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Mesenchymal-epithelial transition (MET) is essential for tissue and organ development and is thought to contribute to cancer by enabling the establishment of metastatic lesions. Despite its importance in both health and disease, there is a lack of in vitro platforms to study MET and little is known about the regulation of MET by mechanical cues. Here, hyaluronic acid-based hydrogels with dynamic and tunable stiffnesses mimicking that of normal and tumorigenic mammary tissue are synthesized. The platform is then utilized to examine the response of mammary epithelial cells and breast cancer cells to dynamic modulation of matrix stiffness. Gradual softening of the hydrogels reduces proliferation and increases apoptosis of breast cancer cells. Moreover, breast cancer cells exhibit temporal changes in cell morphology, cytoskeletal organization, and gene expression that are consistent with mesenchymal-epithelial plasticity as the stiffness of the matrix is reduced. A reduction in matrix stiffness attenuates the expression of integrin-linked kinase, and inhibition of integrin-linked kinase impacts proliferation, apoptosis, and gene expression in cells cultured on stiff and dynamic hydrogels. Overall, these findings reveal intermediate epithelial/mesenchymal states as cells move along a matrix stiffness-mediated MET trajectory and suggest an important role for matrix mechanics in regulating mesenchymal-epithelial plasticity.
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Herein, the origin of interfacial water nanosized channel distributions attached onto Nafion surfaces is investigated. The surface fibrillary hydrophilic and hydrophobic arrangements were observed on AFM images scanned on Nafion surfaces immersed in water. Then, by analyzing the force vs separation curves, it is possible to map arrays of interfacial water channels and their locations. Nafion surface profiles and the water interfacial patterns are then combined using this AFM technique. As there are no reported experimental techniques to measure water nanochannel cross sections, presented measurements report on their dimensions. Water nanochannels characterized by ε < 7 attached to hydrophilic fibrillary sections form aggregated water domains, a highly organized water structure compared with bulk water. Channels are attached to Nafion surface hydrophilic fibrillary domains in confined sites.
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A significant improvement in spatial resolution is reported in Nafion surface maps when compared to previous atomic force microscopy images of the Nafion surface scanned in air. The technique ability is to generate maps showing approximately few nanometer (â¼2-5 nm) patterns to the long fiber length (>2 µm). Atomic force microscopy force vs separation curve profiles registered in water are used to characterize the surface hydrophobic and hydrophilic domains. Initially, Nafion surfaces were imaged in air for comparison and then immersed in water. Nafion surfaces immersed in water display a matrix of hydrophilic and hydrophobic regions with fibrillary structure dimensions of â¼40 nm formed by fiber pairs. Ribbons formed by two pairs with diameters of â¼83 nm are separated by larger channels.
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While there seems to be broad agreement that cluster formation does exist near solid surfaces, its presence at the liquid/vapor interface is controversial. We report experimental studies we have carried out on interfacial water attached on hydrophobic and hydrophilic surfaces. Nanosized steps in the measured force vs distance to the surface curves characterize water cluster profiles. An expansion of the interfacial structure with time is observed; the initial profile extent is typically â¼1 nm, and for longer times expanded structures of â¼70 nm are observed. Our previous results showed that the interfacial water structure has a relative permittivity of ε ≈ 3 at the air/water interface homogeneously increasing to ε ≈ 80 at 300 nm inside the bulk, but here we have shown that the interfacial dielectric permittivity may have an oscillating profile describing the spatial steps in the force vs distance curves. This low dielectric permittivity arrangements of clusters extend the region with ε ≈ 3 inside bulk water and exhibit a behavior similar to that of water networks that expand in time.
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OBJECTIVE: Recent studies indicate that brown adipose tissue, in addition to its role in thermogenesis, has a role in the regulation of whole-body metabolism. Here we characterize the metabolic effects of deleting Rab10, a protein key for insulin stimulation of glucose uptake into white adipocytes, solely from brown adipocytes. METHODS: We used a murine brown adipocyte cell line and stromal vascular fraction-derived in vitro differentiated brown adipocytes to study the role of Rab10 in insulin-stimulated GLUT4 translocation to the plasma membrane and insulin-stimulated glucose uptake. We generated a brown adipocyte-specific Rab10 knockout for in vivo studies of metabolism and thermoregulation. RESULTS: We demonstrate that deletion of Rab10 from brown adipocytes results in a two-fold reduction of insulin-stimulated glucose transport by reducing translocation of the GLUT4 glucose transporter to the plasma membrane, an effect linked to whole-body glucose intolerance and insulin resistance in female mice. This effect on metabolism is independent of the thermogenic function of brown adipocytes, thereby revealing a metabolism-specific role for brown adipocytes in female mice. The reduced glucose uptake induced by Rab10 deletion disrupts ChREBP regulation of de novo lipogenesis (DNL) genes, providing a potential link between DNL in brown adipocytes and whole-body metabolic regulation in female mice. However, deletion of Rab10 from male mice does not induce systemic insulin resistance, although ChREBP regulation is disrupted. CONCLUSIONS: Our studies of Rab10 reveal the role of insulin-regulated glucose transport into brown adipocytes in whole-body metabolic homeostasis of female mice. Importantly, the contribution of brown adipocytes to whole-body metabolic regulation is independent of its role in thermogenesis. It is unclear whether the whole-body metabolic sexual dimorphism is because female mice are permissive to the effects of Rab10 deletion from brown adipocytes or because male mice are resistant to the effect.
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Adipócitos Marrons/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Homeostase , Termogênese , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas rab de Ligação ao GTP/deficiênciaRESUMO
A technique to image ion pairs in solution is reported. We investigated structural and dynamic properties of ion-pair distributions deposited on highly oriented pyrolytic graphite (HOPG) surfaces in electrolyte solutions. Atomic force microscopy images of HOPG immersed in NaCl and KCl solutions display regular arrangements on top of the hexagonal carbon rings forming the HOPG atomic structure. These arrangements are the result of the low value of the aqueous interfacial dielectric constant (εr ≈ 3-11). The measured ion-pair radius is a function of the salt present in the solution; for KCl, the ion-pair radius is equal or smaller than 0.42 nm; for NaCl, the ion-pair radius is 0.36 nm. A comparison of these values with their crystalline lattice dimensions indicates that both KCl and NaCl ion pairs in solution at the HOPG/solution interfacial region exist as tight contact ion pairs in quasistationary distributions. The NaCl ion-pair distribution forms an aligned arrangement, and the KCl distribution is formed by intercalated pairs.
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A clear molecular description of excess hydrated protons and their local hydrogen bond transport network remains elusive. Here, the hydrogen bond network of excess hydrated protons in water bridges was probed by measuring their Raman spectra and comparing them to the spectra of protons in ice and water. The proton vibrational spectrum and the hydrogen bond network translational and librational spectra were recorded. The spectra of the water bridge and water exhibit clear differences, indicating the presence of a structure in water bridges when subjected to an electric field of â¼106 V/m that has not been previously reported. The intermolecular Raman spectrum of the floating water bridge exhibits a hydrogen bond stretching band at 150-250 cm-1, librational bands within the 300-1000 cm-1 spectral range, and a large band at 1500-3000 cm-1, which corresponds to the vibrational signature of excess hydrated protons in the water bridge structure. The excess protons are shown to move predominantly at the air/water interface, and the effect of this distribution is a measurable change in the air/water interfacial tension from â¼80 to â¼32 N/m. Therefore, hydrated protons must have a unique water arrangement that enables them to propagate without sinking into bulk water. This local polarized hydrogen bond network in the interfacial water region is characterized by a translational spectrum similar to that of ice V.
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Excess proton structures in water remain unclear. The motion and nature of excess protons in water were probed using a supported water bridge structure in electric field (E) with an intensity of â¼106 V/m. The experimental setup generated protons that exhibit a long lifetime. The effect of excess protons in water induced a â¼3% variation in the pH for a 300 V overvoltage at the cathode. The current versus voltage curves show a current space-charge-limited operation. By measuring the space-charge distribution in both the cathode and anode and by adjusting the Mott-Gurney law to the measured excess hydrated proton current and the voltage drop in the cationic space-charge region, the protonic mobility was determined to be â¼200 × 10-8 m2/(V·s) (E ≈ 4 × 106 V/m). This measured mobility, which is typically five times larger than the reported mobility for protons in water, is in agreement with the mechanism outlined by Grotthuss in 1805. The measured mid-Raman spectrum covering 1000-3800 cm-1 range indicates the species character. The hydrated excess proton spectral response through the mid-Raman at 1760 and 3200 cm-1 was attributed to the Zundel complex and the region at â¼2000 to â¼2600 cm-1 response is attributed to the Eigen complex, indicating a core structure simultaneously with a Eigen-like and Zundel-like character, suggesting a rapid fluctuation between these two structures or a new specie.
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BACKGROUND: Measurements and models of current flow in the brain during transcranial Direct Current Stimulation (tDCS) indicate stimulation of regions in-between electrodes. Moreover, the folded cortex results in local fluctuations in current flow intensity and direction, and animal studies suggest current flow direction relative to cortical columns determines response to tDCS. METHODS: Here we test this idea by using Transcranial Magnetic Stimulation Motor Evoked Potentials (TMS-MEP) to measure changes in corticospinal excitability following tDCS applied with electrodes aligned orthogonal (across) or parallel to M1 in the central sulcus. RESULTS: Current flow models predicted that the orthogonal electrode montage produces consistently oriented current across the hand region of M1 that flows along cortical columns, while the parallel electrode montage produces non-uniform current directions across the M1 cortical surface. We find that orthogonal, but not parallel, orientated tDCS modulates TMS-MEPs. We also show modulation is sensitive to the orientation of the TMS coil (PA or AP), which is thought to select different afferent pathways to M1. CONCLUSIONS: Our results are consistent with tDCS producing directionally specific neuromodulation in brain regions in-between electrodes, but shows nuanced changes in excitability that are presumably current direction relative to column and axon pathway specific. We suggest that the direction of current flow through cortical target regions should be considered for targeting and dose-control of tDCS.
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Potencial Evocado Motor/fisiologia , Córtex Motor/fisiologia , Estimulação Transcraniana por Corrente Contínua/métodos , Adulto , Vias Aferentes/fisiologia , Axônios/fisiologia , Eletromiografia/métodos , Feminino , Humanos , Masculino , Pensamento/fisiologia , Adulto JovemRESUMO
There are substantial differences across species in the organization and function of the motor pathways. These differences extend to basic electrophysiological properties. Thus, in rat motor cortex, pyramidal cells have long duration action potentials, while in the macaque, some pyramidal neurons exhibit short duration "thin" spikes. These differences may be related to the expression of the fast potassium channel Kv3.1b, which in rat interneurons is associated with generation of thin spikes. Rat pyramidal cells typically lack these channels, while there are reports that they are present in macaque pyramids. Here we made a systematic, quantitative comparison of the Kv3.1b expression in sections from macaque and rat motor cortex, using two different antibodies (NeuroMab, Millipore). As our standard reference, we examined, in the same sections, Kv3.1b staining in parvalbumin-positive interneurons, which show strong Kv3.1b immunoreactivity. In macaque motor cortex, a large sample of pyramidal neurons were nearly all found to express Kv3.1b in their soma membranes. These labeled neurons were identified as pyramidal based either by expression of SMI32 (a pyramidal marker), or by their shape and size, and lack of expression of parvalbumin (a marker for some classes of interneuron). Large (Betz cells), medium, and small pyramidal neurons all expressed Kv3.1b. In rat motor cortex, SMI32-postive pyramidal neurons expressing Kv3.1b were very rare and weakly stained. Thus, there is a marked species difference in the immunoreactivity of Kv3.1b in pyramidal neurons, and this may be one of the factors explaining the pronounced electrophysiological differences between rat and macaque pyramidal neurons.
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Córtex Motor/citologia , Células Piramidais/metabolismo , Canais de Potássio Shaw/metabolismo , Animais , Contagem de Células , Feminino , Filamentos Intermediários/metabolismo , Interneurônios/metabolismo , Macaca mulatta , Masculino , Microscopia Confocal , Parvalbuminas/metabolismo , Ratos , Ratos Wistar , Especificidade da EspécieRESUMO
Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone involved in nutrient homeostasis. GIP receptor (GIPR) is constitutively internalized and returned to the plasma membrane, atypical behavior for a G-protein-coupled receptor (GPCR). GIP promotes GIPR downregulation from the plasma membrane by inhibiting recycling without affecting internalization. This transient desensitization is achieved by altered intracellular trafficking of activated GIPR. GIP stimulation induces a switch in GIPR recycling from a rapid endosomal to a slow trans-Golgi network (TGN) pathway. GPCR kinases and ß-arrestin2 are required for this switch in recycling. A coding sequence variant of GIPR, which has been associated with metabolic alterations, has altered post-activation trafficking characterized by enhanced downregulation and prolonged desensitization. Downregulation of the variant requires ß-arrestin2 targeting to the TGN but is independent of GPCR kinases. The single amino acid substitution in the variant biases the receptor to promote GIP-stimulated ß-arrestin2 recruitment without receptor phosphorylation, thereby enhancing downregulation.
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Polipeptídeo Inibidor Gástrico/genética , Receptores Acoplados a Proteínas G/genética , Receptores dos Hormônios Gastrointestinais/genética , beta-Arrestina 2/genética , Células 3T3-L1 , Animais , Endossomos/metabolismo , Polipeptídeo Inibidor Gástrico/metabolismo , Humanos , Incretinas/genética , Camundongos , Transporte Proteico/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , beta-Arrestina 2/metabolismo , Rede trans-Golgi/genética , Rede trans-Golgi/metabolismoRESUMO
Structures of crystallized deposits formed by the molecular self-assembly of aspartic acid and valine on silicon substrates were imaged by atomic force microscopy. Images of d- and l-aspartic acid crystal surfaces showing extended molecularly flat sheets or regions separated by single molecule thick steps are presented. Distinct orientation surfaces were imaged, which, combined with the single molecule step size, defines the geometry of the crystal. However, single molecule step growth also reveals the crystal chirality, i.e., growth orientations. The imaged ordered lattice of aspartic acid (asp) and valine (val) mostly revealed periodicities corresponding to bulk terminations, but a previously unreported molecular hexagonal lattice configuration was observed for both l-asp and l-val but not for d-asp or d-val. Atomic force microscopy can then be used to identify the different chiral forms of aspartic acid and valine crystals.
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Ácido Aspártico/química , Valina/química , Cristalização , Microscopia de Força Atômica , Estrutura Molecular , Silício/química , EstereoisomerismoRESUMO
OBJECTIVE: Identify the impact of corrective techniques in the mortality rate trends for cardiovascular disease, ischemic heart diseases, and cerebrovascular diseases, in the City of Manaus, State of Amazonas, Brazil, between 1980 and 2007. METHODS: Data were obtained from the Unified Health System's Information Technology Department and the following steps undertaken: (1) proportional redistribution of death records, but without taking into account age and/or sex; (2) redistribution of ill-defined deaths among those whose cause is known; (3) treatment and reallocation of so-called "junk codes" in cardiology; and (4) correction of underrecordings of deaths by indirect techniques. Records treated in steps 1 and 2, and steps 3 and 4 together, generated base rates, adjusted rates, and adjusted and corrected rates, respectively, which were analyzed according to sex and age cohort; subsequently, they were standardized by the direct method. Simple linear regression was used for trend analysis. RESULTS: The Mortality Information System's raw data underestimated death from heart diseases, mainly starting in the year 2000. With regard to the trend over time, the adjusted and corrected rates, compared to the base rates, pointed to the following: attenuation of the downward trend for cardiovascular diseases as a whole; accentuation of the downward trend for ischemic heart diseases; and stability in the rates for cerebrovascular diseases during the period. CONCLUSIONS: This correction made it possible to identify an excess of deaths that had not been computed, generating more reliable adjusted and corrected death rates.
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Doenças Cardiovasculares/mortalidade , Transtornos Cerebrovasculares/mortalidade , Sistemas de Informação em Saúde/organização & administração , Algoritmos , Brasil/epidemiologia , Causas de Morte , Atestado de Óbito , Feminino , Humanos , Classificação Internacional de Doenças , Masculino , Mortalidade/tendências , Melhoria de Qualidade , Sistema de Registros/estatística & dados numéricos , População Urbana/estatística & dados numéricosRESUMO
Objetivo. Identificar o impacto de técnicas corretivas na tendência das taxas de mortalidadepor doenças cardiovasculares, doenças isquêmicas do coração e doenças cerebrovasculares, emManaus (AM), Brasil, no período entre 1980 e 2007.Métodos. Os dados foram obtidos no Departamento de Informática do Sistema Único deSaúde, considerando-se os seguintes passos: (1) redistribuição proporcional dos registros deóbitos com idade e/ou sexo ignorados; (2) redistribuição dos óbitos mal definidos dentre os decausa conhecida; (3) tratamento e realocação dos chamados códigos-lixo em cardiologia; e,(4) correção dos sub-registros de óbitos por técnicas indiretas. Registros tratados nos passos1, 2 e passos 3 e 4 agregados geraram as taxas base, taxas ajustadas e taxas ajustadas ecorrigidas, respectivamente, as quais foram analisadas por sexo e faixa etária; posteriormente,foram padronizadas pelo método direto. A regressão linear simples foi utilizada para análisede tendência.Resultados. Os dados brutos do Sistema de Informação sobre Mortalidade subestimavamas taxas de mortalidade por doenças do coração, principalmente a partir do ano 2000. Quantoà tendência temporal, as taxas ajustadas e corrigidas, comparadas às taxas base, apontam:atenuação da tendência de queda para as doenças cardiovasculares como um todo; acentuaçãoda tendência de queda para as doenças isquêmicas do coração; e estabilidade nas taxas para asdoenças cerebrovasculares no período.Conclusões. Com essa correção, foi possível identificar um excesso de óbitos que não haviasido computado, gerando taxas de mortalidade ajustadas e corrigidas mais confiáveis.
Objective. Identify the impact of corrective techniques in the mortality rate trendsfor cardiovascular disease, ischemic heart diseases, and cerebrovascular diseases, inthe City of Manaus, State of Amazonas, Brazil, between 1980 and 2007.Methods. Data were obtained from the Unified Health Systems InformationTechnology Department and the following steps undertaken: (1) proportionalredistribution of death records, but without taking into account age and/or sex; (2)redistribution of ill-defined deaths among those whose cause is known; (3) treatmentand reallocation of so-called junk codes in cardiology; and (4) correction ofunderrecordings of deaths by indirect techniques. Records treated in steps 1 and 2,and steps 3 and 4 together, generated base rates, adjusted rates, and adjusted and correctedrates, respectively, which were analyzed according to sex and age cohort; subsequently,they were standardized by the direct method. Simple linear regression was used fortrend analysis.Results. The Mortality Information Systems raw data underestimated deathfrom heart diseases, mainly starting in the year 2000. With regard to the trendover time, the adjusted and corrected rates, compared to the base rates, pointed to thefollowing: attenuation of the downward trend for cardiovascular diseases as a whole;accentuation of the downward trend for ischemic heart diseases; and stability in therates for cerebrovascular diseases during the period.Conclusions. This correction made it possible to identify an excess of deaths that hadnot been computed, generating more reliable adjusted and corrected death rates.
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Humanos , Masculino , Feminino , Doenças Cardiovasculares/mortalidade , Transtornos Cerebrovasculares/mortalidade , Sistemas de Informação em Saúde/organização & administração , Algoritmos , Brasil/epidemiologia , Causas de Morte , Atestado de Óbito , Classificação Internacional de Doenças , Mortalidade/tendências , Melhoria de Qualidade , Sistema de Registros/estatística & dados numéricos , População Urbana/estatística & dados numéricosAssuntos
Doenças Cardiovasculares , Sistemas de Informação , Brasil , Sistemas de Informação em Saúde , Classificação Internacional de Doenças , População Urbana , Estatísticas Vitais , Mortalidade , Sub-Registro , Registros de Mortalidade , Doenças Cardiovasculares , Estatísticas Vitais , Mortalidade , Sub-Registro , Registros de Mortalidade , Sistemas de Informação , Transtornos Cerebrovasculares , Algoritmos , Causas de Morte , Atestado de Óbito , Mortalidade , Melhoria de Qualidade , Sistema de Registros , BrasilRESUMO
Water at room temperature is not simply a medium for which uniform properties can always be assumed. Water close to solid hydrophobic or hydrophilic surfaces has elasticity, which is measured by monitoring the quartz crystal microbalance (QCM) resonant frequency and resistance. Small additions of salt are shown to modify this elasticity. Furthermore, near the hydrophobic QCM gold electrode, undersaturated aqueous NaCl solutions present a high concentration of ion pairs, which is confirmed by atomic force microscopy through force versus distance measurements.
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Self-assembled aggregates of amphiphilic surfactant molecules formed on solid surfaces are similar to biological membranes. To understand the formation mechanism of these aggregates, we have studied the formation of self-organized monolayers from low-concentration sodium dodecyl sulfate (SDS) aqueous solutions (concentration below the critical micelle concentration) on gold surfaces. The study has been carried out by using simultaneously quartz crystal microbalance (QCM) and open circuit potential measurements in situ. We have developed a model which explains the variation of the QCM frequency and open circuit potential following SDS additions to water. The dominant growth mechanism during the major part of film formation was demonstrated to be surface diffusion of surfactant molecules.