RESUMO
Understanding cellular remodeling in response to mechanical stimuli is a critical step in elucidating mechanical activation of biochemical signaling pathways. Experimental evidence indicates that external stress-induced subcellular adaptation is accomplished through dynamic cytoskeletal reorganization. To study the interactions between subcellular structures involved in transducing mechanical signals, we combined experimental data and computational simulations to evaluate real-time mechanical adaptation of the actin cytoskeletal network. Actin cytoskeleton was imaged at the same time as an external tensile force was applied to live vascular smooth muscle cells using a fibronectin-functionalized atomic force microscope probe. Moreover, we performed computational simulations of active cytoskeletal networks under an external tensile force. The experimental data and simulation results suggest that mechanical structural adaptation occurs before chemical adaptation during filament bundle formation: actin filaments first align in the direction of the external force by initializing anisotropic filament orientations, then the chemical evolution of the network follows the anisotropic structures to further develop the bundle-like geometry. Our findings present an alternative two-step explanation for the formation of actin bundles due to mechanical stimulation and provide new insights into the mechanism of mechanotransduction.
Assuntos
Citoesqueleto de Actina/fisiologia , Resistência à Tração , Actinas/fisiologia , Animais , Anisotropia , Fenômenos Biomecânicos , Células Cultivadas , Simulação por Computador , Fibronectinas/fisiologia , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Mecanotransdução Celular , Microscopia de Força Atômica , Miócitos de Músculo Liso/metabolismo , Miosinas/fisiologia , Ratos , Estresse MecânicoRESUMO
Numerous studies demonstrating that statistical errors are common in basic science publications have led to calls to improve statistical training for basic scientists. In this article, we sought to evaluate statistical requirements for PhD training and to identify opportunities for improving biostatistics education in the basic sciences. We provide recommendations for improving statistics training for basic biomedical scientists, including: 1. Encouraging departments to require statistics training, 2. Tailoring coursework to the students' fields of research, and 3. Developing tools and strategies to promote education and dissemination of statistical knowledge. We also provide a list of statistical considerations that should be addressed in statistics education for basic scientists.
Assuntos
Bioestatística , Ciência/educaçãoRESUMO
Menin (MEN1) is a tumor-suppressor protein in neuroendocrine tissue. Therefore, we tested the novel hypothesis that menin regulates cholangiocarcinoma proliferation. Menin and miR-24 expression levels were measured in the following intrahepatic and extrahepatic cholangiocarcinoma (CCA) cell lines, Mz-ChA-1, TFK-1, SG231, CCLP, HuCCT-1, and HuH-28, as well as the nonmalignant human intrahepatic biliary line, H69. miR-24 miRNA and menin protein levels were manipulated in vitro in Mz-ChA-1 cell lines. Markers of proliferation and angiogenesis (Ki-67, vascular endothelial growth factors A/C, vascular endothelial growth factor receptors 2/3, angiopoietin 1/2, and angiopoietin receptors 1/2) were evaluated. Mz-ChA-1 cells were injected into the flanks of nude mice and treated with miR-24 inhibitor or inhibitor scramble. Menin expression was decreased in advanced CCA specimens, whereas miR-24 expression was increased in CCA. Menin overexpression decreased proliferation, angiogenesis, migration, and invasion. Inhibition of miR-24 increased menin protein expression while decreasing proliferation, angiogenesis, migration, and invasion. miR-24 was shown to negatively regulate menin expression by luciferase assay. Tumor burden and expression of proliferative and angiogenic markers was decreased in the miR-24 inhibited tumor group compared to controls. Interestingly, treated tumors were more fibrotic than the control group. miR-24-dependent expression of menin may be important in the regulation of nonmalignant and CCA proliferation and may be an additional therapeutic tool for managing CCA progression.
Assuntos
Colangiocarcinoma/genética , Colangiocarcinoma/patologia , MicroRNAs/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Idoso , Indutores da Angiogênese/metabolismo , Animais , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares/patologia , Biópsia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The tumor microenvironment of cholangiocarcinoma (CCA) is composed of numerous cells, including mast cells (MCs). MCs release histamine, which increases CCA progression and angiogenesis. Cholangiocytes secrete stem cell factor, which functions via the MC growth factor receptor c-Kit. Here, we show that cholangiocytes express histidine decarboxylase and its inhibition reduces CCA growth. MC recruitment in the tumor microenvironment increased CCA growth. MC infiltration and MC markers were detected by toluidine blue staining and real-time PCR in human biopsies and in tumors from athymic mice treated with saline, histamine, histidine decarboxylase inhibitor, or cromolyn sodium. Tumor growth, angiogenesis, and epithelial-mesenchymal transition (EMT)/extracellular matrix (ECM) markers were measured in mice treated with cromolyn sodium. In vitro, human CCA cells were treated with MC supernatant fluids before evaluating angiogenesis and EMT/ECM expression. Migration assays were performed with CCA cells treated with the stem cell factor inhibitor. MC supernatant fluids increased CCA histidine decarboxylase, vascular endothelial growth factor, and MC/EMT/ECM expression that decreased with pretreatment of cromolyn sodium. MCs were found in human biopsies. In mice treated with cromolyn sodium, MC infiltration and tumor growth decreased. Inhibition of CCA stem cell factor blocked MC migration and MC/EMT/ECM in CCA. MCs migrate into CCA tumor microenvironment via c-Kit/stem cell factor and increase tumor progression, angiogenesis, EMT switch, and ECM degradation.
Assuntos
Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , Histamina/metabolismo , Mastócitos/metabolismo , Microambiente Tumoral/imunologia , Animais , Neoplasias dos Ductos Biliares/imunologia , Diferenciação Celular/imunologia , Proliferação de Células , Colangiocarcinoma/imunologia , Transição Epitelial-Mesenquimal/imunologia , Imunofluorescência , Xenoenxertos , Humanos , Imuno-Histoquímica , Mastócitos/citologia , Camundongos , Camundongos Nus , Neovascularização Patológica/imunologia , Neovascularização Patológica/patologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Fator de Células-Tronco/metabolismo , Análise Serial de TecidosRESUMO
BACKGROUND & AIMS: Disruption of circadian rhythm is associated with cancer development and progression. MicroRNAs (miRNAs) are a class of small non-coding RNAs that trigger mRNA translation inhibition. We aimed to evaluate the role of Per1 and related miRNAs in cholangiocarcinoma growth. METHODS: The expression of clock genes was evaluated in human cholangiocarcinoma tissue arrays and cholangiocarcinoma lines. The rhythmic expression of clock genes was evaluated in cholangiocarcinoma cells and H69 (non-malignant cholangiocytes) by qPCR. We measured cell proliferation, cell cycle and apoptosis in Mz-ChA-1 cells after Per1 overexpression. We examined tumor growth in vivo after injection of Per1 overexpressing cells. We verified miRNAs that targets Per1. The circadian rhythm of miR-34a was evaluated in cholangiocarcinoma and H69 cells. We evaluated cell proliferation, apoptosis and invasion after inhibition of miR-34a in vitro, and the potential molecular mechanisms by mRNA profiling after overexpression of Per1. RESULTS: Expression of Per1 was decreased in cholangiocarcinoma. The circadian rhythm of Per1 expression was lost in cholangiocarcinoma cells. Decreased cell proliferation, lower G2/M arrest, and enhanced apoptosis were shown in Per1 overexpressing cells. An in vivo study revealed decreased tumor growth, decreased proliferation, angiogenesis and metastasis after overexpressing Per1. Per1 was verified as a target of miR-34a. miR-34a was rhythmically expressed in cholangiocarcinoma cells and H69. The inhibition of miR-34a decreased proliferation, migration and invasion in cholangiocarcinoma cells. mRNA profiling has shown that overexpression of Per1 inhibits cell growth through regulation of multiple cancer-related pathways, such as cell cycle, cell growth and apoptosis pathways. CONCLUSIONS: Disruption of circadian rhythms of clock genes contribute to the malignant phenotypes of human cholangiocarcinoma. LAY SUMMARY: The current study is about how biological clock and its regulators affect the bile duct tumor growth. The disruption of biological clock has a negative impact in different cancers. Per1 is a gene that is involved in maintaining the biological clock and show 24h oscillation. Reduced levels of Per1 and disruption of 24h circadian rhythm was found in bile duct cancer cells. Therefore, a genetic modified bile duct cancer cells was created. It has a higher level of Per1 expression and partially recovered circadian rhythm. Those genetic modified cells also displayed slower cell growth or higher rate of cell death. We also used mice model that lack of immune system to show that our genetic modified bile duct cells form smaller tumor. In addition, we tried to see how Per1 is communicating with other genes in regarding of controlling the tumor growth. We found Per1 is regulated by microRNA-34a, a small non-coding RNA that directly binds to genes and inhibit gene expression. Decreased level of miR-34a has also significantly reduced tumor growth through controlling the cell growth and cell death balance. Therefore bile duct cancer patients may be treated with miR-34a inhibitor or Per1 stimulator in the future.
Assuntos
Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , MicroRNAs/fisiologia , Proteínas Circadianas Period/fisiologia , Animais , Proteínas CLOCK/genética , Linhagem Celular Tumoral , Proliferação de Células , Ritmo Circadiano , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Invasividade NeoplásicaRESUMO
In this study, we examined the ability of vasoactive agonists to induce dynamic changes in vascular smooth muscle cell (VSMC) elasticity and adhesion, and tested the hypothesis that these events are coordinated with rapid remodelling of the cortical cytoskeleton. Real-time measurement of cell elasticity was performed with atomic force microscopy (AFM) and adhesion was assessed with AFM probes coated with fibronectin (FN). Temporal data were analysed using an Eigen-decomposition method. Elasticity in VSMCs displayed temporal oscillations with three components at approximately 0.001, 0.004 and 0.07 Hz, respectively. Similarly, adhesion displayed a similar oscillatory pattern. Angiotensin II (ANG II, 10(-6) M) increased (+100%) the amplitude of the oscillations, whereas the vasodilator adenosine (ADO, 10(-4) M) reduced oscillation amplitude (-30%). To test whether the oscillatory changes were related to the architectural alterations in cortical cytoskeleton, the topography of the submembranous actin cytoskeleton (100-300 nm depth) was acquired with AFM. These data were analysed to compare cortical actin fibre distribution and orientation before and after treatment with vasoactive agonists. The results showed that ANG II increased the density of stress fibres by 23%, while ADO decreased the density of the stress fibres by 45%. AFM data were supported by Western blot and confocal microscopy. Collectively, these observations indicate that VSMC cytoskeletal structure and adhesion to the extracellular matrix are dynamically altered in response to agonist stimulation. Thus, vasoactive agonists probably invoke unique mechanisms that dynamically alter the behaviour and structure of both the VSMC cytoskeleton and focal adhesions to efficiently support the normal contractile behaviour of VSMCs.
Assuntos
Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Vasoconstritores/farmacologia , Actinas/metabolismo , Adenosina/farmacologia , Adenosina/fisiologia , Angiotensina II/farmacologia , Angiotensina II/fisiologia , Animais , Fenômenos Biomecânicos , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/fisiologia , Módulo de Elasticidade/efeitos dos fármacos , Módulo de Elasticidade/fisiologia , Elasticidade/efeitos dos fármacos , Elasticidade/fisiologia , Microscopia de Força Atômica , Microscopia Confocal , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Increased vascular stiffness is fundamental to hypertension, and its complications, including atherosclerosis, suggest that therapy should also be directed at vascular stiffness, rather than just the regulation of peripheral vascular resistance. It is currently held that the underlying mechanisms of vascular stiffness in hypertension only involve the extracellular matrix and endothelium. We hypothesized that increased large-artery stiffness in hypertension is partly due to intrinsic mechanical properties of vascular smooth muscle cells. After confirming increased arterial pressure and aortic stiffness in spontaneously hypertensive rats, we found increased elastic stiffness of aortic smooth muscle cells of spontaneously hypertensive rats compared with Wistar-Kyoto normotensive controls using both an engineered aortic tissue model and atomic force microscopy nanoindentation. Additionally, we observed different temporal oscillations in the stiffness of vascular smooth muscle cells derived from hypertensive and control rats, suggesting that a dynamic component to cellular elastic stiffness is altered in hypertension. Treatment with inhibitors of vascular smooth muscle cell cytoskeletal proteins reduced vascular smooth muscle cell stiffness from hypertensive and control rats, suggesting their participation in the mechanism. This is the first study demonstrating that stiffness of individual vascular smooth muscle cells mediates vascular stiffness in hypertension, a novel concept, which may elucidate new therapies for hypertension and for vascular stiffness.
Assuntos
Hipertensão/fisiopatologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/metabolismo , Rigidez Vascular , Animais , Aorta/metabolismo , Aorta/fisiopatologia , Pressão Arterial , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Elasticidade , Hipertensão/metabolismo , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Fatores de Tempo , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
The aetiological agent of Lyme disease, Borrelia burgdorferi, is transmitted via infected Ixodes spp. ticks. Infection, if untreated, results in dissemination to multiple tissues and significant morbidity. Recent developments in bioluminescence technology allow in vivo imaging and quantification of pathogenic organisms during infection. Herein, luciferase-expressing B. burgdorferi and strains lacking the decorin adhesins DbpA and DbpB, as well as the fibronectin adhesin BBK32, were quantified by bioluminescent imaging to further evaluate their pathogenic potential in infected mice. Quantification of bacterial load was verified by quantitative PCR (qPCR) and cultivation. B. burgdorferi lacking DbpA and DbpB were only seen at the 1 h time point post infection, consistent with its low infectivity phenotype. The bbk32 mutant exhibited a significant decrease in its infectious load at day 7 relative to its parent. This effect was most pronounced at lower inocula and imaging correlated well with qPCR data. These data suggest that BBK32-mediated binding plays an important role in B. burgdorferi colonization. As such, in vivo imaging of bioluminescent Borrelia provides a sensitive means to detect, quantify and temporally characterize borrelial dissemination in a non-invasive, physiologically relevant environment and, more importantly, demonstrated a quantifiable infectivity defect for the bbk32 mutant.
Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/química , Borrelia burgdorferi/metabolismo , Fibronectinas/metabolismo , Medições Luminescentes/métodos , Doença de Lyme/microbiologia , Imagem Molecular/métodos , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Feminino , Humanos , Luciferases/química , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ligação ProteicaRESUMO
BACKGROUND: Chronic kidney disease (CKD) is a major public health problem, and despite continued research in the field, there is still a need to identify both biomarkers of risk and progression, as well as potential therapeutic targets. Structural equation modeling (SEM) is a family of statistical techniques that has been utilized in the fields of sociology and psychology for many years; however, its utilization in the biological sciences is relatively novel. SEM's ability to investigate complex relationships in an efficient, single model could be utilized to understand the progression of CKD, as well as to develop a predictive model to assess kidney status in the patient. METHODS: Fischer 344 rats were fed either an ad libitum diet or a calorically restricted diet, and a time-course study of kidney structure and function was performed. EQS, a SEM software package, was utilized to generate five CKD models of the Fisher 344 rat and identify relationships between measured variables and estimates of kidney damage and kidney function. RESULTS: All models identified strong relationships between a biomarker for CKD, kidney injury molecule-1 (Kim-1) and kidney damage, in the Fischer 344 rat CKD model. Models also indicate a strong relationship between age and renal damage and dysfunction. CONCLUSION: SEM can be used to model CKD and could be useful to examine biomarkers in CKD patients.
Assuntos
Moléculas de Adesão Celular/metabolismo , Nefropatias/metabolismo , Rim/metabolismo , Modelos Estatísticos , Fatores Etários , Animais , Biomarcadores/metabolismo , Nitrogênio da Ureia Sanguínea , Doença Crônica , Creatinina/sangue , Rim/patologia , Rim/fisiopatologia , Nefropatias/patologia , Nefropatias/fisiopatologia , Masculino , Ratos , Ratos Endogâmicos F344 , Método Simples-CegoRESUMO
RATIONALE: Increased aortic stiffness, an important feature of many vascular diseases, eg, aging, hypertension, atherosclerosis, and aortic aneurysms, is assumed because of changes in extracellular matrix (ECM). OBJECTIVE: We tested the hypothesis that the mechanisms also involve intrinsic stiffening of vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Stiffness was measured in vitro both by atomic force microscopy (AFM) and in a reconstituted tissue model, using VSMCs from aorta of young versus old male monkeys (Macaca fascicularis) (n=7/group), where aortic stiffness increases by 200% in vivo. The apparent elastic modulus was increased (P<0.05) in old (41.7+/-0.5 kPa) versus young (12.8+/-0.3 kPa) VSMCs but not after disassembly of the actin cytoskeleton with cytochalasin D. Stiffness of the VSMCs in the reconstituted tissue model was also higher (P<0.05) in old (23.3+/-3.0 kPa) than in young (13.7+/-2.4 kPa). CONCLUSIONS: These data support the novel concept, not appreciated previously, that increased vascular stiffness with aging is attributable not only to changes in ECM but also to intrinsic changes in VSMCs.
Assuntos
Envelhecimento/patologia , Doenças da Aorta/patologia , Músculo Liso Vascular/patologia , Actinas/metabolismo , Fatores Etários , Envelhecimento/metabolismo , Animais , Aorta Torácica/patologia , Doenças da Aorta/etiologia , Células Cultivadas , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Módulo de Elasticidade , Integrina beta1/metabolismo , Macaca fascicularis , Masculino , Microscopia de Força Atômica , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Tubulina (Proteína)/metabolismo , Vimentina/metabolismoRESUMO
Morphological adaptations of vascular smooth muscle cells (VSMC) to the mechanically active environment in which they reside, are mediated by direct interactions with the extracellular matrix (ECM) which induces physiological changes at the intracellular level. This study aimed to analyze the effects of the ECM on RhoA-induced mechanical signaling that controls actin organization and focal adhesion formation. VSMC were transfected with RhoA constructs (wild type, dominant negative or constitutively active) and plated on different ECM proteins used as substrate (fibronectin, collagen IV, collagen I, and laminin) or poly-l-lysine as control. Morphological changes of the VSMC were detected by fluorescence confocal microscopy and total internal reflection fluorescence (TIRF) microscopy, and were independently verified using adhesion assays and Western blot analysis. Our results showed that the ECM has an important role in cell spreading, adhesion and morphology with a direct effect on modulating RhoA signaling. RhoA activity significantly affected the stress fibers and focal adhesions reorganization, but in a context imposed by the ECM. Thus, RhoA activity modulation in VSMC induced an increased activation of stress fibers and FA formation at 5h, while a significant inhibition was recorded at 24h after plating on the different ECM. Our findings provide biophysical evidence that ECM modulates VSMC response to mechanical stimuli inducing intracellular biochemical signaling involved in cellular adaptation to the local microenvironment.
Assuntos
Proteínas da Matriz Extracelular/farmacologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Arteríolas/citologia , Adesões Focais/metabolismo , Microscopia de Fluorescência , Músculo Liso Vascular/citologia , Ratos , Estresse MecânicoRESUMO
Borrelia burgdorferi, the etiological agent of Lyme disease, adapts to unique host environments as a consequence of its complex life cycle that spans both arthropod and mammalian species. In this regard, B. burgdorferi must adapt to various environmental signals, pHs, temperatures, and O(2) and CO(2) levels to establish infectious foci. We hypothesize that the BosR protein functions as a global regulator that is required for both borrelial oxidative homeostasis and pathogenesis. To assess the role of BosR in B. burgdorferi, we constructed an IPTG (isopropyl-beta-d-thiogalactopyranoside)-regulated bosR strain. The selective decrease of bosR resulted in a change in growth when cells were cultured either anaerobically or microaerobically; however, a distinct growth defect was observed for anaerobically grown B. burgdorferi relative to the growth attenuation observed for microaerobically grown B. burgdorferi. B. burgdorferi cells in which BosR levels were reduced were more sensitive to hydrogen peroxide and produced lower levels of NapA (Dps) and SodA, proteins involved in the oxidative stress response. In addition, the levels of OspC and DbpA were also induced coincident with increased BosR levels, suggesting that BosR interfaces with the RpoS regulatory cascade, which is known to modulate virulence gene expression in B. burgdorferi. Taken together, these results indicate that BosR is involved in the resistance of B. burgdorferi to oxidative stressors and affects the expression of genes, either directly or indirectly, whose products are important in borrelial pathogenesis.
Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , Proteínas de Bactérias/genética , Borrelia burgdorferi/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Peróxido de Hidrogênio/farmacologia , Mutação , Estresse OxidativoRESUMO
Summary Borrelia burgdorferi, the Lyme disease spirochete, adapts as it moves between the arthropod and mammalian hosts that it infects. We hypothesize that BosR serves as a global regulator in B. burgdorferi to modulate the oxidative stress response and adapt to mammalian hosts. To test this hypothesis, a bosR mutant in a low-passage B. burgdorferi isolate was constructed. The resulting bosR::kan(R) strain was altered when grown microaerobically or anaerobically suggesting that BosR is required for optimal replication under both growth conditions. The absence of BosR increased the sensitivity of B. burgdorferi to hydrogen peroxide and reduced the synthesis of Cdr and NapA, proteins important for cellular redox balance and the oxidative stress response, respectively, suggesting an important role for BosR in borrelial oxidative homeostasis. For the bosR mutant, the production of RpoS was abrogated and resulted in the loss of OspC and DbpA, suggesting that BosR interfaces with the Rrp2-RpoN-RpoS regulatory cascade. Consistent with the linkage to RpoS, cells lacking bosR were non-infectious in the mouse model of infection. These results indicate that BosR is required for resistance to oxidative stressors and provides a regulatory response that is necessary for B. burgdorferi pathogenesis.
Assuntos
Proteínas de Bactérias/fisiologia , Borrelia burgdorferi/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação Bacteriana da Expressão Gênica , Fator sigma/biossíntese , Estresse Fisiológico , Fatores de Virulência/biossíntese , Estruturas Animais/microbiologia , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Borrelia burgdorferi/efeitos dos fármacos , Borrelia burgdorferi/patogenicidade , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Deleção de Genes , Humanos , Peróxido de Hidrogênio/toxicidade , Doença de Lyme/microbiologia , Camundongos , Camundongos Endogâmicos C3H , Viabilidade Microbiana , Mutagênese Insercional , Estresse Oxidativo , VirulênciaRESUMO
Integrins link the extracellular matrix (ECM) with the intracellular cytoskeleton and other cell adhesion-associated signaling proteins to function as mechanotransducers. However, direct quantitative measurements of the cardiomyocyte mechanical state and its relationship to the interactions between specific ECM proteins and integrins are lacking. The purpose of this study was to characterize the interactions between the ECM protein fibronectin (FN) and integrins in cardiomyocytes and to test the hypothesis that these interactions would vary during contraction and relaxation states in cardiomyocytes. Using atomic force microscopy, we quantified the unbinding force (adhesion force) and adhesion probability between integrins and FN and correlated these measurements with the contractile state as indexed by cell stiffness on freshly isolated mouse cardiomyocytes. Experiments were performed in normal physiological (control), high-K(+) (tonically contracted), or low-Ca(2+) (fully relaxed) solutions. Under control conditions, the initial peak of adhesion force between FN and myocyte alpha(3)beta(1)- and/or alpha(5)beta(1)-integrins was 39.6 +/- 1.3 pN. The binding specificity between FN and alpha(3)beta(1)- and alpha(5)beta(1)-integrins was verified by using monoclonal antibodies against alpha(3)-, alpha(5)-, alpha(3) + alpha(5)-, or beta(1)-integrin subunits, which inhibited binding by 48%, 65%, 70%, or 75%, respectively. Cytochalasin D or 2,3-butanedione monoxime (BDM), to disrupt the actin cytoskeleton or block myofilament function, respectively, significantly decreased the cell stiffness; however, the adhesion force and binding probability were not altered. Tonic contraction with high-K(+) solution increased total cell adhesion (1.2-fold) and cell stiffness (27.5-fold) compared with fully relaxed cells with low-Ca(2+) solution. However, it could be partially prevented by high-K(+) bath solution containing BDM, which suppresses contraction by inhibiting the actin-myosin interactions. Thus, our results demonstrate that integrin binding to FN is modulated by the contractile state of cardiac myocytes.
Assuntos
Fibronectinas/fisiologia , Integrinas/fisiologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Actinas/fisiologia , Animais , Células Cultivadas , Citoesqueleto/fisiologia , Matriz Extracelular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos , Modelos Animais , Miosinas/fisiologiaRESUMO
Atomic force microscopy (AFM) was used to investigate the interaction between alpha5beta1 integrin and fibronectin (FN) in the presence of divalent cations. AFM probes were labeled with FN and used to measure binding strength between alpha5beta1 integrin and FN by quantifying the force required to break single FN-integrin bonds on a physiological range of loading rates (100-10,000 pN/s). The force necessary to rupture single alpha5beta1-FN bond increased twofold over the regime of loading rates investigated. Changes in Mg(2+) and Ca(2+) concentration affected the thermodynamical parameters of the interaction and modulated the binding energy. These data indicate that the external ionic environment in which vascular smooth muscle cells reside, influences the mechanical parameters that define the interaction between the extracellular matrix and integrins. Thus, in a dynamic mechanical environment such as the vascular wall, thermodynamic binding properties between FN and alpha5beta1 integrin vary in relation to locally applied loads and divalent cations concentrations. These changes can be recorded as direct measurements on live smooth muscle cells by using AFM.
Assuntos
Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Integrina alfa5beta1/metabolismo , Magnésio/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Animais , Adesão Celular/fisiologia , Células Cultivadas , Microscopia de Força Atômica/métodos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Ligação Proteica , Ratos , TermodinâmicaRESUMO
Lyme disease, caused by Borrelia burgdorferi, is an inflammatory multistage infection, consisting of localized, disseminated, and persistent disease stages, impacting several organ systems through poorly defined gene regulation mechanisms. The purpose of this study is to further characterize the spatiotemporal transcriptional regulation of B. burgdorferi during mammalian infection of borrelial oxidative stress regulator (bosR) and decorin binding protein (dbpBA) by utilizing bioluminescent B. burgdorferi reporter strains and in vivo imaging. Fluctuating borrelial load was also monitored and used for normalization to evaluate expression levels. bosR transcription is driven by two promoters, Pbb0648 and PbosR, and we focused on the native promoter. bosR expression is low relative to the robustly expressed dbpBA throughout infection. In distal tissues, bosR was the highest in the heart during in the first week whereas dbpBA was readily detectable at all time points with each tissue displaying a distinct expression pattern. This data suggests bosR may have a role in heart colonization and the induction of dbpBA indicates a RpoS independent transcriptional regulation occurring in the mammalian cycle of pathogenesis. These finding demonstrate that B. burgdorferi engages unknown genetic mechanisms to uniquely respond to mammalian tissue environments and/or changing host response over time.
Assuntos
Adesinas Bacterianas/genética , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Regulação Bacteriana da Expressão Gênica , Doença de Lyme/microbiologia , Transcrição Gênica , Adesinas Bacterianas/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Genes Reporter , Medições Luminescentes , Camundongos , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
Aging is a risk factor for chronic kidney disease (CKD) and is itself associated with alterations in renal structure and function. There are no specific interventions to attenuate age-dependent renal dysfunction and the mechanism(s) responsible for these deficits have not been fully elucidated. In this study, male Fischer 344 rats, which develop age-dependent nephropathy, were feed a casein- or soy protein diet beginning at 16 mon (late life intervention) and renal structure and function was assessed at 20 mon. The soy diet did not significantly affect body weight, but was renoprotective as assessed by decreased proteinuria, increased glomerular filtration rate (GFR) and decreased urinary kidney injury molecule-1 (Kim-1). Renal fibrosis, as assessed by hydroxyproline content, was decreased by the soy diet, as were several indicators of inflammation. RNA sequencing identified several candidates for the renoprotective effects of soy, including decreased expression of Twist2, a basic helix-loop-helix transcription factor that network analysis suggest may regulate the expression of several genes associated with renal dysfunction. Twist2 expression is upregulated in the aging kidney and the unilateral ureteral obstruction of fibrosis; the expression is limited to distal tubules of mice. Taken together, these data demonstrate the renoprotective potential of soy protein, putatively by reducing inflammation and fibrosis, and identify Twist2 as a novel mediator of renal dysfunction that is targeted by soy.
RESUMO
BACKGROUND: We introduce Approximate Entropy as a mathematical method of analysis for microarray data. Approximate entropy is applied here as a method to classify the complex gene expression patterns resultant of a clinical sample set. Since Entropy is a measure of disorder in a system, we believe that by choosing genes which display minimum entropy in normal controls and maximum entropy in the cancerous sample set we will be able to distinguish those genes which display the greatest variability in the cancerous set. Here we describe a method of utilizing Approximate Sample Entropy (ApSE) analysis to identify genes of interest with the highest probability of producing an accurate, predictive, classification model from our data set. RESULTS: In the development of a diagnostic gene-expression profile for cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma of the cervix, we identified 208 genes which are unchanging in all normal tissue samples, yet exhibit a random pattern indicative of the genetic instability and heterogeneity of malignant cells. This may be measured in terms of the ApSE when compared to normal tissue. We have validated 10 of these genes on 10 Normal and 20 cancer and CIN3 samples. We report that the predictive value of the sample entropy calculation for these 10 genes of interest is promising (75% sensitivity, 80% specificity for prediction of cervical cancer over CIN3). CONCLUSION: The success of the Approximate Sample Entropy approach in discerning alterations in complexity from biological system with such relatively small sample set, and extracting biologically relevant genes of interest hold great promise.
Assuntos
Perfilação da Expressão Gênica/métodos , Displasia do Colo do Útero/genética , Neoplasias do Colo do Útero/genética , Análise por Conglomerados , Entropia , Feminino , Humanos , Modelos Teóricos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias do Colo do Útero/diagnóstico , Displasia do Colo do Útero/diagnósticoRESUMO
A porcine aortic coarctation model was used to examine regulation of gene expression in early hypertensive vascular remodeling. Aortic segments were collected proximal (high pressure) and distal (low pressure) to the coarctation after 2 wk of sustained hypertension (mean arterial pressure>150 mmHg). Porcine 10K oligoarrays used for gene expression profiling of the two regions of aorta revealed downregulation of cytoskeletal and upregulation of extracellular region genes relative to the whole genome. A genomic database search for transforming growth factor-beta (TGF-beta) control elements showed that 19% of the genes that changed expression due to hypertension contained putative TGF-beta control elements. Real-time RT-PCR and microarray analysis showed no change in expression of TGF-beta1, TGF-beta2, TGF-beta3, or bone morphogenetic proteins-2 and -4, yet immunohistochemical staining for phosphorylated SMAD2, an indicator of TGF-beta signaling, and for phosphorylated SMAD1/5/8, an indicator of signaling through the bone morphogenetic proteins, showed the highest percentage of positively stained cells in the proximal aortic segments of occluded animals. For TGF-beta signaling, this increase was significantly different than for sham-operated controls. Western blot analysis showed no difference in total TGF-beta1 protein levels with respect to treatment or aortic segment. Immunohistochemistry showed that the protein levels of latency-associated peptide was decreased in proximal segments of occluded animals. Collectively, these results suggest that activation of TGF-beta, but not altered expression, may be a major mechanism regulating early hypertensive vascular remodeling.
Assuntos
Aorta/metabolismo , Coartação Aórtica/genética , Hipertensão/genética , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Animais , Aorta/diagnóstico por imagem , Aorta/fisiopatologia , Coartação Aórtica/diagnóstico por imagem , Coartação Aórtica/metabolismo , Coartação Aórtica/fisiopatologia , Western Blotting , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/genética , Bases de Dados Genéticas , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Hipertensão/diagnóstico por imagem , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Imuno-Histoquímica , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Reação em Cadeia da Polimerase , Fluxo Sanguíneo Regional/genética , Proteínas Smad/genética , Proteínas Smad/metabolismo , Suínos , Porco Miniatura , Fator de Crescimento Transformador beta/metabolismo , Ultrassonografia Doppler em CoresRESUMO
Cardiac myosin binding protein-C (cMyBP-C) phosphorylation prevents aging-related cardiac dysfunction. We tested this hypothesis by aging genetic mouse models of hypophosphorylated cMyBP-C, wild-type equivalent, and phosphorylated-mimetic cMyBP-C for 18 to 20 months. Phosphorylated-mimetic cMyBP-C mice exhibited better survival, better preservation of systolic and diastolic functions, and unchanging wall thickness. Wild-type equivalent mice showed decreasing cMyBP-C phosphorylation along with worsening cardiac function and hypertrophy approaching those found in hypophosphorylated cMyBP-C mice. Intact papillary muscle experiments suggested that cMyBP-C phosphorylation increased cross-bridge detachment rates as the underlying mechanism. Thus, phosphorylating cMyBP-C is a novel mechanism with potential to treat aging-related cardiac dysfunction.