Year 2021

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Fonte, M., Tassi, N., Fontinha, D., Bouzón-Arnáiz, I., Ferraz, R., Araújo, M. J., Fernàndez-Busquets, X., Prudêncio, M., Gomes, P., Teixeira, C., (2021). 4,9-Diaminoacridines and 4-aminoacridines as dual-stage antiplasmodial hits ChemMedChem 16, (5), 788-792

Multi-stage drugs have been prioritized in antimalarial drug discovery, as targeting more than one process in the Plasmodium life cycle is likely to increase efficiency, while decreasing the chances of emergence of resistance by the parasite. Herein, we disclose two novel acridine-based families of compounds that combine the structural features of primaquine and chloroquine. Compounds prepared and studied thus far retained the in vitro activity displayed by the parent drugs against the erythrocytic stages of chloroquine-sensitive and -resistant Plasmodium falciparum strains, and against the hepatic stages of Plasmodium berghei, hence acting as dual-stage antiplasmodial hits.

Keywords: Acridines, Antimalarial activity, Blood-stage, Liver-stage, Malaria, Plasmodium, Synthesis

Prischich, Davia, Gomila, Alexandre M. J., Milla-Navarro, Santiago, Sangüesa, Gemma, Diez-Alarcia, Rebeca, Preda, Beatrice, Matera, Carlo, Batlle, Montserrat, Ramírez, Laura, Giralt, Ernest, Hernando, Jordi, Guasch, Eduard, Meana, J. Javier, de la Villa, Pedro, Gorostiza, Pau, (2021). Adrenergic modulation with photochromic ligands Angewandte Chemie International Edition 60, (7), 3625-3631

Adrenoceptors are ubiquitous and mediate important autonomic functions as well as modulating arousal, cognition and pain on a central level. Understanding these physiological processes and their underlying neural circuits requires manipulating adrenergic neurotransmission with high spatio-temporal precision. Here we present a first generation of photochromic ligands (adrenoswitches) obtained via azologization of a class of cyclic amidines related to the known ligand clonidine. Their pharmacology, photochromism, bioavailability and lack of toxicity allow for broad biological applications, as demonstrated by controlling locomotion in zebrafish and pupillary responses in mice.

Keywords: Adrenergic, Neurotransmitters, Azo compounds, Biological activity, Photochromism.

Badia, M., Bolognesi, B., (2021). Assembling the right type of switch: Protein condensation to signal cell death Current Opinion in Cell Biology 69, 55-61

Protein phase transitions are particularly amenable for cell signalling as these highly cooperative processes allow cells to make binary decisions in response to relatively small intracellular changes. The different processes of condensate formation and the distinct material properties of the resulting condensates provide a dictionary to modulate a range of decisions on cell fate. We argue that, on the one hand, the reversibility of liquid demixing offers a chance to arrest cell growth under specific circumstances. On the other hand, the transition to amyloids is better suited for terminal decisions such as those leading to apoptosis and necrosis. Here, we review recent examples of both scenarios, highlighting how mutations in signalling proteins affect the formation of biomolecular condensates with drastic effects on cell survival.

Keywords: Amyloid, Cell death, Deep mutagenesis, LLPS, RNA-binding proteins

Castillo-Escario, Yolanda, Kumru, Hatice, Valls-Solé, Josep, García-Alen, Loreto, Vidal, Joan, Jané, Raimon, (2021). Assessment of trunk flexion in arm reaching tasks with electromyography and smartphone accelerometry in healthy human subjects Scientific Reports 11, (1), 5363

Trunk stability is essential to maintain upright posture and support functional movements. In this study, we aimed to characterize the muscle activity and movement patterns of trunk flexion during an arm reaching task in sitting healthy subjects and investigate whether trunk stability is affected by a startling acoustic stimulus (SAS). For these purposes, we calculated the electromyographic (EMG) onset latencies and amplitude parameters in 8 trunk, neck, and shoulder muscles, and the tilt angle and movement features from smartphone accelerometer signals recorded during trunk bending in 33 healthy volunteers. Two-way repeated measures ANOVAs were applied to examine the effects of SAS and target distance (15 cm vs 30 cm). We found that SAS markedly reduced the response time and EMG onset latencies of all muscles, without changing neither movement duration nor muscle recruitment pattern. Longer durations, higher tilt angles, and higher EMG amplitudes were observed at 30 cm compared to 15 cm. The accelerometer signals had a higher frequency content in SAS trials, suggesting reduced movement control. The proposed measures have helped to establish the trunk flexion pattern in arm reaching in healthy subjects, which could be useful for future objective assessment of trunk stability in patients with neurological affections.

Fernández-Costa, J. M., Fernández-Garibay, X., Velasco-Mallorquí, F., Ramón-Azcón, J., (2021). Bioengineered in vitro skeletal muscles as new tools for muscular dystrophies preclinical studies Journal of Tissue Engineering 12, 1-19

Muscular dystrophies are a group of highly disabling disorders that share degenerative muscle weakness and wasting as common symptoms. To date, there is not an effective cure for these diseases. In the last years, bioengineered tissues have emerged as powerful tools for preclinical studies. In this review, we summarize the recent technological advances in skeletal muscle tissue engineering. We identify several ground-breaking techniques to fabricate in vitro bioartificial muscles. Accumulating evidence shows that scaffold-based tissue engineering provides topographical cues that enhance the viability and maturation of skeletal muscle. Functional bioartificial muscles have been developed using human myoblasts. These tissues accurately responded to electrical and biological stimulation. Moreover, advanced drug screening tools can be fabricated integrating these tissues in electrical stimulation platforms. However, more work introducing patient-derived cells and integrating these tissues in microdevices is needed to promote the clinical translation of bioengineered skeletal muscle as preclinical tools for muscular dystrophies.

Keywords: Biomaterials, Drug screening platforms, Muscular dystrophy, Skeletal muscle, Tissue engineering

Mestre, R., Patiño, T., Sánchez, S., (2021). Biohybrid robotics: From the nanoscale to the macroscale Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology

Biohybrid robotics is a field in which biological entities are combined with artificial materials in order to obtain improved performance or features that are difficult to mimic with hand-made materials. Three main level of integration can be envisioned depending on the complexity of the biological entity, ranging from the nanoscale to the macroscale. At the nanoscale, enzymes that catalyze biocompatible reactions can be used as power sources for self-propelled nanoparticles of different geometries and compositions, obtaining rather interesting active matter systems that acquire importance in the biomedical field as drug delivery systems. At the microscale, single enzymes are substituted by complete cells, such as bacteria or spermatozoa, whose self-propelling capabilities can be used to transport cargo and can also be used as drug delivery systems, for in vitro fertilization practices or for biofilm removal. Finally, at the macroscale, the combinations of millions of cells forming tissues can be used to power biorobotic devices or bioactuators by using muscle cells. Both cardiac and skeletal muscle tissue have been part of remarkable examples of untethered biorobots that can crawl or swim due to the contractions of the tissue and current developments aim at the integration of several types of tissue to obtain more realistic biomimetic devices, which could lead to the next generation of hybrid robotics. Tethered bioactuators, however, result in excellent candidates for tissue models for drug screening purposes or the study of muscle myopathies due to their three-dimensional architecture. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Nanotechnology Approaches to Biology > Nanoscale Systems in Biology. © 2021 Wiley Periodicals LLC.

Keywords: bacteria-bots, biorobots, enzymatic nanomotors, hybrid robotics, muscle-based biorobots

Watt, April C., Cejas, Paloma, DeCristo, Molly J., Metzger-Filho, Otto, Lam, Enid Y. N., Qiu, Xintao, BrinJones, Haley, Kesten, Nikolas, Coulson, Rhiannon, Font-Tello, Alba, Lim, Klothilda, Vadhi, Raga, Daniels, Veerle W., Montero, Joan, Taing, Len, Meyer, Clifford A., Gilan, Omer, Bell, Charles C., Korthauer, Keegan D., Giambartolomei, Claudia, Pasaniuc, Bogdan, Seo, Ji-Heui, Freedman, Matthew L., Ma, Cynthia, Ellis, Matthew J., Krop, Ian, Winer, Eric, Letai, Anthony, Brown, Myles, Dawson, Mark A., Long, Henry W., Zhao, Jean J., Goel, Shom, (2021). CDK4/6 inhibition reprograms the breast cancer enhancer landscape by stimulating AP-1 transcriptional activity Nature Cancer 2, 34-48

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) were designed to induce cancer cell cycle arrest. Recent studies have suggested that these agents also exert other effects, influencing cancer cell immunogenicity, apoptotic responses and differentiation. Using cell-based and mouse models of breast cancer together with clinical specimens, we show that CDK4/6 inhibitors induce remodeling of cancer cell chromatin characterized by widespread enhancer activation, and that this explains many of these effects. The newly activated enhancers include classical super-enhancers that drive luminal differentiation and apoptotic evasion, as well as a set of enhancers overlying endogenous retroviral elements that are enriched for proximity to interferon-driven genes. Mechanistically, CDK4/6 inhibition increases the level of several activator protein-1 transcription factor proteins, which are in turn implicated in the activity of many of the new enhancers. Our findings offer insights into CDK4/6 pathway biology and should inform the future development of CDK4/6 inhibitors.

Keywords: Breast cancer, Cancer, Cancer therapy, Epigenetics

Moya-Andérico, Laura, Admella, Joana, Torrents, Eduard, (2021). A clearing protocol for Galleria mellonella larvae: Visualization of internalized fluorescent nanoparticles New Biotechnology 60, 20-26

Light scattering is a challenge for imaging three-dimensional organisms. A number of new tissue clearing methodologies have been described in recent years, increasing the utilities of clearing techniques to obtain transparent samples. Here, we describe the optimization of a suitable and novel protocol for clearing Galleria mellonella larvae, an alternative infection animal model with a promising potential for the toxicological evaluation of different molecules and materials. This has allowed the visualization of internalised fluorescent nanoparticles using confocal microscopy, opening the door to a wide range of different applications.

Keywords: Nanotoxicology, Tissue clearance

Blanco-Almazan, D., Groenendaal, W., Lozano-Garcia, M., Estrada-Petrocelli, L., Lijnen, L., Smeets, C., Ruttens, D., Catthoor, F., Jané, R., (2021). Combining bioimpedance and myographic signals for the assessment of COPD during loaded breathing IEEE Transactions on Biomedical Engineering 68, (1), 298-307

Chronic Obstructive Pulmonary Disease (COPD) is one of the most common chronic conditions. The current assessment of COPD requires a maximal maneuver during a spirometry test to quantify airflow limitations of patients. Other less invasive measurements such as thoracic bioimpedance and myographic signals have been studied as an alternative to classical methods as they provide information about respiration. Particularly, strong correlations have been shown between thoracic bioimpedance and respiratory volume. The main objective of this study is to investigate bioimpedance and its combination with myographic parameters in COPD patients to assess the applicability in respiratory disease monitoring. We measured bioimpedance, surface electromyography and surface mechanomyography in forty-three COPD patients during an incremental inspiratory threshold loading protocol. We introduced two novel features that can be used to assess COPD condition derived from the variation of bioimpedance and the electrical and mechanical activity during each respiratory cycle. These features demonstrate significant differences between mild and severe patients, indicating a lower inspiratory contribution of the inspiratory muscles to global respiratory ventilation in the severest COPD patients. In conclusion, the combination of bioimpedance and myographic signals provides useful indices to noninvasively assess the breathing of COPD patients.

Keywords: Bioimpedance, Chronic obstructive pulmonary disease, Inspiratory threshold protocol, Myographic signals, Wearables

Low, S. C., Vouloutsi, V., Verschure, P., (2021). Complementary interactions between classical and top-down driven inhibitory mechanisms of attention Cognitive Systems Research 67, 66-72

Selective attention informs decision-making by biasing perceptual processing towards task-relevant stimuli. In experimental and computational literature, this is most often implemented through top-down excitation of selected stimuli. However, physiological and anatomical evidence shows that in certain situations, top-down signals could instead be inhibitory. In this study, we investigated how such an inhibitory mechanism of top-down attention compares with an excitatory one. We did so in a neurorobotics context where the agent was controlled using an established hierarchical architecture. We augmented the architecture with an attentional system that implemented top-down attention biasing as connection gains. We tested four models of top-down attention on the simulated agent performing a foraging task: without top-down biasing, with only excitatory top-down gain, with only inhibitory top-down gain, and with both excitatory and inhibitory top-down gain. We manipulated the reward-distractor ratio that was presented and assessed the agent's performance using accumulated rewards and the latency of the selection. Using these measures, we provide evidence that excitatory and inhibitory mechanisms of attention complement each other.

Keywords: Selective attention, Inhibition, Foraging, Embodied cognition

Wiers, Reinout W., Verschure, Paul, (2021). Curing the broken brain model of addiction: Neurorehabilitation from a systems perspective Addictive Behaviors 112, 106602

The dominant biomedical perspective on addictions has been that they are chronic brain diseases. While we acknowledge that the brains of people with addictions differ from those without, we argue that the “broken brain” model of addiction has important limitations. We propose that a systems-level perspective more effectively captures the integrated architecture of the embodied and situated human mind and brain in relation to the development of addictions. This more dynamic conceptualization places addiction in the broader context of the addicted brain that drives behavior, where the addicted brain is the substrate of the addicted mind, that in turn is situated in a physical and socio-cultural environment. From this perspective, neurorehabilitation should shift from a “broken-brain” to a systems theoretical framework, which includes high-level concepts related to the physical and social environment, motivation, self-image, and the meaning of alternative activities, which in turn will dynamically influence subsequent brain adaptations. We call this integrated approach system-oriented neurorehabilitation. We illustrate our proposal by showing the link between addiction and the architecture of the embodied brain, including a systems-level perspective on classical conditioning, which has been successfully translated into neurorehabilitation. Central to this example is the notion that the human brain makes predictions on future states as well as expected (or counterfactual) errors, in the context of its goals. We advocate system-oriented neurorehabilitation of addiction where the patients' goals are central in targeted, personalized assessment and intervention.

Keywords: Addiction, Brain disease model, Neurorehabilitation, Systems approach

de la Serna, E., Arias-Alpízar, K., Borgheti-Cardoso, L. N., Sanchez-Cano, A., Sulleiro, E., Zarzuela, F., Bosch-Nicolau, P., Salvador, F., Molina, I., Ramírez, M., Fernàndez-Busquets, X., Sánchez-Montalvá, A., Baldrich, E., (2021). Detection of Plasmodium falciparum malaria in 1 h using a simplified enzyme-linked immunosorbent assay Analytica Chimica Acta 1152

Malaria is a parasitic disease caused by protists of the genus Plasmodium, which are transmitted to humans through the bite of infected female Anopheles mosquitoes. Analytical methodologies and efficient drugs exist for the early detection and treatment of malaria, and yet this disease continues infecting millions of people and claiming several hundred thousand lives each year. One of the reasons behind this failure to control the disease is that the standard method for malaria diagnosis, microscopy, is time-consuming and requires trained personnel. Alternatively, rapid diagnostic tests, which have become common for point-of-care testing thanks to their simplicity of use, tend to be insufficiently sensitive and reliable, and PCR, which is sensitive, is too complex and expensive for massive population screening. In this work, we report a sensitive simplified ELISA for the quantitation of Plasmodium falciparum lactate dehydrogenase (Pf-LDH), which is capable of detecting malaria in 45–60 min. Assay development was founded in the selection of high-performance antibodies, implementation of a poly-horseradish peroxidase (polyHRP) signal amplifier, and optimization of whole-blood sample pre-treatment. The simplified ELISA achieved limits of detection (LOD) and quantification (LOQ) of 0.11 ng mL−1 and 0.37 ng mL−1, respectively, in lysed whole blood, and an LOD comparable to that of PCR in Plasmodium in vitro cultures (0.67 and 1.33 parasites μL−1 for ELISA and PCR, respectively). Accordingly, the developed immunoassay represents a simple and effective diagnostic tool for P. falciparum malaria, with a time-to-result of <60 min and sensitivity similar to the reference PCR, but easier to implement in low-resource settings. © 2021 Elsevier B.V.

Keywords: Malaria quantitative diagnosis, Plasmodium culture, Plasmodium LDH, polyHRP signal amplifier, Simplified ELISA

Campo-Pérez, Víctor, Cendra, Maria del Mar, Julián, Esther, Torrents, Eduard, (2021). Easily applicable modifications to electroporation conditions improve the transformation efficiency rates for rough morphotypes of fast-growing mycobacteria New Biotechnology 63, 10-18

Electroporation is the most widely used and efficient method to transform mycobacteria. Through this technique, fast- and slow-growing mycobacteria with smooth and rough morphotypes have been successfully transformed. However, transformation efficiencies differ widely between species and strains. In this study, the smooth and rough morphotypes of Mycobacteroides abscessus and Mycolicibacterium brumae were used to improve current electroporation procedures for fast-growing rough mycobacteria. The focus was on minimizing three well-known and challenging limitations: the mycobacterial restriction-modification systems, which degrade foreign DNA; clump formation of electrocompetent cells before electroporation; and electrical discharges during pulse delivery, which were reduced by using salt-free DNA solution. Herein, different strategies are presented that successfully address these three limitations and clearly improve the electroporation efficiencies over the current procedures. The results demonstrated that combining the developed strategies during electroporation is highly recommended for the transformation of fast-growing rough mycobacteria.

Keywords: Mycobacteria, Clump, Desalted DNA, Electroporation

Oliver-Cervelló, L., Martin-Gómez, H., Reyes, L., Noureddine, F., Ada Cavalcanti-Adam, E., Ginebra, M. P., Mas-Moruno, C., (2021). An Engineered Biomimetic Peptide Regulates Cell Behavior by Synergistic Integrin and Growth Factor Signaling Advanced Healthcare Materials Early View, 2001757

Recreating the healing microenvironment is essential to regulate cell–material interactions and ensure the integration of biomaterials. To repair bone, such bioactivity can be achieved by mimicking its extracellular matrix (ECM) and by stimulating integrin and growth factor (GF) signaling. However, current approaches relying on the use of GFs, such as bone morphogenetic protein 2 (BMP-2), entail clinical risks. Here, a biomimetic peptide integrating the RGD cell adhesive sequence and the osteogenic DWIVA motif derived from the wrist epitope of BMP-2 is presented. The approach offers the advantage of having a spatial control over the single binding of integrins and BMP receptors. Such multifunctional platform is designed to incorporate 3,4-dihydroxyphenylalanine to bind metallic oxides with high affinity in a one step process. Functionalization of glass substrates with the engineered peptide is characterized by physicochemical methods, proving a successful surface modification. The biomimetic interfaces significantly improve the adhesion of C2C12 cells, inhibit myotube formation, and activate the BMP-dependent signaling via p38. These effects are not observed on surfaces displaying only one bioactive motif, a mixture of both motifs or soluble DWIVA. These data prove the biological potential of recreating the ECM and engaging in integrin and GF crosstalk via molecular-based mimics.

Keywords: Biomimetic peptides, Cell adhesion, Cell differentiation, DWIVA, RGD, Surface functionalization

Sotero Chacon, Daisy, de Melo Torres, Taffarel, Bezerra da Silva, Ivanice, Ferreira de Araújo, Thiago, de Araújo Roque, Alan, Ayrton Senna Domingos Pinheiro, Francisco, Selegatto, Denise, Pilon, Alan, Priscila Santos Reginaldo, Fernanda, Tesser da Costa, Cibele, Vilasboa, Johnatan, Freire, Rafael, Luiz Voigt, Eduardo, Angelo Silveira Zuanazzi, José, Libonati, Renata, Rodrigues, Julia A., Santos, Filippe L. M., Castanho Scortecci, Kátia, Peporine Lopes, Norberto, De Santis Ferreira, Leandro, Vieira dos Santos, Leandro, José Cavalheiro, Alberto, Germano Fett-Neto, Arthur, Brandt Giordani, Raquel, (2021). Erythrina velutina Willd. alkaloids: piecing biosynthesis together from transcriptome analysis and metabolite profiling of seeds and leaves Journal of Advanced Research In press,

Natural products of pharmaceutical interest often do not reach the drug market due to the associated low yields and difficult extraction. Knowledge of biosynthetic pathways is a key element in the development of biotechnological strategies for plant specialized metabolite production. The scarce studies regarding non-model plants impair advances in this field. Erythrina spp. are mainly used as central nervous system depressants in folk medicine and are important sources of bioactive tetracyclic benzylisoquinoline alkaloids, which can act on several pathology-related biological targets. Objective: Herein the purpose is to employ combined transcriptome and metabolome analyses (seeds and leaves) of a non-model medicinal Fabaceae species grown in its unique arid natural habitat. The study tries to propose a putative biosynthetic pathway for the bioactive alkaloids by using an omic integrated approach. Methods: The Next Generation Sequencing-based transcriptome (de novo RNA sequencing) was carried out in a Illumina NextSeq 500 platform. Regarding the targeted metabolite profiling, Nuclear Magnetic Resonance and the High-Performance Liquid Chromatography coupled to a micrOTOF-QII, High Resolution Mass Spectrometer, were used. Results: This detailed macro and micromolecular approach applied to seeds and leaves of E. velutina revealed 42 alkaloids by metabolome tools. Based on the combined evidence, 24 gene candidates were put together in a putative pathway leading to the singular alkaloid diversity of this species. Conclusion: These results contribute by indicating potential biotechnological targets erythrina alkaloids biosynthesis as well as to improve molecular databases with omic data from a non-model medicinal plant. Furthermore, they reveal an interesting chemical diversity in Erythrina velutina harvested in Caatinga. Last, but not least, this data may also contribute to tap Brazilian biodiversity in a rational and sustainable fashion, promoting adequate public policies for preservation and protection of sensitive areas within the Caatinga.

Keywords: Transcriptome, Targeted metabolite profile, Benzylisoquinoline Alkaloids, Caatinga

Estrada-Petrocelli, L., Torres, A., Sarlabous, L., Rafols-de-Urquia, M., Ye-Lin, Y., Prats-Boluda, G., Jané, R., Garcia-Casado, J., (2021). Evaluation of respiratory muscle activity by means of concentric ring electrodes IEEE Transactions on Biomedical Engineering 68, (3), 1005 - 1014

Surface electromyography (sEMG) can be used for the evaluation of respiratory muscle activity. Recording sEMG involves the use of surface electrodes in a bipolar configuration. However, electrocardiographic (ECG) interference and electrode orientation represent considerable drawbacks to bipolar acquisition. As an alternative, concentric ring electrodes (CREs) can be used for sEMG acquisition and offer great potential for the evaluation of respiratory muscle activity due to their enhanced spatial resolution and simple placement protocol, which does not depend on muscle fiber orientation. The aim of this work was to analyze the performance of CREs during respiratory sEMG acquisitions. Respiratory muscle sEMG was applied to the diaphragm and sternocleidomastoid muscles using a bipolar and a CRE configuration. Thirty-two subjects underwent four inspiratory load spontaneous breathing tests which was repeated after interchanging the electrode positions. We calculated parameters such as (1) spectral power and (2) median frequency during inspiration, and power ratios of inspiratory sEMG without ECG in relation to (3) basal sEMG without ECG ( Rins/noise ), (4) basal sEMG with ECG ( Rins/cardio ) and (5) expiratory sEMG without ECG ( Rins/exp ). Spectral power, R_ins/noise} and Rins/cardio increased with the inspiratory load. Significantly higher values (p<0.05) of Rins/cardio and significantly higher median frequencies were obtained for CREs. Rins/noise and Rins/exp were higher for the bipolar configuration only in diaphragm sEMG recordings, whereas no significant differences were found in the sternocleidomastoid recordings. Our results suggest that the evaluation of respiratory muscle activity by means of sEMG can benefit from the remarkably reduced influence of cardiac activity, the enhanced detection of the shift in frequency content and the axial isotropy of CREs which facilitates its placement.

Keywords: Concentric ring electrodes, Laplacian potential, Non-invasive respiratory monitoring, Respiratory muscles, Surface electromyography

Seuma, M., Faure, A., Badia, M., Lehner, B., Bolognesi, B., (2021). The genetic landscape for amyloid beta fibril nucleation accurately discriminates familial Alzheimer’s disease mutations eLife 10, e63364

Plaques of the amyloid beta (Aß) peptide are a pathological hallmark of Alzheimer’s disease (AD), the most common form of dementia. Mutations in Aß also cause familial forms of AD (fAD). Here, we use deep mutational scanning to quantify the effects of >14,000 mutations on the aggregation of Aß. The resulting genetic landscape reveals mechanistic insights into fibril nucleation, including the importance of charge and gatekeeper residues in the disordered region outside of the amyloid core in preventing nucleation. Strikingly, unlike computational predictors and previous measurements, the empirical nucleation scores accurately identify all known dominant fAD mutations in Aß, genetically validating that the mechanism of nucleation in a cell-based assay is likely to be very similar to the mechanism that causes the human disease. These results provide the first comprehensive atlas of how mutations alter the formation of any amyloid fibril and a resource for the interpretation of genetic variation in Aß.

Winkler, Pamina M., Campelo, Felix, Giannotti, Marina I., Garcia-Parajo, Maria F., (2021). Impact of glycans on lipid membrane dynamics at the nanoscale unveiled by planar plasmonic nanogap antennas and atomic force spectroscopy The Journal of Physical Chemistry Letters ahead, 1175-1181

Lateral compartmentalization of the plasma membrane is a prominent feature present at multiple spatiotemporal scales that regulates key cellular functions. The extracellular glycocalyx matrix has recently emerged as an important player that modulates the organization of specific receptors and patterns the lipid bilayer itself. However, experimental limitations in investigating its impact on the membrane nanoscale dynamics have hampered detailed studies. Here, we used photonic nanoantenna arrays combined with fluorescence correlation spectroscopy to investigate the influence of hyaluronic acid (HA), a prominent glycosaminoglycan, on the nanoscale organization of mimetic lipid bilayers. Using atomic force microscopy and force spectroscopy, we further correlated our dynamic measurements with the morphology and mechanical properties of bilayers at the nanoscale. Overall, we find that HA has a profound effect on the dynamics, nanoscale organization, and mechanical properties of lipid bilayers that are enriched in sphingolipids and/or cholesterol, such as those present in living cells.

Qamar, B., Solomon, M., Marin, A., Fuerst, T. R., Andrianov, A. K., Muro, S., (2021). Intracellular delivery of active proteins by polyphosphazene polymers Pharmaceutics 13, (2), 249

Achieving intracellular delivery of protein therapeutics within cells remains a significant challenge. Although custom formulations are available for some protein therapeutics, the development of non-toxic delivery systems that can incorporate a variety of active protein cargo and maintain their stability, is a topic of great relevance. This study utilized ionic polyphosphazenes (PZ) that can assemble into supramolecular complexes through non-covalent interactions with different types of protein cargo. We tested a PEGylated graft copolymer (PZ-PEG) and a pyrrolidone containing linear derivative (PZ-PYR) for their ability to intracellularly deliver FITC-avidin, a model protein. In endothelial cells, PZ-PYR/protein exhibited both faster internalization and higher uptake levels than PZ-PEG/protein, while in cancer cells both polymers achieved similar uptake levels over time, although the internalization rate was slower for PZ-PYR/protein. Uptake was mediated by endocytosis through multiple mechanisms, PZ-PEG/avidin colocalized more profusely with endo-lysosomes, and PZ-PYR/avidin achieved greater cytosolic delivery. Consequently, a PZ-PYR-delivered anti-F-actin antibody was able to bind to cytosolic actin filaments without needing cell permeabilization. Similarly, a cell-impermeable Bax-BH3 peptide known to induce apoptosis, decreased cell viability when complexed with PZ-PYR, demonstrating endo-lysosomal escape. These biodegradable PZs were non-toxic to cells and represent a promising platform for drug delivery of protein therapeutics.

Keywords: Cytosolic delivery, Cytotoxicity, Delivery of apoptotic peptides, Endosomal escape, Intracellular delivery of antibody, Intracellular protein delivery, Polyphosphazene polymers

Mesquida-Veny, Francina, Del Río, José Antonio, Hervera, Arnau, (2021). Macrophagic and microglial complexity after neuronal injury Progress in Neurobiology , 101970

Central nervous system (CNS) injuries do not heal properly in contrast to normal tissue repair, in which functional recovery typically occurs. The reason for this dichotomy in wound repair is explained in part by macrophage and microglial malfunction, affecting both the extrinsic and intrinsic barriers to appropriate axonal regeneration. In normal healing tissue, macrophages promote the repair of injured tissue by regulating transitions through different phases of the healing response. In contrast, inflammation dominates the outcome of CNS injury, often leading to secondary damage. Therefore, an understanding of the molecular mechanisms underlying this dichotomy is critical to advance in neuronal repair therapies. Recent studies highlight the plasticity and complexity of macrophages and microglia beyond the classical view of the M1/M2 polarization paradigm. This plasticity represents an in vivo continuous spectrum of phenotypes with overlapping functions and markers. Moreover, macrophage and microglial plasticity affect many events essential for neuronal regeneration after injury, such as myelin and cell debris clearance, inflammation, release of cytokines, and trophic factors, affecting both intrinsic neuronal properties and extracellular matrix deposition. Until recently, this complexity was overlooked in the translation of therapies modulating these responses for the treatment of neuronal injuries. However, recent studies have shed important light on the underlying molecular mechanisms of this complexity and its transitions and effects on regenerative events. Here we review the complexity of macrophages and microglia after neuronal injury and their roles in regeneration, as well as the underlying molecular mechanisms, and we discuss current challenges and future opportunities for treatment.

Keywords: Neuronal injury, Neuroinflammation, Macrophages, Microglia, Chemokines and cytokines, Regeneration

Roki, N., Solomon, M., Casta, L., Bowers, J., Getts, R. C., Muro, S., (2021). A method to improve quantitative radiotracing-based analysis of the in vivo biodistribution of drug carriers Bioengineering and Translational Medicine Early View, e210208

Biodistribution studies are essential in drug carrier design and translation, and radiotracing provides a sensitive quantitation for this purpose. Yet, for biodegradable formulations, small amounts of free‐label signal may arise prior to or immediately after injection in animal models, causing potentially confounding biodistribution results. In this study, we refined a method to overcome this obstacle. First, we verified free signal generation in animal samples and then, mimicking it in a controllable setting, we injected mice intravenously with a radiolabeled drug carrier formulation (125I‐antibody/3DNA) containing a known amount of free radiolabel (125I), or free 125I alone as a control. Corrected biodistribution data were obtained by separating the free radiolabel from blood and organs postmortem, using trichloroacetic acid precipitation, and subtracting the confounding signal from each tissue measurement. Control free 125I‐radiolabel was detected at ≥85% accuracy in blood and tissues, validating the method. It biodistributed very heterogeneously among organs (0.6–39 %ID/g), indicating that any free 125I generated in the body or present in an injected formulation cannot be simply corrected to the free‐label fraction in the original preparation, but the free label must be empirically measured in each organ. Application of this method to the biodistribution of 125I‐antibody/3DNA, including formulations directed to endothelial target ICAM‐1, showed accurate classification of free 125I species in blood and tissues. In addition, this technique rendered data on the in vivo degradation of the traced agents over time. Thus, this is a valuable technique to obtain accurate measurements of biodistribution using 125I and possibly other radiotracers.

Keywords: Biodistribution data correction, Degradation, Drug delivery carriers, Free label, In vivo biodistribution, Radiotracing, Trichloroacetic acid precipitation

Mestre, R., Cadefau, N., Hortelao, A. C., Grzelak, J., Gich, M., Roig, A., Sánchez, S., (2021). Nanorods Based on Mesoporous Silica Containing Iron Oxide Nanoparticles as Catalytic Nanomotors: Study of Motion Dynamics ChemNanoMat 7, (2), 134-140

Self-propelled particles and, in particular, those based on mesoporous silica, have raised considerable interest due to their potential applications in the environmental and biomedical fields thanks to their biocompatibility, tunable surface chemistry and large porosity. Although spherical particles have been widely used to fabricate nano- and micromotors, not much attention has been paid to other geometries, such as nanorods. Here, we report the fabrication of self-propelled mesoporous silica nanorods (MSNRs) that move by the catalytic decomposition of hydrogen peroxide by a sputtered Pt layer, Fe2O3 nanoparticles grown within the mesopores, or the synergistic combination of both. We show that motion can occur in two distinct sub-populations characterized by two different motion dynamics, namely enhanced diffusion or directional propulsion, especially when both catalysts are used. These results open up the possibility of using MSNRs as chassis for the fabrication of self-propelled particles for the environmental or biomedical fields.

Keywords: Mesoporous silica, Nanomotors, Nanorods, Porous materials, Self-propulsion

Enshaei, H., Puiggalí-Jou, A., del Valle, L. J., Turon, P., Saperas, N., Alemán, C., (2021). Nanotheranostic interface based on antibiotic-loaded conducting polymer nanoparticles for real-time monitoring of bacterial growth inhibition Advanced Healthcare Materials

Conducting polymers have been increasingly used as biologically interfacing electrodes for biomedical applications due to their excellent and fast electrochemical response, reversible doping–dedoping characteristics, high stability, easy processability, and biocompatibility. These advantageous properties can be used for the rapid detection and eradication of infections associated to bacterial growth since these are a tremendous burden for individual patients as well as the global healthcare system. Herein, a smart nanotheranostic electroresponsive platform, which consists of chloramphenicol (CAM)-loaded in poly(3,4-ethylendioxythiophene) nanoparticles (PEDOT/CAM NPs) for concurrent release of the antibiotic and real-time monitoring of bacterial growth is presented. PEDOT/CAM NPs, with an antibiotic loading content of 11.9 ± 1.3% w/w, are proved to inhibit the growth of Escherichia coli and Streptococcus sanguinis due to the antibiotic release by cyclic voltammetry. Furthermore, in situ monitoring of bacterial activity is achieved through the electrochemical detection of β-nicotinamide adenine dinucleotide, a redox active specie produced by the microbial metabolism that diffuse to the extracellular medium. According to these results, the proposed nanotheranostic platform has great potential for real-time monitoring of the response of bacteria to the released antibiotic, contributing to the evolution of the personalized medicine.

Keywords: Bacterial detection, Chloramphenicol, Conducting polymers, Drug release, Electrochemical sensors, Electrostimulated release, Polythiophene

Lozano-García, Manuel, Estrada-Petrocelli, Luis, Torres, Abel, Rafferty, Gerrard F., Moxham, John, Jolley, Caroline J., Jané, Raimon, (2021). Noninvasive assessment of neuromechanical coupling and mechanical efficiency of parasternal intercostal muscle during inspiratory threshold loading Sensors 21, (5), 1781

This study aims to investigate noninvasive indices of neuromechanical coupling (NMC) and mechanical efficiency (MEff) of parasternal intercostal muscles. Gold standard assessment of diaphragm NMC requires using invasive techniques, limiting the utility of this procedure. Noninvasive NMC indices of parasternal intercostal muscles can be calculated using surface mechanomyography (sMMGpara) and electromyography (sEMGpara). However, the use of sMMGpara as an inspiratory muscle mechanical output measure, and the relationships between sMMGpara, sEMGpara, and simultaneous invasive and noninvasive pressure measurements have not previously been evaluated. sEMGpara, sMMGpara, and both invasive and noninvasive measurements of pressures were recorded in twelve healthy subjects during an inspiratory loading protocol. The ratios of sMMGpara to sEMGpara, which provided muscle-specific noninvasive NMC indices of parasternal intercostal muscles, showed nonsignificant changes with increasing load, since the relationships between sMMGpara and sEMGpara were linear (R2 = 0.85 (0.75–0.9)). The ratios of mouth pressure (Pmo) to sEMGpara and sMMGpara were also proposed as noninvasive indices of parasternal intercostal muscle NMC and MEff, respectively. These indices, similar to the analogous indices calculated using invasive transdiaphragmatic and esophageal pressures, showed nonsignificant changes during threshold loading, since the relationships between Pmo and both sEMGpara (R2 = 0.84 (0.77–0.93)) and sMMGpara (R2 = 0.89 (0.85–0.91)) were linear. The proposed noninvasive NMC and MEff indices of parasternal intercostal muscles may be of potential clinical value, particularly for the regular assessment of patients with disordered respiratory mechanics using noninvasive wearable and wireless devices.

Keywords: Inspiratory threshold loading, Neuromechanical coupling, Parasternal intercostal muscles, Respiratory pressure, Surface electromyography, Surface mechanomyography

Dhillon, Poonam, Park, Jihwan, Hurtado del Pozo, Carmen, Li, Lingzhi, Doke, Tomohito, Huang, Shizheng, Zhao, Juanjuan, Kang, Hyun Mi, Shrestra, Rojesh, Balzer, Michael S., Chatterjee, Shatakshee, Prado, Patricia, Han, Seung Yub, Liu, Hongbo, Sheng, Xin, Dierickx, Pieterjan, Batmanov, Kirill, Romero, Juan P., Prósper, Felipe, Li, Mingyao, Pei, Liming, Kim, Junhyong, Montserrat, Nuria, Susztak, Katalin, (2021). The nuclear receptor ESRRA protects from kidney disease by coupling metabolism and differentiation Cell Metabolism 33, (2), 379-394.E8

Kidney disease is poorly understood because of the organ’s cellular diversity. We used single-cell RNA sequencing not only in resolving differences in injured kidney tissue cellular composition but also in cell-type-specific gene expression in mouse models of kidney disease. This analysis highlighted major changes in cellular diversity in kidney disease, which markedly impacted whole-kidney transcriptomics outputs. Cell-type-specific differential expression analysis identified proximal tubule (PT) cells as the key vulnerable cell type. Through unbiased cell trajectory analyses, we show that PT cell differentiation is altered in kidney disease. Metabolism (fatty acid oxidation and oxidative phosphorylation) in PT cells showed the strongest and most reproducible association with PT cell differentiation and disease. Coupling of cell differentiation and the metabolism was established by nuclear receptors (estrogen-related receptor alpha [ESRRA] and peroxisomal proliferation-activated receptor alpha [PPARA]) that directly control metabolic and PT-cell-specific gene expression in mice and patient samples while protecting from kidney disease in the mouse model.

Keywords: Single-cell RNA sequencing, Single-cell ATAC sequencing, Kidney, Fibrosis, Organoids, Fatty-acid oxidation, PPARA, ESRRA, Proximal tubule cells, Chronic kidney disease

Abdelrahman, F., Easwaran, M., Daramola, O. I., Ragab, S., Lynch, S., Oduselu, T. J., Khan, F. M., Ayobami, A., Adnan, F., Torrents, E., Sanmukh, S., El-Shibiny, A., (2021). Phage-encoded endolysins Antibiotics 10, (2), 1-31

Due to the global emergence of antibiotic resistance, there has been an increase in research surrounding endolysins as an alternative therapeutic. Endolysins are phage-encoded enzymes, utilized by mature phage virions to hydrolyze the cell wall from within. There is significant evidence that proves the ability of endolysins to degrade the peptidoglycan externally without the assistance of phage. Thus, their incorporation in therapeutic strategies has opened new options for therapeutic application against bacterial infections in the human and veterinary sectors, as well as within the agricultural and biotechnology sectors. While endolysins show promising results within the laboratory, it is important to document their resistance, safety, and immunogenicity for in-vivo application. This review aims to provide new insights into the synergy between endolysins and antibiotics, as well as the formulation of endolysins. Thus, it provides crucial information for clinical trials involving endolysins. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Keywords: Antibiotic resistance, Bacteriophages, Endolysin

Caddeo, C., Gabriele, M., Nácher, A., Fernàndez-Busquets, X., Valenti, D., Maria Fadda, A., Pucci, L., Manconi, M., (2021). Resveratrol and artemisinin eudragit-coated liposomes: A strategy to tackle intestinal tumors International Journal of Pharmaceutics 592

Resveratrol and artemisinin, two naturally occurring compounds with a wide range of biological activities, have been reported to exert antitumor effects against several types of cancer. In this work, Eudragit-coated liposomes were developed to safely transport resveratrol and artemisinin through the gastrointestinal tract and target the intestine. The physico-chemical properties of the Eudragit-coated liposomes were assessed by light scattering and cryogenic transmission electron microscopy. Nanosized (around 100 nm), spherical or elongated, unilamellar vesicles were produced. The protective effect of the Eudragit coating was confirmed by assessing the physical stability of the vesicles in fluids mimicking the gastrointestinal environment. Furthermore, the vesicles were found to exert a pro-oxidant activity in intestinal adenocarcinoma cells, which resulted in a marked mortality due to the generation of reactive oxygen species (ROS). A time- and dose-dependent cell growth inhibitory effect was detected, with elevated ROS levels when resveratrol and artemisinin were combined. Therefore, the proposed formulations may represent a valuable means to counteract intestinal tumor growth. © 2020 Elsevier B.V.

Keywords: Antitumor, Artemisinin, Eudragit, Intestinal delivery, Liposomes, Resveratrol

Garreta, Elena, Kamm, Roger D., Chuva de Sousa Lopes, Susana M., Lancaster, Madeline A., Weiss, Ron, Trepat, Xavier, Hyun, Insoo, Montserrat, Nuria, (2021). Rethinking organoid technology through bioengineering Nature Materials 20, 145-155

In recent years considerable progress has been made in the development of faithful procedures for the differentiation of human pluripotent stem cells (hPSCs). An important step in this direction has also been the derivation of organoids. This technology generally relies on traditional three-dimensional culture techniques that exploit cell-autonomous self-organization responses of hPSCs with minimal control over the external inputs supplied to the system. The convergence of stem cell biology and bioengineering offers the possibility to provide these stimuli in a controlled fashion, resulting in the development of naturally inspired approaches to overcome major limitations of this nascent technology. Based on the current developments, we emphasize the achievements and ongoing challenges of bringing together hPSC organoid differentiation, bioengineering and ethics. This Review underlines the need for providing engineering solutions to gain control of self-organization and functionality of hPSC-derived organoids. We expect that this knowledge will guide the community to generate higher-grade hPSC-derived organoids for further applications in developmental biology, drug screening, disease modelling and personalized medicine.

Hortelao, Ana C., Simó, Cristina, Guix, Maria, Guallar-Garrido, Sandra, Julián, Esther, Vilela, Diana, Rejc, Luka, Ramos-Cabrer, Pedro, Cossíoo, Unai, Gómez-Vallejo, Vanessa, Patiño, Tania, Llop, Jordi, Sánchez, Samuel, (2021). Swarming behavior and in vivo monitoring of enzymatic nanomotors within the bladder Science Robotics 6, (52), eabd2823

Enzyme-powered nanomotors are an exciting technology for biomedical applications due to their ability to navigate within biological environments using endogenous fuels. However, limited studies into their collective behavior and demonstrations of tracking enzyme nanomotors in vivo have hindered progress toward their clinical translation. Here, we report the swarming behavior of urease-powered nanomotors and its tracking using positron emission tomography (PET), both in vitro and in vivo. For that, mesoporous silica nanoparticles containing urease enzymes and gold nanoparticles were used as nanomotors. To image them, nanomotors were radiolabeled with either 124I on gold nanoparticles or 18F-labeled prosthetic group to urease. In vitro experiments showed enhanced fluid mixing and collective migration of nanomotors, demonstrating higher capability to swim across complex paths inside microfabricated phantoms, compared with inactive nanomotors. In vivo intravenous administration in mice confirmed their biocompatibility at the administered dose and the suitability of PET to quantitatively track nanomotors in vivo. Furthermore, nanomotors were administered directly into the bladder of mice by intravesical injection. When injected with the fuel, urea, a homogeneous distribution was observed even after the entrance of fresh urine. By contrast, control experiments using nonmotile nanomotors (i.e., without fuel or without urease) resulted in sustained phase separation, indicating that the nanomotors’ self-propulsion promotes convection and mixing in living reservoirs. Active collective dynamics, together with the medical imaging tracking, constitute a key milestone and a step forward in the field of biomedical nanorobotics, paving the way toward their use in theranostic applications.

Moya-Andérico, L., Vukomanovic, M., Cendra, M. D. M., Segura-Feliu, M., Gil, V., del Río, J. A., Torrents, E., (2021). Utility of Galleria mellonella larvae for evaluating nanoparticle toxicology Chemosphere 266

The use of nanoparticles in consumer products is currently on the rise, so it is important to have reliable methods to predict any associated toxicity effects. Traditional in vitro assays fail to mimic true physiological responses of living organisms against nanoparticles whereas murine in vivo models are costly and ethically controversial. For these reasons, this study aimed to evaluate the efficacy of Galleria mellonella as an alternative, non-rodent in vivo model for examining nanoparticle toxicity. Silver, selenium, and functionalized gold nanoparticles were synthesized, and their toxicity was assessed in G. mellonella larvae. The degree of acute toxicity effects caused by each type of NP was efficiently detected by an array of indicators within the larvae: LD50 calculation, hemocyte proliferation, NP distribution, behavioral changes, and histological alterations. G. mellonella larvae are proposed as a nanotoxicological model that can be used as a bridge between in vitro and in vivo murine assays in order to obtain better predictions of NP toxicity. © 2020 Elsevier Ltd

Keywords: Galleria mellonella, Hemocytes, Nanoparticles, Nanotoxicity, Non-rodent in vivo model, Toxicity screening

Pacheco Estefan, D., Zucca, Riccardo, Arsiwalla, X. D., Principe, A., Zhang, Hui, Rocamora, R., Axmacher, N., Verschure, P., (2021). Volitional learning promotes theta phase coding in the human hippocampus Proceedings of the National Academy of Sciences of the United States of America 118, (10), e2021238118

Electrophysiological studies in rodents show that active navigation enhances hippocampal theta oscillations (4–12 Hz), providing a temporal framework for stimulus-related neural codes. Here we show that active learning promotes a similar phase coding regime in humans, although in a lower frequency range (3–8 Hz). We analyzed intracranial electroencephalography (iEEG) from epilepsy patients who studied images under either volitional or passive learning conditions. Active learning increased memory performance and hippocampal theta oscillations and promoted a more accurate reactivation of stimulus-specific information during memory retrieval. Representational signals were clustered to opposite phases of the theta cycle during encoding and retrieval. Critically, during active but not passive learning, the temporal structure of intracycle reactivations in theta reflected the semantic similarity of stimuli, segregating conceptually similar items into more distant theta phases. Taken together, these results demonstrate a multilayered mechanism by which active learning improves memory via a phylogenetically old phase coding scheme.

Keywords: Active learning, Intracranial EEG, Theta oscillations, Neural phase coding, Hippocampus

Biosca, A., Cabanach, P., Abdulkarim, M., Gumbleton, M., Gómez-Canela, C., Ramírez, M., Bouzón-Arnáiz, I., Avalos-Padilla, Y., Borros, S., Fernàndez-Busquets, X., (2021). Zwitterionic self-assembled nanoparticles as carriers for Plasmodium targeting in malaria oral treatment Journal of Controlled Release 331, 364-375

The current decline in antimalarial drug efficacy due to the evolution of resistant Plasmodium strains calls for new strategies capable of improving the bioavailability of antimalarials, especially of those whose lipophilic character imparts them a low solubility in biological fluids. Here we have designed, synthesized and characterized amphiphilic zwitterionic block copolymers forming nanoparticles capable of penetrating the intestinal epithelium that can be used for oral administration. Poly(butyl methacrylate-co-morpholinoethyl sulfobetaine methacrylate) (PBMA-MESBMA)-based nanoparticles exhibited a specific targeting to Plasmodium falciparum-infected vs. parasite-free red blood cells (74.8%/0.8% respectively), which was maintained upon encapsulation of the lipophilic antimalarial drug curcumin (82.6%/0.3%). The in vitro efficacy of curcumin upon encapsulation was maintained relative to the free compound, with an IC50 around 5 μM. In vivo assays indicated a significantly increased curcumin concentration in the blood of mice one hour after being orally fed PBMA-MESBMA-curcumin in comparison to the administration of free drug (18.7 vs. 2.1 ng/ml, respectively). At longer times, however, plasma curcumin concentration equaled between free and encapsulated drug, which was reflected in similar in vivo antimalarial activities in Plasmodium yoelii yoelii-infected mice. Microscopic analysis in blood samples of fluorescently labeled PBMA-MESBMA revealed the presence of the polymer inside P. yoelii yoelii-parasitized erythrocytes one hour after oral administration to infected animals. © 2021 Elsevier B.V.

Keywords: Curcumin, Drug delivery, Malaria, PBMA-MESBMA, Plasmodium, Zwitterionic block copolymers

Selfa, Idoia Lucía, Gallo, Maria, Montserrat, Nuria, Garreta, Elena, (2021). Directed differentiation of human pluripotent stem sells for the generation of high-order kidney organoids Programmed Morphogenesis: Methods and Protocols (ed. Ebrahimkhani M.R., Hislop J.), Springer US (New York, NY) 2258, 171-192

Our understanding in the inherent properties of human pluripotent stem cells (hPSCs) have made possible the development of differentiation procedures to generate three-dimensional tissue-like cultures, so-called organoids. Here we detail a stepwise methodology to generate kidney organoids from hPSCs. This is achieved through direct differentiation of hPSCs in two-dimensional monolayer culture toward the posterior primitive streak fate, followed by induction of intermediate mesoderm-committed cells, which are further aggregated and cultured in three-dimensions to generate kidney organoids containing segmented nephron-like structures in a process that lasts 20 days. We also provide a concise description on how to assess renal commitment during the time course of kidney organoid generation. This includes the use of flow cytometry and immunocytochemistry analyses for the detection of specific renal differentiation markers.

Blancas, Maria, Valero, Cristina, Vouloutsi, Vasiliki, Mura, Anna, Verschure, P., (2021). Educational robotics: A journey, not a destination Handbook of Research on Using Educational Robotics to Facilitate Student Learning (ed. Papadakis, Stamatios, Kalogiannakis, Michail), IGI Global (Hershey, PA, USA) , 41-67

The aim of this work is two-fold. On the one hand, the authors wish to provide relevant information to educators willing to develop an educational robotics (ER) curriculum. They thus provide the current state of the art in the field of ER and the various approaches reported in the literature. They also provide examples of how computational thinking (CT) can be applied in ER and main theories behind ER: constructivism, constructionism, and inquiry-based learning. As ER requires problem-solving abilities, they discuss the link between CT and metacognition, which is considered one of the required educational improvements of the 21st century (also related to the role of gender in STEM methodologies). On the other hand, they wish to present their methodology to teach coding and ER (coding robots through exploring their affordances – CREA), how it was designed, and its main outcomes. It aims at teaching programming and robotics to children in primary school, focusing not on only the performance of the students, but also the cultivation of collaboration, communication, creativity, and critical thinking.