisado sob diversas condições, que puderam deslocar o equilíbrio de dimerização, e a atividade enzimática foi avaliada em tais condições. Os resultados obtidos sugerem que a espécie ativa para a primeira etapa da reação é o homodímero, e tanto o dímero quanto o monômero se comportam como espécies ativas para a segunda etapa da reação. Propõe-se que mutações clínicas como Y311C, existentes em pacientes com porfiria cutânea tardia, poderiam afetar a estabilidade do dímero e poderiam ser o alvo de futuras terapias gênicas em porfiria cutânea tardia.

Reversibility of glyphosate sorption in pampean loess-derived soil profiles of central Argentina.

There is a growing concern about glyphosate's behavior in the environment. Herbicide behavior in soils greatly depends on adsorption-desorption phenomena, which have shown great variability in soils, although the reversibility of this process has been less examined. The adsorption-desorption behavior of glyphosate was measured on representative soil profiles of the northeast region of Argentinean Pampas, a semi-arid crop cultivating region. Two soil profiles samples (P1 and P2, both Entic Haplustoll) were collected and segmented into depth increments of 0-10, 10-20, 20-40, 40-60, 60-80, and 80-100 cm. Batch adsorption/48 h-desorption isotherms were performed in a controlled setup. Soil samples had a high sand content (77-92%), and a low content of clay (<3%), but markedly differing in the available P content, especially in the upper layers of soil profiles (0-40 cm, P1 range 133-170 ppm; P2 range 7-43 ppm). Adsorption-desorption isotherms showed a similar range of variation, between 150 and 1400 L kg -1for KFads and 450-1400 L kg -1for KFdes, without appreciable evidence of hysteresis (0.95 ± 0.05). Sorption capacity parameters showed a distinct behavior with depth, P1 exhibiting a U-shape with minimum values at intermediate depths (20-60 cm), while P2 decreases sharply between 0 and 40 cm. General linear models considering the specific surface area (SSA) of each sample and the spatial correlation structure for soil profiles showed a main positive association of KFads and KFdes with the soil organic matter, together with a positive association with iron content (KFads), and a negative association of KFdes with available P content. These results indicate high adsorption extents and sorption reversibility of glyphosate to sandy loam soils of the region, which implies the potential for the herbicide to be available for leaching or degradation under a scenario of intensive use.

Top-down modulation impairs priming susceptibility in complex decision-making with social implications.

Could social context variables prime complex decisions? Could top-down processes impair this priming susceptibility? Complex decisions have been mainly studied from economic and moral perspectives, and Dual Process Theories provide evidence of how these processes could be affected. To address these issues from a political perspective, online experiments were conducted. Participants (n = 252) were asked to choose a face from 4 options, each associated with different frequencies (repetition priming) or with phrases with different emotional valence (emotional priming), for an unspecified task (UST group) or an important task (IMT group). The most repeated face was chosen most in the UST group, and was associated with lower response times. Positive faces were equally chosen by both groups. To compare results in a more ecological situation, a social study was conducted during the 2019 Argentine Presidential Election, including online surveys (n = 3673) and analysis of news media mentioning candidates. The familiarity and trust to each candidate explained the voting-probability for most of them, as well as correlated with their frequency of mentions in the news, their positive associations, and election results. Our results suggest complex decision-making is susceptible to priming, depending on top-down modulation.

Rethinking the term "glyphosate effect" through the evaluation of different glyphosate-based herbicide effects over aquatic microbial communities.

Glyphosate-based herbicides (GBH) -the most widely used herbicides in agriculture worldwide-are frequently generalized by the name of "glyphosate". However, GBH encompass a variety of glyphosate salts as active ingredient and different adjuvants, which differ between products. These herbicides reach water bodies and produce diverse impacts over aquatic communities. Yet, the risk assessment assays required for the approval focus mostly on active ingredients. Herein, we compared the effect of five different GBH as well as of monoisopropylamine salt of glyphosate (GIPA) on aquatic microbial communities from natural shallow lakes that were mixed and allowed to evolve in an outdoor pond. We performed an 8-day long assay under indoor control conditions to evaluate the effects of exposure on the structure of nano-plus microphytoplankton (net phytoplankton, with sizes between 2 and 20 µm and >20 µm, respectively) and picoplankton (size ranging between 0.2 and 2 µm) communities through microscopy and flow cytometry, respectively. Significantly different effects were evident on the structure of microbial communities dependent on the GBH, even with herbicides sharing similar active ingredients. Each GBH evoked increases of different magnitude in bacterioplankton abundance. Furthermore, GIPA and a formulation decreased the abundance of a phycocyanin-rich (PC-rich) picocyanobacteria (Pcy) cytometric population and GIPA further altered Pcy composition. Also, two GBH increased net phytoplankton total abundance and, unlike the tested GBH, no apparent effect of GIPA was detected on this community structure. These results demonstrate that GBH effects on aquatic microbial communities should not be summarized as "glyphosate" effects considering that the formulations have effects beyond those exerted by the active ingredients alone. This work intends to alert on the lack of real knowledge regarding the consequences of the variety of GBH on natural aquatic ecosystems. Indeed, the wide use of the term "glyphosate effect" should be thoroughly rethought.

Unraveling the ecological processes modulating the population structure of Escherichia coli in a highly polluted urban stream network.

Escherichia coli dynamics in urban watersheds are affected by a complex balance among external inputs, niche modulation and genetic variability. To explore the ecological processes influencing E. coli spatial patterns, we analyzed its abundance and phylogenetic structure in water samples from a stream network with heterogeneous urban infrastructure and environmental conditions. Our results showed that environmental and infrastructure variables, such as macrophyte coverage, DIN and sewerage density, mostly explained E. coli abundance. Moreover, main generalist phylogroups A and B1 were found in high proportion, which, together with an observed negative relationship between E. coli abundance and phylogroup diversity, suggests that their dominance might be due to competitive exclusion. Lower frequency phylogroups were associated with sites of higher ecological disturbance, mainly involving simplified habitats, higher drainage infrastructure and septic tank density. In addition to the strong negative relationship between phylogroup diversity and dominance, the occurrence of these phylogroups would be associated with increased facilitated dispersal. Nutrients also contributed to explaining phylogroup distribution. Our study proposes the differential contribution of distinct ecological processes to the patterns of E. coli in an urban watershed, which is useful for the monitoring and management of fecal pollution.

Multiple stressors and social-ecological traps in Pampean streams (Argentina): A conceptual model.

Fluvial systems are particularly sensitive to changes in the terrestrial ecosystems where they are embedded, receiving simultaneously the impact of multiple stressors. The design of adequate management policies requires analyzing fluvial systems as social-ecological systems, because the decoupling of natural and social systems can lead to a severe mismatch between maintaining ecological integrity and the pursuit of human well-being. Pampean streams are especially prone to the impact of human activities because they are located in a region that provides almost half of the agricultural production of Argentina and concentrates 66% of the whole population of the country. In the present work, we conceived a general social-ecological framework that links the occurrence of multiple stressors and their impacts on ecosystem services, with changes in environmental perception of streams, which in turn feedback over institutional actions at the watershed's governance. We identified four current key drivers of the dynamics in Pampean streams: a dominant agro-industrial model for the region, a command-and-control governance regime mainly based on an engineering hydraulic perspective, the real estate market speculation of surrounding lands, and the persistence of structural poverty in urban areas. The resulting dynamics resembles the occurrence of different kinds of social-ecological traps, i.e., a highly stable but undesirable state of the system that is difficult to escape. Based on this analysis, we provide a leverage point perspective to avoid this trap. Together, this approach could be applied to other fluvial systems of the world to link the ecological and social domains to multiple stressors analysis, and to improve institutional fit for the sustainability of fluvial social-ecological systems.

Effect of glyphosate on the growth, morphology, ultrastructure and metabolism of Scenedesmus vacuolatus.

The effects of a commercial glyphosate formulation on the oxidative stress parameters and morphology (including the ultrastructure) of the phytoplanktonic green microalga Scenedesmus vacuolatus were evaluated. After 96 h of exposure to increasing herbicide concentrations (0, 4, 6, 8 mg L-1 active ingredient) with the addition of alkyl aryl polyglycol ether surfactant, the growth of the cultures decreased (96 h-IC50- 4.90 mg L-1) and metabolic and morphology alterations were observed. Significant increases in cellular volume (103-353%) and dry weight (105%) and a significant decrease in pigment content (41-48%) were detected. Oxidative stress parameters were significantly affected, showing an increase in the reactive oxygen species (ROS) and reduced glutathione (GSH) contents, oxidative damage to lipids and proteins and a decrease in the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and the detoxifying enzyme glutathione-S-transferase (GST). Cells exposed to glyphosate formulation were larger and showed an increase in vacuole size, bleaching, cell wall thickening and alteration of the stacking pattern of thylakoids. The results of this study showed the participation of oxidative stress in the mechanism of toxic action of the commercial glyphosate formulation on S. vacuolatus and the relation between the biochemical, morphological and ultrastructure alterations.

Neural correlates of perceived confidence in a partial report paradigm.

Confidence judgments are often severely distorted: People may feel underconfident when responding correctly or, conversely, overconfident in erred responses. Our aim here was to identify the timing of brain processes that lead to variations in objective performance and subjective judgments of confidence. We capitalized on the Partial Report Paradigm [Sperling, G. The information available in brief visual presentations. Psychological Monographs: General and Applied, 74, 1, 1960], which allowed us to separate experimentally the moment of encoding of information from that of its retrieval [Zylberberg, A., Dehaene, S., Mindlin, G. B., & Sigman, M. Neurophysiological bases of exponential sensory decay and top-down memory retrieval: A model. Frontiers in Computational Neuroscience, 3, 2009]. We observed that the level of subjective confidence is indexed by two very specific evoked potentials at latencies of about 400 and 600 msec during the retrieval stage and by a stationary measure of intensity of the alpha band during the encoding period. When factoring out the effect of confidence, objective performance shows a weak effect during the encoding and retrieval periods. These results have relevant implications for theories of decision-making and confidence, suggesting that confidence is not constructed online as evidence is accumulated toward a decision. Instead, confidence attributions are more consistent with a retrospective mechanism that monitors the entire decision process.

Distinct patterns of functional brain connectivity correlate with objective performance and subjective beliefs.

The degree of correspondence between objective performance and subjective beliefs varies widely across individuals. Here we demonstrate that functional brain network connectivity measured before exposure to a perceptual decision task covaries with individual objective (type-I performance) and subjective (type-II performance) accuracy. Increases in connectivity with type-II performance were observed in networks measured while participants directed attention inward (focus on respiration), but not in networks measured during states of neutral (resting state) or exogenous attention. Measures of type-I performance were less sensitive to the subjects' specific attentional states from which the networks were derived. These results suggest the existence of functional brain networks indexing objective performance and accuracy of subjective beliefs distinctively expressed in a set of stable mental states.

Parsing a perceptual decision into a sequence of moments of thought.

Theoretical, computational, and experimental studies have converged to a model of decision-making in which sensory evidence is stochastically integrated to a threshold, implementing a shift from an analog to a discrete form of computation. Understanding how this process can be chained and sequenced - as virtually all real-life tasks involve a sequence of decisions - remains an open question in neuroscience. We reasoned that incorporating a virtual continuum of possible behavioral outcomes in a simple decision task - a fundamental ingredient of real-life decision-making - should result in a progressive sequential approximation to the correct response. We used real-time tracking of motor action in a decision task, as a measure of cognitive states reflecting an internal decision process. We found that response trajectories were spontaneously segmented into a discrete sequence of explorations separated by brief stops (about 200 ms) - which remained unconscious to the participants. The characteristics of these stops were indicative of a decision process - a "moment of thought": their duration correlated with the difficulty of the decision and with the efficiency of the subsequent exploration. Our findings suggest that simple navigation in an abstract space involves a discrete sequence of explorations and stops and, moreover, that these stops reveal a fingerprint of moments of thought.

Neurophysiology of perceived confidence.

In a partial report paradigm, subjects observe during a brief presentation a cluttered field and after some time - typically ranging from 100 ms to a second - are asked to report a subset of the presented elements. A vast buffer of information is transiently available to be broadcasted which, if not retrieved in time, fades rapidly without reaching consciousness. An interesting feature of this experiment is that objective performance and subjective confidence is decoupled. This converts this paradigm in an ideal vehicle to understand the brain dynamics of the construction of confidence. Here we report a high-density EEG experiment in which we infer elements of the EEG response which are indicative of subjective confidence. We find that an early response during encoding partially correlates with perceived confidence. However, the bulk of the weight of subjective confidence is determined during a late, N400-like waveform, during the retrieval stage. This shows that we can find markers of access to internal, subjective states, that are uncoupled from objective response and stimulus properties of the task, and we propose that this can be used with decoding methods of EEG to infer subjective mental states.

The spatial and temporal construction of confidence in the visual scene.

Human subjects can report many items of a cluttered field a few hundred milliseconds after stimulus presentation. This memory decays rapidly and after a second only 3 or 4 items can be stored in working memory. Here we compared the dynamics of objective performance with a measure of subjective report and we observed that 1) Objective performance beyond explicit subjective reports (blindsight) was significantly more pronounced within a short temporal interval and within specific locations of the visual field which were robust across sessions 2) High confidence errors (false beliefs) were largely confined to a small spatial window neighboring the cue. The size of this window did not change in time 3) Subjective confidence showed a moderate but consistent decrease with time, independent of all other experimental factors. Our study allowed us to asses quantitatively the temporal and spatial access to an objective response and to subjective reports.

The dynamics of sensory buffers: geometric, spatial, and experience-dependent shaping of iconic memory.

When a stimulus is presented, its sensory trace decays rapidly, lasting for approximately 1000 ms. This brief and labile memory, referred as iconic memory, serves as a buffer before information is transferred to working memory and executive control. Here we explored the effect of different factors--geometric, spatial, and experience--with respect to the access and the maintenance of information in iconic memory and the progressive distortion of this memory. We studied performance in a partial report paradigm, a design wherein recall of only part of a stimulus array is required. Subjects had to report the identity of a letter in a location that was cued in a variable delay after the stimulus onset. Performance decayed exponentially with time, and we studied the different parameters (time constant, zero-delay value, and decay amplitude) as a function of the different factors. We observed that experience (determined by letter frequency) affected the access to iconic memory but not the temporal decay constant. On the contrary, spatial position affected the temporal course of delay. The entropy of the error distribution increased with time reflecting a progressive morphological distortion of the iconic buffer. We discuss our results on the context of a model of information access to executive control and how it is affected by learning and attention.