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Find video protocols related to scientific articles indexed in Pubmed.
DC isoketal-modified proteins activate T cells and promote hypertension.
J. Clin. Invest.
PUBLISHED: 08-04-2014
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Oxidative damage and inflammation are both implicated in the genesis of hypertension; however, the mechanisms by which these stimuli promote hypertension are not fully understood. Here, we have described a pathway in which hypertensive stimuli promote dendritic cell (DC) activation of T cells, ultimately leading to hypertension. Using multiple murine models of hypertension, we determined that proteins oxidatively modified by highly reactive ?-ketoaldehydes (isoketals) are formed in hypertension and accumulate in DCs. Isoketal accumulation was associated with DC production of IL-6, IL-1?, and IL-23 and an increase in costimulatory proteins CD80 and CD86. These activated DCs promoted T cell, particularly CD8+ T cell, proliferation; production of IFN-? and IL-17A; and hypertension. Moreover, isoketal scavengers prevented these hypertension-associated events. Plasma F2-isoprostanes, which are formed in concert with isoketals, were found to be elevated in humans with treated hypertension and were markedly elevated in patients with resistant hypertension. Isoketal-modified proteins were also markedly elevated in circulating monocytes and DCs from humans with hypertension. Our data reveal that hypertension activates DCs, in large part by promoting the formation of isoketals, and suggest that reducing isoketals has potential as a treatment strategy for this disease.
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Inflammatory monocytes orchestrate innate antifungal immunity in the lung.
PLoS Pathog.
PUBLISHED: 02-01-2014
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Aspergillus fumigatus is an environmental fungus that causes invasive aspergillosis (IA) in immunocompromised patients. Although -CC-chemokine receptor-2 (CCR2) and Ly6C-expressing inflammatory monocytes (CCR2?Mo) and their derivatives initiate adaptive pulmonary immune responses, their role in coordinating innate immune responses in the lung remain poorly defined. Using conditional and antibody-mediated cell ablation strategies, we found that CCR2?Mo and monocyte-derived dendritic cells (Mo-DCs) are essential for innate defense against inhaled conidia. By harnessing fluorescent Aspergillus reporter (FLARE) conidia that report fungal cell association and viability in vivo, we identify two mechanisms by which CCR2?Mo and Mo-DCs exert innate antifungal activity. First, CCR2?Mo and Mo-DCs condition the lung inflammatory milieu to augment neutrophil conidiacidal activity. Second, conidial uptake by CCR2?Mo temporally coincided with their differentiation into Mo-DCs, a process that resulted in direct conidial killing. Our findings illustrate both indirect and direct functions for CCR2?Mo and their derivatives in innate antifungal immunity in the lung.
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Matriptase autoactivation is tightly regulated by the cellular chemical environments.
PLoS ONE
PUBLISHED: 01-01-2014
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The ability of cells to rapidly detect and react to alterations in their chemical environment, such as pH, ionic strength and redox potential, is essential for cell function and survival. We present here evidence that cells can respond to such environmental alterations by rapid induction of matriptase autoactivation. Specifically, we show that matriptase autoactivation can occur spontaneously at physiological pH, and is significantly enhanced by acidic pH, both in a cell-free system and in living cells. The acid-accelerated autoactivation can be attenuated by chloride, a property that may be part of a safety mechanism to prevent unregulated matriptase autoactivation. Additionally, the thio-redox balance of the environment also modulates matriptase autoactivation. Using the cell-free system, we show that matriptase autoactivation is suppressed by cytosolic reductive factors, with this cytosolic suppression being reverted by the addition of oxidizing agents. In living cells, we observed rapid induction of matriptase autoactivation upon exposure to toxic metal ions known to induce oxidative stress, including CoCl2 and CdCl2. The metal-induced matriptase autoactivation is suppressed by N-acetylcysteine, supporting the putative role of altered cellular redox state in metal induced matriptase autoactivation. Furthermore, matriptase knockdown rendered cells more susceptible to CdCl2-induced cell death compared to control cells. This observation implies that the metal-induced matriptase autoactivation confers cells with the ability to survive exposure to toxic metals and/or oxidative stress. Our results suggest that matriptase can act as a cellular sensor of the chemical environment of the cell that allows the cell to respond to and protect itself from changes in the chemical milieu.
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Proteasome inhibitors act as bifunctional antagonists of human immunodeficiency virus type 1 latency and replication.
Retrovirology
PUBLISHED: 07-03-2013
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Existing highly active antiretroviral therapy (HAART) effectively controls viral replication in human immunodeficiency virus type 1 (HIV-1) infected individuals but cannot completely eradicate the infection, at least in part due to the persistence of latently infected cells. One strategy that is being actively pursued to eliminate the latent aspect of HIV-1 infection involves therapies combining latency antagonists with HAART. However, discordant pharmacokinetics between these types of drugs can potentially create sites of active viral replication within certain tissues that might be impervious to HAART.
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Isolation of multilineage progenitors from mouse brain.
In Vitro Cell. Dev. Biol. Anim.
PUBLISHED: 01-18-2013
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Stem cells are unique cell populations with the ability to undergo self-renewal and differentiation. These cells have been identified in a wide range of tissues and possess varied differentiation potentials. Tissue-specific stem cells have typically been thought to have limited differentiation capabilities. We show here that fibroblast-like cells isolated from mouse brain possess cross-germ layer differentiation abilities. These cells were found to express typical mesenchymal stem cell markers (CD44, CD29, and CD105) and were able to be passaged more than 50 times. When treated under defined conditions, the brain-derived cells were able to generate many different cell types including adipocytes, osteocytes, astrocytes, neurons, and even hepatocyte-like cells. The hepatocyte-like cells not only expressed liver cell-specific markers, but also exhibited the capacity for glycogen storage and low-density lipoprotein uptake. These results demonstrate the existence of cells in the brain with three-germ-layer differentiation potential.
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Weighted moments estimators of the parameters for the extreme value distribution based on the multiply type II censored sample.
ScientificWorldJournal
PUBLISHED: 01-01-2013
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We propose the weighted moments estimators (WMEs) of the location and scale parameters for the extreme value distribution based on the multiply type II censored sample. Simulated mean squared errors (MSEs) of best linear unbiased estimator (BLUE) and exact MSEs of WMEs are compared to study the behavior of different estimation methods. The results show the best estimator among the WMEs and BLUE under different combinations of censoring schemes.
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Post-transcriptionally regulated expression system in human xenogeneic transplantation models.
Mol. Ther.
PUBLISHED: 05-17-2011
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Cells have developed a mechanism to discriminate between premature termination codons (PTCs) and normal stop codons during translation, sparking vigorous research to develop drugs promoting readthrough at PTCs to treat genetic disorders caused by PTCs. It was posed that this concept could also be applied to regulated gene therapy protocols by incorporating a PTC into a therapeutic gene, so active protein would only be made after administration of a readthrough agent. The strengths of the system are highlighted here by results demonstrating: (i) background expression levels were reduced to 0.01% to 0.0005% of wild type in unselected mass populations of cells depending upon the specific stop codon utilized and its position within the gene; (ii) expression levels responded well to multiple "On" and "Off" regulation cycles in vivo in human xenograft systems; (iii) the level of induction approached three logs using aminoglycoside activators including NB54, a newly synthesized aminoglycoside with significantly reduced toxicity; and (iv) expression levels could be appreciably altered when employing different promoters in a variety of cell types. These results strongly support the contention that this system should have important clinical applications when tight control of gene expression is required.
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A comparison of exogenous promoter activity at the ROSA26 locus using a ?iC31 integrase mediated cassette exchange approach in mouse ES cells.
PLoS ONE
PUBLISHED: 05-11-2011
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The activities of nine ubiquitous promoters (ROSA26, CAG, CMV, CMVd1, UbC, EF1?, PGK, chicken ?-actin and MC1) have been quantified and compared in mouse embryonic stem cells. To avoid the high variation in transgene expression which results from uncontrolled copy number and chromosomal position effects when using random insertion based transgenic approaches, we have adopted a PhiC31 integrase mediated cassette exchange method for the efficient insertion of transgenes at single copy within a defined and well characterized chromosomal position, ROSA26. This has enabled the direct comparison of constructs from within the same genomic context and allows a systematic and quantitative assessment of the strengths of the promoters in comparison with the endogenous ROSA26 promoter. The behavior of these exogenous promoters, when integrated at ROSA26 in both sense and antisense orientations, reveals a large variation in their levels of activity. In addition, a subset of promoters, EF1?, UbC and CAG, show an increased activity in the sense orientation as a consequence of integration. Transient transfection experiments confirmed these observations to reflect integration dependent effects and also revealed significant differences in the behaviour of these promoters when delivered transiently or stably. As well as providing an important reference which will facilitate the choice of an appropriate promoter to achieve the desired level of expression for a specific research question, this study also demonstrates the suitability of the cassette exchange methodology for the robust and reliable expression of multiple variant transgenes in ES cells.
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Efficient co-expression of bicistronic proteins in mesenchymal stem cells by development and optimization of a multifunctional plasmid.
Stem Cell Res Ther
PUBLISHED: 03-14-2011
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Local synthesis of interferon within B16 tumors mediates anti-tumor effects. Based on reports that stem cells are recruited to tumors, and because systemic administration of interferon causes dose-limiting undesirable side effects, we wanted to improve the anti-tumor effects of interferon while simultaneously minimizing its systemic side effects by employing mesenchymal stem cells (MSCs) as tumor-localized ectopic producers of interferon. Many vectors exist to fulfill this purpose, but their transfection efficiency and resulting expression levels vary considerably.
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Defective ubiquitin-mediated degradation of antiapoptotic Bfl-1 predisposes to lymphoma.
Blood
PUBLISHED: 02-25-2010
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The antiapoptotic Bcl-2 family member Bfl-1 is up-regulated in many human tumors in which nuclear factor-kappaB (NF-kappaB) is implicated and contributes significantly to tumor cell survival and chemoresistance. We previously found that NF-kappaB induces transcription of bfl-1 and that the Bfl-1 protein is also regulated by ubiquitin-mediated proteasomal degradation. However, the role that dysregulation of Bfl-1 turnover plays in cancer is not known. Here we show that ubiquitination-resistant mutants of Bfl-1 display increased stability and greatly accelerated tumor formation in a mouse model of leukemia/lymphoma. We also show that tyrosine kinase Lck is up-regulated and activated in these tumors and leads to activation of the IkappaB kinase, Akt, and extracellular signal-regulated protein kinase signaling pathways, which are key mediators in cancer. Coexpression of Bfl-1 and constitutively active Lck promoted tumor formation, whereas Lck knockdown in tumor-derived cells suppressed leukemia/lymphomagenesis. These data demonstrate that ubiquitination is a critical tumor suppression mechanism regulating Bfl-1 function and suggest that mutations in bfl-1 or in the signaling pathways that control its ubiquitination may predispose one to cancer. Furthermore, because bfl-1 is up-regulated in many human hematopoietic tumors, this finding suggests that strategies to promote Bfl-1 ubiquitination may improve therapy.
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Transcriptional control of left-right patterning in cardiac development.
Pediatr Cardiol
PUBLISHED: 01-07-2010
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The heart develops from a simple left-right (L-R) symmetrical tube. Through a complex process of looping and remodelling, it becomes a highly L-R asymmetrical organ with distinct asymmetries in both morphology and function. Abnormal cardiac L-R patterning can result in a spectrum of defects that include, dextrocardia (a malposition of the heart to the right), isomerism of the atria (both atria being morphologically right-sided or left-sided), abnormal ventricular topology (e.g. the morphological left ventricle being dextral to the morphological right ventricle) or mirror-image topology (associated with situs inversus). Intermediate forms include abnormalities such as situs ambiguus and heterotaxia. L-R patterning abnormalities are typically associated with cardiac malformations, and it has become clear that an isolated septal, outflow tract and aortic arch malformation may be the only presenting manifestation of an L-R patterning defect. In the last two decades, there have been seminal advances in our understanding of the mechanisms controlling L-R patterning, and how mutations in L-R patterning genes result in human cardiac malformation. In this review, we provide an overview of the transcriptional mechanisms that result in asymmetric gene activation in mammals, how they receive information from signalling pathways, and how this translates to abnormal cardiac development.
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In vivo post-transcriptional regulation of CD154 in mouse CD4+ T cells.
Eur. J. Immunol.
PUBLISHED: 07-03-2009
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Interactions between CD40 and its ligand CD154 are involved in the progression of both cell mediated and innate immunity. These interactions are brought about by the transient expression of CD154 on activated CD4(+) T cells, which is regulated, in part, at the level of mRNA turnover. Here we have focused on analyzing the pattern of post-transcriptional regulation in mouse CD4(+) T cells in response to activation. Initial experiments identify a region of the murine CD154 mRNA that binds a polypyrimidine tract-binding protein-containing complex (mComplex I), which is activation-dependent and binds to a single CU-rich site within the 3 uTR Subsequent findings demonstrate that in vivo polyclonal activation of T cells leads to a pattern of differential CD154 mRNA stability that is directly dependent on extent of activation. Furthermore, in vitro activation of antigen-primed T cells shows that the CD154 mRNA half-life increases relative to that of unprimed cells. Importantly, this is the first report demonstrating that the regulation of CD154 in vivo is connected to an activation-induced program of mRNA decay and thus provides strong evidence for post-transcriptional mechanisms having a physiological role in regulating CD154 expression during an ongoing immune response.
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Functional significance of SRJ domain mutations in CITED2.
PLoS ONE
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CITED2 is a transcriptional co-activator with 3 conserved domains shared with other CITED family members and a unique Serine-Glycine Rich Junction (SRJ) that is highly conserved in placental mammals. Loss of Cited2 in mice results in cardiac and aortic arch malformations, adrenal agenesis, neural tube and placental defects, and partially penetrant defects in left-right patterning. By screening 1126 sporadic congenital heart disease (CHD) cases and 1227 controls, we identified 19 variants, including 5 unique non-synonymous sequence variations (N62S, R92G, T166N, G180-A187del and A187T) in patients. Many of the CHD-specific variants identified in this and previous studies cluster in the SRJ domain. Transient transfection experiments show that T166N mutation impairs TFAP2 co-activation function and ES cell proliferation. We find that CITED2 is phosphorylated by MAPK1 in vitro at T166, and that MAPK1 activation enhances the coactivation function of CITED2 but not of CITED2-T166N. In order to investigate the functional significance in vivo, we generated a T166N mutation of mouse Cited2. We also used PhiC31 integrase-mediated cassette exchange to generate a Cited2 knock-in allele replacing the mouse Cited2 coding sequence with human CITED2 and with a mutant form deleting the entire SRJ domain. Mouse embryos expressing only CITED2-T166N or CITED2-SRJ-deleted alleles surprisingly show no morphological abnormalities, and mice are viable and fertile. These results indicate that the SRJ domain is dispensable for these functions of CITED2 in mice and that mutations clustering in the SRJ region are unlikely to be the sole cause of the malformations observed in patients with sporadic CHD. Our results also suggest that coding sequence mutations observed in case-control studies need validation using in vivo models and that predictions based on structural conservation and in vitro functional assays, or even in vivo global loss of function models, may be insufficient.
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A cell-autonomous role of Cited2 in controlling myocardial and coronary vascular development.
Eur. Heart J.
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Myocardial development is dependent on concomitant growth of cardiomyocytes and a supporting vascular network. The coupling of myocardial and coronary vascular development is partly mediated by vascular endothelial growth factor (VEGFA) signalling and additional unknown mechanisms. We examined the cardiomyocyte specific role of the transcriptional co-activator Cited2 on myocardial microstructure and vessel growth, in relation to Vegfa expression.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

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.