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One approach to enhance the bioavailability and half-life of peptides in vivo is through N-methylation of one or more of the amino acids within the peptide sequence. However, commercially available Fmoc-N-Me-AA-OHs are limited and often expensive. In this study, a solid-phase synthesis method for Fmoc-N-Me-AA-OH was developed using a 2-chlorotrityl chloride (2-CTC) resin as a temporary protective group for the carboxylic acid strategy. Two strategies for the alkylation step were compared, employing either dimethyl sulfate or methyl iodide in the Biron-Kessler method. In this work we tested the protocol with two amino acids: Fmoc-Thr(tBu)-OH and Fmoc-ßAla-OH. The first one is an alpha amino acid, very hindered and with the amine group directly influenced by the electronic effects of the carboxy group, whereas in Fmoc-ßAla-OH, the presence of a methylene group weakens this influence due to the intervening carbon atoms. The desired amino acids, Fmoc-N-Me-Thr(tBu)-OH and Fmoc-N-Me-ßAla-OH, were synthesized by both strategies with high yield and purity.
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Used in solid-phase peptide synthesis (SPPS) for peptides with an acid termination, the 2-chlorotrityl chloride (2-CTC) resin is highly susceptible to moisture, leading to reduced resin loading and lower synthetic yields. It is therefore recommended that the resin be activated with thionyl chloride (SOCl2) before peptide assembly. Here we present an optimized procedure for resin activation that minimizes the use of SOCl2 as the activation reagent and reduces the activation time. Additionally, we demonstrate the feasibility of reusing the 2-CTC resin when following the activation protocol, achieving comparable results to the first usage of the resin. Moreover, we achieved different degrees of resin activation by varying the amount of SOCl2. For instance, the use of 2% SOCl2 in anhydrous dichloromethane (DCM) allowed up to 44% activation of the resin, thereby making it suitable for the synthesis of longer peptides. Alternatively, employing 25% SOCl2 in anhydrous DCM resulted in up to 80% activation with a reaction time of only 5 min in both cases.
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AIM: To investigate the use of synthetic preimplantation factor (sPIF) as a potential therapeutic tool for improving glucose-stimulated insulin secretion (GSIS), glucose tolerance and insulin sensitivity in the setting of diabetes. MATERIALS AND METHODS: We used a preclinical murine model of type 2 diabetes (T2D) induced by high-fat diet (HFD) feeding for 12 weeks. Saline or sPIF (1 mg/kg/day) was administered to mice by subcutaneously implanted osmotic mini-pumps for 25 days. Glucose tolerance, circulating insulin and C-peptide levels, and GSIS were assessed. In addition, ß-cells (Min-6) were used to test the effects of sPIF on GSIS and insulin-degrading enzyme (IDE) activity in vitro. The effect of sPIF on GSIS was also tested in human islets. RESULTS: GSIS was enhanced 2-fold by sPIF in human islets ex vivo. Furthermore, continuous administration of sPIF to HFD mice increased circulating levels of insulin and improved glucose tolerance, independently of hepatic insulin clearance. Of note, islets isolated from mice treated with sPIF exhibited restored ß-cell function. Finally, genetic (shRNA-IDE) or pharmacological (6bK) inactivation of IDE in Min-6 abolished sPIF-mediated effects on GSIS, showing that both the protein and its protease activity are required for its action. CONCLUSIONS: We conclude that sPIF is a promising secretagogue for the treatment of T2D.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Insulisina , Ilhotas Pancreáticas , Camundongos , Humanos , Animais , Secreção de Insulina , Insulisina/metabolismo , Insulisina/farmacologia , Camundongos Obesos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ilhotas Pancreáticas/metabolismoRESUMO
Developing technological solutions that use yerba mate waste as precursors is key to reducing the environmental impact caused by the lack of treatment and its accumulation in landfills. Due to their physicochemical properties, these residues can be used to develop activated carbons. Activated carbon is a versatile material with a high surface area that can be used for energy storage. In this work, yerba mate residues were valued by producing chemically activated carbon to be used as electrode material in supercapacitors. Activated carbons were developed through chemical activation in two steps with KOH. Variables such as impregnation ratio and activation temperature are studied. The developed carbons were characterized by physicochemical and electrochemical techniques. They were found to have high surface areas, up to 1800 m2 g-1, with a hierarchical porous distribution. A maximum specific capacitance of 644 F g-1 at 0.1 A g-1, and power values of ca 32,000 W kg-1, at 33 A g-1 were found. All the synthesized carbons have excellent electrochemical properties and are suitable for use as active material in supercapacitors.
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Carvão Vegetal , Ilex paraguariensis , Capacitância Elétrica , Eletrodos , PorosidadeRESUMO
Introduction: The development of new materials and tools for radiology is key to the implementation of this diagnostic technique in clinics. In this work, we evaluated the differential accumulation of peptide-functionalized GNRs in a transgenic animal model (APPswe/PSENd1E9) of Alzheimer's disease (AD) by computed tomography (CT) and measured the pharmacokinetic parameters and bioaccumulation of the nanosystem. Methods: The GNRs were functionalized with two peptides, Ang2 and D1, which conferred on them the properties of crossing the blood-brain barrier and binding to amyloid aggregates, respectively, thus making them a diagnostic tool with great potential for AD. The nanosystem was administered intravenously in APPswe/PSEN1dE9 model mice of 4-, 8- and 18-months of age, and the accumulation of gold nanoparticles was observed by computed tomography (CT). The gold accumulation and biodistribution were determined by atomic absorption. Results: Our findings indicated that 18-month-old animals treated with our nanosystem (GNR-D1/Ang2) displayed noticeable differences in CT signals compared to those treated with a control nanosystem (GNR-Ang2). However, no such distinctions were observed in younger animals. This suggests that our nanosystem holds the potential to effectively detect AD pathology. Discussion: These results support the future development of gold nanoparticle-based technology as a more effective and accessible alternative for the diagnosis of AD and represent a significant advance in the development of gold nanoparticle applications in disease diagnosis.
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Doença de Alzheimer , Nanopartículas Metálicas , Nanotubos , Camundongos , Animais , Ouro/química , Bioacumulação , Distribuição Tecidual , Nanopartículas Metálicas/química , Peptídeos/química , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Tomografia Computadorizada por Raios X , Nanotubos/química , Tomografia , Peptídeos beta-Amiloides/metabolismo , Camundongos Transgênicos , Modelos Animais de Doenças , Encéfalo/metabolismoRESUMO
An immunochemical strategy to detect and quantify AIP-IV, the quorum sensing (QS) signaling molecule produced by Staphylococcus aureusagr type IV, is reported here for the first time. Theoretical calculations and molecular modeling studies have assisted on the design and synthesis of a suitable peptide hapten (AIPIVS), allowing to obtain high avidity and specific antibodies toward this peptide despite its low molecular weight. The ELISA developed achieves an IC50 value of 2.80 ± 0.17 and an LOD of 0.19 ± 0.06 nM in complex media such as 1/2 Tryptic Soy Broth. Recognition of other S. aureus AIPs (I-III) is negligible (cross-reactivity below 0.001%), regardless of the structural similarities. A pilot study with a set of clinical isolates from patients with airways infection or colonization demonstrates the potential of this ELISA to perform biomedical investigations related to the role of QS in pathogenesis and the association between dysfunctional agr or the agr type with unfavorable clinical outcomes. The AIP-IV levels could be quantified in the low nanomolar range in less than 1 h after inoculating agr IV-genotyped isolates in the culture broth, while those genotyped as I-III did not show any immunoreactivity after a 48 h growth, pointing to the possibility to use this technology for phenotyping S. aureus. The research strategy here reported can be extended to the rest of the AIP types of S. aureus, allowing the development of powerful multiplexed chips or point-of-care (PoC) diagnostic devices to unequivocally identify its presence and its agr type on samples from infected patients.
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Infecções Estafilocócicas , Staphylococcus aureus , Proteínas de Bactérias/química , Humanos , Peptídeos/química , Projetos Piloto , Infecções Estafilocócicas/diagnósticoRESUMO
The administration and controlled release of drugs over time remains one of the greatest challenges of science today. In the nanomaterials field, anisotropic gold nanoparticles (AuNPs) with plasmon bands centered at the near-infrared region (NIR), such as gold nanorods (AuNRs) and gold nanoprisms (AuNPrs), under laser irradiation, locally increase the temperature, allowing the release of drugs. In this sense, temporally controlled drug delivery could be promoted by external stimuli using thermo-reversible chemical reactions, such as Diels-Alder cycloadditions from a diene and a dienophile fragment (compound a). In this study, an antitumor drug (methotrexate, MTX) was linked to plasmonic AuNPs by a Diels-Alder adduct (compound c), which after NIR suffers a retro-Diels-Alder reaction, producing release of the drug (compound b). We obtained two nanosystems based on AuNRs and AuNPrs. Both nanoconstructs were coated with BSA-r8 (Bovine Serum Albumin functionalized with Arg8, all-D octa arginine) in order to increase the colloidal stability and promote internalization of the nanosystems on HeLa and SK-BR-3 cells. In addition, the presence of BSA allows protecting the cargo from being released on the extracellular environment and promotes the photothermal release of the drug in the presence of glutathione (GSH). The nanosystems' drug release profile was evaluated after NIR irradiation in the presence and absence of glutathione (GSH), showing a considerable increase of drug release when NIR light and glutathione were combined. This work broadens the range of possibilities of using two complementary strategies for the controlled release of an antitumor drug from AuNRs and AuNPrs: the photothermal cleavage of a thermolabile adduct controlled by an external stimulus (laser irradiation), complemented with the use of the intracellular metabolite GSH.
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Nanopartículas Metálicas , Nanotubos , Glutationa , Ouro , Metotrexato/farmacologiaRESUMO
The development of methyl transverse relaxation optimized spectroscopy has greatly facilitated the study of macromolecular assemblies by solution NMR spectroscopy. However, limited sample solubility and stability has hindered application of this technique to ongoing studies of complexes formed on membranes by the neuronal SNAREs that mediate neurotransmitter release and synaptotagmin-1, the Ca2+ sensor that triggers release. Since the 1H NMR signal of a tBu group attached to a large protein or complex can be observed with high sensitivity if the group retains high mobility, we have explored the use of this strategy to analyze presynaptic complexes involved in neurotransmitter release. For this purpose, we attached tBu groups at single cysteines of fragments of synaptotagmin-1, complexin-1 and the neuronal SNAREs by reaction with 5-(tert-butyldisulfaneyl)-2-nitrobenzoic acid (BDSNB), tBu iodoacetamide or tBu acrylate. The tBu resonances of the tagged proteins were generally sharp and intense, although tBu groups attached with BDSNB had a tendency to exhibit somewhat broader resonances that likely result because of the shorter linkage between the tBu and the tagged cysteine. Incorporation of the tagged proteins into complexes on nanodiscs led to severe broadening of the tBu resonances in some cases. However, sharp tBu resonances could readily be observed for some complexes of more than 200 kDa at low micromolar concentrations. Our results show that tagging of proteins with tBu groups provides a powerful approach to study large biomolecular assemblies of limited stability and/or solubility that may be applicable even at nanomolar concentrations.
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Neurônios , Proteínas SNARE , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear BiomolecularRESUMO
It has been reported that DIC can react with OxymaPure to render an oxadiazole compound with the concomitant formation of HCN. Here we demonstrate that this reaction is not a feature of all carbodiimides but rather depends on the alkyl structure that flanks the two N atoms of the carbodiimide. Furthermore, we have identified two carbodiimides, TBEC and EDC·HCl, whose reaction with OxymaPure is exempt from HCN formation.
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BACKGROUND: The variational Free Energy Principle (FEP) establishes that a neural system minimizes a free energy function of their internal state through environmental sensing entailing beliefs about hidden states in their environment. PROBLEM: Because sensations are drastically reduced during sleep, it is still unclear how a self-organizing neural network can modulate free energy during sleep transitions. GOAL: To address this issue, we study how network's state-dependent changes in energy, entropy and free energy connect with changes at the synaptic level in the absence of sensing during a sleep-like transition. APPROACH: We use simulations of a physically plausible, environmentally isolated neuronal network that self-organize after inducing a thalamic input to show that the reduction of non-variational free energy depends sensitively upon thalamic input at a slow, rhythmic Poisson (delta) frequency due to spike timing dependent plasticity. METHODS: We define a non-variational free energy in terms of the relative difference between the energy and entropy of the network from the initial distribution (prior to activity dependent plasticity) to the nonequilibrium steady-state distribution (after plasticity). We repeated the analysis under different levels of thalamic drive - as defined by the number of cortical neurons in receipt of thalamic input. RESULTS: Entraining slow activity with thalamic input induces a transition from a gamma (awake-like state) to a delta (sleep-like state) mode of activity, which can be characterized through a modulation of network's energy and entropy (non-variational free energy) of the ensuing dynamics. The self-organizing response to low and high thalamic drive also showed characteristic differences in the spectrum of frequency content due to spike timing dependent plasticity. CONCLUSIONS: The modulation of this non-variational free energy in a network that self-organizes, seems to be an organizational network principle. This could open a window to new empirically testable hypotheses about state changes in a neural network.
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Entropia , Heurística/fisiologia , Redes Neurais de Computação , Sono/fisiologia , Ritmo Delta/fisiologia , Humanos , Neurônios/fisiologia , Tálamo/fisiologia , Vigília/fisiologiaRESUMO
The development and use of nanosystems is an emerging strategy for the diagnosis and treatment of a broad number of diseases, such as Alzheimer's disease (AD). Here, we developed a neurotheranostic nanosystem based on gold nanorods (GNRs) that works as a therapeutic peptide delivery system and can be detected in vivo for microcomputed tomography (micro-CT), being a diagnostic tool. GNRs functionalized with the peptides Ang2 (a shuttle to the Central Nervous System) and D1 (that binds to the Aß peptide, also inhibiting its aggregation) allowed detecting differences in vivo between wild type and AD mice (APPswe/PSEN1dE9) 15 minutes after a single dose by micro-CT. Moreover, after a recurrent treatment for one month with GNRs-D1/Ang2, we observed a diminution of amyloid load and inflammatory markers in the brain. Thus, this new designed nanosystem exhibits promising properties for neurotheranostics of AD.
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Doença de Alzheimer , Nanotubos , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Ouro , Camundongos , Camundongos Transgênicos , Microtomografia por Raio-XRESUMO
Accurate identification of aquatic organisms and their numerical abundance calculation using echo detection techniques remains a great challenge for marine researchers. A software architecture for echo data processing is presented in this article. Within it, it is discussed how to obtain energetic, morphometric and bathymetric fish school descriptors to accurately identify different fish-species. To accomplish this task it was necessary to have a development platform that allowed reading echo data from a particular echosounder, to detect fish aggregations and then to calculate fish school descriptors that would be used for fish-species identification, in an automatic way. This article also describes thoroughly the digital processing algorithms for this automatic detection and classification, as well as the automatic process required for surface and bottom line detection, which is necessary to determine the exploration range. These algorithms are implemented within the ECOPAMPA software, which is the first Argentinean system for marine species identification. Finally, a comparative result over experimental data of ECOPAMPA against Echoview TM Software Pty Ltd (formerly Myriax Software Pty Ltd), is carefully examined.
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The elapid genus, Micruroides, is considered the sister clade of all New World coral snakes (Genus Micrurus), is monotypic, and is represented by Sonoran Coral Snakes, Micruroides euryxanthus. Coral snakes of the genus Micrurus have been reported to have venoms that are predominantly composed of phospholipases A2 (PLA2) or three finger toxins (3FTx), but the venoms of the genus Micruroides are almost completely unstudied. Here, we present the first description of the venom of M. euryxanthus including identification of some proteins as well as transcriptomic, and biological activity assays. The most abundant components within M. euryxanthus venom are 3FTxs (62.3%) and there was relatively low proportion of PLA2s (14.2%). The venom phenotype supports the hypothesis that the common ancestor of Micrurus and Micruroides had a 3FTx-dominated venom. Within the venom, there were two nearly identical α-neurotoxins (α-Ntx), one of which was designated Eurytoxin, that account for approximately 60% of the venom's lethality to mice. Eurytoxin was cloned, expressed in a soluble and active form, and used to produce rabbit hyperimmune serum. This allowed the analysis of its immunochemical properties, showing them to be different from the recombinant αNTx D.H., present in the venoms of some species of Micrurus. Finally, we observed that the commercial antivenom produced in Mexico for coral snake envenomation is unable to neutralize the lethality from M. euryxanthus venom. This work allowed the classification of Micruroides venom into the 3FTx-predominant group and identified the main components responsible for toxicity to mice.
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Cobras Corais , Venenos Elapídicos , Fosfolipases A2 , Proteínas de Répteis , Animais , Cobras Corais/genética , Cobras Corais/metabolismo , Venenos Elapídicos/biossíntese , Venenos Elapídicos/genética , Fosfolipases A2/biossíntese , Fosfolipases A2/genética , Proteínas de Répteis/biossíntese , Proteínas de Répteis/genética , Especificidade da EspécieRESUMO
Brain natriuretic peptides (BNPs) are well-established cardiovascular disease (CVD) biomarkers that are released from the heart after ventricular wall stress. Particularly, the N-terminal proBNP (NT-proBNP) is a 76 aa long peptide and is recognized as an indicator for the diagnosis of heart failure (HF) and is being used in routine tests in emergency rooms when levels are above 0.4 ng mL-1. Herein, we report a new competitive ELISA for NT-proBNP, which is able to detect this biomarker directly in undiluted human plasma samples. The ELISA has been the result of a rational design of an immunizing peptide hapten and the investigation of different immunochemical conditions, including heterologous competitors and distinct physico-chemical conditions. The developed ELISA is able to detect NT-proBNP with a LOD of 0.40 ± 0.15 ng mL-1 in human plasma samples and quantify this biomarker in the range between 0.97 ± 0.38 and 23.10 ± 9.46 ng mL-1 with good accuracy. The ELISA can simultaneously measure many samples in 1.5 h and has been found to be robust, reproducible and shows great promise in diagnosis of heart failures.
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Insuficiência Cardíaca , Peptídeo Natriurético Encefálico , Biomarcadores , Ensaio de Imunoadsorção Enzimática , Insuficiência Cardíaca/diagnóstico , Humanos , Fragmentos de PeptídeosRESUMO
Scorpion and spider envenomation is treated with the appropriate antivenoms, prepared as described by Césaire Auguste Phisalix and Albert Calmette in 1894. Such treatment requires the acquisition and manipulation of arachnid venoms, both very complicated procedures. Most of the toxins in the venoms of spiders and scorpions are extremely stable cysteine-rich peptide neurotoxins. Many strategies have been developed to obtain synthetic immunogens to facilitate the production of antivenoms against these toxins. For example, whole peptide toxins can be synthesized by solid-phase peptide synthesis (SPPS). Also, epitopes of the toxins can be identified and after the chemical synthesis of these peptide epitopes by SPPS, they can be coupled to protein carriers to develop efficient immunogens. Moreover, multiple antigenic peptides with a polylysine core can be designed and synthesized. This review focuses on the strategies developed to obtain synthetic immunogens for the production of antivenoms against the toxic Cys-rich peptides of scorpions and spiders.
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The venom of Crotalus durissus terrificus (Cdt) is a source of a wide variety of toxins, some of them with interesting pharmacological applications. Of these toxins, the phospholipase A2 (PLA2) subunit of crotoxin (Ctx) has been studied for its potential as an antiviral and antibacterial agent. Peptides have proven useful ligands for the purification of numerous molecules, including antibodies, toxins, enzymes and other proteins. Here, we sought to use a phosphopeptide (P-Lys) as a ligand for PLA2 purification. P-Lys was synthesized in solid phase on Rink-Amide-ChemMatrix resin, immobilized on NHS-agarose, and then evaluated as a chromatographic matrix. Under the best conditions, total protein adsorption reached 39% and only the eluate fraction presented PLA2 activity. Analysis of the eluate by SDS-PAGE showed three bands, one corresponding to the molecular weight of PLA2 (14 kDa). Said bands were analyzed by mass spectrometry and identified as PLA2 and its multimers. The final product showed a purity of over 90%. In addition, slightly changing the process conditions also allowed the isolation of crotamine.
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Cromatografia de Afinidade/métodos , Venenos de Crotalídeos/análise , Fosfolipases A2/análise , Fosfopeptídeos/química , Amidas/química , Animais , Crotalus , Crotoxina/química , Ligantes , Espectrometria de Massas , Sefarose/química , Técnicas de Síntese em Fase Sólida , Succinimidas/químicaRESUMO
Cyclic depsipeptides constitute a fascinating class of natural products. Most of them are characterized by an ester formed between the ß-hydroxy function of Ser/Thr -and related amino acids- and the carboxylic group of the C-terminal amino acid. Less frequent are those where the thiol of Cys is involved rendering a thioester (cyclo thiodepsipeptides) and even less common are the cyclo depsipeptides with a phenyl ester coming from the side-chain of Tyr. Herein, the preparation of the later through a cyclative cleavage using the Fmoc-MeDbz/MeNbz-resin is described. This resin has previously reported for the synthesis of cyclo thiodepsipeptides and homodetic peptides. The use of that resin for the preparation of all these peptides is also summarized.
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The early detection of the amyloid beta peptide aggregates involved in Alzheimer's disease is crucial to test new potential treatments. In this research, we improved the detection of amyloid beta peptide aggregates in vitro and ex vivo by fluorescence combining the use of CRANAD-2 and gold nanorods (GNRs) by the surface enhancement fluorescence effect. We synthetized GNRs and modified their surface with HS-PEG-OMe and HS-PEG-COOH and functionalized them with the D1 peptide, which has the capability to selectively bind to amyloid beta peptide. For an in vitro detection of amyloid beta peptide, we co-incubated amyloid beta peptide aggregates with the probe CRANAD-2 and GNR-PEG-D1 observing an increase in the intensity of the fluorescence signal attributed to surface enhancement fluorescence. Furthermore, the surface enhancement fluorescence effect was observed in brain slices of transgenic mice with Alzheimer´s disease co-incubated with CRANAD-2 and GNR-PEG-D1. An increase in the fluorescence signal was observed allowing the detection of aggregates that cannot be detected with the single use of CRANAD-2. Gold nanoparticles allowed an improvement in the detection of the amyloid aggregated by fluorescence in vitro and ex vivo.
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Intelligent control systems are being developed for the control of plants with complex dynamics. However, the simplicity of the PID (proportional-integrative-derivative) controller makes it still widely used in industrial applications and robotics. This paper proposes an intelligent control system based on a deep reinforcement learning approach for self-adaptive multiple PID controllers for mobile robots. The proposed hybrid control strategy uses an actor-critic structure and it only receives low-level dynamic information as input and simultaneously estimates the multiple parameters or gains of the PID controllers. The proposed approach was tested in several simulated environments and in a real time robotic platform showing the feasibility of the approach for the low-level control of mobile robots. From the simulation and experimental results, our proposed approach demonstrated that it can be of aid by providing with behavior that can compensate or even adapt to changes in the uncertain environments providing a model free unsupervised solution. Also, a comparative study against other adaptive methods for multiple PIDs tuning is presented, showing a successful performance of the approach.
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Cell membrane receptors bind to extracellular ligands, triggering intracellular signal transduction pathways that result in specific cell function. Some receptors require to be associated forming clusters for effective signaling. Increasing evidences suggest that receptor clustering is subjected to spatially controlled ligand distribution at the nanoscale. Herein we present a method to produce in an easy, straightforward process, nanopatterns of biomolecular ligands to study ligandâ»receptor processes involving multivalent interactions. We based our platform in self-assembled diblock copolymers composed of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) that form PMMA nanodomains in a closed-packed hexagonal arrangement. Upon PMMA selective functionalization, biomolecular nanopatterns over large areas are produced. Nanopattern size and spacing can be controlled by the composition of the block-copolymer selected. Nanopatterns of cell adhesive peptides of different size and spacing were produced, and their impact in integrin receptor clustering and the formation of cell focal adhesions was studied. Cells on ligand nanopatterns showed an increased number of focal contacts, which were, in turn, more matured than those found in cells cultured on randomly presenting ligands. These findings suggest that our methodology is a suitable, versatile tool to study and control receptor clustering signaling and downstream cell behavior through a surface-based ligand patterning technique.