Flipped classroom in neurophysiology: performance analysis of a system focusing on intrinsic students' motivation.

Introduction: Teaching methodologies promoting active learning result in higher-order knowledge application, a desirable outcome in health disciplines like Physiology. Flipped-classroom (FC) promotes active learning and engagement in the classroom. Although specialized research keeps accumulating, the advantages of FC for improving academic outcome and ultimately patient care remain controversial and open to further analysis. Objective: This study evaluates the benefits of applying FC to the Neurophysiology module of a Human Physiology course. Methods:We compare final grades of students exposed to standard lecturing (five-years) vs. FC (six-years), and study the FC impact on student motivation, study time and rewards. Differing from conventional FC, we performed no pre-class/in-class assessments, relying on the students' internal motivation to experience our FC model. A printed student workbook was designed as pre-class material for each session. Reading times respect the expected daily study time of students in our system. Results and discussion: Concerning academic performance, our long-term study reports a significant increase in average scores for FC groups. Overall, students get better scores in multiple choice tests than in problem-solving questions. A more detailed analysis uncovers that our FC model helps students to obtain better scores, reducing variability in performance due to assessment methods. Based on our open-ended survey questions, most students rate the FC environment and in-class activities positively and perceive a positive effect of FC on teachers' performance. An objective automatic Sentiment analysis of open-ended answers reveals that FC is positively appreciated by students, associating positive perceptions to their understanding of physiological concepts, and negative evaluations to their time management.

Characterization of Leptoglossus occidentalis Eggs and Egg Glue.

The western conifer seed bug (Leptoglossus occidentalis Heidemann, 1910, Heteroptera: Coreidae) has a significant economic impact due to the reduction in the quality and viability of conifer seed crops; it can feed on up to 40 different species of conifers, showing a clear predilection for Pinus pinea L. in Europe. Its incidence is especially relevant for the pine nut-producing industry, given that the action of this pest insect can reduce the production of pine nuts by up to 25%. As part of ongoing efforts aimed at the design of control strategies for this insect, this work focuses on the characterization (by scanning electron microscopy-energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy, and gas chromatography-mass spectroscopy, GC-MS) of the compounds released by these insects during oviposition, with emphasis on the adhesive secretion that holds L. occidentalis eggs together. Elemental analysis pointed to the presence of significant amounts of compounds with high nitrogen content. Functional groups identified by infrared spectroscopy were compatible with the presence of chitin, scleroproteins, LNSP-like and gelatin proteins, shellac wax analogs, and policosanol. Regarding the chemical species identified by GC-MS, eggs and glue hydromethanolic extracts shared constituents such as butyl citrate, dibutyl itaconate, tributyl aconitate, oleic acid, oleamide, erucamide, and palmitic acid, while eggs also showed stearic and linoleic acid-related compounds. Knowledge of this composition may allow advances in new strategies to address the problem caused by L. occidentalis.

Benchmarking of tools for axon length measurement in individually-labeled projection neurons.

Projection neurons are the commonest neuronal type in the mammalian forebrain and their individual characterization is a crucial step to understand how neural circuitry operates. These cells have an axon whose arborizations extend over long distances, branching in complex patterns and/or in multiple brain regions. Axon length is a principal estimate of the functional impact of the neuron, as it directly correlates with the number of synapses formed by the axon in its target regions; however, its measurement by direct 3D axonal tracing is a slow and labor-intensive method. On the contrary, axon length estimations have been recently proposed as an effective and accessible alternative, allowing a fast approach to the functional significance of the single neuron. Here, we analyze the accuracy and efficiency of the most used length estimation tools-design-based stereology by virtual planes or spheres, and mathematical correction of the 2D projected-axon length-in contrast with direct measurement, to quantify individual axon length. To this end, we computationally simulated each tool, applied them over a dataset of 951 3D-reconstructed axons (from NeuroMorpho.org), and compared the generated length values with their 3D reconstruction counterparts. The evaluated reliability of each axon length estimation method was then balanced with the required human effort, experience and know-how, and economic affordability. Subsequently, computational results were contrasted with measurements performed on actual brain tissue sections. We show that the plane-based stereological method balances acceptable errors (~5%) with robustness to biases, whereas the projection-based method, despite its accuracy, is prone to inherent biases when implemented in the laboratory. This work, therefore, aims to provide a constructive benchmark to help guide the selection of the most efficient method for measuring specific axonal morphologies according to the particular circumstances of the conducted research.

An Evolutionary Perspective of the Lipocalin Protein Family.

The protein family of Lipocalins is ubiquitously present throughout the tree of life, with the exception of the phylum Archaea. Phylogenetic relationships of chordate Lipocalins have been proposed in the past based on protein sequence similarities, but their highly divergent primary structures and a shortage of experimental annotations in genome projects have precluded a well-supported hypothesis for their evolution. In this work we propose a novel topology for the phylogenetic tree of chordate Lipocalins, inferred from multiple amino acid sequence alignments. Sixteen jawed vertebrates with fair coverage by genomic sequencing were compared. The selected species span an evolutionary range of ∼400 million years, allowing for a balanced representation of all major vertebrate clades. A consensus phylogenetic tree is proposed following a comparison of sequence-based maximum-likelihood trees and protein structure dendrograms. This new phylogeny suggests an APOD-like common ancestor in early chordates, which gave rise, via whole-genome or tandem duplications, to the six Lipocalins currently present in fish (APOD, RBP4, PTGDS, AMBP, C8G, and APOM). Further gene duplications of APOM and PTGDS resulted in the altogether 15 Lipocalins found in contemporary mammals. Insights into the functional impact of relevant amino acid residues in early diverging Lipocalins are also discussed. These results should foster the experimental exploration of novel functions alongside the identification of new members of the Lipocalin family.

Effects of Fasting and Feeding on Transcriptional and Posttranscriptional Regulation of Insulin-Degrading Enzyme in Mice.

Insulin-degrading enzyme (IDE) is a highly conserved and ubiquitously expressed Zn2+-metallopeptidase that regulates hepatic insulin sensitivity, albeit its regulation in response to the fasting-to-postprandial transition is poorly understood. In this work, we studied the regulation of IDE mRNA and protein levels as well as its proteolytic activity in the liver, skeletal muscle, and kidneys under fasting (18 h) and refeeding (30 min and 3 h) conditions, in mice fed a standard (SD) or high-fat (HFD) diets. In the liver of mice fed an HFD, fasting reduced IDE protein levels (~30%); whereas refeeding increased its activity (~45%) in both mice fed an SD and HFD. Likewise, IDE protein levels were reduced in the skeletal muscle (~30%) of mice fed an HFD during the fasting state. Circulating lactate concentrations directly correlated with hepatic IDE activity and protein levels. Of note, L-lactate in liver lysates augmented IDE activity in a dose-dependent manner. Additionally, IDE protein levels in liver and muscle tissues, but not its activity, inversely correlated (R2 = 0.3734 and 0.2951, respectively; p < 0.01) with a surrogate marker of insulin resistance (HOMA index). Finally, a multivariate analysis suggests that circulating insulin, glucose, non-esterified fatty acids, and lactate levels might be important in regulating IDE in liver and muscle tissues. Our results highlight that the nutritional regulation of IDE in liver and skeletal muscle is more complex than previously expected in mice, and that fasting/refeeding does not strongly influence the regulation of renal IDE.

Static internal representation of dynamic situations reveals time compaction in human cognition.

INTRODUCTION: The human brain has evolved under the constraint of survival in complex dynamic situations. It makes fast and reliable decisions based on internal representations of the environment. Whereas neural mechanisms involved in the internal representation of space are becoming known, entire spatiotemporal cognition remains a challenge. Growing experimental evidence suggests that brain mechanisms devoted to spatial cognition may also participate in spatiotemporal information processing. OBJECTIVES: The time compaction hypothesis postulates that the brain represents both static and dynamic situations as purely static maps. Such an internal reduction of the external complexity allows humans to process time-changing situations in real-time efficiently. According to time compaction, there may be a deep inner similarity between the representation of conventional static and dynamic visual stimuli. Here, we test the hypothesis and report the first experimental evidence of time compaction in humans. METHODS: We engaged human subjects in a discrimination-learning task consisting in the classification of static and dynamic visual stimuli. When there was a hidden correspondence between static and dynamic stimuli due to time compaction, the learning performance was expected to be modulated. We studied such a modulation experimentally and by a computational model. RESULTS: The collected data validated the predicted learning modulation and confirmed that time compaction is a salient cognitive strategy adopted by the human brain to process time-changing situations. Mathematical modelling supported the finding. We also revealed that men are more prone to exploit time compaction in accordance with the context of the hypothesis as a cognitive basis for survival. CONCLUSIONS: The static internal representation of dynamic situations is a human cognitive mechanism involved in decision-making and strategy planning to cope with time-changing environments. The finding opens a new venue to understand how humans efficiently interact with our dynamic world and thrive in nature.

Machine Learning Representation of Loss of Eye Regularity in a Drosophila Neurodegenerative Model.

The fruit fly compound eye is a premier experimental system for modeling human neurodegenerative diseases. The disruption of the retinal geometry has been historically assessed using time-consuming and poorly reliable techniques such as histology or pseudopupil manual counting. Recent semiautomated quantification approaches rely either on manual region-of-interest delimitation or engineered features to estimate the extent of degeneration. This work presents a fully automated classification pipeline of bright-field images based on orientated gradient descriptors and machine learning techniques. An initial region-of-interest extraction is performed, applying morphological kernels and Euclidean distance-to-centroid thresholding. Image classification algorithms are trained on these regions (support vector machine, decision trees, random forest, and convolutional neural network), and their performance is evaluated on independent, unseen datasets. The combinations of oriented gradient + gaussian kernel Support Vector Machine [0.97 accuracy and 0.98 area under the curve (AUC)] and fine-tuned pre-trained convolutional neural network (0.98 accuracy and 0.99 AUC) yielded the best results overall. The proposed method provides a robust quantification framework that can be generalized to address the loss of regularity in biological patterns similar to the Drosophila eye surface and speeds up the processing of large sample batches.

Control of the neuroprotective Lipocalin Apolipoprotein D expression by alternative promoter regions and differentially expressed mRNA 5' UTR variants.

The Lipocalin Apolipoprotein D (ApoD) is one of the few genes consistently overexpressed in the aging brain, and in most neurodegenerative and psychiatric diseases. Its functions include metabolism regulation, myelin management, neuroprotection, and longevity regulation. Knowledge of endogenous regulatory mechanisms controlling brain disease-triggered ApoD expression is relevant if we want to boost pharmacologically its neuroprotecting potential. In addition to classical transcriptional control, Lipocalins have a remarkable variability in mRNA 5'UTR-dependent translation efficiency. Using bioinformatic analyses, we uncover strong selective pressures preserving ApoD 5'UTR properties, indicating unexpected functional conservation. PCR amplifications demonstrate the production of five 5'UTR variants (A-E) in mouse ApoD, with diverse expression levels across tissues and developmental stages. Importantly, Variant E is specifically expressed in the oxidative stress-challenged brain. Predictive analyses of 5'UTR secondary structures and enrichment in elements restraining translation, point to Variant E as a tight regulator of ApoD expression. We find two genomic regions conserved in human and mouse ApoD: a canonical (α) promoter region and a previously unknown region upstream of Variant E that could function as an alternative mouse promoter (ß). Luciferase assays demonstrate that both α and ß promoter regions can drive expression in cultured mouse astrocytes, and that Promoter ß activity responds proportionally to incremental doses of the oxidative stress generator Paraquat. We postulate that Promoter ß works in association with Variant E 5'UTR as a regulatory tandem that organizes ApoD gene expression in the nervous system in response to oxidative stress, the most common factor in aging and neurodegeneration.

Semantic Knowledge Representation for Strategic Interactions in Dynamic Situations.

Evolved living beings can anticipate the consequences of their actions in complex multilevel dynamic situations. This ability relies on abstracting the meaning of an action. The underlying brain mechanisms of such semantic processing of information are poorly understood. Here we show how our novel concept, known as time compaction, provides a natural way of representing semantic knowledge of actions in time-changing situations. As a testbed, we model a fencing scenario with a subject deciding between attack and defense strategies. The semantic content of each action in terms of lethality, versatility, and imminence is then structured as a spatial (static) map representing a particular fencing (dynamic) situation. The model allows deploying a variety of cognitive strategies in a fast and reliable way. We validate the approach in virtual reality and by using a real humanoid robot.

Rain-Fed Granite Rock Basins Accumulate a High Diversity of Dormant Microbial Eukaryotes.

Rain fed granite rock basins are ancient geological landforms of worldwide distribution and structural simplicity. They support habitats that can switch quickly from terrestrial to aquatic along the year. Diversity of animals and plants, and the connexion between communities in different basins have been widely explored in these habitats, but hardly any research has been carried out on microorganisms. The aim of this study is to provide the first insights on the diversity of eukaryotic microbial communities from these environments. Due to the ephemeral nature of these aquatic environments, we predict that the granitic basins should host a high proportion of dormant microeukaryotes. Based on an environmental DNA diversity survey, we reveal diverse communities with representatives of all major eukaryotic taxonomic supergroups, mainly composed of a diverse pool of low abundance OTUs. Basin communities were very distinctive, with alpha and beta diversity patterns non-related to basin size or spatial distance respectively. Dissimilarity between basins was mainly characterised by turnover of OTUs. The strong microbial eukaryotic heterogeneity observed among the basins may be explained by a complex combination of deterministic factors (diverging environment in the basins), spatial constraints, and randomness including founder effects. Most interestingly, communities contain organisms that cannot coexist at the same time because of incompatible metabolic requirements, thus suggesting the existence of a pool of dormant organisms whose activity varies along with the changing environment. These organisms accumulate in the pools, which turns granitic rock into high biodiversity microbial islands whose conservation and study deserve further attention.

Correction to: Rain-Fed Granite Rock Basins Accumulate a High Diversity of Dormant Microbial Eukaryotes.

The original version of this article contained an erratum of omission in the Acknowledgments section.

Characterization of mammalian Lipocalin UTRs in silico: Predictions for their role in post-transcriptional regulation.

The Lipocalin family is a group of homologous proteins characterized by its big array of functional capabilities. As extracellular proteins, they can bind small hydrophobic ligands through a well-conserved ß-barrel folding. Lipocalins evolutionary history sprawls across many different taxa and shows great divergence even within chordates. This variability is also found in their heterogeneous tissue expression pattern. Although a handful of promoter regions have been previously described, studies on UTR regulatory roles in Lipocalin gene expression are scarce. Here we report a comprehensive bioinformatic analysis showing that complex post-transcriptional regulation exists in Lipocalin genes, as suggested by the presence of alternative UTRs with substantial sequence conservation in mammals, alongside a high diversity of transcription start sites and alternative promoters. Strong selective pressure could have operated upon Lipocalins UTRs, leading to an enrichment in particular sequence motifs that limit the choice of secondary structures. Mapping these regulatory features to the expression pattern of early and late diverging Lipocalins suggests that UTRs represent an additional phylogenetic signal, which may help to uncover how functional pleiotropy originated within the Lipocalin family.

The MTT-formazan assay: Complementary technical approaches and in vivo validation in Drosophila larvae.

The MTT assay was the first widely accepted method to assess cytotoxicity and cell viability. However, there is controversy on whether this indicator is a useful tool. In this work we intend to expand the interpretability of the MTT study by its combination with widely used cellular biology techniques. We propose complementary approaches to the colorimetric assay, based on the use of measurements in three different settings: confocal microscopy, multi-well plate assay and flow cytometry. Using confocal microscopy, we confirmed that MTT uptake and reduction by cells is a time-dependent process, and that formazan accumulates in round-shaped organelles. Quantitative measurements with a multi-well fluorimeter combined with nuclear staining result in a useful method, yielding a ratio between formazan production and cell number that informs about the average cell metabolic state. We also found that flow cytometry is a suitable technique to measure MTT reduction in large cell populations. When assaying the effect of an oxidizing agent such as paraquat (PQ), this approach allows for the distinction of subpopulations of cells with different reducing power. Finally, we prove that it is feasible to monitor MTT reduction in an in vivo model, the Drosophila larvae, without affecting its survival rate. Formazan accumulates exclusively in the larval fat body, confirming its lipid solubility. The methods explored in this work expand the MTT potential as a useful tool to provide information of the physiological state of cells and organisms.

Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress.

Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neurodegeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular functions, critical for the outcome of a wide variety of neurodegenerative diseases. These results open therapeutic opportunities by providing a route of entry and a repair mechanism for lysosomes in pathological situations.

An automated image analysis method to measure regularity in biological patterns: a case study in a Drosophila neurodegenerative model.

The fruitfly compound eye has been broadly used as a model for neurodegenerative diseases. Classical quantitative techniques to estimate the degeneration level of an eye under certain experimental conditions rely either on time consuming histological techniques to measure retinal thickness, or pseudopupil visualization and manual counting. Alternatively, visual examination of the eye surface appearance gives only a qualitative approximation provided the observer is well-trained. Therefore, there is a need for a simplified and standardized analysis of fruitfly eye degeneration extent for both routine laboratory use and for automated high-throughput analysis. We have designed the freely available ImageJ plugin FLEYE, a novel and user-friendly method for quantitative unbiased evaluation of neurodegeneration levels based on the acquisition of fly eye surface pictures. The incorporation of automated image analysis tools and a classification algorithm sustained on a built-in statistical model allow the user to quickly analyze large sample size data with reliability and robustness. Pharmacological screenings or genetic studies using the Drosophila retina as a model system may benefit from our method, because it can be easily implemented in a fully automated environment. In addition, FLEYE can be trained to optimize the image detection capabilities, resulting in a versatile approach to evaluate the pattern regularity of other biological or non-biological samples and their experimental or pathological disruption.