RESUMO
The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an 'experiment of nature' to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent 'domino effect' that impacts stringency of tolerance and B cell fate in the periphery.
Assuntos
Linfócitos B , Proteínas de Ligação a DNA , Proteínas de Homeodomínio , Proteínas Nucleares , Diferenciação Celular , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Humanos , Tolerância Imunológica , Contagem de Linfócitos , Proteínas Nucleares/deficiênciaRESUMO
This study explores the hysteresis phenomenon in DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) monolayers, considering several variables, including temperature, compression and expansion rates, residence time, and subphase content. The investigation focuses on analyzing the influence of these variables on key indicators such as the π-A isotherm curve, loop area, and compression modulus. By employing the Langmuir-Blodgett technique, the findings reveal that all the examined factors significantly affect the aforementioned parameters. Notably, the hysteresis loop, representing dissipated energy, provides valuable insights into the monolayer's viscoelasticity, molecular packing, phase transition changes, and resistance during the isocycle process. These findings contribute to a comprehensive understanding of the structural and dynamic properties of DPPC monolayers, offering insights into their behavior under varying conditions. Moreover, the knowledge gained from this study can aid in the development of precise models and strategies for controlling and manipulating monolayer properties, with potential applications in drug delivery systems, surface coatings, as well as further investigation into air penetration into alveoli and the blinking mechanism.
Assuntos
1,2-Dipalmitoilfosfatidilcolina , Glicerilfosforilcolina , Propriedades de Superfície , 1,2-Dipalmitoilfosfatidilcolina/química , TemperaturaRESUMO
In healthy tissues, cells are in mechanical homeostasis. During cancer progression, this equilibrium is disrupted. Cancer cells alter their mechanical phenotype to a softer and more fluid-like one than that of healthy cells. This is connected to cytoskeletal remodeling, changed adhesion properties, faster cell proliferation and increased cell motility. In this work, we investigated the mechanical properties of breast cancer cells representative of different breast cancer subtypes, using MCF-7, tamoxifen-resistant MCF-7, MCF10A and MDA-MB-231 cells. We derived viscoelastic properties from atomic force microscopy force spectroscopy measurements and showed that the mechanical properties of the cells are associated with cancer cell malignancy. MCF10A are the stiffest and least fluid-like cells, while tamoxifen-resistant MCF-7 cells are the softest ones. MCF-7 and MDA-MB-231 show an intermediate mechanical phenotype. Confocal fluorescence microscopy on cytoskeletal elements shows differences in actin network organization, as well as changes in focal adhesion localization. These findings provide further evidence of distinct changes in the mechanical properties of cancer cells compared to healthy cells and add to the present understanding of the complex alterations involved in tumorigenesis.
Assuntos
Neoplasias da Mama , Citoesqueleto , Humanos , Feminino , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Células MCF-7 , Actinas/metabolismo , Tamoxifeno/farmacologia , Tamoxifeno/metabolismo , Neoplasias da Mama/metabolismo , Microscopia de Força Atômica/métodosRESUMO
Stress-associated changes in the mechanical properties at the single-cell level of Escherichia coli (E. coli) cultures in bioreactors are still poorly investigated. In our study, we compared peptide-producing and non-producing BL21(DE3) cells in a fed-batch cultivation with tightly controlled process parameters. The cell growth, peptide content, and cell lysis were analysed, and changes in the mechanical properties were investigated using atomic force microscopy. Recombinant-tagged somatostatin-28 was expressed as soluble up to 197 ± 11 mg g-1. The length of both cultivated strains increased throughout the cultivation by up to 17.6%, with nearly constant diameters. The peptide-producing cells were significantly softer than the non-producers throughout the cultivation, and respective Young's moduli decreased by up to 57% over time. A minimum Young's modulus of 1.6 MPa was observed after 23 h of the fed-batch. Furthermore, an analysis of the viscoelastic properties revealed that peptide-producing BL21(DE3) appeared more fluid-like and softer than the non-producing reference. For the first time, we provide evidence that the physical properties (i.e., the mechanical properties) on the single-cell level are significantly influenced by the metabolic burden imposed by the recombinant peptide expression and C-limitation in bioreactors.
Assuntos
Reatores Biológicos , Escherichia coli , Proteínas Recombinantes/metabolismo , Ciclo CelularRESUMO
Bacterial cells survive in a wide range of different environments and actively tune their mechanical properties for purposes of growth, movement, division, and nutrition. In Gram-negative bacteria, the cell envelope with its outer membrane and peptidoglycan are the main determinants of mechanical properties and are common targets for the use of antibiotics. The study of bacterial mechanical properties has shown promise in elucidating a structure-function relationship in bacteria, connecting, shape, mechanics, and biochemistry. In this work, we study frequency and time-dependent viscoelastic properties of E. coli cells by atomic force microscopy (AFM). We perform force cycles, oscillatory microrheology, stress relaxation, and creep experiments, and use power law rheology models to fit the experimental results. All data sets could be fitted with the models and provided power law exponents of 0.01 to 0.1 while showing moduli in the range of a few MPa. We provide evidence for the interchangeability of the properties derived from these four different measurement approaches.
Assuntos
Antibacterianos , Escherichia coli , Elasticidade , Microscopia de Força Atômica/métodos , Membrana Celular , ViscosidadeRESUMO
This Special Issue is focused on measuring and characterizing the mechanical and adhesive properties of cells and membranes [...].
Assuntos
Fenômenos Biomecânicos , Adesão Celular , Membrana Celular/metabolismo , Células , Células/citologia , Células/metabolismoRESUMO
Bacillus thuringiensis is a bacterium that produces many insecticidal proteins including cytolytic proteins or Cyt toxins. Although the Cyt toxin shows specific toxicity against Dipteran insect species, the toxin binds directly to the lipid membrane without a specific protein receptor requirement. In this work, we have investigated the interaction of Cyt2Aa2 toxin with lipid bilayers composed of different lipid phases. By means of atomic force microscopy (AFM), lipid phase separation was observed for 1:1 and 4:1â¯M mixtures of DPPC/POPC bilayers. The exposure of Cyt2Aa2 to these lipid bilayers revealed that the toxin selectively bound to Ld lipid bilayer (corresponding to POPC). In turn, it did not bind to the Lo and So phases (corresponding to DPPC). Interestingly, for the bilayer of 4:1 DPPC/POPC mixture, the binding of Cyt2Aa2 was localized at the lipid phase boundary instead of Ld domain as occurred for the 1:1 DPPC/POPC bilayer. In addition, quartz crystal microbalance with dissipation experiments confirmed AFM results. In particular, the measurements showed that amount of protein bound to 1:1 DPPC/POPC (with phase separation) was half of the binding quantified for the Ld phase lipid bilayer (pure POPC and 1:4 DPPC/POPC mixture). These results indicate that the lipid phase (lipid acyl chain) influences the Cyt2Aa2-lipid interaction.
Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Bicamadas Lipídicas/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/análogos & derivados , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Toxinas de Bacillus thuringiensis , Sítios de Ligação , Bicamadas Lipídicas/química , Transição de Fase , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Ligação ProteicaRESUMO
Cytolytic protein (Cyt) is a member of insecticidal proteins produced by Bacillus thuringiensis. Cyt protein has activity against insect cells and mammalian cells, which differ in lipid and cholesterol composition. This study presents the lipid binding behavior of Cyt2Aa2 protein on model membranes containing different levels of cholesterol content by combining Quartz Crystal Microbalance with Dissipation (QCM-D) and Atomic Force Microscopy (AFM). QCM-D results revealed that cholesterol enhances the binding rate of Cyt2Aa2 protein onto lipid bilayers. In addition, the thicker lipid bilayer was observed for the highest cholesterol content. These results were confirmed by AFM. The analysis of protein surface coverage as a function of time showed a slower process for 5:0 and 5:0.2 (POPC:Chol) ratios than for 5:1 and 5:2 (POPC:Chol) ratios. Significantly, the Cyt2Aa2-lipid binding behavior and the proteinâ»lipid layer were different for the 5:3 (POPC:Chol) ratio. Furthermore, AFM images revealed a transformation of Cyt2Aa2/lipid layer structure from strip pattern to ring shape structures (which showed a strong repulsion with AFM tip). In summary, cholesterol increases the binding rate and alters the lipid binding behavior of Cyt2Aa2 protein, although it is not required for Cyt2Aa2 protein binding onto lipid bilayers.
Assuntos
Bacillus thuringiensis/química , Proteínas de Bactérias/metabolismo , Colesterol/metabolismo , Proteínas Hemolisinas/metabolismo , Microscopia de Força Atômica , Ligação ProteicaRESUMO
Bacillus thuringiensis produces cytolytic proteins (Cyt) that show toxicity against dipteran insect larvae acting directly on the cell membrane. Up to now, two different models have been proposed to explain the interaction mechanism of the cytolytic protein Cyt2Aa2 on lipid membranes: pore-forming and detergent-like action. Here we report on the interaction of Cyt2Aa2 with lipid/cholesterol bilayers at early stage (far from equilibrium) as a function of protein concentration. Quartz crystal microbalance with dissipation (QCM-D) measurements showed that the rate of protein adsorption increased with concentration, although the mass of the final protein-lipid was similar after two hours. In addition, the dissipation (compliance of the hybrid lipid/protein layer) increased with decreasing protein concentration. Furthermore, atomic force microscopy (AFM) revealed that the structure of the protein-lipid layer was concentration and time dependent. A rigid hybrid homogeneous layer was observed at protein concentrations of 50 µg/ml and 100 µg/ml after 30 min. At lower concentrations, 10 µg/ml and 17.5 µg/ml, protein adsorption on the lipid layer led to the formation of small aggregates. Interestingly, at 25 µg/ml a transition of a hole-like structure into a homogeneous layer was observed. This suggests that 25 µg/ml is a threshold concentration for the binding mechanism of Cyt2Aa2 on to lipid membranes.
Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Colesterol/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Bicamadas Lipídicas/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/análise , Endotoxinas/análise , Proteínas Hemolisinas/análise , Microscopia de Força Atômica , Agregados Proteicos , Ligação Proteica , Técnicas de Microbalança de Cristal de QuartzoRESUMO
As infectious disease surveillance systems expand to include digital, crowd-sourced, and social network data, public health agencies are gaining unprecedented access to high-resolution data and have an opportunity to selectively monitor informative individuals. Contact networks, which are the webs of interaction through which diseases spread, determine whether and when individuals become infected, and thus who might serve as early and accurate surveillance sensors. Here, we evaluate three strategies for selecting sensors-sampling the most connected, random, and friends of random individuals-in three complex social networks-a simple scale-free network, an empirical Venezuelan college student network, and an empirical Montreal wireless hotspot usage network. Across five different surveillance goals-early and accurate detection of epidemic emergence and peak, and general situational awareness-we find that the optimal choice of sensors depends on the public health goal, the underlying network and the reproduction number of the disease (R0). For diseases with a low R0, the most connected individuals provide the earliest and most accurate information about both the onset and peak of an outbreak. However, identifying network hubs is often impractical, and they can be misleading if monitored for general situational awareness, if the underlying network has significant community structure, or if R0 is high or unknown. Taking a theoretical approach, we also derive the optimal surveillance system for early outbreak detection but find that real-world identification of such sensors would be nearly impossible. By contrast, the friends-of-random strategy offers a more practical and robust alternative. It can be readily implemented without prior knowledge of the network, and by identifying sensors with higher than average, but not the highest, epidemiological risk, it provides reasonably early and accurate information.
Assuntos
Surtos de Doenças , Vigilância em Saúde Pública , Saúde Pública/métodos , Apoio Social , Biologia Computacional , Surtos de Doenças/prevenção & controle , Surtos de Doenças/estatística & dados numéricos , Humanos , Modelos Biológicos , Modelos EstatísticosRESUMO
Bacillus thuringiensis is known by its insecticidal property. The insecticidal proteins are produced at different growth stages, including the cytolytic protein (Cyt2Aa2), which is a bioinsecticide and an antimicrobial protein. However, the binding mechanism (and the interaction) of Cyt2Aa2 on lipid bilayers is still unclear. In this work, we have used quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM) to investigate the interaction between Cyt2Aa2 protein and (cholesterol-)lipid bilayers. We have found that the binding mechanism is concentration dependent. While at 10 µg/mL, Cyt2Aa2 binds slowly on the lipid bilayer forming a compliance protein/lipid layer with aggregates, at higher protein concentrations (100 µg/mL), the binding is fast, and the protein/lipid layer is more rigid including holes (of about a lipid bilayer thickness) in its structure. Our study suggests that the protein/lipid bilayer binding mechanism seems to be carpet-like at low protein concentrations and pore forming-like at high protein concentrations.
Assuntos
Proteínas de Bactérias/química , Bicamadas Lipídicas/química , Técnicas de Microbalança de Cristal de Quartzo , Bacillus thuringiensis/química , Sítios de Ligação , Microscopia de Força Atômica , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Poly(N,N-dimethylacrylamide) (PDMA) brushes with different grafting density and chain length were grown from an ester group-containing initiator using surface-initiated polymerization. Hydrolysis of the PDMA chains from the surface was monitored by measuring thickness of the polymer layer by ellipsometry and extension length by atomic force microscopy. It was found that the initial rate of cleavage of one end-tethered PDMA chains was dependent on the grafting density and chain length; the hydrolysis rate was faster for high grafting density brushes and brushes with higher molecular weights. Additionally, the rate of cleavage of polymer chains during a given experiment changed by up to 1 order of magnitude as the reaction progressed, with a distinct transition to a lower rate as the grafting density decreased. Also, polymer chains undergo selective cleavage, with longer chains in a polydisperse brush being preferentially cleaved at one stage of the hydrolysis reaction. We suggest that the enhanced initial hydrolysis rates seen at high grafting densities and high chain lengths are due to mechanical activation of the ester bond connecting the polymer chains to the surface in association with high lateral pressure within the brush. These results have implications for the preparation of polymers brushes, their stability under harsh conditions, and the analysis of polymer brushes from partial hydrolysates.
Assuntos
Acrilamidas/química , Ésteres , Hidrólise , Microscopia de Força Atômica , Propriedades de SuperfícieRESUMO
Cell disintegration and protein extraction are crucial steps in downstream process development for biopharmaceuticals produced in E. coli. In this study, we explored the extraction mechanism of polyethyleneimine (PEI) at the cellular level and characterized the floc network that is formed upon PEI addition by Focused Beam Reflectance Measurement and Dispersion Analyzer. PEI disintegrates the cells by detachment of the outer membrane allowing protein to diffuse into the interspace of the flocs. Protein release into the supernatant occurs by diffusion out of the floc network. We could show that the type and concentrations of PEIs with varying molecular weight determines the floc properties and thus the extraction efficiency. We could demonstrate why optimal conditions, using 70â¯kDa PEI at 0.25â¯g/g cell dry mass, lead to efficient extraction while at suboptimal conditions extraction is almost negligible. Our findings provide valuable insights into the relationship between floc properties and PEI-driven protein extraction, with potential applications in bioprocessing and biotechnology.
Assuntos
Escherichia coli , Polietilenoimina , Escherichia coli/genética , Peso Molecular , Proteínas de MembranaRESUMO
This manuscript aims to study the reliability of different variables related to performance and acceleration during the golf putt in players with medium-to-high handicaps and to determine the number of attempts necessary to find reliable values for these variables. Eight males and two females [55.67 (13.64) years, 78.4 (11.4) kg, 1.75 (7.95) m] participated in two experimental sessions separated by one week. In these sessions, they performed three blocks of 10 putts trying to stop the golf ball at the center of a dartboard painted 2 m away. The performance was assessed depending on the area of the dartboard where the ball stopped, and the acceleration signals were acquired using the Xsens Dot. The results showed that to evaluate performance, 18 trials were necessary to reach reliable values using the 0-10 scoring system, and 28 trials were necessary for the 0-3 scoring system. Regarding the reliability of the accelerometer-related variables, 7 attempts were necessary to obtain good-to-excellent reliability values for most of the variables. It could be concluded that putting in medium-to-high handicap golf players can be reliably measured using the abovementioned protocol.
Assuntos
Desempenho Atlético , Golfe , Humanos , Masculino , Feminino , Reprodutibilidade dos Testes , Desempenho Atlético/fisiologia , Adulto , Pessoa de Meia-Idade , Idoso , AceleraçãoRESUMO
The sternoclavicular joint dislocation is a very infrequent injury that can put the patient's life at risk if it is not diagnosed and treated properly. This can present as an anterior or posterior dislocation, the latter being less common and more dangerous due to its proximity to visceral structures of the thoracic cavity. Herein, we present the case of a 19-year-old male athlete diagnosed with a posterior dislocation of the right sternoclavicular joint due to indirect trauma during a soccer match, who was successfully treated with a figure-of-eight cerclage with high-resistance sutures plus an InternalBrace technique. After recovery, he has been able to get back to sports with a complete range of motion and experiencing no instability after a two-year follow-up. Figure-of-eight cerclage with high-resistance sutures plus an InternalBrace could be a good technique for surgical treatment of this rare injury, especially in young and physically active patients.
RESUMO
Neurodegenerative diseases represent an increasingly burdensome challenge of the past decade, primarily driven by the global aging of the population. Ongoing efforts focus on implementing diverse strategies to mitigate the adverse effects of neurodegeneration, with the goal of decelerating the pathology progression. Notably, in recent years, it has emerged that the use of nanoparticles (NPs), particularly those obtained through green chemical processes, could constitute a promising therapeutic approach. Green NPs, exclusively sourced from phytochemicals, are deemed safer compared to NPs synthetized through conventional chemical route. In this study, the effects of green chemistry-derived silver NPs (AgNPs) were assessed in neuroblastoma cells, SHSY-5Y, which are considered a pivotal model for investigating neurodegenerative diseases. Specifically, we used two different concentrations (0.5 and 1 µM) of AgNPs and two time points (24 and 48 h) to evaluate the impact on neuroblastoma cells by observing viability reduction and intracellular calcium production, especially using 1 µM at 48 h. Furthermore, investigation using atomic force microscopy (AFM) unveiled an alteration in Young's modulus due to the reorganization of cortical actin following exposure to green AgNPs. This evidence was further corroborated by confocal microscopy acquisitions as well as coherency and density analyses on actin fibers. Our in vitro findings suggest the potential efficacy of green AgNPs against neurodegeneration; therefore, further in vivo studies are imperative to optimize possible therapeutic protocols.
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Receptor-ligand interactions at cell interfaces initiate signaling cascades essential for cellular communication and effector functions. Specifically, T cell receptor (TCR) interactions with pathogen-derived peptides presented by the major histocompatibility complex (pMHC) molecules on antigen-presenting cells are crucial for T cell activation. The binding duration, or dwell time, of TCR-pMHC interactions correlates with downstream signaling efficacy, with strong agonists exhibiting longer lifetimes compared to weak agonists. Traditional surface plasmon resonance (SPR) methods quantify 3D affinity but lack cellular context and fail to account for factors like membrane fluctuations. In the recent years, single-molecule Förster resonance energy transfer (smFRET) has been applied to measure 2D binding kinetics of TCR-pMHC interactions in a cellular context. Here, we introduce a rigorous mathematical model based on survival analysis to determine exponentially distributed receptor-ligand interaction lifetimes, verified through simulated data. Additionally, we developed a comprehensive analysis pipeline to extract interaction lifetimes from raw microscopy images, demonstrating the model's accuracy and robustness across multiple TCR-pMHC pairs. Our new software suite automates data processing to enhance throughput and reduce bias. This methodology provides a refined tool for investigating T cell activation mechanisms, offering insights into immune response modulation.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Receptores de Antígenos de Linfócitos T , Imagem Individual de Molécula , Transferência Ressonante de Energia de Fluorescência/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/química , Ligantes , Humanos , Imagem Individual de Molécula/métodos , Complexo Principal de Histocompatibilidade , Ligação Proteica , Cinética , Linfócitos T/metabolismo , Linfócitos T/imunologiaRESUMO
Amidst the global plastic pollution crisis, the gastrointestinal tract serves as the primary entry point for daily exposure to micro- and nanoplastics. We investigated the complex dynamics between polystyrene micro- and nanoplastics (PS-MNPs) and four distinct human colorectal cancer cell lines (HT29, HCT116, SW480, and SW620). Our findings revealed a significant size- and concentration dependent uptake of 0.25, 1, and 10 µm PS-MNPs across all cell lines, with HCT116 cells exhibiting the highest uptake rates. During cell division, particles were distributed between mother and daughter cells. Interestingly, we observed no signs of elimination from the cells. Short-term exposure to 0.25 µm particles significantly amplified cell migration, potentially leading to pro-metastatic effects. Particles demonstrated high persistence in 2D and 3D cultures, and accumulation in non-proliferating parts of spheroids, without interfering with cell proliferation or division. Our study unveils the disturbing fact of the persistence and bioaccumulation of MNPs in colorectal cancer cell lines, key toxicological traits under REACH (Regulation concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals). Our observations underscore the potential of MNPs as hidden catalysts for tumor progression, particularly through enhancing cell migration and possibly fueling metastasis - a finding that sheds light on a significant and previously underexplored area of concern.
Assuntos
Neoplasias Colorretais , Poluentes Químicos da Água , Humanos , Microplásticos/metabolismo , Plásticos/toxicidade , Poliestirenos/metabolismo , Divisão Celular , Movimento Celular , Poluentes Químicos da Água/metabolismoRESUMO
Fabrication of novel bio-supramolecular structures was achieved by recrystallizing the bacterial surface protein SbpA on amorphous and semicrystalline polylactide derivatives. Differential scanning calorimetry showed that the glass transition temperature (T(g)) for (poly-L-lactide)-PLLA, poly(L,D-lactide)-PDLLA, poly(lactide-co-glycolide)-PLGA and poly(lactide-co-caprolactone)-PLCL was 63 °C, 53 °C, 49 °C and 15 °C, respectively. Tensile stress-strain tests indicated that PLLA, PLGA, and PDLLA had a glassy behaviour when tested below T(g). The obtained Young modulus were 1477 MPa, 1330 MPa, 1306 MPa, and 9.55 MPa for PLLA, PLGA, PDLLA, and PLCL, respectively. Atomic force microscopy results confirmed that SbpA recrystallized on every polymer substrate exhibiting the native S-layer P4 lattice (a = b = 13 nm, γ = 90°). However, the polymer substrate influenced the domain size of the S-protein crystal, with the smallest size for PLLA (0.011 µm(2)), followed by PDLLA (0.034 µm(2)), and PLGA (0.039 µm(2)), and the largest size for PLCL (0.09 µm(2)). quartz crystal microbalance with dissipation monitoring (QCM-D) measurements indicated that the adsorbed protein mass per unit area (~1800 ng cm(-2)) was independent of the mechanical, thermal, and crystalline properties of the polymer support. The slowest protein adsorption rate was observed for amorphous PLCL (the polymer with the weakest mechanical properties and lowest T(g)). QCM-D also monitored protein self-assembly in solution and confirmed that S-layer formation takes place in three main steps: adsorption, self-assembly, and crystal reorganization. Finally, this work shows that biodegradable polylactide derivatives films are a suitable support to form robust biomimetic S-protein layers.
Assuntos
Bacillaceae/química , Proteínas de Bactérias/química , Materiais Biocompatíveis/química , Proteínas de Transporte de Monossacarídeos/química , Poliésteres/química , Materiais Biocompatíveis/síntese química , Cristalização , Poliésteres/síntese química , Temperatura de TransiçãoRESUMO
Cell mechanical properties have been proposed as label free markers for diagnostic purposes in diseases such as cancer. Cancer cells show altered mechanical phenotypes compared to their healthy counterparts. Atomic Force Microscopy (AFM) is a widely utilized tool to study cell mechanics. These measurements often need skilful users, physical modelling of mechanical properties and expertise in data interpretation. Together with the need to perform many measurements for statistical significance and to probe wide enough areas in tissue structures, the application of machine learning and artificial neural network techniques to automatically classify AFM datasets has received interest recently. We propose the use of self-organizing maps (SOMs) as unsupervised artificial neural network applied to mechanical measurements performed via AFM on epithelial breast cancer cells treated with different substances that affect estrogen receptor signalling. We show changes in mechanical properties due to treatments, as estrogen softened the cells, while resveratrol led to an increase in cell stiffness and viscosity. These data were then used as input for SOMs. Our approach was able to distinguish between estrogen treated, control and resveratrol treated cells in an unsupervised manner. In addition, the maps enabled investigation of the relationship of the input variables.