by Keyword: receptors
Camerin, Luisa, Maleeva, Galyna, Gomila, Alexandre M J, Suarez-Pereira, Irene, Matera, Carlo, Prischich, Davia, Opar, Ekin, Riefolo, Fabio, Berrocoso, Esther, Gorostiza, Pau, (2024). Photoswitchable Carbamazepine Analogs for Non-Invasive Neuroinhibition In Vivo Angewandte Chemie (International Ed. Print) 63, e202403636
A problem of systemic pharmacotherapy is off-target activity, which causes adverse effects. Outstanding examples include neuroinhibitory medications like antiseizure drugs, which are used against epilepsy and neuropathic pain but cause systemic side effects. There is a need of drugs that inhibit nerve signals locally and on-demand without affecting other regions of the body. Photopharmacology aims to address this problem with light-activated drugs and localized illumination in the target organ. Here, we have developed photoswitchable derivatives of the widely prescribed antiseizure drug carbamazepine. For that purpose, we expanded our method of ortho azologization of tricyclic drugs to meta/para and to N-bridged diazocine. Our results validate the concept of ortho cryptoazologs (uniquely exemplified by Carbazopine-1) and bring to light Carbadiazocine (8), which can be photoswitched between 400-590 nm light (using violet LEDs and halogen lamps) and shows good drug-likeness and predicted safety. Both compounds display photoswitchable activity in vitro and in translucent zebrafish larvae. Carbadiazocine (8) also offers in vivo analgesic efficacy (mechanical and thermal stimuli) in a rat model of neuropathic pain and a simple and compelling treatment demonstration with non-invasive illumination.
JTD Keywords: Antiepileptic drugs, Anxiet, Azobenzene, Diazocine, Epileps, Ion channels, Neuromodulation, Optical control, Pain, Photopharmacology, Rat, Receptors, Release, Spatiotemporal control, Tricyclic drugs, Zebrafish
Mastrantuono, Elisa, Ghibaudi, Matilde, Matias, Diana, Battaglia, Giuseppe, (2024). The multifaceted therapeutical role of low-density lipoprotein receptor family in high-grade glioma Molecular Oncology ,
The diverse roles of the low-density lipoprotein receptor family (LDLR) have been associated with many processes critical to maintaining central nervous system (CNS) health and contributing to neurological diseases or cancer. In this review, we provide a comprehensive understanding of the LDLR's involvement in common brain tumors, specifically high-grade gliomas, emphasizing the receptors' critical role in the pathophysiology and progression of these tumors due to LDLR's high expression. We delve into LDLR's role in regulating cellular uptake and transport through the brain barrier. Additionally, we highlight LDLR's role in activating several signaling pathways related to tumor proliferation, migration, and invasion, engaging readers with an in-depth understanding of the molecular mechanisms at play. By synthesizing current research findings, this review underscores the significance of LDLR during tumorigenesis and explores its potential as a therapeutic target for high-grade gliomas. The collective insights presented here contribute to a deeper appreciation of LDLR's multifaceted roles and implications for physiological and pathological states, opening new avenues for tumor treatment. The role of LDLR family receptors in mediating the transport of LDL across the blood-brain barrier (BBB), facilitating processes such as survival, proliferation, and invasion in high-grade gliomas. Nanoparticles targeting LDLR can be used for drug delivery, potentially inducing cell death and reducing tumor proliferation and survival in high-grade glioma cells. image
JTD Keywords: Blood-brain-barrier, Cancer, Delivery, Doxorubicin, Expression, Glioblastomas, Low-density lipoprotein receptors, Migratio, Nanoparticles, Proliferation, Targeted therapie, Targeting therapy, Tumor microenvironment
Mughal, Sheeza, Sabater-Arcis, Maria, Artero, Ruben, Ramon-Azcon, Javier, Fernandez-Costa, Juan M, (2024). Taurine activates the AKT-mTOR axis to restore muscle mass and contractile strength in human 3D in vitro models of steroid myopathy Disease Models & Mechanisms 17, dmm050540
Steroid myopathy is a clinically challenging condition exacerbated by prolonged corticosteroid use or adrenal tumors. In this study, we engineered a functional three-dimensional (3D) in vitro skeletal muscle model to investigate steroid myopathy. By subjecting our bioengineered muscle tissues to dexamethasone treatment, we reproduced the molecular and functional aspects of this disease. Dexamethasone caused a substantial reduction in muscle force, myotube diameter and induced fatigue. We observed nuclear translocation of the glucocorticoid receptor (GCR) and activation of the ubiquitin-proteasome system within our model, suggesting their coordinated role in muscle atrophy. We then examined the therapeutic potential of taurine in our 3D model for steroid myopathy. Our findings revealed an upregulation of phosphorylated AKT by taurine, effectively countering the hyperactivation of the ubiquitin- proteasomal pathway. Importantly, we demonstrate that discontinuing corticosteroid treatment was insufficient to restore muscle mass and function. Taurine treatment, when administered concurrently with corticosteroids, notably enhanced contractile strength and protein turnover by upregulating the AKT-mTOR axis. Our model not only identifies a promising therapeutic target, but also suggests combinatorial treatment that may benefit individuals undergoing corticosteroid treatment or those diagnosed with adrenal tumors.
JTD Keywords: 3d bioengineered skeletal muscle tissues, Adrenal cortex hormones, Atroph, Colocalization, Corticosteroids, Dexamethasone, Glucocorticoid-receptor, Humans, Mechanisms, Models, biological, Mtor protein, human, Muscle contraction, Muscle fibers, skeletal, Muscle strength, Muscle, skeletal, Muscular diseases, Organ size, Phosphorylation, Proteasome endopeptidase complex, Proto-oncogene proteins c-akt, Receptors, glucocorticoid, Signal transduction, Skeletal-muscle, Steroid myopathy, Steroids, Supplementation, Taurin, Taurine, Tor serine-threonine kinases, Ubiquitin
Resina, L, Alemán, C, Ferreira, FC, Esteves, T, (2023). Protein-imprinted polymers: How far have "plastic antibodies" come? Biotechnology Advances 68, 108220
Antibodies are highly selective and sensitive, making them the gold standard for recognition affinity tools. However, their production cost is high and their downstream processing is time-consuming. Molecularly imprinted polymers (MIPs) are tailor-made by incorporating specific molecular recognition sites in their structure, thus translating into receptor-like activity mode of action. The interest in molecular imprinting technology, applied to biomacromolecules, has increased in the past decade. MIPs, produced using biomolecules as templates, commonly referred to as "plastic antibodies" or "artificial receptors", have been considered as suitable cheaper and easy to produce alternatives to antibodies. Research on MIPs, designed to recognize proteins or peptides is particularly important, with potential contributions towards biomedical applications, namely biosensors and targeted drug delivery systems. This mini review will cover recent advances on (bio)molecular imprinting technology, where proteins or peptides are targeted or mimicked for sensing and therapeutic applications. Polymerization methods are reviewed elsewhere, being out of the scope of this review. Template selection and immobilization approaches, monomers and applications will be discussed, highlighting possible drawbacks and gaps in research.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.
JTD Keywords: artificial antibodies, assay, biomimetics, biomolecules, biosensors, delivery, diagnostics, drug delivery, electrochemical detection, nanoparticles, receptors, science-and-technology, selective recognition, selective targeting, separation, templates, Artificial antibodies, Biomimetics, Biomolecules, Biosensors, Diagnostics, Drug delivery, Molecularly imprinted polymers, Nanoparticles, Selective targeting, Solid-phase synthesis
Lopes, VR, Birgersson, U, Manivel, VA, Hulsart-Billström, G, Gallinetti, S, Aparicio, C, Hong, J, (2023). Human Whole Blood Interactions with Craniomaxillofacial Reconstruction Materials: Exploring In Vitro the Role of Blood Cascades and Leukocytes in Early Healing Events J Funct Biomater 14, 361
The present study investigated early interactions between three alloplastic materials (calcium phosphate (CaP), titanium alloy (Ti), and polyetheretherketone (PEEK) with human whole blood using an established in vitro slide chamber model. After 60 min of contact with blood, coagulation (thrombin-antithrombin complexes, TAT) was initiated on all test materials (Ti > PEEK > CaP), with a significant increase only for Ti. All materials showed increased contact activation, with the KK-AT complex significantly increasing for CaP (p < 0.001), Ti (p < 0.01), and PEEK (p < 0.01) while only CaP demonstrated a notable rise in KK-C1INH production (p < 0.01). The complement system had significant activation across all materials, with CaP (p < 0.0001, p < 0.0001) generating the most pronounced levels of C3a and sC5b-9, followed by Ti (p < 0.001, p < 0.001) and lastly, PEEK (p < 0.001, p < 0.01). This activation correlated with leukocyte stimulation, particularly myeloperoxidase release. Consequently, the complement system may assume a more significant role in the early stages post implantation in response to CaP materials than previously recognized. Activation of the complement system and the inevitable activation of leukocytes might provide a more favorable environment for tissue remodeling and repair than has been traditionally acknowledged. While these findings are limited to the early blood response, complement and leukocyte activation suggest improved healing outcomes, which may impact long-term clinical outcomes.
JTD Keywords: activation, biomaterial, calcium phosphate, coagulation, complement, cranioplasty, human whole blood, platelets, receptors, surface, titanium, Biomaterials, Calcium phosphate, Coagulation, Complement, Human whole blood, Peroxidase enzymes
Martinez-Torres, S, Mesquida-Veny, F, Del Rio, JA, Hervera, A, (2023). Injury-induced activation of the endocannabinoid system promotes axon regeneration Iscience 26, 106814
Regeneration after a peripheral nerve injury still remains a challenge, due to the limited regenerative potential of axons after injury. While the endocannabinoid system (ECS) has been widely studied for its neuroprotective and analgesic effects, its role in axonal regeneration and during the conditioning lesion remains unexplored. In this study, we observed that a peripheral nerve injury induces axonal regeneration through an increase in the endocannabinoid tone. We also enhanced the regenerative capacity of dorsal root ganglia (DRG) neurons through the inhibition of endocannabinoid degradative enzyme MAGL or a CB1R agonist. Our results suggest that the ECS, via CB1R and PI3K-pAkt pathway activation, plays an important role in promoting the intrinsic regenerative capacity of sensory neurons after injury.© 2023 The Author(s).
JTD Keywords: brain, gene-expression, lesion, nerve, receptors, targets, Clinical neuroscience, Drugs, Endogenous cannabinoid system, Molecular medicine
Riera, R, Archontakis, E, Cremers, G, de Greef, T, Zijlstra, P, Albertazzi, L, (2023). Precision and Accuracy of Receptor Quantification on Synthetic and Biological Surfaces Using DNA-PAINT Acs Sensors 8, 80-93
Characterization of the number and distribution of biological molecules on 2D surfaces is of foremost importance in biology and biomedicine. Synthetic surfaces bearing recognition motifs are a cornerstone of biosensors, while receptors on the cell surface are critical/vital targets for the treatment of diseases. However, the techniques used to quantify their abundance are qualitative or semi-quantitative and usually lack sensitivity, accuracy, or precision. Detailed herein a simple and versatile workflow based on super-resolution microscopy (DNA-PAINT) was standardized to improve the quantification of the density and distribution of molecules on synthetic substrates and cell membranes. A detailed analysis of accuracy and precision of receptor quantification is presented, based on simulated and experimental data. We demonstrate enhanced accuracy and sensitivity by filtering out non-specific interactions and artifacts. While optimizing the workflow to provide faithful counting over a broad range of receptor densities. We validated the workflow by specifically quantifying the density of docking strands on a synthetic sensor surface and the densities of PD1 and EGF receptors (EGFR) on two cellular models.
JTD Keywords: binding, biosensors, cancer, expression, kinetics, localization microscopy, quantification, receptors, single-molecule, super-resolution microscopy, Biosensors, Dna-paint, Quantification, Receptors, Single-molecule, Super-resolution microscopy, Superresolution microscopy
Lagunas, A, Belloir, C, Briand, L, Gorostiza, P, Samitier, J, (2022). Determination of the nanoscale electrical properties of olfactory receptor hOR1A1 and their dependence on ligand binding: Towards the development of capacitance-operated odorant biosensors Biosensors & Bioelectronics 218, 114755
The transduction of odorant binding into cellular signaling by olfactory receptors (ORs) is not understood and knowing its mechanism would enable developing new pharmacology and biohybrid electronic detectors of volatile organic compounds bearing high sensitivity and selectivity. The electrical characterization of ORs in bulk experiments is subject to microscopic models and assumptions. We have directly determined the nanoscale electrical properties of ORs immobilized in a fixed orientation, and their change upon odorant binding, using electrochemical scanning tunneling microscopy (EC-STM) in near-physiological conditions. Recordings of current versus time, distance, and electrochemical potential allows determining the OR impedance parameters and their dependence with odorant binding. Our results allow validating OR structural-electrostatic models and their functional activation processes, and anticipating a novel macroscopic biosensor based on ORs.Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.
JTD Keywords: electrochemical scanning tunneling, electrochemical scanning tunneling microscopy (ec-stm), microscopy (ec-stm), neurons, odorant binding, olfactory receptors (ors), open-circuit voltage(voc), Olfactory receptors (ors), Open-circuit voltage (v(oc)), Transport
Cañellas-Socias, A, Cortina, C, Hernando-Momblona, X, Palomo-Ponce, S, Mulholland, EJ, Turon, G, Mateo, L, Conti, S, Roman, O, Sevillano, M, Slebe, F, Stork, D, Caballé-Mestres, A, Berenguer-Llergo, A, Alvarez-Varela, A, Fenderico, N, Novellasdemunt, L, Jiménez-Gracia, L, Sipka, T, Bardia, L, Lorden, P, Colombelli, J, Heyn, H, Trepat, X, Tejpar, S, Sancho, E, Tauriello, DVF, Leedham, S, Attolini, CSO, Batlle, E, (2022). Metastatic recurrence in colorectal cancer arises from residual EMP1+ cells Nature 611, 603-613
Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years1. Therapies to prevent disease relapse remain an unmet medical need. Here we uncover the identity and features of the residual tumour cells responsible for CRC relapse. An analysis of single-cell transcriptomes of samples from patients with CRC revealed that the majority of genes associated with a poor prognosis are expressed by a unique tumour cell population that we named high-relapse cells (HRCs). We established a human-like mouse model of microsatellite-stable CRC that undergoes metastatic relapse after surgical resection of the primary tumour. Residual HRCs occult in mouse livers after primary CRC surgery gave rise to multiple cell types over time, including LGR5+ stem-like tumour cells2-4, and caused overt metastatic disease. Using Emp1 (encoding epithelial membrane protein 1) as a marker gene for HRCs, we tracked and selectively eliminated this cell population. Genetic ablation of EMP1high cells prevented metastatic recurrence and mice remained disease-free after surgery. We also found that HRC-rich micrometastases were infiltrated with T cells, yet became progressively immune-excluded during outgrowth. Treatment with neoadjuvant immunotherapy eliminated residual metastatic cells and prevented mice from relapsing after surgery. Together, our findings reveal the cell-state dynamics of residual disease in CRC and anticipate that therapies targeting HRCs may help to avoid metastatic relapse.© 2022. The Author(s), under exclusive licence to Springer Nature Limited.
JTD Keywords: colonization, defines, human colon, mutations, plasticity, retrieval, stem-cells, subtypes, underlie, Animal, Animal cell, Animal experiment, Animal model, Animal tissue, Animals, Article, Cancer, Cancer growth, Cancer immunotherapy, Cancer inhibition, Cancer recurrence, Cancer staging, Cell, Cell adhesion, Cell migration, Cell population, Cell surface receptor, Cohort analysis, Colorectal cancer, Colorectal neoplasms, Colorectal tumor, Comprehensive molecular characterization, Controlled study, Crispr-cas9 system, Cytoskeleton, Disease exacerbation, Disease progression, Dynamics, Emp1 gene, Epithelial membrane protein-1, Extracellular matrix, Flow cytometry, Fluorescence intensity, Gene expression, Genetics, Human, Human cell, Humans, Immune response, Immunofluorescence, In situ hybridization, Marker gene, Metastasis potential, Mice, Minimal residual disease, Mouse, Neoplasm proteins, Neoplasm recurrence, local, Neoplasm, residual, Nonhuman, Pathology, Phenotype, Prevention and control, Protein, Receptors, cell surface, Single cell rna seq, Tumor, Tumor protein, Tumor recurrence
Matera, C, Calvé, P, Casadó-Anguera, V, Sortino, R, Gomila, AMJ, Moreno, E, Gener, T, Delgado-Sallent, C, Nebot, P, Costazza, D, Conde-Berriozabal, S, Masana, M, Hernando, J, Casadó, V, Puig, MV, Gorostiza, P, (2022). Reversible Photocontrol of Dopaminergic Transmission in Wild-Type Animals International Journal Of Molecular Sciences 23, 10114
Understanding the dopaminergic system is a priority in neurobiology and neuropharmacology. Dopamine receptors are involved in the modulation of fundamental physiological functions, and dysregulation of dopaminergic transmission is associated with major neurological disorders. However, the available tools to dissect the endogenous dopaminergic circuits have limited specificity, reversibility, resolution, or require genetic manipulation. Here, we introduce azodopa, a novel photoswitchable ligand that enables reversible spatiotemporal control of dopaminergic transmission. We demonstrate that azodopa activates D1-like receptors in vitro in a light-dependent manner. Moreover, it enables reversibly photocontrolling zebrafish motility on a timescale of seconds and allows separating the retinal component of dopaminergic neurotransmission. Azodopa increases the overall neural activity in the cortex of anesthetized mice and displays illumination-dependent activity in individual cells. Azodopa is the first photoswitchable dopamine agonist with demonstrated efficacy in wild-type animals and opens the way to remotely controlling dopaminergic neurotransmission for fundamental and therapeutic purposes.
JTD Keywords: azobenzene, behavior, brainwave, d-1, dopamine, gpcr, in vivo electrophysiology, inhibitors, optogenetics, optopharmacology, photochromism, photopharmacology, photoswitch, stimulation, zebrafish, Animals, Animals, wild, Azobenzene, Behavior, Brainwave, Dopamine, Gpcr, In vivo electrophysiology, Ligands, Mice, Optogenetics, Optopharmacology, Photochromism, Photopharmacology, Photoswitch, Receptors, Synaptic transmission, Zebrafish
Acosta-Gutierrez, S, Matias, D, Avila-Olias, M, Gouveia, VM, Scarpa, E, Forth, J, Contini, C, Duro-Castano, A, Rizzello, L, Battaglia, G, (2022). A Multiscale Study of Phosphorylcholine Driven Cellular Phenotypic Targeting Acs Central Science 8, 891-904
Phenotypic targeting requires the ability of the drug delivery system to discriminate over cell populations expressing a particular receptor combination. Such selectivity control can be achieved using multiplexed-multivalent carriers often decorated with multiple ligands. Here, we demonstrate that the promiscuity of a single ligand can be leveraged to create multiplexed-multivalent carriers achieving phenotypic targeting. We show how the cellular uptake of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-(diisopropylamino)ethyl methacry-late) (PMPC-PDPA) polymersomes varies depending on the receptor expression among different cells. We investigate the PMPC-PDPA polymersome insertion at the single chain/receptor level using all-atom molecular modeling. We propose a theoretical statistical mechanics-based model for polymersome-cell association that explicitly considers the interaction of the polymersome with the cell glycocalyx shedding light on its effect on the polymersome binding. We validate our model experimentally and show that the binding energy is a nonlinear function, allowing us to tune the interaction by varying the radius and degree of polymerization. Finally, we show that PMPC-PDPA polymersomes can be used to target monocytes in vivo due to their promiscuous interaction with SRB1, CD36, and CD81.© 2022 The Authors. Published by American Chemical Society.
JTD Keywords: binding, cd36, multivalency, ph, scavenger receptor, sr-bi, B type-i
Castagna, R, Kolarski, D, Durand-de Cuttoli, R, Maleeva, G, (2022). Orthogonal Control of Neuronal Circuits and Behavior Using Photopharmacology Journal Of Molecular Neuroscience 72, 1433-1442
Over the last decades, photopharmacology has gone far beyond its proof-of-concept stage to become a bona fide approach to study neural systems in vivo. Indeed, photopharmacological control has expanded over a wide range of endogenous targets, such as receptors, ion channels, transporters, kinases, lipids, and DNA transcription processes. In this review, we provide an overview of the recent progresses in the in vivo photopharmacological control of neuronal circuits and behavior. In particular, the use of small aquatic animals for the in vivo screening of photopharmacological compounds, the recent advances in optical modulation of complex behaviors in mice, and the development of adjacent techniques for light and drug delivery in vivo are described.
JTD Keywords: brain circuits, circadian rhythm, in vivo photomodulation, in vivo technology, neuronal receptors, Architecture, Azobenzene photoswitches, Brain circuits, Channels, Circadian rhythm, In vivo photomodulation, In vivo technology, Light, Modulator, Neuronal receptors, Optical control, Optogenetics, Pharmacology, Photopharmacology, Receptors, Systems
Woythe, L, Madhikar, P, Feiner-Gracia, N, Storm, C, Albertazzi, L, (2022). A Single-Molecule View at Nanoparticle Targeting Selectivity: Correlating Ligand Functionality and Cell Receptor Density Acs Nano 16, 3785-3796
Antibody-functionalized nanoparticles (NPs) are commonly used to increase the targeting selectivity toward cells of interest. At a molecular level, the number of functional antibodies on the NP surface and the density of receptors on the target cell determine the targeting interaction. To rationally develop selective NPs, the single-molecule quantitation of both parameters is highly desirable. However, techniques able to count molecules with a nanometric resolution are scarce. Here, we developed a labeling approach to quantify the number of functional cetuximabs conjugated to NPs and the expression of epidermal growth factor receptors (EGFRs) in breast cancer cells using direct stochastic optical reconstruction microscopy (dSTORM). The single-molecule resolution of dSTORM allows quantifying molecules at the nanoscale, giving a detailed insight into the distributions of individual NP ligands and cell receptors. Additionally, we predicted the fraction of accessible antibody-conjugated NPs using a geometrical model, showing that the total number exceeds the accessible number of antibodies. Finally, we correlated the NP functionality, cell receptor density, and NP uptake to identify the highest cell uptake selectivity regimes. We conclude that single-molecule functionality mapping using dSTORM provides a molecular understanding of NP targeting, aiding the rational design of selective nanomedicines.
JTD Keywords: active targeting, active targeting dstorm, antibodies, dstorm, heterogeneity, multivalency, nanomedicine, nanoparticle functionality, size, super-resolution microscopy, surface, Active targeting, Antibodies, Cell membranes, Cell receptors, Cytology, Direct stochastic optical reconstruction microscopy, Dstorm, Heterogeneity, Ligands, Medical nanotechnology, Molecules, Nanomedicine, Nanoparticle functionality, Nanoparticle targeting, Nanoparticles, Optical reconstruction, Single molecule, Stochastic systems, Stochastics, Super-resolution microscopy, Superresolution microscopy
Aydin, O, Passaro, AP, Raman, R, Spellicy, SE, Weinberg, RP, Kamm, RD, Sample, M, Truskey, GA, Zartman, J, Dar, RD, Palacios, S, Wang, J, Tordoff, J, Montserrat, N, Bashir, R, Saif, MTA, Weiss, R, (2022). Principles for the design of multicellular engineered living systems Apl Bioengineering 6, 10903
Remarkable progress in bioengineering over the past two decades has enabled the formulation of fundamental design principles for a variety of medical and non-medical applications. These advancements have laid the foundation for building multicellular engineered living systems (M-CELS) from biological parts, forming functional modules integrated into living machines. These cognizant design principles for living systems encompass novel genetic circuit manipulation, self-assembly, cell–cell/matrix communication, and artificial tissues/organs enabled through systems biology, bioinformatics, computational biology, genetic engineering, and microfluidics. Here, we introduce design principles and a blueprint for forward production of robust and standardized M-CELS, which may undergo variable reiterations through the classic design-build-test-debug cycle. This Review provides practical and theoretical frameworks to forward-design, control, and optimize novel M-CELS. Potential applications include biopharmaceuticals, bioreactor factories, biofuels, environmental bioremediation, cellular computing, biohybrid digital technology, and experimental investigations into mechanisms of multicellular organisms normally hidden inside the “black box” of living cells.
JTD Keywords: cell-fate specification, endothelial-cells, escherichia-coli, extracellular-matrix, gene-expression noise, nuclear hormone-receptors, pluripotent stem-cells, primitive endoderm, transcription factors, Artificial tissues, Assembly cells, Biological parts, Biological systems, Bioremediation, Blood-brain-barrier, Cell engineering, Cell/matrix communication, Design principles, Environmental technology, Functional modules, Fundamental design, Genetic circuits, Genetic engineering, Living machines, Living systems, Medical applications, Molecular biology, Synthetic biology
Pellegrini, P, Hervera, A, Varea, O, Brewer, MK, López-Soldado, I, Guitart, A, Aguilera, M, Prats, N, del Río, JA, Guinovart, JJ, Duran, J, (2022). Lack of p62 Impairs Glycogen Aggregation and Exacerbates Pathology in a Mouse Model of Myoclonic Epilepsy of Lafora Molecular Neurobiology 59, 1214-1229
Lafora disease (LD) is a fatal childhood-onset dementia characterized by the extensive accumulation of glycogen aggregates—the so-called Lafora Bodies (LBs)—in several organs. The accumulation of LBs in the brain underlies the neurological phenotype of the disease. LBs are composed of abnormal glycogen and various associated proteins, including p62, an autophagy adaptor that participates in the aggregation and clearance of misfolded proteins. To study the role of p62 in the formation of LBs and its participation in the pathology of LD, we generated a mouse model of the disease (malinKO) lacking p62. Deletion of p62 prevented LB accumulation in skeletal muscle and cardiac tissue. In the brain, the absence of p62 altered LB morphology and increased susceptibility to epilepsy. These results demonstrate that p62 participates in the formation of LBs and suggest that the sequestration of abnormal glycogen into LBs is a protective mechanism through which it reduces the deleterious consequences of its accumulation in the brain. © 2021, The Author(s).
JTD Keywords: accumulation, astrocytes, autophagy receptors, contributes, deficient mice, epilepsy, glycogen, lafora bodies, lafora disease, malin, metabolism, neurodegeneration, neuroinflammation, p62, polyglucosan bodies, temporal-lobe epilepsy, Epilepsy, Glycogen, Inclusion-body formation, Lafora bodies, Lafora disease, Malin, Neuroinflammation, P62
Leite, DM, Seifi, M, Ruiz-Perez, L, Nguemo, F, Plomann, M, Swinny, JD, Battaglia, G, (2022). Syndapin-2 mediated transcytosis of amyloid-beta across the blood brain barrier Brain Commun 4, fcac093
A deficient transport of amyloid-beta across the blood-brain barrier, and its diminished clearance from the brain, contribute to neurodegenerative and vascular pathologies, such as Alzheimer's disease and cerebral amyloid angiopathy, respectively. At the blood-brain barrier, amyloid-beta efflux transport is associated with the low-density lipoprotein receptor-related protein 1. However, the precise mechanisms governing amyloid-beta transport across the blood-brain barrier, in health and disease, remain to be fully understood. Recent evidence indicates that the low-density lipoprotein receptor-related protein 1 transcytosis occurs through a tuhulation-mediated mechanism stabilized by syndapin-2. Here, we show that syndapin-2 is associated with amyloid-beta clearance via low-density lipoprotein receptor-related protein 1 across the blood-brain barrier. We further demonstrate that risk factors for Alzheimer's disease, amyloid-beta expression and ageing, are associated with a decline in the native expression of syndapin-2 within the brain endothelium. Our data reveals that syndapin-2-mediated pathway, and its balance with the endosomal sorting, are important for amyloid-beta clearance proposing a measure to evaluate Alzheimer's disease and ageing, as well as a target for counteracting amyloid-beta build-up. Moreover, we provide evidence for the impact of the avidity of amyloid-beta assemblies in their trafficking across the brain endothelium and in low-density lipoprotein receptor-related protein 1 expression levels, which may affect the overall clearance of amyloid-beta across the blood-brain barrier.
JTD Keywords: alzheimer’s disease, amyloid-β, blood–brain barrier, syndapin-2, Alzheimer's disease, Alzheimers-disease, Amyloid-beta, Apolipoprotein-j, Blood-brain barrier, Clearance, Expression, Membrane invagination, Peptide, Protein, Rab gtpases, Receptor, Syndapin-2, Transport, Tubular transcytosis
Amil, AF, Verschure, PFMJ, (2021). Supercritical dynamics at the edge-of-chaos underlies optimal decision-making Journal Of Physics-Complexity 2, 45017
Abstract Critical dynamics, characterized by scale-free neuronal avalanches, is thought to underlie optimal function in the sensory cortices by maximizing information transmission, capacity, and dynamic range. In contrast, deviations from criticality have not yet been considered to support any cognitive processes. Nonetheless, neocortical areas related to working memory and decision-making seem to rely on long-lasting periods of ignition-like persistent firing. Such firing patterns are reminiscent of supercritical states where runaway excitation dominates the circuit dynamics. In addition, a macroscopic gradient of the relative density of Somatostatin (SST+) and Parvalbumin (PV+) inhibitory interneurons throughout the cortical hierarchy has been suggested to determine the functional specialization of low- versus high-order cortex. These observations thus raise the question of whether persistent activity in high-order areas results from the intrinsic features of the neocortical circuitry. We used an attractor model of the canonical cortical circuit performing a perceptual decision-making task to address this question. Our model reproduces the known saddle-node bifurcation where persistent activity emerges, merely by increasing the SST+/PV+ ratio while keeping the input and recurrent excitation constant. The regime beyond such a phase transition renders the circuit increasingly sensitive to random fluctuations of the inputs -i.e., chaotic-, defining an optimal SST+/PV+ ratio around the edge-of-chaos. Further, we show that both the optimal SST+/PV+ ratio and the region of the phase transition decrease monotonically with increasing input noise. This suggests that cortical circuits regulate their intrinsic dynamics via inhibitory interneurons to attain optimal sensitivity in the face of varying uncertainty. Hence, on the one hand, we link the emergence of supercritical dynamics at the edge-of-chaos to the gradient of the SST+/PV+ ratio along the cortical hierarchy, and, on the other hand, explain the behavioral effects of the differential regulation of SST+ and PV+ interneurons by neuromodulators like acetylcholine in the presence of input uncertainty.
JTD Keywords: attractor model, cortex, cortical networks, edge-of-chaos, model, nmda receptors, Attractor model, Cortical hierarchies, Decision making, Dynamics, Edge of chaos, Edge-of-chaos, High-order, Higher-order, Inhibitory interneurons, Neurons, Optimal decision making, Persistent activities, Persistent activity, Supercritical, Supercriticality
Barbero-Castillo, A, Riefolo, F, Matera, C, Caldas-Martínez, S, Mateos-Aparicio, P, Weinert, JF, Garrido-Charles, A, Claro, E, Sanchez-Vives, MV, Gorostiza, P, (2021). Control of Brain State Transitions with a Photoswitchable Muscarinic Agonist Advanced Science 8, 2005027
The ability to control neural activity is essential for research not only in basic neuroscience, as spatiotemporal control of activity is a fundamental experimental tool, but also in clinical neurology for therapeutic brain interventions. Transcranial-magnetic, ultrasound, and alternating/direct current (AC/DC) stimulation are some available means of spatiotemporal controlled neuromodulation. There is also light-mediated control, such as optogenetics, which has revolutionized neuroscience research, yet its clinical translation is hampered by the need for gene manipulation. As a drug-based light-mediated control, the effect of a photoswitchable muscarinic agonist (Phthalimide-Azo-Iper (PAI)) on a brain network is evaluated in this study. First, the conditions to manipulate M2 muscarinic receptors with light in the experimental setup are determined. Next, physiological synchronous emergent cortical activity consisting of slow oscillations-as in slow wave sleep-is transformed into a higher frequency pattern in the cerebral cortex, both in vitro and in vivo, as a consequence of PAI activation with light. These results open the way to study cholinergic neuromodulation and to control spatiotemporal patterns of activity in different brain states, their transitions, and their links to cognition and behavior. The approach can be applied to different organisms and does not require genetic manipulation, which would make it translational to humans.
JTD Keywords: brain states, light-mediated control, muscarinic acetylcholine receptors, neuromodulation, Activation, Alternating/direct currents, Basal forebrain, Brain, Brain states, Clinical research, Clinical translation, Controlled drug delivery, Cortex, Forebrain cholinergic system, Genetic manipulations, Higher frequencies, Hz oscillation, Light‐, Light-mediated control, Mediated control, Muscarinic acetylcholine receptors, Muscarinic agonists, Muscarinic receptor, Neurology, Neuromodulation, Neurons, Noradrenergic modulation, Parvalbumin-positive interneurons, Photopharmacology, Receptor-binding, Slow, Spatiotemporal control, Spatiotemporal patterns
Riefolo, F, Sortino, R, Matera, C, Claro, E, Preda, B, Vitiello, S, Traserra, S, Jimenez, M, Gorostiza, P, (2021). Rational Design of Photochromic Analogues of Tricyclic Drugs Journal Of Medicinal Chemistry 64, 9259-9270
Tricyclic chemical structures are the core of many important drugs targeting all neurotransmitter pathways. These medicines enable effective therapies to treat from peptic ulcer disease to psychiatric disorders. However, when administered systemically, they cause serious adverse effects that limit their use. To obtain localized and on-demand pharmacological action using light, we have designed photoisomerizable ligands based on azobenzene that mimic the tricyclic chemical structure and display reversibly controlled activity. Pseudo-analogues of the tricyclic antagonist pirenzepine demonstrate that this is an effective strategy in muscarinic acetylcholine receptors, showing stronger inhibition upon illumination both in vitro and in cardiac atria ex vivo. Despite the applied chemical modifications to make pirenzepine derivatives sensitive to light stimuli, the most potent candidate of the set, cryptozepine-2, maintained a moderate but promising M-1 vs M-2 subtype selectivity. These photoswitchable crypto-azologs of tricyclic drugs might open a general way to spatiotemporally target their therapeutic action while reducing their systemic toxicity and adverse effects.
JTD Keywords: Binding, Dose-response relationship, drug, Drug design, Humans, M1, Molecular structure, Muscarinic antagonists, Pirenzepine, Rat-brain, Receptor, Receptors, muscarinic, Structure-activity relationship
Prischich, D, Gomila, AMJ, Milla-Navarro, S, Sanguesa, G, Diez-Alarcia, R, Preda, B, Matera, C, Batlle, M, Ramírez, L, Giralt, E, Hernando, J, Guasch, E, Meana, JJ, de la Villa, P, Gorostiza, P, (2021). Adrenergic Modulation With Photochromic Ligands Angewandte Chemie (International Ed. Print) 60, 3625-3631
© 2020 Wiley-VCH GmbH 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.
JTD Keywords: adrenergic receptors, azo compounds, neurotransmitters, photochromism, Adrenergic agents, Adrenergic receptors, Animals, Azo compounds, Chromogenic compounds, Ligands, Mice, Mice, nude, Molecular structure, Neurotransmitters, Photochromism, Photopharmacology, Receptors, adrenergic, Zebrafish
Maleeva, G, Nin-Hill, A, Rustler, K, Petukhova, E, Ponomareva, D, Mukhametova, E, Gomila, AMJ, Wutz, D, Alfonso-Prieto, M, König, B, Gorostiza, P, Bregestovski, P, (2021). Subunit-specific photocontrol of glycine receptors by azobenzene-nitrazepam photoswitcher Eneuro 8, 0294-20.2020
© 2021 Maleeva et al. Photopharmacology is a unique approach that through a combination of photochemistry methods and advanced life science techniques allows the study and control of specific biological processes, ranging from intracellular pathways to brain circuits. Recently, a first photochromic channel blocker of anion-selective GABAA receptors, the azobenzene-nitrazepam-based photochromic compound (Azo-NZ1), has been described. In the present study, using patch-clamp technique in heterologous system and in mice brain slices, site-directed mutagenesis and molecular modeling we provide evidence of the interaction of Azo-NZ1 with glycine receptors (GlyRs) and determine the molecular basis of this interaction. Glycinergic synaptic neurotransmission determines an important inhibitory drive in the vertebrate nervous system and plays a crucial role in the control of neuronal circuits in the spinal cord and brain stem. GlyRs are involved in locomotion, pain sensation, breathing, and auditory function, as well as in the development of such disorders as hyperekplexia, epilepsy, and autism. Here, we demonstrate that Azo-NZ1 blocks in a UV-dependent manner the activity of a2 GlyRs (GlyR2), while being barely active on a1 GlyRs (GlyR1). The site of Azo-NZ1 action is in the chloride-selective pore of GlyR at the 2’ position of transmembrane helix 2 and amino acids forming this site determine the difference in Azo-NZ1 blocking activity between GlyR2 and GlyR1. This subunit-specific modulation is also shown on motoneurons of brainstem slices from neonatal mice that switch during development from expressing “fetal” GlyR2 to “adult” GlyR1 receptors.
JTD Keywords: brain slices, glycine receptors, hypoglossal motoneurons, molecular modelling, patch-clamp, photopharmacology, Brain slices, Glycine receptors, Hypoglossal motoneurons, Molecular modelling, Patch-clamp, Photopharmacology
Delcanale, P., Porciani, D., Pujals, S., Jurkevich, A., Chetrusca, A., Tawiah, K. D., Burke, D. H., Albertazzi, L., (2020). Aptamers with tunable affinity enable single-molecule tracking and localization of membrane receptors on living cancer cells Angewandte Chemie - International Edition 59, (42), 18546-18555
Tumor cell-surface markers are usually overexpressed or mutated protein receptors for which spatiotemporal regulation differs between and within cancers. Single-molecule fluorescence imaging can profile individual markers in different cellular contexts with molecular precision. However, standard single-molecule imaging methods based on overexpressed genetically encoded tags or cumbersome probes can significantly alter the native state of receptors. We introduce a live-cell points accumulation for imaging in nanoscale topography (PAINT) method that exploits aptamers as minimally invasive affinity probes. Localization and tracking of individual receptors are based on stochastic and transient binding between aptamers and their targets. We demonstrated single-molecule imaging of a model tumor marker (EGFR) on a panel of living cancer cells. Affinity to EGFR was finely tuned by rational engineering of aptamer sequences to define receptor motion and/or native receptor density.
JTD Keywords: Aptamers, Cell-surface receptors, Live-cell imaging, PAINT, Single-molecule tracking
Gomila, Alexandre M. J., Rustler, Karin, Maleeva, Galyna, Nin-Hill, Alba, Wutz, Daniel, Bautista-Barrufet, Antoni, Rovira, Xavier, Bosch, Miquel, Mukhametova, Elvira, Petukhova, Elena, Ponomareva, Daria, Mukhamedyarov, Marat, Peiretti, Franck, Alfonso-Prieto, Mercedes, Rovira, Carme, König, Burkhard, Bregestovski, Piotr, Gorostiza, Pau, (2020). Photocontrol of endogenous glycine receptors in vivo Cell Chemical Biology 27, (11), 1425-1433.e7
Glycine receptors (GlyRs) are indispensable for maintaining excitatory/inhibitory balance in neuronal circuits that control reflexes and rhythmic motor behaviors. Here we have developed Glyght, a GlyR ligand controlled with light. It is selective over other Cys-loop receptors, is active in vivo, and displays an allosteric mechanism of action. The photomanipulation of glycinergic neurotransmission opens new avenues to understanding inhibitory circuits in intact animals and to developing drug-based phototherapies.
JTD Keywords: Glycine receptors, Photopharmacology, Optopharmacology, Inhibitory neurotransmission, CNS, Photoswitch
Maleeva, Galyna, Nin-Hill, Alba, Rustler, Karin, Petukhova, Elena, Ponomareva, Daria, Mukhametova, Elvira, Gomila-Juaneda, Alexandre, Wutz, Daniel, Alfonso-Prieto, Mercedes, König, Burkhard, Gorostiza, Pau, Bregestovski, Piotr, (2020). Subunit-specific photocontrol of glycine receptors by azobenzene-nitrazepam photoswitcher eneuro 8, (1), 0294-20
Photopharmacology is a unique approach that through a combination of photochemistry methods and advanced life science techniques allows the study and control of specific biological processes, ranging from intracellular pathways to brain circuits. Recently, a first photochromic channel blocker of anion-selective GABAA receptors, Azo-NZ1, has been described. In the present study using patch-clamp technique in heterologous system and in mice brain slices, site-directed mutagenesis and molecular modelling we provide evidence of the interaction of Azo-NZ1 with glycine receptors (GlyRs) and determine the molecular basis of this interaction. Glycinergic synaptic neurotransmission determines an important inhibitory drive in the vertebrate nervous system and plays a crucial role in the control of neuronal circuits in the spinal cord and brain stem. GlyRs are involved in locomotion, pain sensation, breathing and auditory function, as well as in the development of such disorders as hyperekplexia, epilepsy and autism. Here we demonstrate that Azo-NZ1 blocks in a UV dependent manner the activity of alpha2 GlyRs (GlyR2), while being barely active on alpha1 GlyRs (GlyR1). The site of Azo-NZ1 action is in the chloride-selective pore of GlyR at the 2’ position of transmembrane helix 2 and amino acids forming this site determine the difference in Azo-NZ1 blocking activity between GlyR2 and GlyR1. This subunit specific modulation is also shown on motoneurons of brainstem slices from neonatal mice that switch during development from expressing "foetal" GlyR2 to "adult" GlyR1 receptors.
Significance Statement Photochromic molecules are becoming widely used for studying and modulating various biological processes. Successful application of these compounds, whose activity can be controlled with light, potentially provides a promising tool for future therapeutic approaches. The main advantage of such compounds is their precise spatial and temporal selectivity, a property that favours specific drug action and diminishes their side effects. In the present study, we describe in detail the interaction of the novel azobenzene-nitrazepam-based photochromic compound (Azo-NZ1) with glycine receptors (GlyRs) and determine its subunit-specific blocking activity in the Cl-selective pore of GlyRs. This compound offers a new strategy for specific control of glycinergic circuits and stepping stone for design of new GlyR-active drugs.
JTD Keywords: Brain slices, Glycine receptors, Hypoglossal motoneurons, Molecular modelling, Patch-clamp, Photopharmacology
Garcia-Esparcia, Paula, López-González, Irene, Grau-Rivera, Oriol, García-Garrido, María Francisca, Konetti, Anusha, Llorens, Franc, Zafar, Saima, Carmona, Margarita, del Rio, José Antonio, Zerr, Inga, Gelpi, Ellen, Ferrer, Isidro, (2017). Dementia with Lewy Bodies: Molecular pathology in the frontal cortex in typical and rapidly progressive forms Frontiers in Neurology 8, Article 89
Objectives: The goal of this study was to assess mitochondrial function, energy, and purine metabolism, protein synthesis machinery from the nucleolus to the ribosome, inflammation, and expression of newly identified ectopic olfactory receptors (ORs) and taste receptors (TASRs) in the frontal cortex of typical cases of dementia with Lewy bodies (DLB) and cases with rapid clinical course (rpDLB: 2 years or less) compared with middle-aged non-affected individuals, in order to learn about the biochemical abnormalities underlying Lewy body pathology. Methods: Real-time quantitative PCR, mitochondrial enzymatic assays, and analysis of β-amyloid, tau, and synuclein species were used. Results: The main alterations in DLB and rpDLB, which are more marked in the rapidly progressive forms, include (i) deregulated expression of several mRNAs and proteins of mitochondrial subunits, and reduced activity of complexes I, II, III, and IV of the mitochondrial respiratory chain; (ii) reduced expression of selected molecules involved in energy metabolism and increased expression of enzymes involved in purine metabolism; (iii) abnormal expression of nucleolar proteins, rRNA18S, genes encoding ribosomal proteins, and initiation factors of the transcription at the ribosome; (iv) discrete inflammation; and (v) marked deregulation of brain ORs and TASRs, respectively. Severe mitochondrial dysfunction involving activity of four complexes, minimal inflammatory responses, and dramatic altered expression of ORs and TASRs discriminate DLB from Alzheimer’s disease. Altered solubility and aggregation of α-synuclein, increased β-amyloid bound to membranes, and absence of soluble tau oligomers are common in DLB and rpDLB. Low levels of soluble β-amyloid are found in DLB. However, increased soluble β-amyloid 1–40 and β-amyloid 1–42, and increased TNFα mRNA and protein expression, distinguish rpDLB. Conclusion: Molecular alterations in frontal cortex in DLB involve key biochemical pathways such as mitochondria and energy metabolism, protein synthesis, purine metabolism, among others and are accompanied by discrete innate inflammatory response.
JTD Keywords: Dementia with Lewy bodies, Alzheimer’s disease, α-synuclein, Mitochondria, Protein synthesis, Inflammation, β-amyloid, Olfactory receptors
Quadri, M., Matera, C., Silnovi, Pismataro, M. C., Horenstein, N. A., Stokes, C., Papke, R. L., Dallanoce, C., (2017). Identification of α7 nicotinic acetylcholine receptor silent agonists based on the spirocyclic quinuclidine-Δ2-isoxazoline scaffold: Synthesis and electrophysiological evaluation ChemMedChem XXIV National Meeting in Medicinal Chemistry (NMMC 2016) , Wiley Online Library (Perugia, Spain) 12, (16), 1335-1348
Compound 11 (3-(benzyloxy)-1′-methyl-1′-azonia-4H-1′-azaspiro[isoxazole-5,3′-bicyclo[2.2.2]octane] iodide) was selected from a previous set of nicotinic ligands as a suitable model compound for the design of new silent agonists of α7 nicotinic acetylcholine receptors (nAChRs). Silent agonists evoke little or no channel activation but can induce the α7 desensitized Ds state, which is sensitive to a type II positive allosteric modulator, such as PNU-120596. Introduction of meta substituents into the benzyloxy moiety of 11 led to two sets of tertiary amines and quaternary ammonium salts based on the spirocyclic quinuclidinyl-Δ2-isoxazoline scaffold. Electrophysiological assays performed on Xenopus laevis oocytes expressing human α7 nAChRs highlighted four compounds that are endowed with a significant silent-agonism profile. Structure–activity relationships of this group of analogues provided evidence of the crucial role of the positive charge at the quaternary quinuclidine nitrogen atom. Moreover, the present study indicates that meta substituents, in particular halogens, on the benzyloxy substructure direct specific interactions that stabilize a desensitized conformational state of the receptor and induce silent activity
JTD Keywords: Agonists, Cycloaddition, Nitrogen heterocycles, Receptors, Spiro compounds
Sanmartí-Espinal, M., Galve, R., Iavicoli, P., Persuy, M. A., Pajot-Augy, E., Marco, M. P., Samitier, J., (2016). Immunochemical strategy for quantification of G-coupled olfactory receptor proteins on natural nanovesicles Colloids and Surfaces B: Biointerfaces 139, 269-276
Cell membrane proteins are involved in a variety of biochemical pathways and therefore constitute important targets for therapy and development of new drugs. Bioanalytical platforms and binding assays using these membrane protein receptors for drug screening or diagnostic require the construction of well-characterized liposome and lipid bilayer arrays that act as support to prevent protein denaturation during biochip processing. Quantification of the protein receptors in the lipid membrane arrays is a key issue in order to produce reproducible and well-characterized chips. Herein, we report a novel immunochemical analytical approach for the quantification of membrane proteins (i.e., G-protein-coupled receptor, GPCR) in nanovesicles (NVs). The procedure allows direct determination of tagged receptors (i.e., c-myc tag) without any previous protein purification or extraction steps. The immunochemical method is based on a microplate ELISA format and quantifies this tag on proteins embedded in NVs with detectability in the picomolar range, using protein bioconjugates as reference standards. The applicability of the method is demonstrated through the quantification of the c-myc-olfactory receptor (OR, c-myc-OR1740) in the cell membrane NVs. The reported method opens the possibility to develop well-characterized drug-screening platforms based on G-coupled proteins embedded on membranes.
JTD Keywords: Bioelectronic nose, Competitive ELISA, G-protein-coupled receptors quantification, Natural vesicles, Olfactory receptors, Transmembrane proteins
Castaño, O., Sachot, N., Xuriguera, E., Engel, E., Planell, J. A., Park, J. H., Jin, G. Z., Kim, T. H., Kim, J. H., Kim, H. W., (2014). Angiogenesis in bone regeneration: Tailored calcium release in hybrid fibrous scaffolds ACS Applied Materials & Interfaces 6, (10), 7512-7522
In bone regeneration, silicon-based calcium phosphate glasses (Bioglasses) have been widely used since the 1970s. However, they dissolve very slowly because of their high amount of Si (SiO2 > 45%). Recently, our group has found that calcium ions released by the degradation of glasses in which the job of silicon is done by just 5% of TiO2 are effective angiogenic promoters, because of their stimulation of a cell-membrane calcium sensing receptor (CaSR). Based on this, other focused tests on angiogenesis have found that Bioglasses also have the potential to be angiogenic promoters even with high contents of silicon (80%); however, their slow degradation is still a problem, as the levels of silicon cannot be decreased any lower than 45%. In this work, we propose a new generation of hybrid organically modified glasses, ormoglasses, that enable the levels of silicon to be reduced, therefore speeding up the degradation process. Using electrospinning as a faithful way to mimic the extracellular matrix (ECM), we successfully produced hybrid fibrous mats with three different contents of Si (40, 52, and 70%), and thus three different calcium ion release rates, using an ormoglass–polycaprolactone blend approach. These mats offered a good platform to evaluate different calcium release rates as osteogenic promoters in an in vivo subcutaneous environment. Complementary data were collected to complement Ca2+ release analysis, such as stiffness evaluation by AFM, ζ-potential, morphology evaluation by FESEM, proliferation and differentiation analysis, as well as in vivo subcutaneous implantations. Material and biological characterization suggested that compositions of organic/inorganic hybrid materials with a Si content equivalent to 40%, which were also those that released more calcium, were osteogenic. They also showed a greater ability to form blood vessels. These results suggest that Si-based ormoglasses can be considered an efficient tool for calcium release modulation, which could play a key role in the angiogenic promoting process.
JTD Keywords: Biological materials, Blood vessels, Calcium, Electrospinning, Glass, Hybrid materials, Silicon oxides, Sol-gel process, Sol-gels, Angiogenesis, Biological characterization, Calcium phosphate glass, Calcium-sensing receptors, Degradation process, Extracellular matrices, Organic/inorganic hybrid materials, ormoglasses, Silicon
Llorens, F., Del Rio, J. A., (2012). Unraveling the neuroprotective mechanisms of PrPC in excitotoxicity Prion , 6, (3), 245-251
Knowledge of the natural roles of cellular prion protein (PrPC) is essential to an understanding of the molecular basis of prion pathologies. This GPIanchored protein has been described in synaptic contacts, and loss of its synaptic function in complex systems may contribute to the synaptic loss and neuronal degeneration observed in prionopathy. In addition, Prnp knockout mice show enhanced susceptibility to several excitotoxic insults, GABAA receptor-mediated fast inhibition was weakened, LTP was modified and cellular stress increased. Although little is known about how PrPC exerts its function at the synapse or the downstream events leading to PrPCmediated neuroprotection against excitotoxic insults, PrPC has recently been reported to interact with two glutamate
receptor subunits (NR2D and GluR6/7). In both cases the presence of PrPC blocks the neurotoxicity induced by NMDA and Kainate respectively. Furthermore, signals for seizure and neuronal cell death in response to Kainate in Prnp knockout mouse are associated with JNK3 activity, through enhancing the interaction of GluR6 with PSD-95. In combination with previous data, these results shed light on the molecular mechanisms behind the role of PrPC in excitotoxicity. Future experimental approaches are suggested and discussed.
JTD Keywords: Prion protein, Excitotoxicity, Neuroprotection, Glutamate receptors, Synapse, prionopathy
van Zanten, Thomas S., Lopez-Bosque, M. J . , Garcia-Parajo, M. F., (2010). Imaging individual proteins and nanodomains on intact cell membranes with a probe-based optical antenna Small 6, (2), 270-275
Optical antennas that confine and enhance electromagnetic fields in a nanometric region hold great potential for nanobioimaging and biosensing. Probe-based monopole optical antennas are fabricated to enhance fields localized to <30 nm near the antenna apex in aqueous conditions. These probes are used under appropriate excitation antenna conditions to image individual antibodies with an unprecedented resolution of 26 ± 4 nm and virtually no surrounding background. On intact cell membranes in physiological conditions, the obtained resolution is 30 ± 6 nm. Importantly, the method allows individual proteins to be distinguished from nanodomains and the degree of clustering to be quantified by directly measuring physical size and intensity of individual fluorescent spots. Improved antenna geometries should lead to true live cell imaging below 10-nm resolution with position accuracy in the subnanometric range.
JTD Keywords: Cell membranes, Cell receptors, Focused ion beam milling, Nanodomains, Optical antennas
Sanmarti, M., Iavicoli, P., Pajot-Augy, E., Gomila, G., Samitier, J., (2010). Human olfactory receptors immobilization on a mixed self assembled monolayer for the development of a bioelectronic nose Procedia Engineering (EUROSENSOR XXIV CONFERENCE) 24th Eurosensor Conference (ed. Jakoby, B., Vellekoop, M.J.), Elsevier Science (Linz, Austria) 5, 786-789
The present work focuses on the development of an immunosensing surface to build a portable olfactory system for the detection of complex mixture of odorants. Homogeneous cell derived vesicles expressing the olfactory receptors were produced and immobilized with efficiency onto a gold substrate through an optimized surface functionalization method.
JTD Keywords: Bioelectronic noses, Biosensors, Nanoproteoliposomes, Nanosomes, Olfactory receptors, SAMs
de Bakker, Barbel I., Bodnar, Andrea, van Dijk, Erik M. H. P., Vamosi, Gyorgy, Damjanovich, Sandor, Waldmann, Thomas A., van Hulst, Niek F., Jenei, Attila, Garcia-Parajo, M. F., (2008). Nanometer-scale organization of the alpha subunits of the receptors for IL2 and IL15 in human T lymphoma cells Journal of Cell Science 121, (5), 627-633
Interleukin 2 and interleukin 15 (IL2 and IL15, respectively) provide quite distinct contributions to T-cell-mediated immunity, despite having similar receptor composition and signaling machinery. As most of the proposed mechanisms underlying this apparent paradox attribute key significance to the individual {alpha}-chains of IL2 and IL15 receptors, we investigated the spatial organization of the receptors IL2R{alpha} and IL15R{alpha} at the nanometer scale expressed on a human CD4+ leukemia T cell line using single-molecule-sensitive near-field scanning optical microscopy (NSOM). In agreement with previous findings, we here confirm clustering of IL2R{alpha} and IL15R{alpha} at the submicron scale. In addition to clustering, our single-molecule data reveal that a non-negligible percentage of the receptors are organized as monomers. Only a minor fraction of IL2R{alpha} molecules reside outside the clustered domains, whereas [~]30% of IL15R{alpha} molecules organize as monomers or small clusters, excluded from the main domain regions. Interestingly, we also found that the packing densities per unit area of both IL2R{alpha} and IL15R{alpha} domains remained constant, suggesting a `building block' type of assembly involving repeated structures and composition. Finally, dual-color NSOM demonstrated co-clustering of the two {alpha}-chains. Our results should aid understanding the action of the IL2R-IL15R system in T cell function and also might contribute to the more rationale design of IL2R- or IL15R-targeted immunotherapy agents for treating human leukemia.
JTD Keywords: Near-field scanning optical microscopy (NSOM), Interleukin receptors IL2R, IL15R, Single-molecule detection, Nanometer-scale membrane organization
De Bakker, B. I., De Lange, F., Cambi, A., Korterik, J. P., Van Dijk, E. M. H. P., Van Hulst, N. F., Figdor, C. G., Garcia-Parajo, M. F., (2007). Nanoscale organization of the pathogen receptor DC-SIGN mapped by single-molecule high-resolution fluorescence microscopy ChemPhysChem , 8, (10), 1473-1480
DC-SIGN, a C-type lectin exclusively expressed on dendritic cells (DCs), plays an important role in pathogen recognition by binding with high affinity to a large variety of microorganisms. Recent experimental evidence points to a direct relation between the function of DC-SIGN as a viral receptor and its spatial arrangement on the plasma membrane. We have investigated the nanoscale organization of fluorescently labeled DC-SIGN on intact isolated DCs by means of near-field scanning optical microscopy (NSOM) combined with single-molecule detection. Fluorescence spots of different intensity and size have been directly visualized by optical means with a spatial resolution of less than 100 nm. Intensity- and size-distribution histograms of the DC-SIGN fluorescent spots confirm that approximately 80% of the receptors are organized in nanosized domains randomly distributed on the cell membrane. Intensity-size correlation analysis revealed remarkable heterogeneity in the molecular packing density of the domains. Furthermore, we have mapped the intermolecular organization within a dense cluster by means of sequential NSOM imaging combined with discrete single-molecule photobleaching. In this way we have determined the spatial coordinates of 13 different individual dyes, with a localization accuracy of 6 nm. Our experimental observations are all consistent with an arrangement of DC-SIGN designed to maximize its chances of binding to a wide range of microorganisms. Our data also illustrate the potential of NSOM as an ultrasensitive, high-resolution technique to probe nanometer-scale organization of molecules on the cell membrane.
JTD Keywords: High-resolution optical microscopy, Lectins, Membranes, Receptors, Single-molecule studies