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Find video protocols related to scientific articles indexed in Pubmed.
Selective microbial genomic DNA isolation using restriction endonucleases.
PLoS ONE
PUBLISHED: 01-01-2014
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To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including ?-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment.
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Staphylococcus aureus TargetArray: comprehensive differential essential gene expression as a mechanistic tool to profile antibacterials.
Antimicrob. Agents Chemother.
PUBLISHED: 06-14-2010
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The widespread emergence of antibiotic-resistant bacteria and a lack of new pharmaceutical development have catalyzed a need for new and innovative approaches for antibiotic drug discovery. One bottleneck in antibiotic discovery is the lack of a rapid and comprehensive method to identify compound mode of action (MOA). Since a hallmark of antibiotic action is as an inhibitor of essential cellular targets and processes, we identify a set of 308 essential genes in the clinically important pathogen Staphylococcus aureus. A total of 446 strains differentially expressing these genes were constructed in a comprehensive platform of sensitized and resistant strains. A subset of strains allows either target underexpression or target overexpression by heterologous promoter replacements with a suite of tetracycline-regulatable promoters. A further subset of 236 antisense RNA-expressing clones allows knockdown expression of cognate targets. Knockdown expression confers selective antibiotic hypersensitivity, while target overexpression confers resistance. The antisense strains were configured into a TargetArray in which pools of sensitized strains were challenged in fitness tests. A rapid detection method measures strain responses toward antibiotics. The TargetArray antibiotic fitness test results show mechanistically informative biological fingerprints that allow MOA elucidation.
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In vitro pharmacodynamics of levofloxacin and other aerosolized antibiotics under multiple conditions relevant to chronic pulmonary infection in cystic fibrosis.
Antimicrob. Agents Chemother.
PUBLISHED: 10-05-2009
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The inhalational administration of antibiotics can provide high concentrations locally in the lungs of cystic fibrosis patients and, thus, can be useful for the treatment of chronic bacterial infections. The present study evaluated the in vitro activities of levofloxacin, ciprofloxacin, tobramycin, amikacin, and aztreonam against clinical isolates of Pseudomonas aeruginosa, Burkholderia cepacia complex, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and Staphylococcus aureus from cystic fibrosis patients. Levofloxacin was the most potent antibiotic against all cystic fibrosis isolates tested, with MIC(90)s ranging from 8 to 32 microg/ml. Levofloxacin was more potent than the aminoglycosides and aztreonam against P. aeruginosa biofilms. Time-kill assays with drug concentrations achievable in sputum following aerosol administration showed that levofloxacin had the most rapid rate of killing among mucoid and nonmucoid isolates of P. aeruginosa. In contrast to tobramycin, the bactericidal activity of levofloxacin was not affected by sputum from cystic fibrosis patients. The results of the study show that the high concentrations of levofloxacin readily achievable in the lung following aerosol delivery may be useful for the management of pulmonary infections in patients with cystic fibrosis.
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Effect of oxygen limitation on the in vitro activity of levofloxacin and other antibiotics administered by the aerosol route against Pseudomonas aeruginosa from cystic fibrosis patients.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 06-19-2009
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Studies have demonstrated that thickened mucous layers in the lungs of cystic fibrosis (CF) patients contain areas of low oxygen tension. These microaerophilic environments may reduce the activity of aerosol antibiotics used in the management of chronic infection in CF. The aim of this study was to compare the MICs of levofloxacin, tobramycin, amikacin, and aztreonam against Pseudomonas aeruginosa under reference and anaerobic conditions and evaluate the in vitro pharmacodynamics of levofloxacin under aerobic and hypoxic testing conditions. The MICs for 114 isolates of P. aeruginosa from CF patients were determined in cation-adjusted Mueller Hinton broth alone or supplemented with 1% potassium nitrate for anaerobic testing. Levofloxacin time-kill curves were performed under aerobic and hypoxic conditions using strains of P. aeruginosa with elevated efflux pump overexpression and/or target mutations. The MICs of nonmucoid or mucoid P. aeruginosa isolates to levofloxacin incubated under aerobic and anaerobic conditions were similar. In contrast, anaerobic incubation resulted in higher MICs for tobramycin, amikacin, and aztreonam among nonmucoid or mucoid isolates, with > or =4-fold increase in MICs for over 40% of the isolates. Time-kill curves performed in aerobic and hypoxic environments with levofloxacin concentrations attained in CF sputum demonstrated similar activity, approaching a maximum bactericidal effect within 10 min of exposure. Together, these results indicate that the activity of some antibiotics against P. aeruginosa is significantly reduced under conditions relevant to the CF lung environment. In contrast, levofloxacin maintains activity against P. aeruginosa under anaerobic or hypoxic conditions similar to those found in CF microaerophilic environments.
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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.