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Find video protocols related to scientific articles indexed in Pubmed.
Cooperativity Among Rev-Associated Nuclear Export Signals Regulates HIV-1 Gene Expression and is a Determinant of Virus Species Tropism.
J. Virol.
PUBLISHED: 10-01-2014
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Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the CRM1 (mCRM1) nuclear export receptor, and rescued by expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions. We show that Rev is rapidly exported from the nucleus by mCRM1 despite only weak contributions to HIV-1's post-transcriptional stages. Indeed, Rev preferentially accumulates in the cytoplasm of murine 3T3 cells with or without hCRM1 expression, in contrast to human HeLa cells where Rev exhibits striking en masse transitions between the nuclear and cytoplasmic compartments. Efforts to bias Rev's trafficking either into or out of the nucleus revealed that Rev encoding a second CRM1 binding domain (Rev-2xNES), or Rev-dependent viral gag-pol mRNAs bearing tandem RREs (GP-2xRRE), rescue virus particle production in murine cells even in the absence of hCRM1. Combined, these results suggest a model wherein Rev-associated nuclear export signals cooperate to regulate the number or quality of CRM1's interactions with viral Rev/RRE ribonucleoprotein complexes in the nucleus. This mechanism regulates CRM1-dependent viral gene expression and is a determinant of HIV-1's capacity to produce virions in non-human cell types.
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Distinct characteristics of e13a2 versus e14a2 BCR-ABL1 driven chronic myeloid leukemia under first-line therapy with imatinib.
Haematologica
PUBLISHED: 05-16-2014
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The vast majority of chronic myeloid leukemia patients express a BCR-ABL1 fusion gene mRNA encoding a 210 kDa tyrosine kinase which promotes leukemic transformation. A possible differential impact of the corresponding BCR-ABL1 transcript variants e13a2 ("b2a2") and e14a2 ("b3a2") on disease phenotype and outcome is still a subject of debate. A total of 1105 newly diagnosed imatinib-treated patients were analyzed according to transcript type at diagnosis (e13a2, n=451; e14a2, n=496; e13a2+e14a2, n=158). No differences regarding age, sex, or Euro risk score were observed. A significant difference was found between e13a2 and e14a2 when comparing white blood cells (88 vs. 65 × 10(9)/L, respectively; P<0.001) and platelets (296 vs. 430 × 10(9)/L, respectively; P<0.001) at diagnosis, indicating a distinct disease phenotype. No significant difference was observed regarding other hematologic features, including spleen size and hematologic adverse events, during imatinib-based therapies. Cumulative molecular response was inferior in e13a2 patients (P=0.002 for major molecular response; P<0.001 for MR4). No difference was observed with regard to cytogenetic response and overall survival. In conclusion, e13a2 and e14a2 chronic myeloid leukemia seem to represent distinct biological entities. However, clinical outcome under imatinib treatment was comparable and no risk prediction can be made according to e13a2 versus e14a2 BCR-ABL1 transcript type at diagnosis. (clinicaltrials.gov identifier:00055874).
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Kelch-like ECT2-interacting protein KLEIP regulates late-stage pulmonary maturation via Hif-2? in mice.
Dis Model Mech
PUBLISHED: 05-01-2014
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Respiratory distress syndrome (RDS) caused by preterm delivery is a major clinical problem with limited mechanistic insight. Late-stage embryonic lung development is driven by hypoxia and the hypoxia-inducible transcription factors Hif-1? and Hif-2?, which act as important regulators for lung development. Expression of the BTB-and kelch-domain-containing (BTB-kelch) protein KLEIP (Kelch-like ECT2-interacting protein; also named Klhl20) is controlled by two hypoxia response elements, and KLEIP regulates stabilization and transcriptional activation of Hif-2?. Based on the available data, we hypothesized an essential role for KLEIP in murine lung development and function. Therefore, we have performed a functional, histological, mechanistic and interventional study in embryonic and neonatal KLEIP(-/-) mice. Here, we show that about half of the KLEIP(-/-) neonates die due to respiratory failure that is caused by insufficient aeration, reduced septal thinning, reduced glycogenolysis, type II pneumocyte immaturity and reduced surfactant production. Expression analyses in embryonic day (E) 18.5 lungs identified KLEIP in lung capillaries, and showed strongly reduced mRNA and protein levels for Hif-2? and VEGF; such reduced levels are associated with embryonic endothelial cell apoptosis and lung bleedings. Betamethasone injection in pregnant females prevented respiratory failure in KLEIP(-/-) neonates, normalized lung maturation, vascularization, aeration and function, and increased neonatal Hif-2? expression. Thus, the experimental study shows that respiratory failure in KLEIP(-/-) neonates is determined by insufficient angiocrine Hif-2?-VEGF signaling and that betamethasone activates this newly identified signaling cascade in late-stage embryonic lung development.
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Equivalence of BCR-ABL transcript levels with complete cytogenetic remission in patients with chronic myeloid leukemia in chronic phase.
J. Cancer Res. Clin. Oncol.
PUBLISHED: 04-29-2014
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Chronic myeloid leukemia (CML) patients are monitored by both cytogenetic and molecular assessments, although present guidelines appear to switch from cytogenetic to molecular criteria. Due to the increasing use of molecular measurements, it was the aim of this work to identify a BCR-ABL level according to the international scale (BCR-ABL(IS)) as an equivalent substitute for complete cytogenetic remission (CCyR).
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Controlling Polymerization Initiator Concentration in Mesoporous Silica Thin Films.
Langmuir
PUBLISHED: 12-23-2013
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We present a strategy toward controlled polymer density in mesopores by specifically adjusting the local amount of polymerization initiator at the pore wall. The polymerization initiator concentration as well as the polymer functionalization has a direct impact on mesoporous membrane properties such as ionic permselectivity. Mesoporous silica-based thin films were prepared with specifically adjusted amount of polymerization initiator (4-(3-triethoxysilyl)propoxybenzophenone (BPSilane)) or initiator binding functions ((3-aminopropyl)triethoxysilane (APTES)), directly and homogeneously incorporated into the silica wall pursuing a sol-gel-based co-condensation approach. The amount of polymerization initiator was adjusted by varying its concentration in the sol-gel precursor solution. The surface chemistry, porosity, pore accessibility, and reactivity of the surface functional groups were investigated by using infrared spectroscopy, X-ray photoelectron spectroscopy, X-ray reflectometry, ellipsometry, atomic force microscopy, and transmission electron microscopy. We could gradually modify the amount of reactive polymerization initiators in these mesoporous membranes. Mesopores were maintained for APTES containing films for all tested ratios up to 25 mol % and for BPSilane containing films up to 15 mol %. These films showed accessible and charge-dependent ionic permselectivity and an increasing degree of functionalization with increasing precursor ratio. This approach can directly result in control of polymer grafting density in mesoporous films and thus has a direct impact on applications such as the control of ionic transport through mesoporous silica membranes.
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Cube-octameric silsesquioxane-mediated cargo peptide delivery into living cancer cells.
Org. Biomol. Chem.
PUBLISHED: 09-26-2013
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Cube octameric silsesquioxanes (COSS) are among the smallest nanoparticles known to date with a diameter of only 0.7 nm. We describe a COSS-based delivery system which allows for the drug targeting in human cells. It comprises a siloxane core with seven pendant aminopropyl groups and a fluorescently labeled peptidic ligand attached to one cage corner via a reversible disulfide bond to ensure its intracellular release. Bimodal amplitude-modulated atomic force microscopy (AFM) experiments revealed the formation of dendritic COSS structures by a self-assembly of single particles on negatively charged surfaces. Nuclear targeting was demonstrated in HeLa cells by selective binding of released p21(Cip1/Waf1)-derived cargo peptide to PCNA, a protein involved in DNA replication and repair.
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Phylogeography and postglacial recolonization of Europe by Rhinolophus hipposideros: evidence from multiple genetic markers.
Mol. Ecol.
PUBLISHED: 04-23-2013
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The demographic history of Rhinolophus hipposideros (lesser horseshoe bat) was reconstructed across its European, North African and Middle-Eastern distribution prior to, during and following the most recent glaciations by generating and analysing a multimarker data set. This data set consisted of an X-linked nuclear intron (Bgn; 543 bp), mitochondrial DNA (cytb-tRNA-control region; 1630 bp) and eight variable microsatellite loci for up to 373 individuals from 86 localities. Using this data set of diverse markers, it was possible to determine the species demography at three temporal stages. Nuclear intron data revealed early colonization into Europe from the east, which pre-dates the Quaternary glaciations. The mtDNA data supported multiple glacial refugia across the Mediterranean, the largest of which were found in the Ibero-Maghreb region and an eastern location (Anatolia/Middle East)-that were used by R. hipposideros during the most recent glacial cycles. Finally, microsatellites provided the most recent information on these species movements since the Last Glacial Maximum and suggested that lineages that had diverged into glacial refugia, such as in the Ibero-Maghreb region, have remained isolated. These findings should be used to inform future conservation management strategies for R. hipposideros and show the power of using a multimarker data set for phylogeographic studies.
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90K, an interferon-stimulated gene product, reduces the infectivity of HIV-1.
Retrovirology
PUBLISHED: 04-16-2013
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In response to viral infections, interferons induce the transcription of several hundred genes in mammalian cells. Specific antiviral functions, however, have only been attributed to a few of them. 90K/LGALS3BP has been reported to be an interferon-stimulated gene that is upregulated in individuals with cancer or HIV-1 infection.
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Nanomechanical coupling enables detection and imaging of 5 nm superparamagnetic particles in liquid.
Nanotechnology
PUBLISHED: 02-17-2011
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We demonstrate that a force microscope operated in a bimodal mode enables the imaging and detection of superparamagnetic particles down to 5 nm. The bimodal method exploits the nanomechanical coupling of the excited modes to enhance the sensitivity of the higher mode to detect changes in material properties. The coupling requires the presence of nonlinear forces. Remarkably, bimodal operation enables us to identify changes of slowly varying forces (quasi-linear) in the presence of a stronger nonlinear force. Thus, unambiguous identification of single apoferritin (non-magnetic) and ferritin (magnetic) molecules in air and liquid is accomplished.
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The Rac1 regulator ELMO1 controls vascular morphogenesis in zebrafish.
Circ. Res.
PUBLISHED: 05-13-2010
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Angiogenesis is regulated by the small GTPase Rac1. The ELMO1/DOCK180 complex forms a guanine nucleotide exchange factor for Rac1, regulating its activation during cell migration in different biological systems. Objective: To investigate the function of ELMO1/DOCK180 in vascular development.
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Phylogeography of the greater horseshoe bat, Rhinolophus ferrumequinum: contrasting results from mitochondrial and microsatellite data.
Mol. Ecol.
PUBLISHED: 02-05-2009
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Phylogeographical studies are typically based on haplotype data, occasionally on nuclear markers such as microsatellites, but rarely combine both. This is unfortunate because the use of markers with contrasting modes of inheritance and rates of evolution might provide a more accurate and comprehensive understanding of a species history. Here we present a detailed study of the phylogeography of the greater horseshoe bat, Rhinolophus ferrumequinum, using 1098 bp of the mitochondrial ND2 gene from 45 localities from across its Palaearctic range to infer population history. In addition, we re-analysed a large microsatellite data set available for this species and compared the results of both markers to infer population relationships and the historical processes influencing them. We show that mtDNA, the most popular marker in phylogeography studies, yielded a misleading result, and would have led us to conclude erroneously that a single expansion had taken place in Europe. Only by combining the mitochondrial and microsatellite data sets are we able to reconstruct the species history and show two colonization events in Europe, one before the Last Glacial Maximum (LGM) and one after it. Combining markers also revealed the importance of Asia Minor as an ancient refugium for this species and a source population for the expansion of the greater horseshoe bat into Europe before the LGM.
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Inhibition of Rho-dependent kinases ROCK I/II activates VEGF-driven retinal neovascularization and sprouting angiogenesis.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 01-30-2009
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Vascular endothelial growth factor (VEGF) is an endothelial-specific growth factor that activates the small GTPase RhoA. While the role of RhoA for VEGF-driven endothelial migration and angiogenesis has been studied in detail, the function of its target proteins, the Rho-dependent kinases ROCK I and II, are controversially discussed. Using the mouse model of oxygen-induced proliferative retinopathy, ROCK I/II inhibition by H-1152 resulted in increased angiogenesis. This enhanced angiogenesis, however, was completely blocked by the VEGF-receptor antagonist PTK787/ZK222584. Loss-of-function experiments in endothelial cells revealed that inhibition of ROCK I/II using the pharmacological inhibitor H-1152 and ROCK I/II-specific small-interfering RNAs resulted in a rise of VEGF-driven sprouting angiogenesis. These functional data were biochemically substantiated by showing an enhanced VEGF-receptor kinase insert domain receptor phosphorylation and extracellular signal-regulated kinase 1/2 activation after inhibition of ROCK I/II. Thus our data identify that the inhibition of Rho-dependent kinases ROCK I/II activates angiogenesis both, in vitro and in vivo.
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Repulsive bimodal atomic force microscopy on polymers.
Beilstein J Nanotechnol
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Bimodal atomic force microscopy can provide high-resolution images of polymers. In the bimodal operation mode, two eigenmodes of the cantilever are driven simultaneously. When examining polymers, an effective mechanical contact is often required between the tip and the sample to obtain compositional contrast, so particular emphasis was placed on the repulsive regime of dynamic force microscopy. We thus investigated bimodal imaging on a polystyrene-block-polybutadiene diblock copolymer surface and on polystyrene. The attractive operation regime was only stable when the amplitude of the second eigenmode was kept small compared to the amplitude of the fundamental mode. To clarify the influence of the higher eigenmode oscillation on the image quality, the amplitude ratio of both modes was systematically varied. Fourier analysis of the time series recorded during imaging showed frequency mixing. However, these spurious signals were at least two orders of magnitude smaller than the first two fundamental eigenmodes. Thus, repulsive bimodal imaging of polymer surfaces yields a good signal quality for amplitude ratios smaller than A(01)/A(02) = 10:1 without affecting the topography feedback.
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From pico to nano: biofunctionalization of cube-octameric silsesquioxanes by peptides and miniproteins.
Org. Biomol. Chem.
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Polyhedral silsesquioxanes are considered valuable conjugation scaffolds. Nevertheless, only a few examples of silsesquioxane-assembled peptide oligomers have been reported to date. We developed a new bioorthogonal cube-octameric silsesquioxane (COSS) scaffold bearing eight aminooxy coupling sites allowing for the conjugation of diverse peptides via oxime ligation. We found that the coupling efficacy depends on the ligand in view of steric hindrance and electrostatic repulsion. For the first time scaffold-based conjugation of cystine-knot miniproteins having a backbone of about thirty amino acids was successfully accomplished without loss of bioactivity. Atomic force microscopy (AFM) provided further knowledge on the size of COSS verifying them as picoscaffolds growing upon bioconjugation to nano-dimension.
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KLEIP deficiency in mice causes progressive corneal neovascular dystrophy.
Invest. Ophthalmol. Vis. Sci.
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The BTB-kelch protein KLEIP/KLHL20 is an actin binding protein that regulates cell-cell contact formation and cell migration. The aim of our study was to characterize KLEIPs function in ocular health and disease in mice.
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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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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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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.