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| 2. |
- Hernandez, Frank J, et al.
(författare)
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Degradation of nuclease-stabilized RNA oligonucleotides in Mycoplasma-contaminated cell culture media
- 2012
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Ingår i: Nucleic Acid Therapeutics. - : Mary Ann Liebert. - 2159-3337 .- 2159-3345. ; 22:1, s. 58-68
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Tidskriftsartikel (refereegranskat)abstract
- Artificial RNA reagents such as small interfering RNAs (siRNAs) and aptamers often must be chemically modified for optimal effectiveness in environments that include ribonucleases. Mycoplasmas are common bacterial contaminants of mammalian cell cultures that are known to produce ribonucleases. Here we describe the rapid degradation of nuclease-stabilized RNA oligonucleotides in a human embryonic kidney 293 (HEK) cell culture contaminated with Mycoplasma fermentans, a common species of mycoplasma. RNA with 2′-fluoro-or 2′-O-methyl-modified pyrimidines was readily degraded in conditioned media from this culture, but was stable in conditioned media from uncontaminated HEK cells. RNA completely modified with 2′-O-methyls was not degraded in the mycoplasma-contaminated media. RNA zymogram analysis of conditioned culture media and material centrifuged from the media revealed several distinct protein bands (ranging from 30 to 68kDa) capable of degrading RNA with 2′-fluoro-or 2′-O-methyl-modified pyrimidines. Finally, the mycoplasma-associated nuclease was detected in material centrifuged from the contaminated culture supernatants in as little as 15 minutes with an RNA oligo-containing 2′-O-methyl-modified pyrimidines and labeled with a 5′-fluorescein amidite (FAM) and 3′-quencher. These results suggest that mycoplasma contamination may be a critical confounding variable for cell culture experiments involving RNA-based reagents, with particular relevance for applications involving naked RNA (e.g., aptamer-siRNA chimeras). © 2012 Mary Ann Liebert, Inc.
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| 3. |
- Hernandez, Frank J, et al.
(författare)
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Noninvasive imaging of Staphylococcus aureus infections with a nuclease-activated probe
- 2014
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Ingår i: Nature Medicine. - : Nature Publishing Group. - 1078-8956 .- 1546-170X. ; 20:3, s. 301-306
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Tidskriftsartikel (refereegranskat)abstract
- Technologies that enable the rapid detection and localization of bacterial infections in living animals could address an unmet need for infectious disease diagnostics. We describe a molecular imaging approach for the specific, noninvasive detection of S. aureus based on the activity of the S. aureus secreted nuclease, micrococcal nuclease (MN). Several short synthetic oligonucleotides, rendered resistant to mammalian serum nucleases by various chemical modifications and flanked with a fluorophore and quencher, were activated upon degradation by purified MN and in S. aureus culture supernatants. A probe consisting of a pair of deoxythymidines flanked by several 2′-O-methyl-modified nucleotides was activated in culture supernatants of S. aureus but not in culture supernatants of several other pathogenic bacteria. Systemic administration of this probe to mice bearing S. aureus muscle infections resulted in probe activation at the infection sites in an MN-dependent manner. This new bacterial imaging approach has potential clinical applicability for infections with S. aureus and several other medically important pathogens. © 2014 Nature America, Inc.
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| 4. |
- Kiedrowski, Megan R., et al.
(författare)
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Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease
- 2014
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Ingår i: PLOS ONE. - San Francisco, United States : Public Library of Science. - 1932-6203. ; 9:4
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Tidskriftsartikel (refereegranskat)abstract
- Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc) enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2) that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET) assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme. © 2014 Kiedrowski et al.
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| 5. |
- Kwiecinski, Jakub, 1985, et al.
(författare)
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Biofilm formation by Staphylococcus aureus clinical isolates correlates with the infection type.
- 2019
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Ingår i: Infectious diseases. - : Informa UK Limited. - 2374-4243 .- 2374-4235. ; 51:6, s. 446-451
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Tidskriftsartikel (refereegranskat)abstract
- Biofilms are involved in many Staphylococcus aureus infections, but relation of biofilm formation and the infection types or the clinical outcomes remain unclear.We measured biofilm formation, with a microtiter plate assay, of a collection of methicillin-sensitive clinical isolates from 159 invasive S. aureus infections, encompassing all cases occurring within a hospital catchment area during two years, and from additional 49 non-invasive skin infections from the same region. Results were related to available clinical and microbiological documentation.Isolates from medical device infections (intravenous line-associated and prosthetic joint infections), as well as isolates from superficial skin infections, were particularly proficient in forming biofilms. No increased biofilm-forming capacity was seen in isolates from endocarditis, osteomyelitis, or other infections. There was also a correlation of biofilm formation with the agr type of the isolates. Thicker biofilms were more resistant to antibiotic treatment in vitro. No correlation between biofilm formation and clinical outcomes was noted.S. aureus isolates from 'classical' biofilm-related infections, but also from superficial skin infections, are especially proficient in forming biofilms. There is, however, no obvious relation of biofilm-forming capacity of isolates and the clinical outcome of the infection, and more studies on this issue are needed.
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