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Find video protocols related to scientific articles indexed in Pubmed.
Treatment with the reactive oxygen species scavenger EUK-207 reduces lung damage and increases survival during 1918 influenza virus infection in mice.
Free Radic. Biol. Med.
PUBLISHED: 03-21-2013
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The 1918 influenza pandemic caused over 40 million deaths worldwide, with 675,000 deaths in the United States alone. Studies in several experimental animal models showed that 1918 influenza virus infection resulted in severe lung pathology associated with dysregulated immune and cell death responses. To determine if reactive oxygen species produced by host inflammatory responses play a central role in promoting severity of lung pathology, we treated 1918 influenza virus-infected mice with the catalytic catalase/superoxide dismutase mimetic, salen-manganese complex EUK-207 beginning 3 days postinfection. Postexposure treatment of mice infected with a lethal dose of the 1918 influenza virus with EUK-207 resulted in significantly increased survival and reduced lung pathology without a reduction in viral titers. In vitro studies also showed that EUK-207 treatment did not affect 1918 influenza viral replication. Immunohistochemical analysis showed a reduction in the detection of the apoptosis marker cleaved caspase-3 and the oxidative stress marker 8-oxo-2-deoxyguanosine in lungs of EUK-207-treated animals compared to vehicle controls. High-throughput sequencing and RNA expression microarray analysis revealed that treatment resulted in decreased expression of inflammatory response genes and increased lung metabolic and repair responses. These results directly demonstrate that 1918 influenza virus infection leads to an immunopathogenic immune response with excessive inflammatory and cell death responses that can be limited by treatment with the catalytic antioxidant EUK-207.
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Francisella tularensis subsp. tularensis induces a unique pulmonary inflammatory response: role of bacterial gene expression in temporal regulation of host defense responses.
PLoS ONE
PUBLISHED: 01-01-2013
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Pulmonary exposure to Francisella tularensis is associated with severe lung pathology and a high mortality rate. The lack of induction of classical inflammatory mediators, including IL1-? and TNF-?, during early infection has led to the suggestion that F. tularensis evades detection by host innate immune surveillance and/or actively suppresses inflammation. To gain more insight into the host response to Francisella infection during the acute stage, transcriptomic analysis was performed on lung tissue from mice exposed to virulent (Francisella tularensis ssp tularensis SchuS4). Despite an extensive transcriptional response in the lungs of animals as early as 4 hrs post-exposure, Francisella tularensis was associated with an almost complete lack of induction of immune-related genes during the initial 24 hrs post-exposure. This broad subversion of innate immune responses was particularly evident when compared to the pulmonary inflammatory response induced by other lethal (Yersinia pestis) and non-lethal (Legionella pneumophila, Pseudomonas aeruginosa) pulmonary infections. However, the unique induction of a subset of inflammation-related genes suggests a role for dysregulation of lymphocyte function and anti-inflammatory pathways in the extreme virulence of Francisella. Subsequent activation of a classical inflammatory response 48 hrs post-exposure was associated with altered abundance of Francisella-specific transcripts, including those associated with bacterial surface components. In summary, virulent Francisella induces a unique pulmonary inflammatory response characterized by temporal regulation of innate immune pathways correlating with altered bacterial gene expression patterns. This study represents the first simultaneous measurement of both host and Francisella transcriptome changes that occur during in vivo infection and identifies potential bacterial virulence factors responsible for regulation of host inflammatory pathways.
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Lethal synergism of 2009 pandemic H1N1 influenza virus and Streptococcus pneumoniae coinfection is associated with loss of murine lung repair responses.
MBio
PUBLISHED: 01-01-2011
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Secondary bacterial infections increase disease severity of influenza virus infections and contribute greatly to increased morbidity and mortality during pandemics. To study secondary bacterial infection following influenza virus infection, mice were inoculated with sublethal doses of 2009 seasonal H1N1 virus (NIH50) or pandemic H1N1 virus (Mex09) followed by inoculation with Streptococcus pneumoniae 48 h later. Disease was characterized by assessment of weight loss and survival, titration of virus and bacteria by quantitative reverse transcription-PCR (qRT-PCR), histopathology, expression microarray, and immunohistochemistry. Mice inoculated with virus alone showed 100% survival for all groups. Mice inoculated with Mex09 plus S. pneumoniae showed severe weight loss and 100% mortality with severe alveolitis, denuded bronchiolar epithelium, and widespread expression of apoptosis marker cleaved caspase 3. In contrast, mice inoculated with NIH50 plus S. pneumoniae showed increased weight loss, 100% survival, and slightly enhanced lung pathology. Mex09-S. pneumoniae coinfection also resulted in increased S. pneumoniae replication in lung and bacteremia late in infection. Global gene expression profiling revealed that Mex09-S. pneumoniae coinfection did not induce significantly more severe inflammatory responses but featured significant loss of epithelial cell reproliferation and repair responses. Histopathological examination for cell proliferation marker MCM7 showed significant staining of airway epithelial cells in all groups except Mex09-S. pneumoniae-infected mice. This study demonstrates that secondary bacterial infection during 2009 H1N1 pandemic virus infection resulted in more severe disease and loss of lung repair responses than did seasonal influenza viral and bacterial coinfection. Moreover, this study provides novel insights into influenza virus and bacterial coinfection by showing correlation of lethal outcome with loss of airway basal epithelial cells and associated lung repair responses.
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Image-based feedback control for real-time sorting of microspheres in a microfluidic device.
Lab Chip
PUBLISHED: 06-30-2010
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We describe a control system to automatically distribute antibody-functionalized beads to addressable assay chambers within a PDMS microfluidic device. The system used real-time image acquisition and processing to manage the valve states required to sort beads with unit precision. The image processing component of the control system correctly counted the number of beads in 99.81% of images (2689 of 2694), with only four instances of an incorrect number of beads being sorted to an assay chamber, and one instance of inaccurately counted beads being improperly delivered to waste. Post-experimental refinement of the counting script resulted in one counting error in 2694 images of beads (99.96% accuracy). We analyzed a range of operational variables (flow pressure, bead concentration, etc.) using a statistical model to characterize those that yielded optimal sorting speed and efficiency. The integrated device was able to capture, count, and deliver beads at a rate of approximately four per minute so that bead arrays could be assembled in 32 individually addressable assay chambers for eight analytical measurements in duplicate (512 beads total) within 2.5 hours. This functionality demonstrates the successful integration of a robust control system with precision bead handling that is the enabling technology for future development of a highly multiplexed bead-based analytical device.
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Bright field microscopy as an alternative to whole cell fluorescence in automated analysis of macrophage images.
PLoS ONE
PUBLISHED: 08-28-2009
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Fluorescence microscopy is the standard tool for detection and analysis of cellular phenomena. This technique, however, has a number of drawbacks such as the limited number of available fluorescent channels in microscopes, overlapping excitation and emission spectra of the stains, and phototoxicity.
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Resolving cell population heterogeneity: real-time PCR for simultaneous multiplexed gene detection in multiple single-cell samples.
PLoS ONE
PUBLISHED: 06-09-2009
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Decoding the complexity of multicellular organisms requires analytical procedures to overcome the limitations of averaged measurements of cell populations, which obscure inherent cell-cell heterogeneity and restrict the ability to distinguish between the responses of individual cells within a sample. For example, defining the timing, magnitude and the coordination of cytokine responses in single cells is critical for understanding the development of effective immunity. While approaches to measure gene expression from single cells have been reported, the absolute performance of these techniques has been difficult to assess, which likely has limited their wider application. We describe a straightforward method for simultaneously measuring the expression of multiple genes in a multitude of single-cell samples using flow cytometry, parallel cDNA synthesis, and quantification by real-time PCR. We thoroughly assess the performance of the technique using mRNA and DNA standards and cell samples, and demonstrate a detection sensitivity of approximately 30 mRNA molecules per cell, and a fractional error of 15%. Using this method, we expose unexpected heterogeneity in the expression of 5 immune-related genes in sets of single macrophages activated by different microbial stimuli. Further, our analyses reveal that the expression of one pro-inflammatory cytokine is not predictive of the expression of another pro-inflammatory cytokine within the same cell. These findings demonstrate that single-cell approaches are essential for studying coordinated gene expression in cell populations, and this generic and easy-to-use quantitative method is applicable in other areas in biology aimed at understanding the regulation of cellular responses.
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A microfluidic device for multiplexed protein detection in nano-liter volumes.
Anal. Biochem.
PUBLISHED: 01-10-2009
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We describe a microfluidic immunoassay device that permits sensitive and quantitative multiplexed protein measurements on nano-liter-scale samples. The device exploits the combined power of integrated microfluidics and optically encoded microspheres to create an array of approximately 100-microm(2) sensors functionalized with capture antibodies directed against distinct targets. This strategy overcomes the need for performing biochemical coupling of affinity reagents to the device substrate, permits multiple proteins to be detected in a nano-liter-scale sample, is scalable to large numbers of samples, and has the required sensitivity to measure the abundance of proteins derived from single mammalian cells. The sensitivity of the device is sufficient to detect 1000 copies of tumor necrosis factor (TNF) in a volume of 4.7nl.
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Integrative analysis of N-linked human glycoproteomic data sets reveals PTPRF ectodomain as a novel plasma biomarker candidate for prostate cancer.
J. Proteome Res.
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In an attempt to identify prostate cancer biomarkers with greater diagnostic and prognostic capabilities, we have developed an integrative proteomic discovery workflow focused on N-linked glycoproteins that refines the target selection process. In this work, hydrazide-based chemistry was used to identify N-linked glycopeptides from 22Rv1 prostate cancer cells cultured in vitro, which were compared with glycopeptides identified from explanted 22Rv1 murine tumor xenografts. One hundred and four human glycoproteins were identified in the former analysis and 75 in the latter, with 40 proteins overlapping between data sets. Of the 40 overlapping proteins, 80% have multiple literature references to the neoplastic process and ?40% to prostatic neoplasms. These include a number of well-known prostate cancer-associated biomarkers, such as prostate-specific membrane antigen (PSMA). By integrating gene expression data and available literature, we identified members of the overlap data set that deserve consideration as potential prostate cancer biomarkers. Specifically, the identification of the extracellular domain of protein tyrosine phosphatase receptor type F (PTPRF) was of particular interest due to the direct involvement of PTPRF in the control of ?-catenin signaling, as well as dramatically elevated gene expression levels in the prostate compared to other tissues. In this investigation, we demonstrate that the PTPRF E-subunit is more abundant in human prostate tumor tissue compared to normal control and also detectable in murine plasma by immunoblot and ELISA. Specifically, PTPRF distinguishes between animals xenografted with the 22Rv1 cells and control animals as early as 14 days after implantation. This result suggests that the ectodomain of PTPRF has the potential to function as a novel plasma or tissue-based biomarker for prostate cancer. The workflow described adds to the literature of potential biomarker candidates for prostate cancer and demonstrates a pathway to developing new diagnostic assays.
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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.