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1.
Soft Matter ; 14(2): 216-227, 2018 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-29227498

RESUMO

In this study, we experimentally observed and characterized soft elastic particle deformation in confined flow in a microchannel with a rectangular cross-section. Hydrogel microparticles of pNIPAM were produced using two different concentrations of crosslinker. This resulted in particles with two different shear moduli of 13.3 ± 5.5 Pa and 32.5 ± 15.7 Pa and compressive moduli of 66 ± 10 Pa and 79 ± 15 Pa, respectively, as measured by capillary micromechanics. Under flow, the particle shapes transitioned from circular to egg, triangular, arrowhead, and ultimately parachute shaped with increasing shear rate. The shape changes were reversible, and deformed particles relaxed back to circular/spherical in the absence of flow. The thresholds for each shape transition were quantified using a non-dimensional radius of curvature at the tip, particle deformation, circularity, and the depth of the concave dimple at the trailing edge. Several of the observed shapes were distinct from those previously reported in the literature for vesicles and capsules; the elastic particles had a narrower leading tip and a lower circularity. Due to variations in the shear moduli between particles within a batch of particles, each flow rate corresponded to a small but finite range of capillary number (Ca) and resulted in a series of shapes. By arranging the images on a plot of Ca versus circularity, a direct correlation was developed between shape and Ca and thus between particle deformation and shear modulus. As the shape was very sensitive to differences in shear modulus, particle deformation in confined flow may allow for better differentiation of microparticle shear modulus than other methods.

2.
Soft Matter ; 13(34): 5785-5794, 2017 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-28766673

RESUMO

Monodisperse particles of varying size, shape, and deformability were produced using two microfluidic strategies. For both strategies, monodisperse emulsion droplets of a crosslinkable solution were generated via flow-focusing. Subsequently, droplets were crosslinked either on chip or in an external bath. On-chip gelation resulted in spherical particles; varying the degree of crosslinking varied the deformability systematically. The optimized flow-focusing device design separated the production of monodisperse aqueous alginate droplets and the on-chip introduction of crosslinking ions. Two features were then adapted to target softer particles: the dispersed phase design and the polymer choice. The alternative design used a sheathed dispersed phase, with the polymer solution surrounding an unreactive viscous core, which generated alginate particles with a softer core. Poly(N-isopropylacrylamide) (pNIPAM) allowed access to a broad range of moduli. The resulting spherical particles were characterized using capillary micromechanics to determine the shear (G) and compressive (K) moduli. Particles with G = 0.013 kPa to 26 kPa and K = 0.221 kPa to 34.9 kPa were obtained; the softest particles are an order of magnitude softer than those previously reported. The second approach, based on earlier work by Hu et al., produced axisymmetric, non-spherical particles with fore-aft asymmetry. Alginate drops were again formed in a flow-focusing device but were crosslinked off-chip in an external gelation bath. By changing the bath viscosity, crosslinker concentration, and outlet height, the falling droplets deformed differently during gelation, resulting in a variety of shapes, such as teardrop, mushroom, and bowl shapes.

3.
Biomicrofluidics ; 11(3): 034112, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28580045

RESUMO

Two size-based separation strategies are evaluated for suspensions consisting of giant unilamellar vesicles with a broad, continuous distribution of diameters. Microfluidic devices were designed to separate an initial suspension into larger and smaller particles via either filtration or inertial focusing. These separation mechanisms were tested with suspensions of vesicles and suspensions of rigid spheres separately to illustrate the effect of deformability on separation ability. We define several separation metrics to assess the separation ability and to enable comparison between separation strategies. The filtration device significantly reduced the polydispersity of the separated vesicle fractions relative to the starting suspension and displayed an ability to separate vesicle suspensions at high throughputs. The device that utilized inertial focusing exhibited adequate polydispersity reduction and performed best with diluted vesicle suspensions. The inertial device had fewer issues with debris and trapped air, leading to short device preparation times and indicating a potential for continuous separation operation.

4.
Biophys J ; 111(9): 2039-2050, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27806284

RESUMO

The quantification of cellular mechanical properties is of tremendous interest in biology and medicine. Recent microfluidic technologies that infer cellular mechanical properties based on analysis of cellular deformations during microchannel traversal have dramatically improved throughput over traditional single-cell rheological tools, yet the extraction of material parameters from these measurements remains quite complex due to challenges such as confinement by channel walls and the domination of complex inertial forces. Here, we describe a simple microfluidic platform that uses hydrodynamic forces at low Reynolds number and low confinement to elongate single cells near the stagnation point of a planar extensional flow. In tandem, we present, to our knowledge, a novel analytical framework that enables determination of cellular viscoelastic properties (stiffness and fluidity) from these measurements. We validated our system and analysis by measuring the stiffness of cross-linked dextran microparticles, which yielded reasonable agreement with previously reported values and our micropipette aspiration measurements. We then measured viscoelastic properties of 3T3 fibroblasts and glioblastoma tumor initiating cells. Our system captures the expected changes in elastic modulus induced in 3T3 fibroblasts and tumor initiating cells in response to agents that soften (cytochalasin D) or stiffen (paraformaldehyde) the cytoskeleton. The simplicity of the device coupled with our analytical model allows straightforward measurement of the viscoelastic properties of cells and soft, spherical objects.


Assuntos
Elasticidade , Dispositivos Lab-On-A-Chip , Animais , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Glioblastoma/patologia , Camundongos , Células NIH 3T3 , Viscosidade
5.
Soft Matter ; 12(16): 3787-96, 2016 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-26984509

RESUMO

Vesicles provide an attractive model system to understand the deformation of living cells in response to mechanical forces. These simple, enclosed lipid bilayer membranes are suitable for complementary theoretical, numerical, and experimental analysis. A recent study [Narsimhan, Spann, Shaqfeh, J. Fluid Mech., 2014, 750, 144] predicted that intermediate-aspect-ratio vesicles extend asymmetrically in extensional flow. Upon infinitesimal perturbation to the vesicle shape, the vesicle stretches into an asymmetric dumbbell with a cylindrical thread separating the two ends. While the symmetric stretching of high-aspect-ratio vesicles in extensional flow has been observed and characterized [Kantsler, Segre, Steinberg, Phys. Rev. Lett., 2008, 101, 048101] as well as recapitulated in numerical simulations by Narsimhan et al., experimental observation of the asymmetric stretching has not been reported. In this work, we present results from microfluidic cross-slot experiments observing this instability, along with careful characterization of the flow field, vesicle shape, and vesicle bending modulus. The onset of this shape transition depends on two non-dimensional parameters: reduced volume (a measure of vesicle asphericity) and capillary number (ratio of viscous to bending forces). We observed that every intermediate-reduced-volume vesicle that extends forms a dumbbell shape that is indeed asymmetric. For the subset of the intermediate-reduced-volume regime we could capture experimentally, we present an experimental phase diagram for asymmetric vesicle stretching that is consistent with the predictions of Narsimhan et al.


Assuntos
Bicamadas Lipídicas/química , Lipossomos/química , Modelos Químicos
6.
Soft Matter ; 12(4): 1051-61, 2016 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-26575011

RESUMO

In this paper, we use rheometry and flow visualization to study the dynamics of the interface between shear bands in a wormlike micellar solution sheared between concentric cylinders, i.e., in a Taylor-Couette (TC) cell, and to evaluate the stress diffusion coefficient and the stress correlation length in the Johnson-Segalman model. Two wormlike micellar solutions are studied: an aqueous solution of CTAB-NaNO3 and a solution of CPCl-NaSal in brine. These systems are highly elastic, exhibit Maxwellian behavior in linear viscoelasticity experiments, and shear banding in nonlinear experiments [S. Lerouge, et al., Soft Matter, 2008, 4, 1808-1819, M. A. Fardin, et al., Soft Matter, 2012, 8(39), 10072-10089, P. Ballesta, et al., J. Rheol., 2007, 51, 1047]. A large, custom-built, computer controlled TC cell allows us to rotate both cylinders independently and to visualize the flow in the r-z plane using a CCD camera. At low shear rates, the flow is stable and the fluid appears homogeneous throughout the gap between the cylinders. Above a critical shear rate, a shear banding transition occurs. This manifests itself in the formation of two distinct bands in the r-z plane, with an interface between the two bands. For sufficiently high ramp speeds, multiple steps of interface evolution are identified, as noted by Radulescu, Lerouge, and others [O. Redulescu, et al., Europhys. Lett., 2003, 62, 230, S. Lerouge, et al., Soft Matter, 2008, 4, 1808-1819]. We quantify the interface travel using direct visualization and use this measure, as well as superposition rheometry [P. Ballesta, et al., J. Rheol., 2007, 51, 1047], to determine the stress diffusion coefficient D and the stress correlation length ζ in the Johnson-Segalman model. These parameters are evaluated at different temperatures, shear rates, and gap sizes. We find that the stress diffusion coefficient and the stress correlation length exhibit a strong dependence on the gap of the Taylor-Couette cell for both shear-banding systems. For the CTAB-NaNO3 system, we report a linear dependence of the stress diffusion coefficient on temperature for the parameter range considered. In addition, we find that for this system, the stress diffusion coefficient is independent of shear rate. For the CPCl-NaSal system, we observe the same color changes in the sample reported by others on extended light exposure; however, we find that different histories of light exposure do not affect the measured stress diffusion coefficient.

7.
Biomicrofluidics ; 9(5): 054102, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26392834

RESUMO

The flow of λ-DNA solutions in a gradual micro-contraction was investigated using direct measurement techniques. The effects on DNA transport in microscale flows are significant because the flow behavior is influenced by macromolecular conformations, both viscous and elastic forces dominate inertial forces at this length scale, and the fully extended length of the molecule approaches the characteristic channel length wc (L/wc ∼ 0.13). This study examines the flow of semi-dilute and entangled DNA solutions in a gradual planar micro-contraction for low Reynolds numbers (3.7 × 10(-6 )< Re < 3.1 × 10(-1)) and high Weissenberg numbers (0.4 < Wi < 446). The semi-dilute DNA solutions have modest elasticity number, El = Wi/Re = 55, and do not exhibit viscoelastic behavior. For the entangled DNA solutions, we access high elasticity numbers (7.9 × 10(3 )< El < 6.0 × 10(5)). Video microscopy and streak images of entangled DNA solution flow reveal highly elastic behavior evidenced by the presence of large, stable vortices symmetric about the centerline and upstream of the channel entrance. Micro-particle image velocimetry measurements are used to obtain high resolution, quantitative velocity measurements of the vortex growth in this micro-contraction flow. These direct measurements provide a deeper understanding of the underlying physics of macromolecular transport in microfluidic flow, which will enable the realization of enhanced designs of lab-on-a-chip systems.

8.
Annu Rev Chem Biomol Eng ; 6: 293-317, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26134738

RESUMO

Microfluidic systems are attracting increasing interest for the high-throughput measurement of cellular biophysical properties and for the creation of engineered cellular microenvironments. Here we review recent applications of microfluidic technologies to the mechanics of living cells and synthetic cell-mimetic systems. We begin by discussing the use of microfluidic devices to dissect the mechanics of cellular mimics, such as capsules and vesicles. We then explore applications to circulating cells, including erythrocytes and other normal blood cells, and rare populations with potential disease diagnostic value, such as circulating tumor cells. We conclude by discussing how microfluidic devices have been used to investigate the mechanics, chemotaxis, and invasive migration of adherent cells. In these ways, microfluidic technologies represent an increasingly important toolbox for investigating cellular mechanics and motility at high throughput and in a format that lends itself to clinical translation.


Assuntos
Técnicas Analíticas Microfluídicas/métodos , Animais , Células Artificiais/citologia , Fenômenos Biomecânicos , Movimento Celular , Técnicas Citológicas/instrumentação , Técnicas Citológicas/métodos , Desenho de Equipamento , Humanos , Técnicas Analíticas Microfluídicas/instrumentação
9.
Lab Chip ; 12(3): 647-51, 2012 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-22173785

RESUMO

Polymer-monovalent salt-induced single-molecule DNA compaction/condensation in a microfluidic stagnation point flow was studied by analyzing both DNA compaction images and time trajectories. For the whole DNA compaction process we observed three successive steps: Step I, a relaxation process of the stretched DNA that occurs slowly along the whole DNA chain, Step II, nucleus formation and growth, and Step III, corresponding to a rapid compaction of the chain. A memory effect was observed between Steps I and III, and a new (intruder-induced) nucleation mode was observed for the first time. This study extends the use of the microfluidic stagnation point flow, which we have previously used for sequence detection and to measure enzyme kinetics site-specifically.


Assuntos
DNA/química , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/instrumentação , DNA/isolamento & purificação , Dispositivos Lab-On-A-Chip , Nanotecnologia , Polímeros/metabolismo , Sais
10.
Nanoscale ; 3(8): 3027-39, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21734993

RESUMO

This review is a practical guide for experimentalists interested in specifically labeling internal sequences on double-stranded (ds) DNA molecules for single-molecule experiments. We describe six labeling approaches demonstrated in a single-molecule context and discuss the merits and drawbacks of each approach with particular attention to the amount of specialized training and reagents required. By evaluating each approach according to criteria relevant to single-molecule experiments, including labeling yield and compatibility with cofactors such as Mg(2+), we provide a simple reference for selecting a labeling method for given experimental constraints. Intended for non-specialists seeking accessible solutions to DNA labeling challenges, the approaches outlined emphasize simplicity, robustness, suitability for use by non-biologists, and utility in diverse single-molecule experiments.


Assuntos
DNA/química , Técnicas Genéticas , Sondas Moleculares , DNA/análise , DNA/metabolismo , Metiltransferases/química , Metiltransferases/metabolismo , Ácidos Nucleicos Peptídicos/análise , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/metabolismo
11.
Lab Chip ; 11(3): 435-42, 2011 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-21072428

RESUMO

We demonstrate the feasibility of a single-molecule microfluidic approach to both sequence detection and obtaining kinetic information for restriction endonucleases on dsDNA. In this method, a microfluidic stagnation point flow is designed to trap, hold, and linearize double-stranded (ds) genomic DNA to which a restriction endonuclease has been pre-bound sequence-specifically. By introducing the cofactor magnesium, we determine the binding location of the enzyme by the cleavage process of dsDNA as in optical restriction mapping, however here the DNA need not be immobilized on a surface. We note that no special labeling of the enzyme is required, which makes it simpler than our previous scheme using stagnation point flows for sequence detection. Our accuracy in determining the location of the recognition site is comparable to or better than other single molecule techniques due to the fidelity with which we can control the linearization of the DNA molecules. In addition, since the cleavage process can be followed in real time, information about the cleavage kinetics, and subtle differences in binding and cleavage frequencies among the recognition sites, may also be obtained. Data for the five recognition sites for the type II restriction endonuclease EcoRI on λ-DNA are presented as a model system. While the roles of the varying fluid velocity and tension along the chain backbone on the measured kinetics remain to be determined, we believe this new method holds promise for a broad range of studies of DNA-protein interactions, including the kinetics of other DNA cleavage processes, the dissociation of a restriction enzyme from the cleaved substrate, and other macromolecular cleavage processes.


Assuntos
Desoxirribonuclease EcoRI/química , Microfluídica/métodos , DNA/química , Cinética , Modelos Lineares , Magnésio/química , Técnicas Analíticas Microfluídicas , Ligação Proteica , Especificidade por Substrato
12.
Nano Lett ; 10(11): 4697-701, 2010 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-20923183

RESUMO

The ability to strongly and sequence-specifically attach modifications such as fluorophores and haptens to individual double-stranded (ds) DNA molecules is critical to a variety of single-molecule experiments. We propose using modified peptide nucleic acids (PNAs) for this purpose and implement them in two model single-molecule experiments where individual DNA molecules are manipulated via microfluidic flow and optical tweezers, respectively. We demonstrate that PNAs are versatile and robust sequence-specific tethers.


Assuntos
Micromanipulação/métodos , Técnicas de Sonda Molecular , Sondas Moleculares/isolamento & purificação , Pinças Ópticas , Ácidos Nucleicos Peptídicos/isolamento & purificação , Análise de Sequência de DNA/métodos , Sequência de Bases , Sondas Moleculares/química , Dados de Sequência Molecular , Ácidos Nucleicos Peptídicos/química
13.
Proc Natl Acad Sci U S A ; 107(32): 14170-5, 2010 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-20660732

RESUMO

Periostin is considered to be a matricellular protein with expression typically confined to cells of mesenchymal origin. Here, by using in situ hybridization, we show that periostin is specifically up-regulated in bronchial epithelial cells of asthmatic subjects, and in vitro, we show that periostin protein is basally secreted by airway epithelial cells in response to IL-13 to influence epithelial cell function, epithelial-mesenchymal interactions, and extracellular matrix organization. In primary human bronchial epithelial cells stimulated with periostin and epithelial cells overexpressing periostin, we reveal a function for periostin in stimulating the TGF-beta signaling pathway in a mechanism involving matrix metalloproteinases 2 and 9. Furthermore, conditioned medium from the epithelial cells overexpressing periostin caused TGF-beta-dependent secretion of type 1 collagen by airway fibroblasts. In addition, mixing recombinant periostin with type 1 collagen in solution caused a dramatic increase in the elastic modulus of the collagen gel, indicating that periostin alters collagen fibrillogenesis or cross-linking and leads to stiffening of the matrix. Epithelial cell-derived periostin in asthma has roles in TGF-beta activation and collagen gel elasticity in asthma.


Assuntos
Asma/metabolismo , Brônquios/patologia , Moléculas de Adesão Celular/fisiologia , Colágeno Tipo I/biossíntese , Células Epiteliais/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Asma/patologia , Brônquios/citologia , Células Cultivadas , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Elasticidade , Géis , Humanos , Metaloproteinases da Matriz
14.
Lab Chip ; 10(12): 1543-9, 2010 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-20358051

RESUMO

We demonstrate the use of a microfluidic stagnation point flow to trap and extend single molecules of double-stranded (ds) genomic DNA for detection of target sequences along the DNA backbone. Mutant EcoRI-based fluorescent markers are bound sequence-specifically to fluorescently labeled ds lambda-DNA. The marker-DNA complexes are introduced into a microfluidic cross slot consisting of flow channels that intersect at ninety degrees. Buffered solution containing the marker-DNA complexes flows in one channel of the cross slot, pure buffer flows in the opposing channel at the same flow rate, and fluid exits the two channels at ninety degrees from the inlet channels. This creates a stagnation point at the center of a planar extensional flow, where marker-DNA complexes may be trapped and elongated along the outflow axis. The degree of elongation can be controlled using the flow strength (i.e., a non-dimensional flow rate) in the device. Both the DNA backbone and the markers bound along the stretched DNA are observed directly using fluorescence microscopy, and the location of the markers along the DNA backbone is measured. We find that our method permits detection of each of the five expected target site positions to within 1.5 kb with standard deviations of <1.5 kb. We compare the method's precision and accuracy at molecular extensions of 68% and 88% of the contour length to binding distributions from similar data obtained via molecular combing. We also provide evidence that increased mixing of the sample during binding of the marker to the DNA improves binding to internal target sequences of dsDNA, presumably by extending the DNA and making the internal binding sites more accessible.


Assuntos
Técnicas Analíticas Microfluídicas , Análise de Sequência de DNA/instrumentação , DNA/genética , DNA/metabolismo , Desoxirribonuclease EcoRI/metabolismo , Corantes Fluorescentes/metabolismo , Genômica , Distribuição Normal
15.
Biomacromolecules ; 11(3): 643-7, 2010 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-20131893

RESUMO

Monodisperse microspheres of reconstituted silkworm cocoon silk were produced using a glass capillary-based microfluidic system and by identifying an appropriate solvent/nonsolvent fluid system. The microspheres can be produced to a range of different diameters depending on the system flow rates and have a nearly homogeneous size distribution. The silk microspheres exhibit a unique core--shell architecture and have a largely beta-sheet structure, as measured by infrared spectroscopy. Mechanical characterization was performed with AFM nanoindentation and indicates that the microspheres are unexpectedly soft for a silk material. Because silk is well established as biocompatible and biodegradable, we anticipate that these silk microspheres could have particular utility in drug delivery and controlled release.


Assuntos
Microfluídica , Microesferas , Seda , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Espectroscopia de Infravermelho com Transformada de Fourier
16.
Anal Chem ; 81(24): 10049-54, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19908852

RESUMO

We have created a fluorescent marker using a mutant EcoRI restriction endonuclease (K249C) that enables prolonged, direct visualization of specific sequences on genomic lengths of double-stranded (ds) DNA. The marker consists of a biotinylated enzyme, attached through the biotin-avidin interaction to a fluorescent nanosphere. Control over biotin position with respect to the enzyme's binding pocket is achieved by biotinylating the mutant EcoRI at the mutation site. Biotinylated enzyme is incubated with dsDNA and NeutrAvidin-coated, fluorescent nanospheres under conditions that allow enzyme binding but prevent cleavage. Marker-laden DNA is then fluorescently stained and stretched on polylysine-coated glass slides so that the positions of the bound markers along individual DNA molecules can be measured. We demonstrate the marker's ability to bind specifically to its target sequence using both bulk gel-shift assays and single-molecule methods.


Assuntos
DNA/análise , DNA/genética , Nanoestruturas/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Avidina/metabolismo , Sequência de Bases , Biotina/metabolismo , Biotinilação , Cristalografia por Raios X , Fluorescência , Modelos Moleculares , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Especificidade por Substrato
17.
Am J Respir Crit Care Med ; 180(3): 203-10, 2009 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-19423716

RESUMO

RATIONALE: Airway mucus plugs, composed of mucin glycoproteins mixed with plasma proteins, are an important cause of airway obstruction in acute severe asthma, and they are poorly treated with current therapies. OBJECTIVES: To investigate mechanisms of airway mucus clearance in health and in acute severe asthma. METHODS: We collected airway mucus from patients with asthma and nonasthmatic control subjects, using sputum induction or tracheal aspiration. We used rheological methods complemented by centrifugation-based mucin size profiling and immunoblotting to characterize the physical properties of the mucus gel, the size profiles of mucins, and the degradation products of albumin in airway mucus. MEASUREMENTS AND MAIN RESULTS: Repeated ex vivo measures of size and entanglement of mucin polymers in airway mucus from nonasthmatic control subjects showed that the mucus gel is normally degraded by proteases and that albumin inhibits this degradation. In airway mucus collected from patients with asthma at various time points during acute asthma exacerbation, protease-driven mucus degradation was inhibited at the height of exacerbation but was restored during recovery. In immunoblots of human serum albumin digested by neutrophil elastase and in immunoblots of airway mucus, we found that albumin was a substrate of neutrophil elastase and that products of albumin degradation were abundant in airway mucus during acute asthma exacerbation. CONCLUSIONS: Rheological methods complemented by centrifugation-based mucin size profiling of airway mucins in health and acute asthma reveal that mucin degradation is inhibited in acute asthma, and that an excess of plasma proteins present in acute asthma inhibits the degradation of mucins in a protease-dependent manner. These findings identify a novel mechanism whereby plasma exudation may impair airway mucus clearance.


Assuntos
Asma/metabolismo , Mucinas/análise , Depuração Mucociliar/efeitos dos fármacos , Inibidor Secretado de Peptidases Leucocitárias/farmacologia , Inibidores de Serino Proteinase/farmacologia , Escarro/química , Doença Aguda , Adulto , Idoso , Asma/tratamento farmacológico , Elasticidade , Eletroforese em Gel Bidimensional , Feminino , Seguimentos , Humanos , Immunoblotting , Masculino , Pessoa de Meia-Idade , Peso Molecular , Escarro/efeitos dos fármacos , Viscosidade , Adulto Jovem
18.
Biotechnol Prog ; 25(2): 417-23, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19334285

RESUMO

Enzymes and other biomolecules are often immobilized in a matrix to improve their stability or to improve their ability to be reused. Performing a polycondensation reaction in the presence of a biomolecule of interest relies on random entrapment events during polymerization and may not ensure efficient, homogeneous, or complete biomolecule encapsulation. To overcome these limitations, we have developed a method of incorporating autosilification activity into proteins without affecting enzymatic functionality. The unmodified R5 silaffin peptide from Cylindrotheca fusiformis is capable of initiating silica polycondensation in vitro at ambient temperatures and pressures in aqueous solution. In this study, translational fusion proteins between R5 and various functional proteins (phosphodiesterase, organophosphate hydrolase, and green fluorescent protein) were produced in Escherichia coli. Each of the fusion proteins initiated silica polycondensation, and enzymatic activity (or fluorescence) was retained in the resulting silica spheres. Under certain circumstances, the enzymatically-active biosilica displayed improved stability relative to free enzyme at elevated temperatures.


Assuntos
Enzimas Imobilizadas/química , Peptídeos/química , Dióxido de Silício/química , Cápsulas/química , Catálise , Estabilidade Enzimática , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Peptídeos/genética , Peptídeos/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Temperatura
19.
Biomacromolecules ; 10(1): 49-57, 2009 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-19053289

RESUMO

Spiders and silkworms employ the complex flow of highly concentrated silk solution as part of silk fiber spinning. To understand the role of fluidic forces in this process, the flow of silk solution in the spider major ampullate and silkworm silk glands was investigated using numerical simulation. Our simulations demonstrate significant differences between flow in the spider and silkworm silk glands. In particular, shear flow effects are shown to be much greater in the spider than the silkworm, the silkworm gland exhibits a much different flow extension profile than the spider gland, and the residence time within the spider gland is eight times greater than in the silkworm gland. Lastly, simulations on the effect of spinning speed on the flow of silk solution suggest that a critical extension rate is the initiating factor for fiber formation from silk solution. These results provide new insight into silk spinning processes and will guide the future development of novel fiber spinning technologies.


Assuntos
Bombyx/fisiologia , Glândulas Exócrinas/metabolismo , Proteínas de Insetos/biossíntese , Proteínas de Insetos/química , Seda/biossíntese , Seda/metabolismo , Aranhas/fisiologia , Animais , Bombyx/anatomia & histologia , Glândulas Exócrinas/anatomia & histologia , Análise de Elementos Finitos , Seda/química , Software , Soluções , Aranhas/anatomia & histologia , Viscosidade
20.
Phys Rev E Stat Nonlin Soft Matter Phys ; 78(3 Pt 2): 036314, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18851150

RESUMO

Secondary flows that are absent in Newtonian flows are found for semidilute lambda -DNA solutions in abrupt planar 90 degrees microbends at modest levels of elasticity. Flow visualization and microparticle image velocimetry experiments show that a vortex, which is present in the inner, upstream corner of the bend, grows with increasing Reynolds and Weissenberg number (9.9x10;{-7}

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