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Currently, there is a great need for the development of three-dimensional (3D) in vitro lung models. Particularly, the production of a suitable 3D model of pulmonary epithelium for understanding the pathophysiology of diseases such as the COVID-19 must consider the tissue architecture and presence, for example, of the angiotensin-converting enzyme-2 (ACE-2) in the cells. Different polymeric membranes are being used to support cell culturing, especially of lung cells, however, there is still no information about the culture of these cells onto bacterial nanocellulose (BNC) matrices. We have used the BNC matrix CellFate® as a support for the assembly of a 3D in vitro model of lung epithelium, composed of human lung fibroblasts (HLF) and lung adenocarcinoma cells (CALU-3). CellFate® matrices were made from bacterial fermentation resulting in a natural and biocompatible biopolymer. Cells were cultured onto CellFate® and maintained in a 5% CO2 humidified atmosphere at 37°C. Cell viability was assessed by the resazurin method The samples were, then, exposed to the air-liquid interface (ALI), and histologically analyzed. ACE-2 activity was verified on the hydrolyze of the fluorogenic substrate Mca-APK(Dnp)-OH, and its presence was evaluated by flow cytometry. The expression of the anionic transporter SLCO3A1 was evaluated by qPCR. Cell viability analysis indicates that CellFate® was not toxic to these cells. By flow cytometry, the presence of the ACE-2 was identified in the CALU-3 cells surface corroborating the results obtained from enzymatic activity analysis. The SLCO3A1 transporter expression was identified in cells cultured onto CellFate®, but not in cells cultured onto the transwell (control). CALU-3 cells cultivated onto CellFate® resulted in a pseudostratified organization, a typical morphology of the human respiratory tract epithelium. The current model opens perspectives for studies involving physiological characterization, improving its relevance for the understanding of the pathophysiology of diseases as well as the response to drugs.

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Parkinson's disease (PD) is a complex and multifaceted neurodegenerative disorder that results from multiple environmental factors and multicellular interactions. Although several PD neuropathologies have been identified and described, the thorough understanding of PD pathophysiology and research has been largely limited by the absence of reliablein vitromodels that truly recapitulate PD microenvironments. Here, we propose a neuroimmune co-culture system that models PD neuropathologies by combining relevant multicellular interactions with environments that mimic the brain. This system is composed of: (i) 3D bioprinted cultures of mature human dopaminergic (DA) neurons grown on extracellular matrix (ECM)-derived scaffolds doped with electroconductive nanostructures, and (ii) a direct co-culture of human astrocytes and differentiated monocytes that models neuroinflammatory responses. When co-cultured in a transwell format, these two compartments recreate relevant multicellular environments that model PD pathologies after exposure to the neurotoxin A53Tα-synuclein. With immunofluorescent staining and gene expression analyses, we show that functional and mature DA 3D networks are generated within our ECM-derived scaffolds with superior performance to standard 2D cultures. Moreover, by analyzing cytokine secretion, cell surface markers, and gene expression, we define a human monocyte differentiation scheme that allows the appearance of both monocyte-derived macrophages and dendritic cell phenotypes, as well as their optimal co-culture ratios with human astrocytes to recreate synergistic neuroinflammatory responses. We show that the combined response of both compartments to A53Tα-synuclein stimulates the formation of intracellularα-synuclein aggregates, induces progressive mitochondrial dysfunction and reactive oxygen species production, downregulates the expression of synaptic, DA, and mitophagy-related genes, and promotes the initiation of apoptotic processes within the DA networks. Most importantly, these intracellular pathologies were comparable or superior to those generated with a rotenone-stimulated 2D control that represents the current standard forin vitroPD models and showed increased resilience towards these neurotoxic insults, allowing the study of disease progression over longer time periods than current models. Taken together, these results position the proposed model as a superior alternative to current 2D models for generating PD-related pathologiesin vitro.

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We created the "3-dimensional synaptic puzzle" (3Dsp) as an educational resource for the physiology teaching of synaptic transmission (ST). In this study, we aimed to apply and evaluate the use of 3Dsp. For this, we divided 175 university students from public and private universities into two groups: 1) control (CT; students that were only exposed to traditional class or video lessons about ST), and; 2) test (3Dsp; students that were exposed to the 3Dsp practical class in addition to the traditional theoretical class). ST knowledge of students was evaluated before, immediately after, and 15 days after interventions. Additionally, students completed a questionnaire about their perception of teaching-learning methods used in physiology classes and their self-perception of engagement in the physiology content. The CT groups improved their ST knowledge score from pretest to immediate (P < 0.0001 for all groups) and late posttest (P < 0.0001 for all groups). 3Dsp groups also enhanced their score from pretest to immediate (P = 0.029 for public university students; P < 0.0001 for private university students) and late posttest (P < 0.0001 for all groups). We also observed improvement from the immediate to late posttest in the 3Dsp group from private universities (P < 0.001). Both private groups performed better in general ST and specific electrical synapse questions in the pretest and immediate posttest compared to the public CT group (P < 0.05 for all comparisons). More than 90% of the students from both universities affirmed that the 3Dsp contributed to their physiology comprehension and that they would recommend the use of the 3-D models to other teachers in their classes.NEW & NOTEWORTHY We included a 3-dimensional puzzle (3Dsp) of electrical and chemical synapses in the physiology of synaptic transmission (ST) teaching. After a traditional or video lesson class, students from private and public universities were oriented to use the educational resource. More than 90% of the students affirmed that the 3Dsp improved their comprehension of ST content.

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Facial nerve stimulation (FNS) is a potential complication which may affect the auditory performance of children with cochlear implants (CIs). We carried out an exploratory prospective observational study to investigate the effects of the electrical stimulation pattern on FNS reduction in young children with CI. Ten ears of seven prelingually deafened children with ages up to 6 years old who undergone a unilateral or bilateral CI surgery were included in this study. Electromyographic (EMG) action potentials from orbicularis oculi muscle were recorded using monopolar biphasic stimulation (ST1) and multi-mode monophasic stimulation with capacitive discharge (ST2). Presence of EMG responses, facial nerve stimulation thresholds (T-FNS) and EMG amplitudes were compared between ST1 and ST2. Intra-cochlear electrodes placement, cochlear-nerve and electrode-nerve distances were also estimated to investigate their effects on EMG responses. The use of ST2 significantly reduced the presence of intraoperative EMG responses compared to ST1. Higher stimulation levels were required to elicit FNS with ST2, with smaller amplitudes, compared to ST1. No and weak correlation was observed between cochlea-nerve and electrode-nerve distances and EMG responses, respectively. ST2 may reduce FNS in young children with CI. Differently from the electrical stimulation pattern, the cochlea-nerve and electrode-nerve distances seem to have limited effects on FNS in this population.

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Objectives: To investigate the outcomes of cochlear re-implantation using multi-mode grounding stimulation associated with anodic monophasic pulses to manage abnormal facial nerve stimulation (AFNS) in cochlear implant (CI) recipients. Methods: Retrospective case report. An adult CI recipient with severe AFNS and decrease in auditory performance was re-implanted with a new CI device to change the pulse shape and stimulation mode. Patient's speech perception scores and AFNS were compared before and after cochlear re-implantation, using monopolar stimulation associated with cathodic biphasic pulses and multi-mode stimulation mode associated to anodic monophasic pulses, respectively. The insertion depth angle and the electrode-nerve distances were also investigated, before and after cochlear re-implantation. Results: AFNS was resolved, and the speech recognition scores rapidly increased in the first year after cochlear re-implantation while remaining stable. After cochlear re-implantation, the e15 and e20 electrodes showed shorter electrode-nerve distances compared to their correspondent e4 and e7 electrodes, which induced AFNS in the first implantation. Conclusions: Cochlear re-implantation with multi-mode grounding stimulation associated with anodic monophasic pulses was an effective strategy for managing AFNS. The patient's speech perception scores rapidly improved and AFNS was not detected four years after cochlear re-implantation.

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Aquatic biota is increasingly being exposed to chemical pollutants due to human activities and the relationship between the level of environmental pollution and fish reproduction is a continuously ongoing issue. The vitellogenin (Vtg) protein synthesis can be induced in the liver of juvenile and male fish after stimulation of the estrogen receptor and therefore, Vtg has been used as a biomarker of xenoestrogen exposure in several fish species. The current study reported the first physicochemical characterization of Vtg from Oreochromis niloticus. Adult male fish were exposed to 17α-ethinylestradiol for Vtg induction. Purified vitellogenin from plasma showed low stability at 25 and 4 °C in saline conditions, and good stability in acidic (low pH) or in heated conditions. The 3D modeling provided useful information on the structure of O. niloticus Vtg showing conserved structural features. According to bioinformatics and experimental results, there are important structural differences between the two chemical forms of Vtg (VtgAb and VtgC) in a phylogenetic context. The present results add information about the development of ecotoxicological immunoassays to study the endocrine disruption in O. niloticus improving the Vtg performance as a biomarker of reproduction in fish.

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Nuclear receptors are ligand-activated transcription factors capable of regulating the expression of complex gene networks. The family includes seven subfamilies of protein with a wide phylogenetic distribution. A novel subfamily with two DNA-binding domains (2DBDs) has been first reported in Schistosoma mansoni (Platyhelminth, Trematoda). Employing an ab initio protocol and homology modeling methods, the full-length 3D structure of the Eg2DBDα.1 nuclear receptor from Echinococcus granulosus (Platyhelminth, Cestoda) was generated. The model analysis reveals the presence of the conserved three-layered alpha-helical sandwich structure in the ligand binding domain, and a particularly long and flexible hinge region. Molecular dynamics simulations were performed previous to dock a conformational library of fatty acids and retinoic acids. Our results indicate that oleic and linoleic acids are suitable ligands to this receptor. The ligand-protein complex is stabilized mainly by hydrogen bonds and hydrophobic interactions. The fact that 2DBD nuclear receptors have not been identified in vertebrates confers particular interest to these nuclear receptors, not only concerning their structure and function but as targets of new anthelmintic drugs.Communicated by Ramaswamy H. Sarma.

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As hypoxic tumors show resistance to several clinical treatments, photosynthetic microorganisms have been recently suggested as a promising safe alternative for oxygenating the tumor microenvironment. The relationship between organisms and the effect microalgae have on tumors is still largely unknown, evidencing the need for a simple yet representative model for studying photosynthetic tumor oxygenation in a reproducible manner. Here, we present a 3D photosynthetic tumor model composed of human melanoma cells and the microalgae Chlamydomonas reinhardtii, both seeded into a collagen scaffold, which allows for the simultaneous study of both cell types. This work focuses on the biocompatibility and cellular interactions of the two cell types, as well as the study of photosynthetic oxygenation of the tumor cells. It is shown that both cell types are biocompatible with one another at cell culture conditions and that a 10:1 ratio of microalgae to cells meets the metabolic requirement of the tumor cells, producing over twice the required amount of oxygen. This 3D tumor model provides an easy-to-use in vitro resource for analyzing the effects of photosynthetically produced oxygen on a tumor microenvironment, thus opening various potential research avenues.

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Increasing studies on vasculogenic mimicry (VM) have shown that the hypoxic microenvironment and the presence of endothelial cell play an important role in regulating tumor phenotype and aggressiveness. Thus, the representation of these factors in vitro becomes necessary to mimic VM. This chapter provides a protocol for mimicking VM in vitro in a more robust 3D model that includes the presence of 3D matrix, melanoma cells, a hypoxia-inducing agent, and endothelial cells.

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Macroautophagy/autophagy is a fundamental catabolic pathway that maintains cellular homeostasis in eukaryotic cells by forming double-membrane-bound vesicles named autophagosomes. The autophagy family genes remain largely unexplored except in some model organisms. Legumes are a large family of economically important crops, and knowledge of their important cellular processes is essential. Here, to first address the knowledge gaps, we identified 17 ATG families in Phaseolus vulgaris, Medicago truncatula and Glycine max based on Arabidopsis sequences and elucidated their phylogenetic relationships. Second, we dissected ATG18 in subfamilies from early plant lineages, chlorophytes to higher plants, legumes, which included a total of 27 photosynthetic organisms. Third, we focused on the ATG18 family in P. vulgaris to understand the protein structure and developed a 3D model for PvATG18b. Our results identified ATG homologs in the chosen legumes and differential expression data revealed the nitrate-responsive nature of ATG genes. A multidimensional scaling analysis of 280 protein sequences from 27 photosynthetic organisms classified ATG18 homologs into three subfamilies that were not based on the BCAS3 domain alone. The domain structure, protein motifs (FRRG) and the stable folding conformation structure of PvATG18b revealing the possible lipid-binding sites and transmembrane helices led us to propose PvATG18b as the functional homolog of AtATG18b. The findings of this study contribute to an in-depth understanding of the autophagy process in legumes and improve our knowledge of ATG18 subfamilies.

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El análisis de cuestionarios respondidos por alumnos de la disciplina Sistemas Sensoriales del curso de Fonoaudiología mostró que había dificultades para entender la histología de las regiones anatómicas del oído. Se estableció que la compresión de la histología demandaba una actividad didáctica complementaria. Por ello, se decidió crear una clase teórico-práctica diferenciada (CTP) usando un modelo tridimensional (3D) del oído humano y preparaciones histológicas de sus regiones. El modelo es una réplica anatómica muy aumentada del oído humano que permite la manipulación exploratoria de modo más eficaz que con piezas anatómicas clásicas. La CTP fue implementada en los dos semestres de 2014. La evaluación de la CTP incluyó un cuestionario de opiniones, más un cuaderno de actividades. Los resultados mostraron medias finales de mejores desempeños en ese año respecto al 2013, donde no fue dada este tipo de clase teórico-práctica. Se concluye que la CTP, asociada al modelo tridimensional y las explicaciones teóricas-prácticas de las preparaciones histológicas, permitió una mejor comprensión del contenido enseñado e introdujo una metodología más eficiente para el proceso de enseñanza-aprendizaje de un tema tan complejo así como importante para la formación académica de los estudiantes de Fonoaudiología. Adicionalmente, a partir de esta experiencia teórico-práctica se ha demostrado, una vez más, la importancia de la relación personal y profesional entre el profesor, preparador estudiantil y los alumnos para la calidad del trabajo académico.

We designed a differentiated theoretical-practical class (CTP) after analyzing questionnaires (Qs) answered by Speech-therapy students that attended the course "Sensory Systems". The Qs showed difficulties related with understanding the practical histology of the sensory systems, as well as problems to correlate it with the learning of the anatomy. We therefore established the to implement an auxiliary didactic activity (CTP). We used a three-dimensional model (3D) AS-001, of the University Extension Project (3D Museum). AS-001, is an enlarged anatomical replica of the human ear which is much more efficient for exploratory manipulation much than anatomical modeling. The assessment of CTP included a questionnaire and a notebook of activities. The CTP was implemented in both semesters during the year 2014. Results showed improvements when compared to 2013's data, when this type of theoretical-practical contents were not implemented. We conclude that the association of the three -dimensional model along with histological analysis of the parts of the ear effectively improved students' understanding of the subject. We therefore consider that CTP is a suitable methodology for the teaching-learning process of a complex, important subject for the academic training of Speech-therapy undergraduate students. Our experience allowed us to determine how important the personal and professional relationship between teachers and students is to improve academic work.

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Introducción: la fibrilación auricular (AF), es la más común de la arritmia cardiaca sostenida y un factor de riesgo para el accidente cerebro vascular y otras morbilidades, si no es tratada. Estudios epidemiológicos muestran que la AF tiende a perpetuarse con el tiempo, generando cambios electrofisiológicos y anatómicos denominados: remodelados auriculares. Se ha demostrado que estos cambios provocan variaciones de la velocidad de conducción (CV), en el tejido auricular. Objetivo: estudiar el efecto del remodelado de gap junctions en la propagación del potencial de acción, implementando un modelo 3D de aurícula humana altamente realista. Materiales y Métodos: se incorporaron los cambios generados por el remodelado eléctrico a un modelo de potencial de acción (AP) de miocito auricular, acoplado con un modelo tridimensional anatómicamente realista de aurícula humana dilatada. Mediante simulaciones de la propagación del AP en condiciones de remodelado eléctrico y anatómico, y de remodelado de gap junctions, se midieron las ventanas vulnerables de generación de reentradas en la base de las venas pulmonares izquierdas de la aurícula. Resultados: los resultados obtenidos indican que la ventana vulnerable en el remodelado de gap junctions, se desplazó 38 ms con relación al modelo dilatado, lo que nos muestra el impacto de la dilatación con remodelado de gap junction. Conclusiones: el remodelado eléctrico generó una disminución del 70 % en la duración del potencial de acción y una disminución de las velocidades de conducción entre un 14.6 y un 26 %, que fueron medidas en diferentes regiones de la aurícula dilatada. El foco disparado en la base de las venas pulmonares izquierdas, generó un frente de onda que mantiene una actividad reentrante debido a la anatomía subyacente de las venas pulmonares.

Introduction: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a significant risk factor for cerebrovascular accident and other morbidities if left untreated. Epidemiological studies show that AF tends to persist over time, creating electrophysiological and anatomical changes called atrial remodeling. It has been shown that these changes result in variations in conduction velocity (CV) in the atrial tissue. Objective: to study the effect of remodeling of gap junctions in the propagation of the action potential by implementing a highly realistic 3D human atrial model. Materials and methods: the changes caused by electrical remodeling were incorporated in an atrial myocyte action potential (AP) model coupled with an anatomically realistic three-dimensional model of dilated human atria. Through simulations of the AP spread in variations of anatomical and electrical remodeling and of gap junctions remodeling, vulnerable windows of reentry generation were measured at the base of the atrium left pulmonary veins. Results: the results obtained indicate that vulnerable window in the gap junctions remodeling moved 38 ms in relation with the expanded model which shows the impact of the dilatation gap junction remodeling. Conclusions: the electrical remodeling produced 70% decrease in action potential duration and decreased conduction velocities between 14.6 and 26 %, which were measured in different regions of the dilated atrium. The focus shot at the base of the left pulmonary veins created a wave which maintains a reentering activity due to the underlying anatomy of the pulmonary veins.