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1.
Proc Natl Acad Sci U S A ; 121(2): e2304135120, 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38147542

RESUMO

Active hydroponic substrates that stimulate on demand the plant growth have not been demonstrated so far. Here, we developed the eSoil, a low-power bioelectronic growth scaffold that can provide electrical stimulation to the plants' root system and growth environment in hydroponics settings. eSoil's active material is an organic mixed ionic electronic conductor while its main structural component is cellulose, the most abundant biopolymer. We demonstrate that barley seedlings that are widely used for fodder grow within the eSoil with the root system integrated within its porous matrix. Simply by polarizing the eSoil, seedling growth is accelerated resulting in increase of dry weight on average by 50% after 15 d of growth. The effect is evident both on root and shoot development and occurs during the growth period after the stimulation. The stimulated plants reduce and assimilate NO3- more efficiently than controls, a finding that may have implications on minimizing fertilizer use. However, more studies are required to provide a mechanistic understanding of the physical and biological processes involved. eSoil opens the pathway for the development of active hydroponic scaffolds that may increase crop yield in a sustainable manner.


Assuntos
Fenômenos Biológicos , Plântula , Plântula/metabolismo , Hidroponia/métodos , Raízes de Plantas/metabolismo
2.
Int J Mol Sci ; 24(15)2023 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-37569350

RESUMO

Nanotechnology offers new possibilities in molecular diagnostics, with nanoparticles gaining attention as biosensor upgrades. This study evaluates gold-coated silver nanoplates coated with PEG for enhanced protection, aiming to detect Spike protein with higher sensitivity, and emphasizes the importance of considering complex environments and appropriate controls for specific binding and accurate analysis. The sensitivity of antibody-coated PEGAuTSNPs as tools for immunoassays is demonstrated through fibronectin (Fn)- anti-fibronectin binding within an isolated extracellular matrix as a complex and native environment of Fn. Moreover, the optimal functionalization volume of Spike protein was determined (4 µg/mL of PEGAuTSNP). Anti-Spike was added to confirm binding, while the TJP1 protein was used as a negative control. The same experiment was used in the presence of horse serum to simulate a complex environment. According to Localized Surface Plasmon Resonance analysis and Dynamic Light Scattering size measurements, anti-Spike exhibited a stronger affinity for the nanoplates, causing TJP1 to be replaced by the antibody on the nanoplates' surface. Future research will involve exploring alternative complex environments, filtering larger molecules, and the optimization of immunoassay performance.

3.
Mol Microbiol ; 114(6): 1019-1037, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32808689

RESUMO

In this paper, we explored the presence of GATA in Entamoeba histolytica and their function as regulators of phagocytosis-related genes. Bioinformatics analyses evidenced a single 579 bp sequence encoding for a protein (EhGATA), smaller than GATA factors of other organisms. EhGATA appeared phylogenetically close to Dictyostelium discoideum and Schistosoma mansoni GATA proteins. Its sequence predicts the presence of a zinc-finger DNA binding domain and an AT-Hook motif; it also has two nuclear localization signals. By transmission electron and confocal microscopy, anti-EhGATA antibodies revealed the protein in the cytoplasm and nucleus, and 65% of nuclear signal was in the heterochromatin. EhGATA recombinant protein and trophozoites nuclear extracts bound to GATA-DNA consensus sequence. By in silico scrutiny, 1,610 gene promoters containing GATA-binding sequences appeared, including Ehadh and Ehvps32 promoters, whose genes participate in phagocytosis. Chromatin immunoprecipitation assays showed that EhGATA interact with Ehadh and Ehvps32 promoters. In EhGATA-overexpressing trophozoites (NeoGATA), the Ehadh and Ehvps32 mRNAs amount was modified, strongly supporting that EhGATA could regulate their transcription. NeoGATA trophozoites exhibited rounded shapes, high proliferation rates, and diminished erythrophagocytosis. Our results provide new insights into the role of EhGATA as a noncanonical transcription factor, regulating genes associated with phagocytosis.


Assuntos
Entamoeba histolytica/metabolismo , Fatores de Transcrição GATA/metabolismo , Fagocitose , Proteínas de Protozoários/metabolismo , Trofozoítos/metabolismo , Motivos de Aminoácidos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Entamoeba histolytica/genética , Fatores de Transcrição GATA/genética , Regulação da Expressão Gênica , Filogenia , Regiões Promotoras Genéticas , Proteínas de Protozoários/genética , Proteínas Recombinantes/metabolismo , Trofozoítos/citologia
4.
Small ; 15(43): e1902189, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31513355

RESUMO

Electronic control of biological processes with bioelectronic devices holds promise for sophisticated regulation of physiology, for gaining fundamental understanding of biological systems, providing new therapeutic solutions, and digitally mediating adaptations of organisms to external factors. The organic electronic ion pump (OEIP) provides a unique means for electronically-controlled, flow-free delivery of ions, and biomolecules at cellular scale. Here, a miniaturized OEIP device based on glass capillary fibers (c-OEIP) is implanted in a biological organism. The capillary form factor at the sub-100 µm scale of the device enables it to be implanted in soft tissue, while its hyperbranched polyelectrolyte channel and addressing protocol allows efficient delivery of a large aromatic molecule. In the first example of an implantable bioelectronic device in plants, the c-OEIP readily penetrates the leaf of an intact tobacco plant with no significant wound response (evaluated up to 24 h) and effectively delivers the hormone abscisic acid (ABA) into the leaf apoplast. OEIP-mediated delivery of ABA, the phytohormone that regulates plant's tolerance to stress, induces closure of stomata, the microscopic pores in leaf's epidermis that play a vital role in photosynthesis and transpiration. Efficient and localized ABA delivery reveals previously unreported kinetics of ABA-induced signal propagation.


Assuntos
Ácido Abscísico/farmacologia , Eletrônica , Bombas de Íon/metabolismo , Nicotiana/fisiologia , Reguladores de Crescimento de Plantas/farmacologia , Estômatos de Plantas/fisiologia , Estômatos de Plantas/efeitos dos fármacos , Nicotiana/efeitos dos fármacos
5.
PLoS Pathog ; 12(12): e1006089, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28002502

RESUMO

Entamoeba histolytica, the highly phagocytic protozoan causative of human amoebiasis lacks the machinery to synthesize cholesterol. Here, we investigated the presence of NPC1 and NPC2 proteins in this parasite, which are involved in cholesterol trafficking in mammals. Bioinformatics analysis revealed one Ehnpc1 and two Ehnpc2 genes. EhNPC1 appeared as a transmembrane protein and both EhNPC2 as peripheral membrane proteins. Molecular docking predicted that EhNPC1 and EhNPC2 bind cholesterol and interact with each other. Genes and proteins were identified in trophozoites. Serum pulse-chase and confocal microscopy assays unveiled that after trophozoites sensed the cholesterol source, EhNPC1 and EhNPC2 were organized around the plasma membrane in a punctuated pattern. Vesicles emerged and increased in number and size and some appeared full of cholesterol with EhNPC1 or EhNPC2 facing the extracellular space. Both proteins, but mostly EhNPC2, were found out of the cell associated with cholesterol. EhNPC1 and cholesterol formed networks from the plasma membrane to the nucleus. EhNPC2 appeared in erythrocytes that were being ingested by trophozoites, co-localizing with cholesterol of erythrocytes, whereas EhNPC1 surrounded the phagocytic cup. EhNPC1 and EhNPC2 co-localized with EhSERCA in the endoplasmic reticulum and with lysobisphosphatidic acid and EhADH (an Alix protein) in phagolysosomes. Immunoprecipitation assays confirmed the EhNPC1 and EhNPC2 association with cholesterol, EhRab7A and EhADH. Serum starved and blockage of cholesterol trafficking caused a low rate of phagocytosis and incapability of trophozoites to produce damage in the mouse colon. Ehnpc1 and Ehnpc2 knockdown provoked in trophozoites a lower intracellular cholesterol concentration and a diminished rate of phagocytosis; and Ehnpc1 silencing also produced a decrease of trophozoites movement. Trafficking of EhNPC1 and EhNPC2 during cholesterol uptake and phagocytosis as well as their association with molecules involved in endocytosis strongly suggest that these proteins play a key role in cholesterol uptake.


Assuntos
Colesterol/metabolismo , Entamoeba histolytica/metabolismo , Entamebíase/metabolismo , Proteínas de Protozoários/metabolismo , Trofozoítos/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Simulação de Acoplamento Molecular , Fagocitose/fisiologia , Filogenia , Reação em Cadeia da Polimerase , Transporte Proteico/fisiologia , Homologia de Sequência de Aminoácidos , Virulência/fisiologia
6.
Small ; 13(2)2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27753200

RESUMO

The electronic spin filtering capability of a single chiral helical peptide is measured. A ferromagnetic electrode source is employed to inject spin-polarized electrons in an asymmetric single-molecule junction bridging an α-helical peptide sequence of known chirality. The conductance comparison between both isomers allows the direct determination of the polarization power of an individual chiral molecule.


Assuntos
Peptídeos/química , Marcadores de Spin , Sequência de Aminoácidos , Condutividade Elétrica , Eletrodos , Elétrons , Ouro/química , Níquel/química , Estereoisomerismo
7.
Exp Cell Res ; 339(1): 67-80, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26453937

RESUMO

The lack of a successful treatment for triple-negative breast cancer demands the study of the heterogeneity of cells that constitute these tumors. With this aim, two clones from triple negative breast MDA-MB-231 cancer cells were isolated: One with fibroblast-like appearance (F) and another with semi-epithelial (SE) morphology. Cells of the F clone have a higher migration and tumorigenesis capacity than SE cells, suggesting that these cells are in a more advanced stage of epithelial to mesenchymal transformation. In agreement, F cells have a diminished expression of the tight junction proteins claudins 1 and 4, and an increased content of ß-catenin. The latter is due to an augmented activity of the canonical Wnt route and of the EGFR/PI3K/mTORC2/AKT pathway favoring the cytoplasmic accumulation of ß-catenin and its transcriptional activity. In addition, F cells display increased phosphorylation of ß-catenin at Tyr654 by Src. These changes favor in F cells, the over-expression of Snail that promotes EMT. Finally, we observe that both F and SE cells display markers of cancer stem cells, which are more abundant in the F clone.


Assuntos
Receptores ErbB/metabolismo , Complexos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Apoptose , Western Blotting , Proliferação de Células , Quimiotaxia , Transição Epitelial-Mesenquimal , Feminino , Imunofluorescência , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Fosforilação , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Cell Calcium ; 121: 102891, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38772195

RESUMO

The TMEM16A channel, a member of the TMEM16 protein family comprising chloride (Cl-) channels and lipid scramblases, is activated by the free intracellular Ca2+ increments produced by inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release after GqPCRs or Ca2+ entry through cationic channels. It is a ubiquitous transmembrane protein that participates in multiple physiological functions essential to mammals' lives. TMEM16A structure contains two identical 10-segment monomers joined at their transmembrane segment 10. Each monomer harbours one independent hourglass-shaped pore gated by Ca2+ ligation to an orthosteric site adjacent to the pore and controlled by two gates. The orthosteric site is created by assembling negatively charged glutamate side chains near the pore´s cytosolic end. When empty, this site generates an electrostatic barrier that controls channel rectification. In addition, an isoleucine-triad forms a hydrophobic gate at the boundary of the cytosolic vestibule and the inner side of the neck. When the cytosolic Ca2+ rises, one or two Ca2+ ions bind to the orthosteric site in a voltage (V)-dependent manner, thus neutralising the electrostatic barrier and triggering an allosteric gating mechanism propagating via transmembrane segment 6 to the hydrophobic gate. These coordinated events lead to pore opening, allowing the Cl- flux to ensure the physiological response. The Ca2+-dependent function of TMEM16A is highly regulated. Anions with higher permeability than Cl- facilitate V dependence by increasing the Ca2+ sensitivity, intracellular protons can replace Ca2+ and induce channel opening, and phosphatidylinositol 4,5-bisphosphate bound to four cytosolic sites likely maintains Ca2+ sensitivity. Additional regulation is afforded by cytosolic proteins, most likely by phosphorylation and protein-protein interaction mechanisms.


Assuntos
Anoctamina-1 , Cálcio , Humanos , Animais , Anoctamina-1/metabolismo , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Ativação do Canal Iônico
9.
Adv Healthc Mater ; 12(27): e2301194, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37171457

RESUMO

Tumor-derived extracellular vesicles (TEVs) induce the epithelial-to-mesenchymal transition (EMT) in nonmalignant cells to promote invasion and cancer metastasis, representing a novel therapeutic target in a field severely lacking in efficacious antimetastasis treatments. However, scalable technologies that allow continuous, multiparametric monitoring for identifying metastasis inhibitors are absent. Here, the development of a functional phenotypic screening platform based on organic electrochemical transistors (OECTs) for real-time, noninvasive monitoring of TEV-induced EMT and screening of antimetastatic drugs is reported. TEVs derived from the triple-negative breast cancer cell line MDA-MB-231 induce EMT in nonmalignant breast epithelial cells (MCF10A) over a nine-day period, recapitulating a model of invasive ductal carcinoma metastasis. Immunoblot analysis and immunofluorescence imaging confirm the EMT status of TEV-treated cells, while dual optical and electrical readouts of cell phenotype are obtained using OECTs. Further, heparin, a competitive inhibitor of cell surface receptors, is identified as an effective blocker of TEV-induced EMT. Together, these results demonstrate the utility of the platform for TEV-targeted drug discovery, allowing for facile modeling of the transient drug response using electrical measurements, and provide proof of concept that inhibitors of TEV function have potential as antimetastatic drug candidates.


Assuntos
Neoplasias da Mama , Vesículas Extracelulares , Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Detecção Precoce de Câncer , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Transição Epitelial-Mesenquimal/genética , Movimento Celular , Melanoma Maligno Cutâneo
10.
Adv Sci (Weinh) ; 9(2): e2101711, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34741447

RESUMO

There is increasing demand for direct in situ metabolite monitoring from cell cultures and in vivo using implantable devices. Electrochemical biosensors are commonly preferred due to their low-cost, high sensitivity, and low complexity. Metabolite detection, however, in cultured cells or sensitive tissue is rarely shown. Commonly, glucose sensing occurs indirectly by measuring the concentration of hydrogen peroxide, which is a by-product of the conversion of glucose by glucose oxidase. However, continuous production of hydrogen peroxide in cell media with high glucose is toxic to adjacent cells or tissue. This challenge is overcome through a novel, stacked enzyme configuration. A primary enzyme is used to provide analyte sensitivity, along with a secondary enzyme which converts H2 O2 back to O2 . The secondary enzyme is functionalized as the outermost layer of the device. Thus, production of H2 O2 remains local to the sensor and its concentration in the extracellular environment does not increase. This "biostack" is integrated with organic electrochemical transistors to demonstrate sensors that monitor glucose concentration in cell cultures in situ. The "biostack" renders the sensors nontoxic for cells and provides highly sensitive and stable detection of metabolites.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Glucose/análise , Glucose/metabolismo , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/metabolismo , Células Cultivadas , Humanos
11.
Exp Cell Res ; 316(19): 3124-39, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20868680

RESUMO

With the aim of discovering new molecular interactions of the tight junction protein ZO-2, a two-hybrid screen was performed on a human kidney cDNA library using as bait the middle segment of ZO-2. Through this assay we identified a 24-kDa novel protein herein named ZASP for ZO-2 associated speckle protein. ZO-2/ZASP interaction further confirmed by pull down and immunoprecipitation experiments, requires the presence of the intact PDZ binding motif SQV of ZASP and the third PDZ domain of ZO-2. ZASP mRNA and protein are present in the kidney and in several epithelial cell lines. Endogenous ZASP is expressed primarily in nuclear speckles in co-localization with splicing factor SC-35. Nocodazole treatment and wash out reveals that ZASP disappears from the nucleus during mitosis in accordance with speckle disassembly during metaphase. ZASP amino acid sequence exhibits a canonical nuclear exportation signal and in agreement the protein exits the nucleus through a process mediated by exportin/CRM1. ZASP over-expression blocks the inhibitory activity of ZO-2 on cyclin D1 gene transcription and protein expression. The identification of ZASP helps to unfold the complex nuclear molecular arrays that form on ZO-2 scaffolds.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Antígenos CD1/metabolismo , Sequência de Bases , Ciclo Celular , Linhagem Celular , Núcleo Celular/metabolismo , Clonagem Molecular , Ciclina D1/genética , Cães , Regulação da Expressão Gênica , Humanos , Imunoprecipitação , Carioferinas/metabolismo , Rim/metabolismo , Proteínas com Domínio LIM , Proteínas de Membrana/química , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína da Zônula de Oclusão-2
12.
Ann Hepatol ; 10(4): 486-92, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21911890

RESUMO

OBJECTIVE: To evaluate the efficacy of low carbohydrate diet (LCD) as compared with low fat diet (LFD) to decrease aminotransferase levels in obese women with nonalcoholic fatty liver disease. MATERIAL AND METHODS: A total of 59 women were randomly enrolled in a non-controlled clinical intervention study to receive either LCD or LFD during six months. Apparently healthy non-pregnant obese women aged 20 to 65 years were eligible to participate. Previous diagnosis of hepatic disease, serum creatinine level ≥ 1.5 mg/dL, severe life-limiting medical illness, pregnancy, active participation in other dietary program, use of weight loss drugs, or alcohol consumption ≥ 30 g per day were exclusion criteria. RESULTS: A total of 31 obese women who received LCD were compared with 28 women allocated in the LFD group. There were 3 (LCD group) and 2 (LFD group) women with lost of follow-up. No differences in the proportion of type 2 diabetes, hypertension and hyperlipidemia were noted between women in the LCD and LFD groups. At end of follow-up, there were not significant statistical differences in the anthropometric and biochemical characteristics between women in both groups. The weight loss was 5.7 and 5.5% for women in the LCD LFD groups. Although the decrease of AST (31.7 and 22.4%) and ALT (41 and 33.3%) levels was more elevated in the women of LCD group, as compared with the LFD group, there were not significant statistical differences. CONCLUSIONS: Our results show that weight loss, irrespective of the type of diet, reduces aminotransferase levels in obese women with NAFLD.


Assuntos
Alanina Transaminase/sangue , Aspartato Aminotransferases/sangue , Dieta com Restrição de Carboidratos , Dieta com Restrição de Gorduras , Fígado Gorduroso/etiologia , Obesidade/dietoterapia , Redução de Peso , Adulto , Biomarcadores/sangue , Distribuição de Qui-Quadrado , Regulação para Baixo , Fígado Gorduroso/sangue , Feminino , Humanos , México , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica , Obesidade/sangue , Obesidade/complicações , Fatores de Tempo , Resultado do Tratamento
13.
ACS Biomater Sci Eng ; 7(12): 5585-5597, 2021 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-34802228

RESUMO

Cancer-derived exosomes (cEXOs) facilitate transfer of information between tumor and human primary stromal cells, favoring cancer progression. Although the mechanisms used during this information exchange are still not completely understood, it is known that binding is the initial contact established between cEXOs and cells. Hence, studying binding and finding strategies to block it are of great therapeutic value. However, such studies are challenging for a variety of reasons, including the need for human primary cell culture, the difficulty in decoupling and isolating binding from internalization and cargo delivery, and the lack of techniques to detect these specific interactions. In this work, we created a supported biomimetic stem cell membrane incorporating membrane components from human primary adipose-derived stem cells (ADSCs). We formed the supported membrane on glass and on multielectrode arrays to offer the dual option of optical or electrical detection of cEXO binding to the membrane surface. Using our platform, we show that cEXOs bind to the stem cell membrane and that binding is blocked when an antibody to integrin ß1, a component of ADSC surface, is exposed to the membrane surface prior to cEXOs. To test the biological outcome of blocking this interaction, we first confirm that adding cEXOs to cultured ADSCs leads to the upregulation of vascular endothelial growth factor, a measure of proangiogenic activity. Next, when ADSCs are first blocked with anti-integrin ß1 and then exposed to cEXOs, the upregulation of proangiogenic activity and cell proliferation are significantly reduced. This biomimetic membrane platform is the first cell-free label-free in vitro platform for the recapitulation and study of cEXO binding to human primary stem cells with potential for therapeutic molecule screening as it is compatible with scale-up and multiplexing.


Assuntos
Exossomos , Neoplasias , Biomimética , Humanos , Células-Tronco , Fator A de Crescimento do Endotélio Vascular
14.
Ann Hepatol ; 9(2): 144-9, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20526006

RESUMO

OBJECTIVE: To determine the clinical characteristics of NAFLD in asymptomatic obese women. METHODS: A total of 457 asymptomatic obese women were enrolled in a cross-sectional study and allocated into groups with and without NAFLD. Irrespective of ALT levels, diagnosis of NAFLD was established by ultrasonographic findings; irrespective of fibrosis, NASH was defined by hepatic histological changes. RESULTS: One hundred ninety five (42.7%) women had elevated ALT levels. Diagnosis of NAFLD was established in 228 (49.9%) women; among women with NAFLD, 34 (14.9%) have ALT levels within the normal range. On the other hand, based on the healthy range for ALT levels (19 UI/L), 336 (73.5%) women had elevated ALT, but only 2 (0.9%) women with NAFLD exhibited ALT levels within normal healthy values. Furthermore, 93 (41%) women who had AST/ALT levels (3) 1 underwent liver biopsy; of these, 90 (96.8%) had diagnosis of NASH and 3 (3.2%) of hepatic cirrhosis. Women with NAFLD were more obese and have higher fasting plasma glucose, triglycerides, ALT, and AST levels than obese women without NAFLD. Seventy six (16.6%) women had diagnosis of diabetes; of these 47 (61.8) in the NAFLD group. CONCLUSIONS: Results of this study support the statement that women with NAFLD have an adverse metabolic profile. Furthermore, our results show that hyperglycemia, hypertriglyceridemia and markers of liver injury such as AST/ALT > or = 1 may be useful for early recognition of NAFLD.


Assuntos
Fígado Gorduroso/etiologia , Cirrose Hepática/etiologia , Obesidade/complicações , Adulto , Alanina Transaminase/sangue , Aspartato Aminotransferases/sangue , Biomarcadores/sangue , Biópsia por Agulha , Glicemia/metabolismo , Índice de Massa Corporal , Ensaios Enzimáticos Clínicos , Estudos Transversais , Diabetes Mellitus/sangue , Diabetes Mellitus/etiologia , Progressão da Doença , Diagnóstico Precoce , Fígado Gorduroso/sangue , Fígado Gorduroso/diagnóstico por imagem , Feminino , Humanos , Hiperglicemia/sangue , Hiperglicemia/etiologia , Hipertrigliceridemia/sangue , Hipertrigliceridemia/etiologia , Cirrose Hepática/sangue , Cirrose Hepática/diagnóstico por imagem , México , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/diagnóstico por imagem , Valor Preditivo dos Testes , Fatores de Risco , Fatores Sexuais , Triglicerídeos/sangue , Ultrassonografia
15.
Mol Biol Cell ; 18(12): 4826-36, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17881732

RESUMO

Recent reports have indicated the participation of tight junction (TJ) proteins in the regulation of gene expression and cell proliferation. Here, we have studied the role of zona occludens (ZO)-2, a TJ peripheral protein, in the regulation of cyclin D1 transcription. We found that ZO-2 down-regulates cyclin D1 transcription in a dose-dependent manner. To understand how ZO-2 represses cyclin D1 promoter activity, we used deletion analyses and found that ZO-2 negatively regulates cyclin D1 transcription via an E box and that it diminishes cell proliferation. Because ZO-2 does not associate directly with DNA, electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay were used to identify the transcription factors mediating the ZO-2-repressive effect. c-Myc was found to bind the E box present in the cyclin D1 promoter, and the overexpression of c-Myc augmented the inhibition generated by ZO-2 transfection. The presence of ZO-2 and c-Myc in the same complex was further demonstrated by immunoprecipitation. ChIP and reporter gene assays using histone deacetylases (HDACs) inhibitors demonstrated that HDACs are necessary for ZO-2 repression and that HDAC1 is recruited to the E box. We conclude that ZO-2 down-regulates cyclin D1 transcription by interacting with the c-Myc/E box element and by recruiting HDAC1.


Assuntos
Ciclina D1/genética , Ciclina D1/metabolismo , Regulação para Baixo/genética , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Animais , Linhagem Celular , Cães , Elementos E-Box , Histona Desacetilases/metabolismo , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição/genética , Proteína da Zônula de Oclusão-2
16.
Artigo em Inglês | MEDLINE | ID: mdl-30327757

RESUMO

Entamoeba histolytica is the etiologic agent of human amoebiasis, disease that causes 40,000 to 100,000 deaths annually worldwide. The cytopathic activity as well as the growth and differentiation of this microorganism is dependent on both, extracellular and free cytoplasmic calcium. However, few is known about the proteins that regulate the calcium flux in this parasite. In many cells, the calcium extrusion from the cytosol is performed by plasma membrane Ca2+-ATPases and calcium/cation exchangers. The aim of this work was to identify a calcium/cation exchanger of E. histolytica and to analyze its possible role in some cellular processes triggered by calcium flux, such as the programmed cell death and in vitro virulence. By searching putative calcium/cation exchangers in the genome database of E. histolyica we identified a protein belonging to the CCX family (EhCCX). We generated a specific antibody against EhCCX, which showed that this protein was expressed in higher levels in E. histolytica than its orthologous in the non-pathogenic amoeba E. dispar. In addition, the expression of EhCCX was increased in trophozoites incubated with hydrogen peroxide. This E. histolytica exchanger was localized in the plasma membrane and in the membrane of some cytoplasmic vesicles. However, after 10 min of erythrophagocytosis, EhCCX was found predominantly in the plasma membrane of the trophozoites. On the other hand, the parasites that overexpress this exchanger contained higher cytosolic calcium levels than control, but the extrusion of calcium after the addition of hydrogen peroxide was more efficient in EhCCX-overexpressing trophozoites; consequently, the programmed cell death was retarded in these parasites. Interestingly, the overexpression of EhCCX increased the in vitro virulence of trophozoites. These results suggest that EhCCX plays important roles in the programmed cell death and in the in vitro virulence of E. histolytica.


Assuntos
Antiporters/metabolismo , Apoptose , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Cátions/metabolismo , Entamoeba histolytica/enzimologia , Antiporters/genética , ATPases Transportadoras de Cálcio/genética , Membrana Celular/enzimologia , Vesículas Citoplasmáticas/enzimologia , Entamoeba histolytica/patogenicidade , Entamoeba histolytica/fisiologia , Perfilação da Expressão Gênica , Virulência
17.
J Mater Chem B ; 3(47): 9140-9147, 2015 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-32263128

RESUMO

Protein conformational changes are detected both in vitro and for the first time in the presence of living cells using versatile plasmonic nanoplates. Au-edge-coated triangular silver nanoplates (AuTSNP) exhibit some of the highest refractive index sensitivity values recorded to date and exhibit a strong spectral response to surface biomolecular interactions. Large spectral shifts of over 30 nm distinguish between pH induced compact and extended conformations of the ubiquitous extracellular matrix protein fibronectin (Fn). Conformational transition of Fn from compact to extended is accompanied by a red spectral shift of 27 nm while a corresponding blue spectral shift of 25 nm accompanies the reverse conformational transition. Cleavage of Fn by cathepsin B, which plays an important role in cellular functions and in cancer metastasis is characterised by a blue spectral shift with detection in serum using a straightforward no-wash assay demonstrated. Spectral monitoring of nanoplates decorated with Fn and incubated with MDCK II cells shows extensive shifts of 156 nm and cellular morphological re-arrangement as Fn uncoils from a compact format to from fibrils within the extracellular matrix.

18.
Sci Rep ; 5: 11613, 2015 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-26112429

RESUMO

Impedance sensing of biological systems allows for monitoring of cell and tissue properties, including cell-substrate attachment, layer confluence, and the "tightness" of an epithelial tissue. These properties are critical for electrical detection of tissue health and viability in applications such as toxicological screening. Organic transistors based on conducting polymers offer a promising route to efficiently transduce ionic currents to attain high quality impedance spectra, but collection of complete impedance spectra can be time consuming (minutes). By applying uniform white noise at the gate of an organic electrochemical transistor (OECT), and measuring the resulting current noise, we are able to dynamically monitor the impedance and thus integrity of cultured epithelial monolayers. We show that noise sourcing can be used to track rapid monolayer disruption due to compounds which interfere with dynamic polymerization events crucial for maintaining cytoskeletal integrity, and to resolve sub-second alterations to the monolayer integrity.


Assuntos
Técnicas Eletroquímicas/métodos , Fenômenos Eletrofisiológicos/fisiologia , Polímeros/química , Transistores Eletrônicos , Animais , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Cães , Impedância Elétrica , Eletricidade , Técnicas Eletroquímicas/instrumentação , Eletrodos , Células Madin Darby de Rim Canino , Ruído , Reprodutibilidade dos Testes , Fatores de Tempo
19.
Adv Mater ; 26(41): 7083-90, 2014 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-25179835

RESUMO

A planar, conducting-polymer-based transistor for combined optical and electronic monitoring of live cells provides a unique platform for monitoring the health of cells in vitro. Monitoring of MDCK-I epithelial cells over several days is shown, along with a demonstration of the device for toxicology studies, of use in future drug discovery or diagnostics applications.


Assuntos
Equipamentos e Provisões Elétricas , Células Epiteliais/citologia , Dispositivos Ópticos , Poliestirenos/química , Tiofenos/química , Transistores Eletrônicos , Animais , Cães , Células Madin Darby de Rim Canino
20.
Adv Healthc Mater ; 3(7): 1053-60, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24497469

RESUMO

Ion flow across polarized epithelia is a tightly regulated process. Measurement of the transepithelial resistance is a highly relevant parameter for assessing the function or health of the tissue. Dynamic, electrical measurements of transepithelial ion flow are preferred as they provide the most accurate snapshot of effects of external stimuli. Enteric pathogens such as Salmonella typhimurium are known to disrupt ion flow in gastrointestinal epithelia. Here, for the first time, the use of organic transistors as a powerful potential alternative for front-line, disposable, high-throughput diagnostics of enteric pathogens is demonstrated. The transistors' ability to detect early and subtle changes in transepithelial ion flow is capitalized upon to develop a highly sensitive detector of epithelial integrity. Stable operation of the organic devices under physiological conditions is shown, followed by dynamic, pathogen-specific diagnosis of infection of epithelia. Further, operation of the device is possible in complex matrices, showing particular promise for food and safety applications.


Assuntos
Técnicas Eletroquímicas/métodos , Epitélio/microbiologia , Interações Hospedeiro-Patógeno/fisiologia , Infecções por Salmonella/fisiopatologia , Salmonella typhimurium/fisiologia , Animais , Células CACO-2 , Técnicas Eletroquímicas/instrumentação , Eletrodos , Desenho de Equipamento , Humanos , Cinética , Leite/microbiologia
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