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Find video protocols related to scientific articles indexed in Pubmed.
Defect Removal in the Course of Directed Self-Assembly is Facilitated in the Vicinity of the Order-Disorder Transition.
Phys. Rev. Lett.
PUBLISHED: 10-15-2014
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The stability of prototypical defect morphologies in thin films of symmetric diblock copolymers on chemically patterned substrates is investigated by self-consistent field theory. The excess free energy of defects and barriers of defect-removal mechanisms are obtained by computing the minimum free-energy path. Distinct defect-removal mechanisms are illustrated demonstrating that (i) defects will become unstable at a characteristic value of incompatibility ?N_{*} above the order-disorder transition and (ii) the kinetics is accelerated at weak segregation. Numerical findings are placed in the context of physical mechanisms, and implications for directed self-assembly are discussed.
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Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material.
Hum. Gene Ther.
PUBLISHED: 10-03-2014
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Abstract Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.
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Characterization of Variants of the Pore-Forming Toxin ClyA from Escherichia coli Controlled by a Redox Switch.
Biochemistry
PUBLISHED: 09-30-2014
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The ?-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic phenotype of several Escherichia coli and Salmonella enterica strains. ClyA is a soluble, 34 kDa monomer that assembles into a dodecameric pore complex in the presence of membranes or detergent. The comparison of the X-ray structures of monomeric ClyA and the ClyA protomer in the pore complex revealed one of the largest conformational transitions observed so far in proteins, involving the structural rearrangement of more than half of all residues, which is consistent with the finding that conversion from the monomer to the assembly competent protomer is rate-limiting for pore assembly. Here, we introduced artificial disulfide bonds at two distinct sites into the ClyA monomer that both prevent a specific structural rearrangement required for protomer formation. Using electron microscopy and hemolytic activity assays, we show that the engineered disulfides indeed trap these ClyA variants in an assembly incompetent state. Assembly of the variants into functional pore complexes can be completely recovered by disulfide reduction. The assembly kinetics of the ClyA variants recorded with circular dichroism and fluorescence spectroscopy revealed the same mechanism of protomer formation that was observed for wild-type ClyA, proceeding via an intermediate with decreased secondary structure content.
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CXCR3 modulates glial accumulation and activation in cuprizone-induced demyelination of the central nervous system.
J Neuroinflammation
PUBLISHED: 05-15-2014
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The functional state of glial cells, like astrocytes and microglia, critically modulates the course of neuroinflammatory and neurodegenerative diseases and can have both detrimental and beneficial effects. Glial cell function is tightly controlled by cellular interactions in which cytokines are important messengers. Recent studies provide evidence that in particular chemokines are important modulators of glial cell function. During the course of CNS diseases like multiple sclerosis or Alzheimer's disease, and in the corresponding animal models, the chemokines CXCL9 and CXCL10 are abundantly expressed at sites of glial activation, arguing for an important role of these chemokines and their corresponding receptor CXCR3 in glial activation. To clarify the role of this chemokine system in glial cell activation, we characterized the impact of CXCR3 on glial activation in a model of toxic demyelination in which glial activation without a prominent influx of hematogenous cells is prototypical.
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Directed transport of polymer drops on vibrating superhydrophobic substrates: a molecular dynamics study.
Soft Matter
PUBLISHED: 05-08-2014
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Using molecular dynamics simulations of a coarse-grained polymer liquid we investigate the transport of droplets on asymmetrically structured (saw-tooth shaped), vibrating substrates. Due to a continuous supply of power by substrate vibrations and the asymmetry of its topography, the droplets are driven in a preferred direction. We study this directed motion as a function of the size of the droplets, the linear dimensions of the substrate corrugation, and the period of vibrations. Two mechanisms of driven transport are identified: (i) one that relies on the droplet's contact lines and (ii) in a range of vibration periods, the entire contact area contributes to the driving. In this latter regime, the set-up may be used in experiments for sorting droplets according to their size. Additionally, we show that the linear dimension of the substrate corrugation affects the flux inside the droplet. While on a substrate with a fine corrugation droplets mostly slide, on a more coarsely corrugated substrate the flux may exhibit an additional rotation pattern.
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Assessing cisplatin-induced ototoxicity and otoprotection in whole organ culture of the mouse inner ear in simulated microgravity.
Toxicol. Lett.
PUBLISHED: 03-25-2014
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Cisplatin is a widely used anti-cancer drug. Ototoxicity is a major dose-limiting side-effect. A reproducible mammalian in-vitro model of cisplatin ototoxicity is required to screen and validate otoprotective drug candidates. We utilized a whole organ culture system of the postnatal mouse inner ear in a rotating wall vessel bioreactor under "simulated microgravity" culture conditions. As previously described this system allows whole organ culture of the inner ear and quantitative assessment of ototoxic effects of aminoglycoside induced hair cell loss. Here we demonstrate that this model is also applicable to the assessment of cisplatin induced ototoxicity. In this model cisplatin induced hair cell loss was dose and time dependent. Increasing exposure time of cisplatin led to decreasing EC50 concentrations. Outer hair cells were more susceptible than inner hair cells, and hair cells in the cochlear base were more susceptible than hair cells in the cochlear apex. Initial cisplatin dose determined the final extent of hair cell loss irrespective if the drug was withdrawn or continued. Dose dependant otoprotection was demonstrated by co-administration of the antioxidant agent N-acetyl l-cysteine. The results support the use of this inner ear organ culture system as an in vitro assay and validation platform for inner ear toxicology and the search for otoprotective compounds.
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Mechanics of membrane fusion/pore formation.
Chem. Phys. Lipids
PUBLISHED: 02-27-2014
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Lipid bilayers play a fundamental role in many biological processes, and a considerable effort has been invested in understanding their behavior and the mechanism of topological changes like fusion and pore formation. Due to the time- and length-scale on which these processes occur, computational methods have proven to be an especially useful tool in their study. With their help, a number of interesting findings about the shape of fusion intermediates could be obtained, and novel hypotheses about the mechanism of topological changes and the involvement of peptides therein were suggested. In this work, we try to present a summary of these developments together with some hitherto unpublished results, featuring, among others, the shape of stalks and fusion pores, possible modes of action of the influenza HA fusion peptide and the SNARE protein complex, the mechanism of supported lipid bilayer formation by vesicle spreading, and the free energy and transition pathway of the fusion process.
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Raft formation in lipid bilayers coupled to curvature.
Biophys. J.
PUBLISHED: 02-07-2014
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We present computer simulations of a membrane in which the local composition is coupled to the local membrane curvature. At high temperatures (i.e., above the temperature of macroscopic phase separation), finite-sized transient domains are observed, reminiscent of lipid rafts. The domain size is in the range of hundred nanometers, and set by the membrane elastic properties. These findings are in line with the notion of the membrane as a curvature-induced microemulsion. At low temperature, the membrane phase separates. The transition to the phase-separated regime is continuous and belongs to the two-dimensional Ising universality class when the coupling to curvature is weak, but becomes first-order for strong curvature-composition coupling.
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Therapeutic inflammatory monocyte modulation using immune-modifying microparticles.
Sci Transl Med
PUBLISHED: 01-17-2014
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Inflammatory monocyte-derived effector cells play an important role in the pathogenesis of numerous inflammatory diseases. However, no treatment option exists that is capable of modulating these cells specifically. We show that infused negatively charged, immune-modifying microparticles (IMPs), derived from polystyrene, microdiamonds, or biodegradable poly(lactic-co-glycolic) acid, were taken up by inflammatory monocytes, in an opsonin-independent fashion, via the macrophage receptor with collagenous structure (MARCO). Subsequently, these monocytes no longer trafficked to sites of inflammation; rather, IMP infusion caused their sequestration in the spleen through apoptotic cell clearance mechanisms and, ultimately, caspase-3-mediated apoptosis. Administration of IMPs in mouse models of myocardial infarction, experimental autoimmune encephalomyelitis, dextran sodium sulfate-induced colitis, thioglycollate-induced peritonitis, and lethal flavivirus encephalitis markedly reduced monocyte accumulation at inflammatory foci, reduced disease symptoms, and promoted tissue repair. Together, these data highlight the intricate interplay between scavenger receptors, the spleen, and inflammatory monocyte function and support the translation of IMPs for therapeutic use in diseases caused or potentiated by inflammatory monocytes.
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Emergency medical care of multiple sclerosis patients: primary data from the mount sinai resource utilization in multiple sclerosis project.
J Clin Neurol
PUBLISHED: 01-16-2014
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There has been no systematic analysis of emergency department (ED) utilization in the multiple sclerosis (MS) population. We investigated the acute-care needs of MS patients using ED as a route for entry into healthcare services.
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Effector molecules released by Th1 but not Th17 cells drive an M1 response in microglia.
Brain Behav. Immun.
PUBLISHED: 01-02-2014
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Microglia act as sensors of inflammation in the central nervous system (CNS) and respond to many stimuli. Other key players in neuroinflammatory diseases are CD4+ T helper cell (Th) subsets that characteristically secrete IFN-? (Th1) or IL-17 (Th17). However, the potential of a distinct cytokine milieu generated by these effector T cell subsets to modulate microglial phenotype and function is poorly understood. We therefore investigated the ability of factors secreted by Th1 and Th17 cells to induce microglial activation. In vitro experiments wherein microglia were cultured in the presence of supernatants derived from polarized Th1 or Th17 cultures, revealed that Th1-associated factors could directly activate and trigger a proinflammatory M1-type gene expression profile in microglia that was cell-cell contact independent, whereas Th17 cells or its associated factors did not have any direct influence on microglia. To assess the effects of the key Th17 effector cytokine IL-17A in vivo we used transgenic mice in which IL-17A is specifically expressed in astrocytes. Flow cytometric and histological analysis revealed only subtle changes in the phenotype of microglia suggesting only minimal effects of constitutively produced IL-17A on microglia in vivo. Neither IL-23 signaling nor addition of GM-CSF, a recently described effector molecule of Th17 cells, changed the incapacity of Th17 cells to activate microglia. These findings demonstrate a potent effect of Th1 cells on microglia, however, the mechanism of how Th17 cells achieve their effect in CNS inflammation remains unclear.
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The bacteriostatic protein lipocalin 2 is induced in the central nervous system of mice with west nile virus encephalitis.
J. Virol.
PUBLISHED: 10-30-2013
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Lipocalin 2 (Lcn2) is a bacteriostatic factor produced during the innate immune response to bacterial infection. Whether Lcn2 has a function in viral infection is unknown. We investigated the regulation and function of Lcn2 in the central nervous system (CNS) of mice during West Nile virus (WNV) encephalitis. Lcn2 mRNA and protein were induced in the brain by day 5, and this induction increased further by day 7 postinfection but was delayed compared with the induction of the toll-like receptor 3 (TLR3) gene, retinoic acid-inducible gene 1 (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) gene. The Lcn2 mRNA and protein were both found at high levels in the choroid plexus, vascular endothelium, macrophage/microglia, and astrocytes. However, some neuronal subsets contained Lcn2 protein but no detectable mRNA. In Lcn2 knockout (KO) mice, with the exception of CXC motif chemokine 5 (CXCL5), which was significantly more downregulated than in wild-type (WT) mice, expression levels of a number of other host response genes were similar in the two genotypes. The brain from Lcn2 and WT mice with WNV encephalitis contained similar numbers of infiltrating macrophages, granulocytes, and T cells. Lcn2 KO and WT mice had no significant difference in tissue viral loads or survival after infection with different doses of WNV. We conclude that Lcn2 gene expression is induced to high levels in a time-dependent fashion in a variety of cells and regions of the CNS of mice with WNV encephalitis. The function of Lcn2 in the host response to WNV infection remains largely unknown, but our data indicate that it is dispensable as an antiviral or immunoregulatory factor in WNV encephalitis.
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Mechanisms of vesicle spreading on surfaces: coarse-grained simulations.
Langmuir
PUBLISHED: 03-21-2013
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Exposition of unilamellar vesicles to attractive surfaces is a frequently used way to create supported lipid bilayers. Although this approach is known to produce continuous supported bilayer coatings, the mechanism of their formation and its dependence on factors like surface interaction and roughness or membrane tension as well as the interplay between neighboring vesicles or the involvement of preadsorbed bilayer patches are not well understood. Using dissipative particle dynamics simulations, we assess different mechanisms of vesicle spreading on attractive surfaces, placing special emphasis on the orientation of the resulting bilayer. Making use of the universality of collective phenomena in lipid membranes, we employed a solvent-free coarse-grained model, enabling us to cover the relatively large system sizes and time scales required. Our results indicate that one can control the mechanism of vesicle spreading by tuning the strength and range of the interactions with the substrate as well as the surfaces roughness, resulting in a switch from a predominant inside-up to an outside-up orientation of the created supported bilayer.
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Parameter passing between molecular dynamics and continuum models for droplets on solid substrates: the static case.
J Chem Phys
PUBLISHED: 02-22-2013
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We study equilibrium properties of polymer films and droplets on a solid substrate employing particle-based simulation techniques (molecular dynamics) and a continuum description. Parameter-passing techniques are explored that facilitate a detailed comparison of the two models. In particular, the liquid-vapor, solid-liquid, and solid-vapor interface tensions, and the Derjaguin or disjoining pressure are determined by molecular dynamics simulations. This information is then introduced into continuum descriptions accounting for (i) the full curvature and (ii) a long-wave approximation of the curvature (thin film model). A comparison of the dependence of the contact angle on droplet size indicates that the theories agree well if the contact angles are defined in a compatible manner.
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CNS-targeted production of IL-17A induces glial activation, microvascular pathology and enhances the neuroinflammatory response to systemic endotoxemia.
PLoS ONE
PUBLISHED: 01-20-2013
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Interleukin-17A (IL-17A) is a key cytokine modulating the course of inflammatory diseases. Whereas effector functions of IL-17A like induction of antimicrobial peptides and leukocyte infiltration could clearly be demonstrated for peripheral organs, CNS specific effects are not well defined and appear controversial. To further clarify the functional significance of IL-17A in the CNS, we generated a transgenic mouse line with astrocyte-restricted expression of the IL-17A gene. GFAP/IL-17A transgenic mice develop normally and do not show any signs of neurological dysfunction. However, histological characterization revealed astrocytosis and activation of microglia. Demyelination, neurodegeneration or prominent tissue damage was not observed but a vascular pathology mimicking microangiopathic features was evident. Histological and flow cytometric analysis demonstrated the absence of parenchymal infiltration of immune cells into the CNS of GFAP/IL-17A transgenic mice. In GFAP/IL-17A mice, LPS-induced endotoxemia led to a more pronounced microglial activation with expansion of a distinct CD45(high)/CD11b(+) population and increased induction of proinflammatory cytokines compared with controls. Our data argues against a direct role of IL-17A in mediating tissue damage during neuroinflammation. More likely IL-17A acts as a modulating factor in the network of induced cytokines. This novel mouse model will be a very useful tool to further characterize the role of IL-17A in neuroinflammatory disease models.
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Monte-Carlo simulation of ternary blends of block copolymers and homopolymers.
J Chem Phys
PUBLISHED: 09-29-2011
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We perform a theoretically informed coarse grain Monte-Carlo simulation in the nPT-ensemble and the Gibbs ensemble on symmetric ternary mixtures of AB-diblock copolymers with the corresponding homopolymers. We study the lamellar period by varying the length and amount of homopolymers. The homopolymer distribution within the lamellar morphology is determined as is the maximum amount of homopolymer within the lamellae. Gibbs ensemble simulations are used to locate the three-phase coexistence between two homopolymer-rich phases and a lamellar phase.
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Speeding up intrinsically slow collective processes in particle simulations by concurrent coupling to a continuum description.
Phys. Rev. Lett.
PUBLISHED: 08-15-2011
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The difficulty to study intrinsically slow collective processes by computer simulation of particle models stems from multiple disparate time scales (e.g., stiff bonded interactions versus soft nonbonded interactions). Continuum models, which describe the system by collective variables rather than the coordinates of the individual molecular constituents, often do not suffer from this time-scale problem because the stiff microscopic degrees of freedom have been integrated out. We propose to concurrently couple these two descriptions by a heterogeneous multiscale method. We illustrate the technique by studying the Lifshitz-Slyozov coarsening mechanism in a binary polymer blend using a soft coarse-grained particle model and a Landau-Ginzburg-de Gennes free energy functional, respectively. A speedup of up to two orders of magnitudes is achieved.
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Serum from interferon-?-1b-treated patients with early multiple sclerosis stabilizes the blood-brain barrier in vitro.
Mult. Scler.
PUBLISHED: 08-15-2011
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Interferon-? (IFN-?) stabilizes the blood-brain barrier (BBB) in vitro. Here we investigated the effect of serum from 15 IFN-?-1b-treated multiple sclerosis (MS) patients on the permeability read-outs of small solutes in an in vitro BBB model consisting of human brain microvascular endothelial cells in co-culture with rat astrocytes. The addition of sera from IFN-?-treated patients resulted in a significantly (p?
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Lipocalin 2 in the central nervous system host response to systemic lipopolysaccharide administration.
J Neuroinflammation
PUBLISHED: 07-14-2011
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Lipocalin 2 (Lcn2) is a bacteriostatic factor that may also modulate cellular function, however, little is known concerning the expression or role of Lcn2 in CNS inflammation. Therefore, here we investigated the regulation and possible function of Lcn2 in the CNS following peripheral lipopolysaccharide (LPS) injection in mice.
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Curvature-dependent elastic properties of liquid-ordered domains result in inverted domain sorting on uniaxially compressed vesicles.
Phys. Rev. Lett.
PUBLISHED: 04-05-2011
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Using a coarse-grained molecular model we study the spatial distribution of lipid domains on a 20-nm-sized vesicle. The lipid mixture laterally phase separates into a raftlike, liquid-ordered (l(o)) phase and a liquid-disordered phase. As we uniaxially compress the mixed vesicle keeping the enclosed volume constant, we impart tension onto the membrane. The vesicle adopts a barrel shape, which is composed of two flat contact zones and a curved edge. The l(o) domain, which exhibits a higher bending rigidity, segregates to the highly curved edge. This inverted domain sorting switches to normal domain sorting, where the l(o) domain prefers the flat contact zone, when we release the contents of the vesicle. We rationalize this domain sorting by a pronounced reduction of the bending rigidity and area compressibility of the l(o) phase upon bending.
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Polymer-solid contacts described by soft, coarse-grained models.
Phys Chem Chem Phys
PUBLISHED: 03-22-2011
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The ability of soft, coarse-grained models to describe the narrow interface of a nearly incompressible polymer melt in contact with a solid is explored by numerical self-consistent field calculations and Monte-Carlo simulations. We investigate the effect of the discreteness of the bead-spring architecture by quantitatively comparing the results of a bead-spring model with different number of beads, N, but identical end-to-end distance, R(e), and a continuous Gaussian-thread model. If the width, ?, of the narrow polymer-solid contact is smaller or comparable to the length of a statistical segment, b=R(e)/?N-1, strong differences in the interface tension and the density profiles between the two models are observed, and strategies for compensating the discrete nature of the bead-spring model are investigated. Compensating the discretization of the chain contour in the bead-spring model by applying an external segment-solid potential, we simultaneously adjust the interface tension and the density profile to the predictions of the Gaussian-thread model. We suggest that the geometry of the polymer-solid contact and the interface tension are relevant characteristics that a coarse-grained model of polymer-solid contacts must reproduce in order to establish a quantitative relationship to an experimental system.
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Opposing roles for CXCR3 signaling in central nervous system versus ocular inflammation mediated by the astrocyte-targeted production of IL-12.
Am. J. Pathol.
PUBLISHED: 01-11-2011
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CXCR3 and its ligands are important for the trafficking of activated CD4(+) T(H)1 T cells, CD8(+) T cells, and natural killer cells during inflammation. Recent functional studies demonstrate a more diverse role of CXCR3 in inflammatory diseases of the central nervous system (CNS). We examined the impact of CXCR3 on a less complex interferon-?-dependent, type 1 cell-mediated immune response in the CNS, induced in mice by the transgenic production of glial fibrillary acidic protein IL-12 (GF-IL12) by astrocytes and retinal Müller cells. GF-IL12 mice develop ataxia because of severe cerebellar inflammation but have little overt ocular disease. Surprisingly, CXCR3-deficient GF-IL12 mice (GF-IL12/CXCR3KO) have drastically reduced ataxia but developed cataracts, severe ocular inflammation, and eye atrophy. Most GF-IL12/CXCR3KO mice had minimal cerebellar inflammation but severe retinal disorganization, loss of photoreceptors, and lens destruction in the eye. The number of CD3(+), CD11b(+), and natural killer 1.1(+) cells were reduced in the CNS but highly increased in the eyes of GF-IL12/CXCR3KO compared with GF-IL12 mice. High levels of interferon-?, IL-1, tumor necrosis factor ?, CXCL9, CXCL10, and CCL5 were found in GF-IL12 cerebelli and GF-IL12/CXCR3KO eyes. Our findings demonstrate key but paradoxical functions for CXCR3 in IL-12-induced immune disease in the CNS, promoting inflammation in the brain yet restricting it in the eye. We conclude that the function of CXCR3 in cellular immune disease is driven by a common trigger and is controlled by tissue-specific factors.
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Three-phase coexistence with sequence partitioning in symmetric random block copolymers.
Phys Rev E Stat Nonlin Soft Matter Phys
PUBLISHED: 01-10-2011
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We inquire into the possible coexistence of macroscopic and microstructured phases in random Q-block copolymers built of incompatible monomer types A and B with equal average concentrations. In our microscopic model, one block comprises M identical monomers. The block-type sequence distribution is Markovian and characterized by the correlation ?. Upon increasing the incompatibility ? (by decreasing temperature) in the disordered state, the known ordered phases form: for ?>?(c), two coexisting macroscopic A- and B-rich phases, for ?
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On the longevity of resident endoneurial macrophages in the peripheral nervous system: a study of physiological macrophage turnover in bone marrow chimeric mice.
J. Peripher. Nerv. Syst.
PUBLISHED: 12-28-2010
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Macrophages are intimately involved in the pathogenesis of peripheral nervous system (PNS) disorders. Recently, we characterized a resident endoneurial macrophage population, which contributes rapidly to the endoneurial macrophage response in PNS diseases. Unlike microglial cells, resident macrophages undergo a physiological turnover of 50% in the sciatic nerve and 80% in dorsal root ganglia (DRG) within 12 weeks. Further information about the dynamics of this turnover is not available. This study examined the macrophage turnover in the sciatic nerve and DRGs over a longer period and addresses the question whether the turnover of resident macrophages is complete or whether there is a truly resident endoneurial macrophage population. We used chimeric mice carrying GFP(+) bone marrow and immunohistochemistry to detect hematogenous (GFP(+)) endoneurial macrophages after turnover. Non-exchanged, resident macrophages were GFP(-). Quantification of GFP(+) and GFP(-) macrophages revealed a maximal turnover of 75%, reached in DRGs after 12 weeks and in sciatic nerves after 36 weeks. GFP(-) long-term resident macrophages were further characterized after sciatic nerve injury, where they participated in the early macrophage response of Wallerian degeneration. Our results point toward a small but truly resident PNS macrophage population. These macrophages are an interesting target for further characterization and might have a distinct role in peripheral nerve disease.
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Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material.
Hum. Gene Ther.
PUBLISHED: 09-16-2010
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A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18?x?10¹¹ particles/ml; 95% confidence interval [CI], 7.89?x?10¹¹ to 1.05?x?10¹² particles/ml), vector genomes ({X}, 3.28?x?10¹? vector genomes/ml; 95% CI, 2.70?x?10¹? to 4.75?x?10¹? vector genomes/ml), transducing units ({X}, 5.09?x?10? transducing units/ml; 95% CI, 2.00?x?10? to 9.60?x?10? transducing units/ml), and infectious units ({X}, 4.37?x?10? TCID?? IU/ml; 95% CI, 2.06?x?10? to 9.26?x?10? TCID?? IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.
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The cell-specific induction of CXC chemokine ligand 9 mediated by IFN-gamma in microglia of the central nervous system is determined by the myeloid transcription factor PU.1.
J. Immunol.
PUBLISHED: 06-28-2010
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The IFN-gamma-inducible chemokines CXCL9 and CXCL10 are implicated in the pathogenesis of T cell-mediated immunity in the CNS. However, in various CNS immune pathologies the cellular localization of these chemokines differs, with CXCL9 produced by macrophage/microglia whereas CXCL10 is produced by both macrophage/microglia and astrocytes. In this study, we determined the mechanism for the microglial cell-restricted expression of the Cxcl9 gene induced by IFN-gamma. In cultured glial cells, the induction of the CXCL9 (in microglia) and CXCL10 (in microglia and astrocytes) mRNAs by IFN-gamma was not inhibited by cycloheximide. Of various transcription factors involved with IFN-gamma-mediated gene regulation, PU.1 was identified as a constitutively expressed NF in microglia but not in astrocytes. STAT1 and PU.1 bound constitutively to the Cxcl9 gene promoter in microglia, and this increased significantly following IFN-gamma treatment with IFN regulatory factor-8 identified as an additional late binding factor. However, in astrocytes, STAT1 alone bound to the Cxcl9 gene promoter. STAT1 was critical for IFN-gamma induction of both the Cxcl9 and Cxcl10 genes in microglia and in microglia and astrocytes, respectively. The small interfering RNA-mediated knockdown of PU.1 in microglia markedly impaired IFN-gamma-induced CXCL9 but not STAT1 or IFN regulatory factor-8. Cells of the D1A astrocyte line showed partial reprogramming to a myeloid-like phenotype posttransduction with PU.1 and, in addition to the expression of CD11b, acquired the ability to produce CXCL9 in response to IFN-gamma. Thus, PU.1 not only is crucial for the induction of CXCL9 by IFN-gamma in microglia but also is a key determinant factor for the cell-specific expression of this chemokine by these myeloid cells.
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A physiological frequency-position map of the chinchilla cochlea.
Hear. Res.
PUBLISHED: 05-12-2010
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Accumulating evidence indicates that mammalian cochlear frequency-position maps (location of maximum vibration of the basilar membrane as a function of frequency) depend on the physiological condition of the inner ear. Cochlear damage desensitizes the ear, after the damage the original location of maximum vibration is tuned to a lower sound frequency. This suggests that frequency-position maps, derived from such desensitized ears, are shifted to lower frequencies, corresponding to a shift of the basilar membrane vibration pattern towards the base for a given stimulus frequency. To test this hypothesis, we re-mapped the cochlear frequency-position map in the chinchilla. We collected frequency-position data from chinchillas in normal physiological condition ("physiological map") and compared these to data previously established from sound overexposed ears ("anatomical map"). The characteristic frequency (CF) of neurons in the cochlear nucleus was determined. Horse-radish peroxidase (HRP) or biocytin (BCT) were injected iontophoretically to trace auditory nerve fibers towards their innervation site in the organ of Corti. The relationship between distance from the base (d, percent) and frequency (f, kHz) was described best by a simple exponential function: d = 61.2 - 42.2 x log(f). The slope of the function was 2.55 mm/octave. Compared to the "anatomical map", the "physiological map" was shifted by about 0.3 octaves to higher frequencies corresponding to a shift of the basilar membrane vibration pattern of 0.8 mm towards the apex for a given stimulus frequency. Our findings affirm that frequency-position maps in the mammalian cochlea depend on the condition of the inner ear. Damage-induced desensitization in mammalian inner ears results in similar shifts of CF (about 0.5 octaves) but different shifts of the maximum of the vibration pattern towards the base at given frequencies, dependent on the mapping constant of the species, longer basilar membranes showing a larger basal shift. Furthermore, the results substantiate the notion that "crowding" at lower frequencies appears to be a specialization rather than a general feature.
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Directed assembly of supramolecular copolymers in thin films: thermodynamic and kinetic advantages.
Phys. Rev. Lett.
PUBLISHED: 05-07-2010
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Using computer simulation of a coarse-grained model for supramolecular polymers, we investigate the potential of quasiblock copolymers (QBCPs) assembled on chemically patterned substrates for creating device-oriented nanostructures. QBCPs are comprised of AB diblock copolymers and supramolecular B segments that can reversibly bond to any available B terminus, on either the copolymers or the B oligomers, creating a polydisperse blend of B homopolymers, and AB and ABA copolymers. We demonstrate the defect-free replication of patterns with perpendicularly crossing, A-preferential lines, where the same QBCP can simultaneously replicate patterns differing by up to 50% in their length scales. We demonstrate how the pattern affects the distribution of molecular architectures and the key role of supramolecular associations for replicating patterns with different length scales.
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Main phase transition in lipid bilayers: Phase coexistence and line tension in a soft, solvent-free, coarse-grained model.
J Chem Phys
PUBLISHED: 04-29-2010
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We devise a soft, solvent-free, coarse-grained model for lipid bilayer membranes. The nonbonded interactions take the form of a weighted-density functional, which allows us to describe the thermodynamics of self-assembly and packing effects of the coarse-grained beads in terms of a density expansion of the equation of state and weighting functions that regularize the microscopic bead densities, respectively. Identifying the length and energy scales via the bilayer thickness and the thermal energy scale, k(B)T, the model qualitatively reproduces key characteristics (e.g., bending rigidity, area per molecule, and compressibility) of lipid membranes. We employ this model to study the main phase transition between the fluid and the gel phase of the bilayer membrane. We accurately locate the phase coexistence using free energy calculations and also obtain estimates for the bare and the thermodynamic line tension.
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Measuring excess free energies of self-assembled membrane structures.
Faraday Discuss.
PUBLISHED: 02-18-2010
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Using computer simulation of a solvent-free, coarse-grained model for amphiphilic membranes, we study the excess free energy of hourglass-shaped connections (i.e., stalks) between two apposed bilayer membranes. In order to calculate the free energy by simulation in the canonical ensemble, we reversibly transfer two apposed bilayers into a configuration with a stalk in three steps. First, we gradually replace the intermolecular interactions by an external, ordering field. The latter is chosen such that the structure of the non-interacting system in this field closely resembles the structure of the original, interacting system in the absence of the external field. The absence of structural changes along this path suggests that it is reversible; a fact which is confirmed by expanded-ensemble simulations. Second, the external, ordering field is changed as to transform the non-interacting system from the apposed bilayer structure to two-bilayers connected by a stalk. The final external field is chosen such that the structure of the non-interacting system resembles the structure of the stalk in the interacting system without a field. On the third branch of the transformation path, we reversibly replace the external, ordering field by non-bonded interactions. Using expanded-ensemble techniques, the free energy change along this reversible path can be obtained with an accuracy of 10(-3)k(B)T per molecule in the n VT-ensemble. Calculating the chemical potential, we obtain the free energy of a stalk in the grandcanonical ensemble, and employing semi-grandcanonical techniques, we calculate the change of the excess free energy upon altering the molecular architecture. This computational strategy can be applied to compute the free energy of self-assembled phases in lipid and copolymer systems, and the excess free energy of defects or interfaces.
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Simulations of theoretically informed coarse grain models of polymeric systems.
Faraday Discuss.
PUBLISHED: 02-18-2010
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Simulations of theoretically informed coarse grain models, where the interaction energy is given by a functional of the local density, are discussed in the context of polymeric melts. Two different implementations are presented by addressing two examples. The first relies on a grid-based representation of non-bonded interactions and focuses on the concept of density multiplication in block copolymer lithography. Monte Carlo simulations are used in a high-throughput manner to explore the parameter space, and to identify morphologies amenable to lithographic fabrication. In the second example, which focuses on the order-disorder transition of block copolymers, the constraints imposed by a grid are removed, thereby enabling simulations in arbitrary ensembles and direct calculation of local stresses and free energies.
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Emerging applications of stimuli-responsive polymer materials.
Nat Mater
PUBLISHED: 01-22-2010
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Responsive polymer materials can adapt to surrounding environments, regulate transport of ions and molecules, change wettability and adhesion of different species on external stimuli, or convert chemical and biochemical signals into optical, electrical, thermal and mechanical signals, and vice versa. These materials are playing an increasingly important part in a diverse range of applications, such as drug delivery, diagnostics, tissue engineering and smart optical systems, as well as biosensors, microelectromechanical systems, coatings and textiles. We review recent advances and challenges in the developments towards applications of stimuli-responsive polymeric materials that are self-assembled from nanostructured building blocks. We also provide a critical outline of emerging developments.
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Theoretically informed coarse grain simulations of polymeric systems.
J Chem Phys
PUBLISHED: 09-04-2009
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A Monte Carlo formalism for the study of polymeric melts is described. The model is particle-based, but the interaction is derived from a local density functional that appears in the field-based model. The method enables Monte Carlo simulations in the nVT, nPT, semigrandcanonical and Gibbs ensembles, and direct calculation of free energies. The approach is illustrated in the context of two examples. In the first, we consider the phase separation of a binary homopolymer blend and present results for the phase diagram and the critical point. In the second, we address the microphase separation of a symmetric diblock copolymer, examine the distribution of local stresses in lamellae, and determine the order-disorder transition temperature.
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Microphase separation of mixed polymer brushes: dependence of the morphology on grafting density, composition, chain-length asymmetry, solvent quality, and selectivity.
J Phys Chem B
PUBLISHED: 09-02-2009
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Microphase separation of binary mixed A/B polymer brushes exposed to different solvents is studied using Single-Chain-in-Mean-Field simulations. Effects of solvent quality and selectivity, grafting density, composition, and chain-length asymmetry are systematically investigated, and diagrams of morphologies in various solvents are constructed as a function of grafting density and composition or chain-length asymmetry. The structure of the microphase segregated morphologies lacks long-range periodic order, and it is analyzed quantitatively using Minkowski measures.
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Molecular aspects of tinnitus.
Hear. Res.
PUBLISHED: 07-15-2009
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Molecular changes caused by sensory trauma and subsequent structural alterations of the central nervous system are only beginning to be identified. In most cases, the generation of tinnitus can be linked to damage of the peripheral auditory system, probably even in cases where hearing impairment cannot be assessed by audiometry. Within a common view, acoustic trauma and salicylate induce abnormal excitability at the level of the brainstem, subcortical and cortical level that may be related to tinnitus. The present review summarizes studies emphasizing a crucial role of molecular events that occur in the cochlea exhibiting the potential to alter the network activity in distinct areas of the brain, including the limbic system. We proceed from the inner ear to the auditory cortex and discuss the recent molecular findings in the central auditory system as a secondary step of previous neuronal changes in the periphery.
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Site-specific production of IL-6 in the central nervous system retargets and enhances the inflammatory response in experimental autoimmune encephalomyelitis.
J. Immunol.
PUBLISHED: 07-13-2009
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IL-6 is crucial for the induction of many murine models of autoimmunity including experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. To establish the role of site-specific production of IL-6 in autoimmunity, we examined myelin oligodendrocyte glycoprotein immunization-induced EAE in transgenic mice (GFAP-IL6) with IL-6 production restricted to the cerebellum. Myelin oligodendrocyte glycoprotein-immunized (Mi-) GFAP-IL6 mice developed severe ataxia but no physical signs of spinal cord involvement, which was in sharp contrast to Mi-wild type (WT) animals that developed classical EAE with ascending paralysis. Immune pathology and demyelination were nearly absent from the spinal cord, but significantly increased in the cerebellum of Mi-GFAP-IL6 mice. Tissue damage in the cerebellum in the Mi-GFAP-IL6 mice was accompanied by increased total numbers of infiltrating leukocytes and increased proportions of both neutrophils and B-cells. With the exception of IL-17 mRNA, which was elevated in both control immunized and Mi-GFAP-IL6 cerebellum, the level of other cytokine and chemokine mRNAs were comparable with Mi-WT cerebellum whereas significantly higher levels of IFN-gamma and TNF-alpha mRNA were found in Mi-WT spinal cord. Thus, site-specific production of IL-6 in the cerebellum redirects trafficking away from the normally preferred antigenic site the spinal cord and acts as a leukocyte "sink" that markedly enhances the inflammatory cell accumulation and disease. The mechanisms underlying this process likely include the induction of specific chemokines, activation of microglia, and activation and loss of integrity of the blood-brain barrier present in the cerebellum of the GFAP-IL6 mice before the induction of EAE.
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Monte carlo simulation of coarse grain polymeric systems.
Phys. Rev. Lett.
PUBLISHED: 05-12-2009
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We introduce a particle-based Monte Carlo formalism for the study of polymeric melts, where the interaction energy is given by a local density functional, as is done in traditional field-theoretic models. The method enables Monte Carlo simulations in arbitrary ensembles and direct calculation of free energies. We present results for the phase diagram and the critical point of a binary homopolymer blend. For a symmetric diblock copolymer, we compute the distribution of local stress in lamellae and locate the order-disorder transition.
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Nuclear and membrane expression of the angiogenesis regulator delta-like ligand 4 (DLL4) in normal and malignant human tissues.
Histopathology
PUBLISHED: 05-06-2009
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Delta-like ligand 4 (DLL4) is one of five known Notch ligands in mammals and interacts predominantly with Notch 1. DLL4 is induced by vascular endothelial growth factor (VEGF) and acts downstream of VEGF as a brake on VEGF-induced vessel growth, forming an autoregulatory negative feedback loop inactivating VEGF. This action was believed to occur only in vascular development, raising hopes that DLL4 could be a specific drug target for controlling vessel growth in tumours and other pathological conditions. Our aim was to pursue this by raising a monoclonal antibody to the internal domain of DLL4 and assess its distribution in normal and malignant tissues in comparison with antibodies against the external domain of DLL4.
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Radiation therapy for early stages of morbus Ledderhose.
Strahlenther Onkol
PUBLISHED: 05-03-2009
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To evaluate the efficacy of radiation therapy (RT) in the treatment of early stages of benign plantar fibromatosis (Morbus Ledderhose [ML]).
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Nucleation in A/B/AB blends: interplay between microphase assembly and macrophase separation.
J Chem Phys
PUBLISHED: 04-25-2009
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We study the interplay between microphase assembly and macrophase separation in A/B/AB ternary polymer blends by examining the free energy of localized fluctuation structures (micelles or droplets), with emphasis on the thermodynamic relationship between swollen micelles (microemulsion) and the macrophase-separated state, using self-consistent field theory and an extended capillary model. Upon introducing homopolymer B into a micelle-forming binary polymer blend A/AB, micelles can be swollen by B. A small amount of component B (below the A-rich binodal of macrophase coexistence) will not affect the stability of the swollen micelles. A large excess of homopolymer, B, will induce a microemulsion failure and lead to a macrophase separation. Between the binodal and the microemulsion failure concentration, macrophase separation in A/B/AB occurs by a two-step nucleation mechanism via a metastable microemulsion droplet of finite size. Our results illustrate a recently proposed argument that the two-step nucleation via a metastable intermediate is a general phenomenon in systems involving short-range attraction and long-range repulsion.
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A modified and automated version of the Fluorimetric Detection of Alkaline DNA Unwinding method to quantify formation and repair of DNA strand breaks.
BMC Biotechnol.
PUBLISHED: 04-23-2009
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Formation and repair of DNA single-strand breaks are important parameters in the assessment of DNA damage and repair occurring in live cells. The Fluorimetric Detection of Alkaline DNA Unwinding (FADU) method [Birnboim HC, Jevcak JJ. Cancer Res (1981) 41:1889-1892] is a sensitive procedure to quantify DNA strand breaks, yet it is very tedious to perform.
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Deafness in TRbeta mutants is caused by malformation of the tectorial membrane.
J. Neurosci.
PUBLISHED: 02-27-2009
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Thyroid hormone receptor beta (TRbeta) dysfunction leads to deafness in humans and mice. Deafness in TRbeta(-/-) mutant mice has been attributed to TRbeta-mediated control of voltage- and Ca(2+)-activated K(+) (BK) channel expression in inner hair cells (IHCs). However, normal hearing in young constitutive BKalpha(-/-) mutants contradicts this hypothesis. Here, we show that mice with hair cell-specific deletion of TRbeta after postnatal day 11 (P11) have a delay in BKalpha expression but normal hearing, indicating that the origin of hearing loss in TRbeta(-/-) mutant mice manifested before P11. Analyzing the phenotype of IHCs in constitutive TRbeta(-/-) mice, we found normal Ca(2+) current amplitudes, exocytosis, and shape of compound action potential waveforms. In contrast, reduced distortion product otoacoustic emissions and cochlear microphonics associated with an abnormal structure of the tectorial membrane and enhanced tectorin levels suggest that disturbed mechanical performance is the primary cause of deafness resulting from TRbeta deficiency.
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Computing free energies of interfaces in self-assembling systems.
Phys Chem Chem Phys
PUBLISHED: 02-03-2009
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Using molecular simulation of a coarse-grained model for a symmetric diblock copolymer melt, we calculate the free energy of interfaces between lamellar morphologies with different orientations. Two examples are considered: (a) a T-junction between two lamellar structures with perpendicular orientation and (b) a surface reconstruction that arises when lamella-forming diblock copolymers assemble on a stripe-patterned surface, where the pattern period is significantly larger than the lamellar spacing in the bulk. The computational scheme relies on reversibly relating the defect structure to a reference state by an external ordering field. Free energy differences are accurately obtained by using a combination of expanded-ensemble and replica-exchange Monte Carlo techniques.
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Accuracy of velocity distortion product otoacoustic emissions for estimating mechanically based hearing loss.
Hear. Res.
PUBLISHED: 02-02-2009
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Distortion product otoacoustic emissions (DPOAEs) measured as vibration of the human eardrum have been successfully used to estimate hearing threshold. The estimates have proved more accurate than similar methods using sound-pressure DPOAEs. Nevertheless, the estimation accuracy of the new technique might have been influenced by endogenous noise, such as heart beat, breathing and swallowing. Here, we investigate in an animal model to what extent the accuracy of the threshold estimation technique using velocity-DPOAEs might be improved by reducing noise sources. Velocity-DPOAE I/O functions were measured in normal and hearing-impaired anaesthetized guinea pigs. Hearing loss was either conductive or induced by furosemide injection. The estimated distortion product threshold (EDPT) obtained by extrapolation of the I/O function to the abscissa was found to linearly correlate with the compound action potential threshold at the f(2) frequency, provided that furosemide data were excluded. The standard deviation of the linear regression fit was 6 dB as opposed to 8 dB in humans, suggesting that this accuracy should be achievable in humans with appropriate improvement of signal-to-noise ratio. For the furosemide animals, the CAP threshold relative to the regression line provided an estimate of the functional loss of the inner hair cell system. For mechanical losses in the middle ear and/or cochlear amplifier, DPOAEs measured as velocity of the umbo promise an accuracy of hearing threshold estimation comparable to classical audiometry.
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Introducing bio- and micro-technology into undergraduate thermal-fluids courses: investigating pipe pressure loss via atomic force microscopy.
Conf Proc IEEE Eng Med Biol Soc
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To introduce bio- and micro-technologies into general undergraduate thermal-fluids classes, a hands-on interdisciplinary in-class demonstration is described that juxtaposes classical pressure loss pipe flow experiments against a modern micro-characterization technique, AFM profilometry. Both approaches measure surface roughness and can segue into classroom discussions related to material selection and design of bio-medical devices to handle biological fluids such as blood. Appealing to the range of engineering students populating a general thermal-fluids course, a variety of pipe/hose/tube materials representing a spectrum of disciplines can be tested using both techniques. This in-class demonstration relies on technical content already available in standard thermal-fluids textbooks, provides experimental juxtaposition between classical and micro-technology-enabled approaches to the same experiment, and can be taught by personnel with no specialized micro- or bio-technology expertise.
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Targeted blockade in lethal West Nile virus encephalitis indicates a crucial role for very late antigen (VLA)-4-dependent recruitment of nitric oxide-producing macrophages.
J Neuroinflammation
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Infiltration of Ly6C(hi) monocytes from the blood is a hallmark of viral encephalitis. In mice with lethal encephalitis caused by West Nile virus (WNV), an emerging neurotropic flavivirus, inhibition of Ly6C(hi) monocyte trafficking into the brain by anti-very late antigen (VLA)-4 integrin antibody blockade at the time of first weight loss and leukocyte influx resulted in long-term survival of up to 60% of infected mice, with subsequent sterilizing immunity. This treatment had no effect on viral titers but appeared to be due to inhibition of Ly6C(hi) macrophage immigration. Although macrophages isolated from the infected brain induced WNV-specific CD4(+) T-cell proliferation, T cells did not directly contribute to pathology, but are likely to be important in viral control, as antibody-mediated T-cell depletion could not reproduce the therapeutic benefit of anti-VLA-4. Instead, 70% of infiltrating inflammatory monocyte-derived macrophages were found to be making nitric oxide (NO). Furthermore, aminoguanidine-mediated inhibition of induced NO synthase activity in infiltrating macrophages significantly prolonged survival, indicating involvement of NO in the immunopathology. These data show for the first time the therapeutic effects of temporally targeting pathogenic NO-producing macrophages during neurotropic viral encephalitis.
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Translationally invariant slip-spring model for entangled polymer dynamics.
Phys. Rev. Lett.
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The topological effect of noncrossability of long flexible macromolecules is effectively described by a slip-spring model, which represents entanglements by local, pairwise, translationally invariant interactions that do not alter any equilibrium properties. We demonstrate that the model correctly describes many aspects of the dynamical and rheological behavior of entangled polymer liquids, such as segmental mean-square displacements and shear thinning, in a computationally efficient manner. Furthermore, the model can account for the reduction of entanglements under shear.
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Transition path from two apposed membranes to a stalk obtained by a combination of particle simulations and string method.
Phys. Rev. Lett.
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The formation of an hourglass-shaped passage (stalk) connecting two apposed membranes is an essential initial step in membrane fusion. The most probable transition path from two separate membranes to a stalk, i.e., the minimum free-energy path (MFEP), is constructed using a combination of particle simulations and string method. For the reversible transition path in the coarse-grained membrane model, a collective order parameter, m, can be identified as the local difference of hydrophilic and hydrophobic densities. In particle simulations, the free energy F[m] as a functional of m is not readily available. This difficulty is overcome by an equation-free approach, where the morphology and the excess free energy along the MFEP are obtained by an on-the-fly string method. The transition state is confirmed by diagonalization of order-parameter fluctuations and by the probability of reaching either stalk or bilayer morphology from different positions along the MFEP.
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Geometry-controlled interface localization-delocalization transition in block copolymers.
Phys. Rev. Lett.
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Lamellar copolymers confined into a film of thickness D by two stripe-patterned surfaces, which are rotated against each other by a twist angle ?, form lamellar domains that register and align with the respective chemical surface patterns. The two domains of thickness x and D-x are separated by an interface that resembles a twist grain boundary. At small twist angles ? or strong selectivity of the surface patterns, this interface fluctuates around the middle of the film, x?D/2, while the interface is localized at one of the surfaces, x?0 or x?D, in the opposite limit. These two morphologies are separated by an interface localization-delocalization transition (ILDT) that can be controlled by the twist angle ?. For thin films, we find a second-order ILDT while the ILDT is first-order for large D values. A phenomenological interface Hamiltonian is used to relate the findings to the ILDT of symmetric mixtures, and the predictions are confirmed by molecular simulation.
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Colloidal nanocrystals embedded in macrocrystals: robustness, photostability, and color purity.
Nano Lett.
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The incorporation of colloidal quantum dots (QDs) into ionic crystals of various salts (NaCl, KCl, KBr, etc.) is demonstrated. The resulting mixed crystals of various shapes and beautiful colors preserve the strong luminescence of the incorporated QDs. Moreover, the ionic salts appear to be very tight matrices, ensuring the protection of the QDs from the environment and as a result providing them with extraordinary high photo- and chemical stability. A prototype of a white light-emitting diode (WLED) with a color conversion layer consisting of this kind of mixed crystals is demonstrated. These materials may also find applications in nonlinear optics and as luminescence standards.
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Fluoroscopy-based computer-assisted navigation for implant placement and hip resurfacing arthroplasty in the proximal femur: the zero-dose C-arm navigation approach.
Biomed Tech (Berl)
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Abstract In the proximal femur, a high accuracy of implant placement reduces the risk of mechanical failure. We have tested a new computer-assisted planning and navigation system based on two-dimensional fluoroscopy using the so-called zero-dose C-arm navigation approach to optimise implant placement in fracture fixation and hip resurfacing. The aim of this review is to compare the results of this system with the current literature. Use of the novel system enables a minimally invasive approach to the hip and results in enhanced accuracy of implant placement compared with conventional techniques. Its precision is comparable to navigation systems currently in the market. The new system reduces irradiation but requires more operation time in comparison with established navigation systems. We believe zero-dose C-arm navigation can effectively be used to support surgeons in modern orthopaedic and trauma surgery departments, and can sufficiently serve the demands of both sections, especially at a time focusing on saving costs.
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Co-localisation of K(ir)4.1 and AQP4 in rat and human cochleae reveals a gap in water channel expression at the transduction sites of endocochlear K(+) recycling routes.
Cell Tissue Res.
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Sensory transduction in the cochlea depends on perilymphatic-endolymphatic potassium (K(+)) recycling. It has been suggested that the epithelial supporting cells (SCs) of the cochlear duct may form the intracellular K(+) recycling pathway. Thus, they must be endowed with molecular mechanisms that facilitate K(+) uptake and release, along with concomitant osmotically driven water movements. As yet, no molecules have been described that would allow for volume-equilibrated transepithelial K(+) fluxes across the SCs. This study describes the subcellular co-localisation of the K(ir)4.1 K(+) channel (K(ir)4.1) and the aquaporin-4 water channel (AQP4) in SCs, on the basis of immunohistochemical double-labelling experiments in rat and human cochleae. The results of this study reveal the expression of K(ir)4.1 in the basal or basolateral membranes of the SCs in the sensory domain of the organ of Corti that are adjacent to hair cells and in the non-sensory domains of the inner and outer sulci that abut large extracellular fluid spaces. The SCs of the inner sulcus (interdental cells, inner sulcus cells) and the outer sulcus (Hensens cells, outer sulcus cells) display the co-localisation of K(ir)4.1 and AQP4 expression. However, the SCs in the sensory domain of the organ of Corti reveal a gap in the expression of AQP4. The outer pillar cell is devoid of both K(ir)4.1 and AQP4. The subcellular co-localisation of K(ir)4.1 and AQP4 in the SCs of the cochlea described in this study resembles that of the astroglia of the central nervous system and the glial Mueller cells in the retina.
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Line-tension controlled mechanism for influenza fusion.
PLoS ONE
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Our molecular simulations reveal that wild-type influenza fusion peptides are able to stabilize a highly fusogenic pre-fusion structure, i.e. a peptide bundle formed by four or more trans-membrane arranged fusion peptides. We rationalize that the lipid rim around such bundle has a non-vanishing rim energy (line-tension), which is essential to (i) stabilize the initial contact point between the fusing bilayers, i.e. the stalk, and (ii) drive its subsequent evolution. Such line-tension controlled fusion event does not proceed along the hypothesized standard stalk-hemifusion pathway. In modeled influenza fusion, single point mutations in the influenza fusion peptide either completely inhibit fusion (mutants G1V and W14A) or, intriguingly, specifically arrest fusion at a hemifusion state (mutant G1S). Our simulations demonstrate that, within a line-tension controlled fusion mechanism, these known point mutations either completely inhibit fusion by impairing the peptides ability to stabilize the required peptide bundle (G1V and W14A) or stabilize a persistent bundle that leads to a kinetically trapped hemifusion state (G1S). In addition, our results further suggest that the recently discovered leaky fusion mutant G13A, which is known to facilitate a pronounced leakage of the target membrane prior to lipid mixing, reduces the membrane integrity by forming a super bundle. Our simulations offer a new interpretation for a number of experimentally observed features of the fusion reaction mediated by the prototypical fusion protein, influenza hemagglutinin, and might bring new insights into mechanisms of other viral fusion reactions.
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Water channel proteins in the inner ear and their link to hearing impairment and deafness.
Mol. Aspects Med.
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The inner ear is a fluid-filled sensory organ that transforms mechanical stimuli into the senses of hearing and balance. These neurosensory functions depend on the strict regulation of the volume of the two major extracellular fluid domains of the inner ear, the perilymph and the endolymph. Water channel proteins, or aquaporins (AQPs), are molecular candidates for the precise regulation of perilymph and endolymph volume. Eight AQP subtypes have been identified in the membranous labyrinth of the inner ear. Similar AQP subtypes are also expressed in the kidney, where they function in whole-body water regulation. In the inner ear, AQP subtypes are ubiquitously expressed in distinct cell types, suggesting that AQPs have an important physiological role in the volume regulation of perilymph and endolymph. Furthermore, disturbed AQP function may have pathophysiological relevance and may turn AQPs into therapeutic targets for the treatment of inner ear diseases. In this review, we present the currently available knowledge regarding the expression and function of AQPs in the inner ear. We give special consideration to AQP subtypes AQP2, AQP4 and AQP5, which have been studied most extensively. The potential functions of AQP2 and AQP5 in the resorption and secretion of endolymph and of AQP4 in the equilibration of cell volume are described. The pathophysiological implications of these AQP subtypes for inner ear diseases, that appear to involve impaired fluid regulation, such as Menières disease and Sjögrens syndrome, are discussed.
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Entry of herpes simplex virus type 1 (HSV-1) into the distal axons of trigeminal neurons favors the onset of nonproductive, silent infection.
PLoS Pathog.
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Following productive, lytic infection in epithelia, herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection in sensory neurons that is interrupted by episodes of reactivation. In order to better understand what triggers this lytic/latent decision in neurons, we set up an organotypic model based on chicken embryonic trigeminal ganglia explants (TGEs) in a double chamber system. Adding HSV-1 to the ganglion compartment (GC) resulted in a productive infection in the explants. By contrast, selective application of the virus to distal axons led to a largely nonproductive infection that was characterized by the poor expression of lytic genes and the presence of high levels of the 2.0-kb major latency-associated transcript (LAT) RNA. Treatment of the explants with the immediate-early (IE) gene transcriptional inducer hexamethylene bisacetamide, and simultaneous co-infection of the GC with HSV-1, herpes simplex virus type 2 (HSV-2) or pseudorabies virus (PrV) helper virus significantly enhanced the ability of HSV-1 to productively infect sensory neurons upon axonal entry. Helper-virus-induced transactivation of HSV-1 IE gene expression in axonally-infected TGEs in the absence of de novo protein synthesis was dependent on the presence of functional tegument protein VP16 in HSV-1 helper virus particles. After the establishment of a LAT-positive silent infection in TGEs, HSV-1 was refractory to transactivation by superinfection of the GC with HSV-1 but not with HSV-2 and PrV helper virus. In conclusion, the site of entry appears to be a critical determinant in the lytic/latent decision in sensory neurons. HSV-1 entry into distal axons results in an insufficient transactivation of IE gene expression and favors the establishment of a nonproductive, silent infection in trigeminal neurons.
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Stemness of the organ of Corti relates to the epigenetic status of Sox2 enhancers.
PLoS ONE
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In the adult mammalian auditory epithelium, the organ of Corti, loss of sensory hair cells results in permanent hearing loss. The underlying cause for the lack of regenerative response is the depletion of otic progenitors in the cell pool of the sensory epithelium. Here, we show that an increase in the sequence-specific methylation of the otic Sox2 enhancers NOP1 and NOP2 is correlated with a reduced self-renewal potential in vivo and in vitro; additionally, the degree of methylation of NOP1 and NOP2 is correlated with the dedifferentiation potential of postmitotic supporting cells into otic stem cells. Thus, the stemness the organ of Corti is related to the epigenetic status of the otic Sox2 enhancers. These observations validate the continued exploration of treatment strategies for dedifferentiating or reprogramming of differentiated supporting cells into progenitors to regenerate the damaged organ of Corti.
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Directed assembly of non-equilibrium ABA triblock copolymer morphologies on nanopatterned substrates.
ACS Nano
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The majority of past work on directed assembly of block copolymers on chemically nanopatterned surfaces (or chemical patterns) has focused on AB diblock copolymers, and the resulting morphologies have generally corresponded to equilibrium states. Here we report a study on directed assembly of ABA triblock copolymers. Directed assembly of thin films of symmetric poly(methyl methacrylate-b-styrene-b-methyl methacrylate) (PMMA-b-PS-b-PMMA) triblock copolymers is shown to be capable of achieving a high degree of perfection, registration, and accuracy on striped patterns having periods, L(s), commensurate with the bulk period of the copolymer, L(o). When L(s) is incommensurate with L(o), the triblock copolymer domains can reach dimensions up to 55% larger or 13% smaller than L(o). The range over which triblock copolymers tolerate departures from a commensurate L(s) is significantly larger than that accessible with the corresponding diblock copolymer material on analogous directed assembly systems. The assembly kinetics of the triblock copolymer is approximately 3 orders of magnitude slower than observed in the diblock system. Theoretically informed simulations are used to interpret our experimental observations; a thermodynamic analysis reveals that triblocks can form highly ordered, non-equilibrium metastable structures that do not arise in the diblock.
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Tailoring substrates for long-term organotypic culture of adult neuronal tissue.
Adv. Mater. Weinheim
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Organotypic tissue cultures are highly promising for performing in vivo type studies in vitro. Currently, however, very limited survival times of only a few days for adult tissue often severely limit their application. Here, superhydrophilic nanostructured substrates with ideal material properties ensure tissue adhesion, essential for organotypic culture, while migration of single cells out of the tissue is hampered. Tuning substrate properties, for the first time, adult neuronal tissue could be cultured for 14 days with no indications of degeneration.
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Three-dimensional computer-assisted navigation for the placement of cannulated hip screws. A pilot study.
Int Orthop
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Medial femoral neck fractures are common, and closed reduction and internal fixation by three cannulated screws is an accepted method for the surgical treatment. Computer navigation for screw placement may reduce fluoroscopy time, the number of guidewire passes and optimise screw placement.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.