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1.
J Biomed Mater Res A ; 110(12): 1944-1952, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35950648

RESUMO

Small animal models have demonstrated the efficacy of ex vivo regional gene therapy using scaffolds loaded with BMP-2-expressing mesenchymal stem cells (MSCs). Prior to clinical translation, optimization of seeding techniques of the transduced cells will be important to minimize time and resource expenditure, while maximizing cell delivery and BMP-2 production. No prior studies have investigated cell-seeding techniques in the setting of transduced cells for gene therapy applications. Using BMP-2-expressing transduced adipose-derived MSCs and a porous ceramic scaffold, this study compared previously described static and dynamic seeding techniques with respect to cell seeding efficiency, uniformity of cell distribution, and in vitro BMP-2 production. Static and negative pressure seeding techniques demonstrated the highest seeding efficiency, while orbital shaking was associated with the greatest increases in BMP-2 production per cell. Low density cell suspensions were associated with the highest seeding efficiency and uniformity of cell distribution, and the greatest increases in BMP-2 production from 2 to 7 days after seeding. Our results highlight the potential for development of an optimized cell density and seeding technique that could greatly reduce the number of MSCs needed to produce therapeutic BMP-2 levels in clinical situations. Further studies are needed to investigate in vivo effects of cell seeding techniques on bone healing.


Assuntos
Proteína Morfogenética Óssea 2 , Células-Tronco Mesenquimais , Animais , Proteína Morfogenética Óssea 2/farmacologia , Contagem de Células , Cerâmica , Terapia Genética/métodos , Humanos , Osteogênese , Porosidade , Tecidos Suporte
2.
Development ; 148(23)2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34738615

RESUMO

The development of the first synapse of the visual system between photoreceptors and bipolar cells in the outer plexiform layer (OPL) of the human retina is crucial for visual processing but poorly understood. By studying the maturation state and spatial organization of photoreceptors, depolarizing bipolar cells and horizontal cells in the human fetal retina, we establish a pseudo-temporal staging system for OPL development that we term OPL-Stages 0 to 4. This was validated through quantification of increasingly precise subcellular localization of bassoon to the OPL with each stage (P<0.0001). By applying these OPL staging criteria to human retinal organoids (HROs) derived from human embryonic and induced pluripotent stem cells, we observed comparable maturation from OPL-Stage 0 at day 100 in culture up to OPL-Stage 3 by day 160. Quantification of presynaptic protein localization confirmed progression from OPL-Stage 0 to 3 (P<0.0001). Overall, this study defines stages of human OPL development through mid-gestation and establishes HROs as a model system that recapitulates key aspects of human photoreceptor-bipolar cell synaptogenesis in vitro.


Assuntos
Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Organoides/metabolismo , Retina/metabolismo , Linhagem Celular , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/citologia , Retina/citologia
3.
Res Sq ; 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34312617

RESUMO

Increasing evidence of new-onset diabetes during the COVID19 pandemic indicates that the SARS-CoV2 virus may drive beta-cell dysfunction leading to diabetes, but it is unclear if it is a primary or secondary effect. Here, we present evidence of SARS-CoV-2 infection of pancreatic beta cells in vivo using a robust and reproducible non-human primates model of mild to moderate COVID19 pathogenesis. Pancreas from SARS-CoV-2 infected subjects were positive for the SARS-CoV2 spike protein by immunohistochemistry and structures indicative of viral replication were evident by electron microscopy. Total beta cell area was decreased in SARS-CoV-2-infected pancreas, attributable to beta cell atrophy. Beta cell granularity was decreased. These histologic phenotypes persisted beyond the duration of the clinical disease course. Detailed electron microscopy of SARS-CoV-2 infected beta-cells revealed ultrastructural hallmarks of beta cell stress that are seen in islets of patients with Type 2 diabetes, including disrupted mitochondria and dilated endoplasmic reticulum. To assess the metabolic status of beta cells from SARS-CoV-2-infected subjects, we used fluorescence life-time imaging to measure the ratio of free and bound NADH as a surrogate of glycolytic and oxidative metabolism. We report an increase in free NADH levels, suggesting that beta cells from SARS-CoV-2-infected subjects adopt a more glycolytic metabolic profile. Taken together, we conclude that SARS-CoV-2 infection induces beta cell stress that may compromise beta-cell function beyond the duration of the disease course. This raises the possibility that the beta cell stress and injury may have clinical implications of the long-term future health of patients that have recovered from COVID19.

4.
J Biol Chem ; 295(41): 13981-13993, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32817337

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) have long been viewed as mere housekeeping proteins and have therefore often been overlooked in drug discovery. However, recent findings have revealed that many aaRSs have noncanonical functions, and several of the aaRSs have been linked to autoimmune diseases, cancer, and neurological disorders. Deciphering these roles has been challenging because of a lack of tools to enable their study. To help solve this problem, we have generated recombinant high-affinity antibodies for a collection of thirteen cytoplasmic and one mitochondrial aaRSs. Selected domains of these proteins were produced recombinantly in Escherichia coli and used as antigens in phage display selections using a synthetic human single-chain fragment variable library. All targets yielded large sets of antibody candidates that were validated through a panel of binding assays against the purified antigen. Furthermore, the top-performing binders were tested in immunoprecipitation followed by MS for their ability to capture the endogenous protein from mammalian cell lysates. For antibodies targeting individual members of the multi-tRNA synthetase complex, we were able to detect all members of the complex, co-immunoprecipitating with the target, in several cell types. The functionality of a subset of binders for each target was also confirmed using immunofluorescence. The sequences of these proteins have been deposited in publicly available databases and repositories. We anticipate that this open source resource, in the form of high-quality recombinant proteins and antibodies, will accelerate and empower future research of the role of aaRSs in health and disease.


Assuntos
Aminoacil-tRNA Sintetases , Anticorpos de Cadeia Única , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/imunologia , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia
5.
Sci Adv ; 4(10): eaau5501, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30345365

RESUMO

Domain walls (DWs) are singularities in an ordered medium that often host exotic phenomena such as charge ordering, insulator-metal transition, or superconductivity. The ability to locally write and erase DWs is highly desirable, as it allows one to design material functionality by patterning DWs in specific configurations. We demonstrate such capability at room temperature in a charge density wave (CDW), a macroscopic condensate of electrons and phonons, in ultrathin 1T-TaS2. A single femtosecond light pulse is shown to locally inject or remove mirror DWs in the CDW condensate, with probabilities tunable by pulse energy and temperature. Using time-resolved electron diffraction, we are able to simultaneously track anti-synchronized CDW amplitude oscillations from both the lattice and the condensate, where photoinjected DWs lead to a red-shifted frequency. Our demonstration of reversible DW manipulation may pave new ways for engineering correlated material systems with light.

6.
J R Soc Interface ; 15(139)2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29467259

RESUMO

This study investigates the structural basis for the red, silver and black coloration of the theridiid spider, Phoroncidia rubroargentea (Berland, 1913) from Madagascar. Specimens of this species can retain their colour after storage in ethanol for decades, whereas most other brightly pigmented spider specimens fade under identical preservation conditions. Using correlative optical, structural and chemical analysis, we identify the colour-generating structural elements and characterize their optical properties. The prominent silvery appearance of the spider's abdomen results from regularly arranged guanine microplatelets, similar to those found in other spiders and fish. The microplatelets are composed of a doublet structure twinned about the [[Formula: see text]] axis, as suggested by electron diffraction. The red coloration originates from chambered microspheres (approx. 1 µm in diameter), which contain structured fluorescent material. Co-localization of the red microparticles on top of the reflective guanine microplatelets appears to enhance the red coloration. The spider's thick cuticular layer, which encases its abdomen, varies in its optical properties, being transparent in regions where only guanine reflectors are present, and tanned, exhibiting light absorption where the red microspheres are found. Moreover, colour degradation in some preserved spider specimens that had suffered damage to the cuticular layer suggests that this region of the exoskeleton may play an important role in the stabilization of the red coloration.


Assuntos
Pigmentação/fisiologia , Aranhas/fisiologia , Animais , Madagáscar
7.
PLoS One ; 12(12): e0188772, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29228002

RESUMO

The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development.


Assuntos
Ciclo Celular/genética , Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , Algoritmos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias/genética , Neoplasias/patologia
8.
Science ; 350(6259): 404-9, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-26449474

RESUMO

The NLR family apoptosis inhibitory proteins (NAIPs) bind conserved bacterial ligands, such as the bacterial rod protein PrgJ, and recruit NLR family CARD-containing protein 4 (NLRC4) as the inflammasome adapter to activate innate immunity. We found that the PrgJ-NAIP2-NLRC4 inflammasome is assembled into multisubunit disk-like structures through a unidirectional adenosine triphosphatase polymerization, primed with a single PrgJ-activated NAIP2 per disk. Cryo-electron microscopy (cryo-EM) reconstruction at subnanometer resolution revealed a ~90° hinge rotation accompanying NLRC4 activation. Unlike in the related heptameric Apaf-1 apoptosome, in which each subunit needs to be conformationally activated by its ligand before assembly, a single PrgJ-activated NAIP2 initiates NLRC4 polymerization in a domino-like reaction to promote the disk assembly. These insights reveal the mechanism of signal amplification in NAIP-NLRC4 inflammasomes.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas de Bactérias/química , Proteínas de Ligação ao Cálcio/química , Imunidade Inata , Inflamassomos/imunologia , Proteína Inibidora de Apoptose Neuronal/química , Sequência de Aminoácidos , Animais , Apoptose , Proteínas Adaptadoras de Sinalização CARD/química , Caspase 1/química , Microscopia Crioeletrônica , Camundongos , Dados de Sequência Molecular , Polimerização , Mapas de Interação de Proteínas , Estrutura Terciária de Proteína
9.
Mol Microbiol ; 88(3): 510-22, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23489252

RESUMO

Borrelia burgdorferi gene product BB0323 is required for cell fission and pathogen persistence in vivo. Here, we show that BB0323, which is conserved among globally prevalent infectious strains, supports normal spirochaete growth and morphology even at early phases of cell division. We demonstrate that native BB0323 undergoes proteolytic processing at the C-terminus, at a site after the first 202 N-terminal amino acids. We further identified a periplasmic BB0323 binding protein in B. burgdorferi, annotated as BB0104, having serine protease activity responsible for the primary cleavage of BB0323 to produce discrete N- and C-terminal polypeptides. These two BB0323 polypeptides interact with each other, and either individually or as a complex, are associated with multiple functions in spirochaete biology and infectivity. While N-terminal BB0323 is adequate to support cell fission, the C-terminal LysM domain is dispensable for this process, despite its ability to bind B. burgdorferi peptidoglycan. However, the LysM domain or the precisely processed BB0323 product is essential for mammalian infection. As BB0323 is a membrane protein crucial for B. burgdorferi survival in vivo, exploring its function may suggest novel ways to interrupt infection while enhancing our understanding of the intricate spirochaete fission process.


Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/patogenicidade , Peptídeos/metabolismo , Fenótipo , Processamento de Proteína Pós-Traducional , Animais , Proteínas de Bactérias/genética , Western Blotting , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , Clonagem Molecular , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C3H , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
J Neurosci ; 32(48): 17306-20, 2012 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-23197722

RESUMO

Glial cell line-derived neurotrophic factor (GDNF) and its receptor GFRα1 are prominently expressed in the olfactory epithelium (OE) and olfactory bulb (OB), but their importance for olfactory system development is completely unknown. We have investigated the consequences of GFRα1 deficiency for mouse olfactory system development and function. In the OE, GFRα1 was expressed in basal precursors, immature olfactory sensory neurons (OSNs), and olfactory ensheathing cells (OECs), but was excluded from mature OSNs. The OE of newborn Gfra1 knock-out mice was thinner and contained fewer OSNs, but more dividing precursors, suggesting deficient neurogenesis. Immature OSN axon bundles were enlarged and associated OECs increased, indicating impaired migration of OECs and OSN axons. In the OB, GFRα1 was expressed in immature OSN axons and OECs of the nerve layer, as well as mitral and tufted cells, but was excluded from GABAergic interneurons. In newborn knock-outs, the nerve layer was dramatically reduced, exhibiting fewer axons and OECs. Bulbs were smaller and presented fewer and disorganized glomeruli and a significant reduction in mitral cells. Numbers of tyrosine hydroxylase-, calbindin-, and calretinin-expressing interneurons were also reduced in newborn mice lacking Gfra1. At birth, the OE and OB of Gdnf knock-out mice displayed comparable phenotypes. Similar deficits were also found in adult heterozygous Gfra1(+/-) mutants, which in addition displayed diminished responses in behavioral tests of olfactory function. We conclude that GFRα1 is critical for the development and function of the main olfactory system, contributing to the development and allocation of all major classes of neurons and glial cells.


Assuntos
Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Neuroglia/metabolismo , Bulbo Olfatório/metabolismo , Condutos Olfatórios/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Animais , Axônios/metabolismo , Comportamento Animal/fisiologia , Diferenciação Celular , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Camundongos , Camundongos Knockout , Bulbo Olfatório/crescimento & desenvolvimento , Mucosa Olfatória/crescimento & desenvolvimento , Mucosa Olfatória/metabolismo , Condutos Olfatórios/crescimento & desenvolvimento , Percepção Olfatória/fisiologia , Neurônios Receptores Olfatórios/crescimento & desenvolvimento , Olfato/fisiologia , Ácido gama-Aminobutírico/metabolismo
11.
Nano Lett ; 12(10): 5403-6, 2012 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-22928701

RESUMO

Cell-biomaterial interactions can be controlled by modifying the surface chemistry or nanotopography of the material, to induce cell proliferation and differentiation if desired. Here we combine both approaches in forming silk nanofibers (SNFs) containing gold nanoparticles (AuNPs) and subsequently chemically modifying the fibers. Silk fibroin mixed with gold seed nanoparticles was electrospun to form SNFs doped with gold seed nanoparticles (SNF(seed)). Following gold reduction, there was a 2-fold increase in particle diameter confirmed by the appearance of a strong absorption peak at 525 nm. AuNPs were dispersed throughout the AuNP-doped silk nanofibers (SNFs(Au)). The Young's modulus of the SNFs(Au) was almost 70% higher than that of SNFs. SNFs(Au) were modified with the arginine-glycine-aspartic acid (RGD) peptide. Human mesenchymal stem cells that were cultured on RGD-modified SNF(Au) had a more than 2-fold larger cell area compared to the cells cultured on bare SNFs; SNF(Au) also increased cell size. This approach may be used to alter the cell-material interface in tissue engineering and other applications.


Assuntos
Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Nanocompostos/química , Nanocompostos/ultraestrutura , Tamanho Celular , Células Cultivadas , Módulo de Elasticidade , Ouro , Humanos , Células-Tronco Mesenquimais/citologia , Microscopia Eletrônica de Varredura , Nanotecnologia , Oligopeptídeos , Seda , Engenharia Tecidual
12.
FEMS Microbiol Ecol ; 81(2): 407-18, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22432637

RESUMO

Marine sponges can harbor dense and diverse bacterial communities, yet we have a limited understanding of important aspects of this symbiosis. We developed an experimental methodology that permits manipulating the composition of the microbial community. Specifically, we evaluated sponge cell aggregates (SCA) from Clathria prolifera that had been treated with different classes of antibiotics to determine whether this system might offer novel experimental approaches to the study of sponge/bacterial symbioses. Microscopic analysis of the SCA demonstrated that two distinct morphological types of microbiota existed on the external surface vs. the internal regions of the SCA. Denaturing gradient gel electrophoresis and sequence analysis of 16S rRNA gene clone libraries indicated that we were unable to create entirely aposymbiotic SCA but that different classes of antibiotics produced distinctive shifts in the SCA-associated bacterial community. After exposure to antibiotics, some bacterial species were 'revealed', thus uncovering novel components of the sponge-associated community. The antibiotic treatments used here had little discernible effect on the formation of SCA or subsequent development of the adult. The experimental approach we describe offers empirical options for studying the role symbionts play in sponge growth and development and for ascertaining relationships among bacterial species in communities residing in sponges.


Assuntos
Bactérias/classificação , Metagenoma , Poríferos/microbiologia , Simbiose , Animais , Antibacterianos/farmacologia , Cultura Axênica , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Biodiversidade , DNA Bacteriano/genética , Eletroforese em Gel de Gradiente Desnaturante , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
13.
J Neurosci ; 30(27): 9271-9, 2010 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-20610762

RESUMO

The odor response properties of a mammalian olfactory sensory neuron (OSN) are determined by the tightly regulated expression of a single member of a very large family of odorant receptors (ORs). The OR also plays an important role in focusing the central projections of all OSNs expressing that particular receptor to a pair of stereotypic locations (glomeruli) in each olfactory bulb (OB), thus creating a spatial map of odor responses in the brain. Here we show that when initiated late in neural development, transgenic expression of one OR in almost all OSNs has little influence on the architecture of the OB in mice. In contrast, early OR-transgene expression (mediated by the Ggamma8-promoter) in 50-70% of OSNs grossly distorts the morphology of glomeruli and alters the projection patterns of many residual OSNs not expressing the transgene. Interestingly, this disruption of targeting persists in adult animals despite the downregulation of Ggamma8 and transgenic OR expression that occurs as olfactory neurogenesis declines. Indeed, functional imaging studies reveal a dramatic decrease in the complexity of responses to odorants in adult Ggamma8-transgenic OR mice. Thus, we show that initiation of transgenic OR expression early in the development of OSNs, rather than just the extent of transgene expression, determines its effectiveness at modifying OB anatomy and function. Together, these data imply that OR-expression timing needs to be very tightly controlled to achieve the precise wiring and function of the mammalian olfactory system.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Rede Nervosa/metabolismo , Condutos Olfatórios/metabolismo , Receptores Odorantes/metabolismo , Animais , Animais Recém-Nascidos , Embrião de Mamíferos , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Rede Nervosa/embriologia , Rede Nervosa/crescimento & desenvolvimento , Odorantes , Proteína de Marcador Olfatório/genética , Proteína de Marcador Olfatório/metabolismo , Condutos Olfatórios/anatomia & histologia , Condutos Olfatórios/embriologia , Condutos Olfatórios/crescimento & desenvolvimento , Receptores Odorantes/classificação , Receptores Odorantes/genética , beta-Galactosidase/metabolismo
14.
Langmuir ; 26(1): 560-9, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-19678633

RESUMO

The distance dependence and kinetics of the heterogeneous electron transfer (ET) reaction for the redox protein azurin adsorbed to an electrode modified with a gold nanoparticle film are investigated using cyclic voltammetry. The nanoparticle films are comprised of nonaqueous nanoparticles, known as monolayer-protected clusters (MPCs), which are covalently networked with dithiol linkers. The MPC film assembly serves as an alternative adsorption platform to the traditional alkanethiolate self-assembled monolayer (SAM) modified electrodes that are commonly employed to study the ET kinetics of immobilized redox proteins, a strategy known as protein monolayer electrochemistry. Voltammetric analysis of the ET kinetics for azurin adsorbed to SAMs of increasing chain length results in quasi-reversible voltammetry with significant peak splitting. We observed rate constants (k degrees (ET)) of 12-20 s(-1) for the protein at SAMs of shorter alkanethiolates that decays exponentially (beta = 0.9/CH(2) or 0.8/A) at SAMs of longer alkanethiolates (9-11 methylene units) or an estimated distance of 1.23 nm and is representative of classical electronic tunneling behavior over increasing distance. Azurin adsorbed to the MPC film platforms of increasing thickness results in reversible voltammetry with very little voltammetric peaks splitting and nearly negligible decay of the ET rate over significant distances up to 20 nm. The apparent lack of distance dependence for heterogeneous ET reactions at MPC film assemblies is attributed to a two-step mechanism involving extremely fast electronic hopping through the MPC film architecture. These results suggest that MPC platforms may be used in protein monolayer electrochemistry to create adsorption platforms of higher architecture that can accommodate greater than monolayer protein coverage and increase the Faradaic signal, a finding with significant implications for amperometric biosensor design and development.


Assuntos
Azurina/química , Nanopartículas Metálicas/química , Adsorção , Azurina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ácidos Carboxílicos/química , Eletroquímica , Transporte de Elétrons , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Cinética , Modelos Moleculares , Conformação Proteica , Pseudomonas aeruginosa , Eletricidade Estática
15.
Macromol Rapid Commun ; 31(11): 1003-9, 2010 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-21590850

RESUMO

We have used a combinatorial gradient technique to map precisely how the terrace structure and microdomain lattice alignment in a thin film of a sphere-forming diblock copolymer are affected by both the thickness of the copolymer film and the height of a series of parallel step edges fabricated on the substrate. We find that for film thicknesses slightly incommensurate with integer numbers of sphere layers, the step edges act as nucleation sites for regions with one more or one fewer layers of spheres. We also find that for our system, the hexagonal lattice formed by a single layer of spheres on the low side of a step edge is aligned along the direction of the step edge only where the film on the high side is sufficiently thin to support only a wetting layer of copolymer material. This work will guide the tuning of film thickness and step height in future studies and applications of graphoepitaxy in block copolymer films.

16.
Mol Cell Biol ; 28(11): 3790-803, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18285456

RESUMO

Hypoxia-inducible transcription factor 1 (HIF-1) and HIF-2alpha regulate the expression of an expansive array of genes associated with cellular responses to hypoxia. Although HIF-regulated genes mediate crucial beneficial short-term biological adaptations, we hypothesized that chronic activation of the HIF pathway in cardiac muscle, as occurs in advanced ischemic heart disease, is detrimental. We generated mice with cardiac myocyte-specific deletion of the von Hippel-Lindau protein (VHL), an essential component of an E3 ubiquitin ligase responsible for suppressing HIF levels during normoxia. These mice were born at expected frequency and thrived until after 3 months postbirth, when they developed severe progressive heart failure and premature death. VHL-null hearts developed lipid accumulation, myofibril rarefaction, altered nuclear morphology, myocyte loss, and fibrosis, features seen for various forms of human heart failure. Further, nearly 50% of VHL(-/-) hearts developed malignant cardiac tumors with features of rhabdomyosarcoma and the capacity to metastasize. As compelling evidence for the mechanistic contribution of HIF-1alpha, the concomitant deletion of VHL and HIF-1alpha in the heart prevented this phenotype and restored normal longevity. These findings strongly suggest that chronic activation of the HIF pathway in ischemic hearts is maladaptive and contributes to cardiac degeneration and progression to heart failure.


Assuntos
Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hipóxia/genética , Hipóxia/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Animais , Capilares/crescimento & desenvolvimento , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Receptores ErbB/metabolismo , Deleção de Genes , Técnicas de Transferência de Genes , Insuficiência Cardíaca/metabolismo , Neoplasias Cardíacas/genética , Neoplasias Cardíacas/metabolismo , Neoplasias Cardíacas/patologia , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metabolismo dos Lipídeos/genética , Lipídeos/análise , Camundongos , Camundongos Knockout , Miocárdio/química , Neovascularização Fisiológica/genética , Fosforilação , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas ras/metabolismo
17.
Cell ; 131(5): 1009-17, 2007 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-18045541

RESUMO

Mammalian odorant receptors (ORs) are crucial for establishing the functional organization of the olfactory system, but the mechanisms controlling their expression remain largely unexplained. Here, we utilized a transgenic approach to explore OR gene regulation. We determined that although olfactory sensory neurons (OSNs) are capable of supporting expression of multiple functional ORs, several levels of control ensure that each neuron normally expresses only a single odorant receptor. Surprisingly, this regulation extends beyond endogenous ORs even preventing expression of transgenes consisting of OR-coding sequences driven by synthetic promoters. Thus, part of the intrinsic feedback system must rely on elements present in the OR-coding sequence. Notably, by expressing the same transgenic ORs precociously in immature neurons, we have overcome this suppression and established a generic method to express any OR in approximately 90% of OSNs. These results provide important insights into the hierarchy of OR gene expression and the vital role of the OR-coding sequence in this regulation.


Assuntos
Fases de Leitura Aberta/genética , Receptores Odorantes/genética , Alelos , Animais , Sequência de Bases/fisiologia , Regulação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Neurônios Receptores Olfatórios/metabolismo , Fases de Leitura Aberta/fisiologia , Regiões Promotoras Genéticas/fisiologia , Receptores Odorantes/metabolismo , Receptores Odorantes/fisiologia
18.
J Neurosci ; 26(44): 11257-66, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17079653

RESUMO

In mammals, each olfactory bulb contains two mirror-symmetric glomerular maps. Isofunctional glomeruli within each bulb are specifically linked through a set of reciprocal intrabulbar projections (IBPs) to form an intrabulbar map. We injected neural tracers into the glomerular layer on one side of the bulb and examined the resulting projection on the opposite side. In adult mice, the size of the projection tuft is directly proportional to the size of the injected region. Using this ratio as a measure of IBP maturity, we find an immature 5:1 projection to injection ratio at 1 week of age that gradually refines to a mature 1:1 by 7 weeks. Moreover, whereas the glomerular map is able to form despite the elimination of odorant-induced activity, the intrabulbar map shows clear activity dependence for its precise formation. Here we show through experiments with both naris-occluded and anosmic mice that odorant-induced activity is not required to establish IBPs but is crucial for projection refinement. In contrast, increased glomerular activation through exposure to distinct odorants during map development can accelerate the refinement of projections associated with the activated glomeruli. These findings illustrate a clear role for odorant-induced activity in shaping the internal circuitry of the bulb. Interestingly, activity deprivation can alter the organization of both the developing and the mature map to the same degree, demonstrating that intrabulbar map plasticity is maintained into adulthood with no discernible critical period.


Assuntos
Mapeamento Encefálico/métodos , Plasticidade Neuronal/fisiologia , Bulbo Olfatório/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Odorantes , Bulbo Olfatório/química , Bulbo Olfatório/crescimento & desenvolvimento , Condutos Olfatórios/química , Condutos Olfatórios/crescimento & desenvolvimento , Condutos Olfatórios/fisiologia , Olfato/fisiologia
19.
J Cell Biol ; 170(2): 317-25, 2005 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-16027225

RESUMO

Viruses have often been observed in association with the dense microvilli of polarized epithelia as well as the filopodia of nonpolarized cells, yet whether interactions with these structures contribute to infection has remained unknown. Here we show that virus binding to filopodia induces a rapid and highly ordered lateral movement, "surfing" toward the cell body before cell entry. Virus cell surfing along filopodia is mediated by the underlying actin cytoskeleton and depends on functional myosin II. Any disruption of virus cell surfing significantly reduces viral infection. Our results reveal another example of viruses hijacking host machineries for efficient infection by using the inherent ability of filopodia to transport ligands to the cell body.


Assuntos
Actinas/fisiologia , Vírus da Leucose Aviária/fisiologia , Vírus da Leucemia Murina/fisiologia , Miosinas/fisiologia , Pseudópodes/fisiologia , Animais , Vírus da Leucose Aviária/efeitos dos fármacos , Vírus da Leucose Aviária/ultraestrutura , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Membrana Celular/virologia , Citocalasina D/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Vírus da Leucemia Murina/efeitos dos fármacos , Vírus da Leucemia Murina/ultraestrutura , Camundongos , Microscopia Eletrônica , Pseudópodes/ultraestrutura , Pseudópodes/virologia
20.
Brain Res Bull ; 66(2): 114-9, 2005 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-15982527

RESUMO

Sleep frequently fragmented or disrupted for prolonged periods can result in mood changes and impaired mental ability and performance. Sleep deprivation is defined as depriving a person or organism of sleep for various periods of fixed durations. Sleep disruption (SD) occurs when a person is awakened at any time when they would normally be sleeping; sometimes on a schedule but usually unexpectedly. It seems as if any disruption of an entrained sleep pattern can induce learning and memory impairment; and mood changes including irritability and aggression. Because memory is impaired under these conditions several studies have been conducted recently to examine changes in long term potentiation (LTP) in hippocampal brain slices following various periods of sleep deprivation in rats. Results of the present study show clearly that LTP is also decreased following SD but to a greater extent than that observed following sleep deprivation. The purpose of the present study was to measure dentate granule cell LTP in anesthetized rats following 1-, 2-, or 3-day schedules of SD using a modified flower pot procedure. Results showed that a single disruption of 3 h reduced LTP from a normal 38.7-7.6%; that endured for at least 14 h; and 9 h reduced it completely. Easy to handle animals become irritable, hyperactive, and aggressive following SD. Results are discussed in terms of stress related effects of SD and changes in synaptic plasticity.


Assuntos
Giro Denteado/patologia , Potenciação de Longa Duração/fisiologia , Neurônios/fisiologia , Privação do Sono/fisiopatologia , Análise de Variância , Animais , Estimulação Elétrica/métodos , Depressão Sináptica de Longo Prazo/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
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