Sequence length dependence in arginine/phenylalanine oligopeptides: Implications for self-assembly and cytotoxicity

We present a detailed study on the self-assembly and cytotoxicity of arginine-rich fragments with general form [RF]n (n = 1–5). These highly simplified sequences, containing only two l-amino acids, provide suitable models for exploring both structure and cytotoxicity features of arginine-based oligopeptides. The organization of the sequences is revealed over a range of length scales, from the nanometer range down to the level of molecular packing, and their cytotoxicity toward C6 rat glioma and RAW264.7 macrophage cell lines is investigated. We found that the polymorphism is dependent on peptide length, with a progressive increase in crystalline ordering upon increasing the number of [RF] pairs along the backbone. A dependence on length was also found for other observables, including critical aggregation concentrations, formation of chiral assemblies and half maximum inhibitory concentrations (IC50). Whereas shorter peptides self-assemble into fractal-like aggregates, clear fibrillogenic capabilities are identified for longer sequences with octameric and decameric chains exhibiting crystalline phases organized into cross-β structures. Cell viability assays revealed dose-dependent cytotoxicity profiles with very similar behavior for both glioma and macrophage cell lines, which has been interpreted as evidence for a nonspecific mechanism involved in toxicity. We propose that structural organization of [RF]n peptides plays a paramount role regarding toxicity due to strong increase of local charge density induced by self-assemblies rich in cationic groups when interacting with cell membranes

Synthesis optimization and development of polymeric nanoparticulated and liposomal systems for central nervous systems cell-derived applications.

O presente trabalho tem como intuito avaliar e otimizar o processo de confecção e as propriedades de nanopartículas poliméricas (NPs) e de lipossomos (LPs) para o transporte de substâncias de interesse visando o tratamento e prevenção de doenças que atingem o sistema nervoso central. As NPs de PLGA com polissorbato-80 (T-80) adsorvido foram otimizadas quanto ao tipo de tensoativo, regime de sonicação, volume de solução de T-80 e tipo de agitador sobre o tamanho, polidispersão e potencial zeta das NPs; ao passo que LPs funcionalizados com anticorpo anti-transferrina também tiveram sua confecção otimizada e suas propriedades foram avaliadas para diferentes misturas de lipídeos contendo DPPC ou DOPC. Após a otimização dos nanocarreadores, ensaios celulares em células Neuro-2a e SH-SY5Y foram realizados de modo comparar a citotoxicidade e a cinética de internalização de tais sistemas. Demonstra-se que o processo de confecção dos carreadores para aplicação no sistema nervoso central requer investigação pormenorizada para melhores resultados na aplicação biológica.This study aims at analyzing and optimizing the obtainment process and properties of polymeric nanoparticles (NPs) and liposomes (LPs) for the transport of substance of interest with the purpose of treatment and prevention of various central nervous system diseases. PLGA NPs coated with polissorbate-80 (T-80) were optimized regarding the type of surfactant, sonication regime, T-80 solution dispersion volume and size of stirrer on the size, polidispersity and zeta potencial of NPs; whilst anti-transferrin antibody functionalized LPs also had their synthesis optimized and their properties were also compared for different lipid mixtures containing DPPC or DOPC. After optimization, the nanocarriers were submitted to in vitro assays with Neuro-2a and SH-SY5Y cells in order to compare their cytotoxicity and internalization kinetics. It is demonstrated that the process to obtain nanocarriers for central nervous system applications require detailed analysis when seeking the most favorable biological results

Synthesis optimization and development of polymeric nanoparticulated and liposomal systems for central nervous systems cell-derived applications.

O presente trabalho tem como intuito avaliar e otimizar o processo de confecção e as propriedades de nanopartículas poliméricas (NPs) e de lipossomos (LPs) para o transporte de substâncias de interesse visando o tratamento e prevenção de doenças que atingem o sistema nervoso central. As NPs de PLGA com polissorbato-80 (T-80) adsorvido foram otimizadas quanto ao tipo de tensoativo, regime de sonicação, volume de solução de T-80 e tipo de agitador sobre o tamanho, polidispersão e potencial zeta das NPs; ao passo que LPs funcionalizados com anticorpo anti-transferrina também tiveram sua confecção otimizada e suas propriedades foram avaliadas para diferentes misturas de lipídeos contendo DPPC ou DOPC. Após a otimização dos nanocarreadores, ensaios celulares em células Neuro-2a e SH-SY5Y foram realizados de modo comparar a citotoxicidade e a cinética de internalização de tais sistemas. Demonstra-se que o processo de confecção dos carreadores para aplicação no sistema nervoso central requer investigação pormenorizada para melhores resultados na aplicação biológica.This study aims at analyzing and optimizing the obtainment process and properties of polymeric nanoparticles (NPs) and liposomes (LPs) for the transport of substance of interest with the purpose of treatment and prevention of various central nervous system diseases. PLGA NPs coated with polissorbate-80 (T-80) were optimized regarding the type of surfactant, sonication regime, T-80 solution dispersion volume and size of stirrer on the size, polidispersity and zeta potencial of NPs; whilst anti-transferrin antibody functionalized LPs also had their synthesis optimized and their properties were also compared for different lipid mixtures containing DPPC or DOPC. After optimization, the nanocarriers were submitted to in vitro assays with Neuro-2a and SH-SY5Y cells in order to compare their cytotoxicity and internalization kinetics. It is demonstrated that the process to obtain nanocarriers for central nervous system applications require detailed analysis when seeking the most favorable biological results

Glipicam-1 no glioblastoma humano: implicações na tumorigênese e na quimioterapia

Introduction: Glioblastoma is one of the most common malignant brain tumors, in which patients have a mean survival of 24 months. The scientific literature has revealed evidence that glypican-1 is overexpressed in human glioblastoma and is negatively correlated with patient’s survival. Glypican-1 is a membrane-bound heparan sulfate proteoglycan, and the heparan sulfate GAG chains are classically known to interact with morphogens frequently associated with cancer. Objective: This study aims to investigate how glypican-1 influences the tumoral profile of human glioblastoma using in vitro cell line models. Methods: Glypican-1 was knocked-down from U-251 MG cells using shRNA followed by clonal selection. The glypican-1 silencing effects were assessed by profiling transcriptional discrepancies using RT-qPCR as well as other techniques such as flow cytometry, cell viability, migration, proliferation, adhesion, clonogenicity, and susceptibility to temozolomide. Confocal microscopy was used to investigate glypican1 localization in lipid rafts or its association with syndecan-4 and glypican-3. Results: Glypican-1 was associated with the expression of other heparan sulfate proteoglycans and metalloproteases. By downregulating glypican-1, the cellular growth rate was reduced by up to 71% and proliferation up to 54%, in which cells were significantly shifted towards G0 as opposed to G1 phases. Cellular migration was severely affected and may be almost abolished by negatively modulating glypican-1. Glioblastoma U-251 MG cells poorly adhered to laminin but showed high affinity to type IV collagen and vitronectin. However, glypican-1 solely affected the affinity towards laminin binding of glioblastoma U-251 MG cells, although slower kinetic adhesion profiles were observed when the proteoglycan was knocked down. This proteoglycan was highly prevalent in glioblastoma cells, being primarily localized in the cellular membrane and extracellular vesicles, occasionally with glypican-3. Glypican-1 could also be found in cell-cell junctions with syndecan-4 but was not identified in lipid rafts in this study. Glypican-1 was also revealed to be a probable mediator of temozolomide resistance mechanisms in glioblastoma, as glypican-1-silenced cells were much more susceptible to this alkylating agent than in U-251 MG itself. Conclusion: We present evidence not only to support facts that glypican-1 is an elementary macromolecule in glioblastoma tumoral microenvironment, but also to introduce this proteoglycan as a promising therapeutic target for this highly malignant tumor.Introdução: O glioblastoma é um dos tumores cerebrais malignos mais comuns, em que os pacientes possuem uma sobrevida média de 24 meses. A literatura científica revelou evidências de que o glipicam-1 é superexpresso no glioblastoma humano e está negativamente correlacionado com a sobrevivência desses pacientes. O glipicam-1 é um proteoglicano de heparam sulfato de membrana, cujas cadeias de heparam sulfato são classicamente conhecidas por se associar a morfógenos, frequentemente associados ao câncer. Objetivo: Este estudo tem como objetivo investigar como o glipicam-1 influencia o perfil tumoral no glioblastoma humano por meio de modelos com linhagens celulares in vitro. Métodos: O glipicam-1 foi silenciado das células U-251 MG utilizando um shRNA específico, seguido de uma seleção clonal. Os efeitos do silenciamento foram avaliados pela análise de discrepâncias transcricionais avaliadas por meio de RTqPCR, bem como por outras técnicas, como citometria de fluxo, atividade metabólica, migração, proliferação, adesão, e clonogenicidade celulares; e ensaio de susceptibilidade à temozolomida. A microscopia confocal foi empregada para investigar a localização de glipicam-1 em balsas lipídicas ou sua associação com sindecam-4 e glipicam-3. Resultados: O glipicam-1 mostrou estar associado à expressão de outros proteoglicanos de heparam sulfato e determinadas metaloproteases. Pelo silenciamento de glipicam-1, a taxa de crescimento celular foi reduzida em até 71% e a proliferação em 54%, sendo que os clones celulares indicaram estar preferencialmente na fase G0 do ciclo celular em vez de G1. A migração celular foi severamente afetada pela depleção do proteoglicano. Células U-251 MG aderiram mal à laminina, mas tiveram alta afinidade ao colágeno do tipo IV e à vitronectina. No entanto, o glipican-1 apenas influenciou diretamente a afinidade de ligação das células U-251 MG à laminina, embora uma cinética de adesão mais lenta foi observada mediante o knock-down. Glipicam-1 revelou ser altamente prevalente nas células de glioblastoma, primariamente residindo na membrana celular e em vesículas extracelulares, ocasionalmente associado ao glipicam-3. O glipicam-1 também pôde ser identificado em junções célula-célula juntamente com sindecam-4, mas não foi identificado em balsas lipídicas neste estudo. Este proteoglicano também revelou indícios de ser um potencial mediador dos mecanismos de resistência à temozolomida no glioblastoma, uma vez que células silenciadas para glipicam-1 foram mais susceptíveis a este agente alquilante em relação à linhagem controle U-251 MG. Conclusão: Neste trabalho, apresentamos dados que evidenciam a importância do glipicam-1 como uma macromolécula fundamental no microambiente tumoral do glioblastoma humano, e que também representa um alvo terapêutico promissor para este tumor altamente maligno.Dados abertos - Sucupira - Teses e dissertações (2019

Development and validation of the dystonia-pain classification System : a multicenter study

Abstract: Background: Dystonia is associated with disabling nonmotor symptoms like chronic pain (CP), which is prevalent in dystonia and significantly impacts the quality of life (QoL). There is no validated tool for assessing CP in dystonia, which substantially hampers pain management. Objective: The aim was to develop a CP classification and scoring system for dystonia. Methods: A multidisciplinary group was established to develop the Dystonia-Pain Classification System (Dystonia- PCS). The classification of CP as related or unrelated to dystonia was followed by the assessment of pain severity score, encompassing pain intensity, frequency, and impact on daily living. Then, consecutive patients with inherited/idiopathic dystonia of different spatial distribution were recruited in a cross-sectional multicenter validation study. Dystonia-PCS was compared to validated pain, mood, QoL, and dystonia scales (Brief Pain Inventory, Douleur Neuropathique-4 questionnaire, European QoL-5 Dimensions-3 Level Version, and Burke–Fahn–Marsden Dystonia Rating Scale). Results: CP was present in 81 of 123 recruited patients, being directly related to dystonia in 82.7%, aggravated by dystonia in 8.8%, and nonrelated to dystonia in 7.5%. affected by this disorder. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. Dystonia-PCS had excellent intra-rater (Intraclass Correlation Coefficient - ICC: 0.941) and inter-rater (ICC: 0.867) reliability. In addition, pain severity score correlated with European QoL-5 Dimensions-3 Level Version’s pain subscore (r = 0.635, P < 0.001) and the Brief Pain Inventory’s severity and interference scores (r = 0.553, P < 0.001 and r = 0.609, P < 0.001, respectively). Conclusions: Dystonia-PCS is a reliable tool to categorize and quantify CP impact in dystonia and will help improve clinical trial design and management of CP in patients affected by this disorder