| 1. |
- Brundin, Malin, et al.
(författare)
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DNA Binding to hydroxyapatite : a potential mechanism for preservation of microbial DNA
- 2013
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Ingår i: Journal of Endodontics. - : Elsevier. - 0099-2399 .- 1878-3554. ; 39:2, s. 211-216
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Molecular methods are increasingly being deployed for analysis of the microbial flora in the root canal. Such methods are based on the assumption that recovered DNA is associated with the active endodontic infection, yet paleomicrobiology research is based on the recovery of ancient DNA from centuriesold tooth and bone samples, which points to considerable longevity of the DNA molecule in these tissues. The main component of dentin and bone is the mineral hydroxyapatite. This study assessed DNA binding to hydroxyapatite and whether thiS binding affinity stabilizes the DNA molecule in various media.Methods: DNA was extracted from Fusobacterium nucleatum and added to ceramic hydroxyapatite for 90 minutes. The DNA-bound hydroxyapatite was incubated in different media (ie, water, sera, and DNase I) for up to 3 months. At predetermined intervals, the recovery of detectable DNA was assessed by releasing the DNA from the hydroxyapatite using EDTA and evaluating the presence of DNA by gel electrophoresis and polymerase chain reaction (PCR) amplification.Results: When incubated with hydroxyapatite, nonamplified DNA was detectable after 3 months in water, sera, and DNase I. In contrast, DNA incubated in the same media (without hydroxyapatite) decomposed to levels below the detection level of PCR within 3 weeks, with the exception of DNA in sera in which PCR revealed a weak positive amplification product.Conclusions: These results confirm a specific binding affinity of hydroxyapatite for DNA. Hydroxyapatite-bound DNA is more resistant to decay and less susceptible to degradation by serum and nucleases, which may account for the long-term persistence of DNA in bone and tooth.
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| 4. |
- Brundin, Malin, et al.
(författare)
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Preservation of bacterial dna by human dentin
- 2014
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Ingår i: Journal of Endodontics. - : Elsevier BV. - 0099-2399 .- 1878-3554. ; 40:2, s. 241-245
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Tidskriftsartikel (refereegranskat)abstract
- INTRODUCTION: The capacity of dentin and collagen to bind DNA and protect against spontaneous and nuclease-induced degradation was evaluated individually and by the incubation of DNA with nuclease-producing bacteria in a mixed culture.METHODS: Extracted Fusobacterium nucleatum DNA was incubated with dentin shavings or collagen for 90 minutes. The DNA-bound substrates were incubated in different media (water, sera, and DNase I) for up to 3 months. Amplifiable DNA was released from dentin using EDTA,or from collagen using proteinase K, and evaluated by polymerase chain reaction (PCR). The stability of dentin-bound DNA was also assessed in a mixed culture (Parvimonas micra and Pseudoramibacter alactolyticus) containing a DNase-producing species, Prevotella intermedia. Samples were analyzed for amplifiable DNA.RESULTS: In water, dentin-bound DNA was recoverable by PCR at 3 months compared with no detectable DNA after 4 weeks in controls (no dentin). DNA bound to collagen was detectable by PCR after 3 months of incubation in water. In 10% human sera, amplifiable DNA was detectable at 3 months when dentin bound and in controls (no dentin). In mixed bacterial culture, dentin-bound DNA was recoverable throughout the experimental period (3 months), compared with no recoverable F. nucleatum DNA within 24 hours in controls (no dentin).CONCLUSIONS: There is a strong binding affinity between DNA and dentin, and between DNA and serum proteins or collagen. These substrates preserve DNA against natural decomposition and protect DNA from nuclease activity, factors that may confound molecular analysis of the endodontic microbiota yet favor paleomicrobiological studies of ancient DNA.
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| 5. |
- Brundin, Malin, 1972-, et al.
(författare)
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Preservation of Fusobacterium nucleatum and Peptostreptococcus anaerobius DNA after loss of cell viability
- 2015
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Ingår i: International Endodontic Journal. - : Wiley-Blackwell. - 0143-2885 .- 1365-2591. ; 48:1, s. 37-45
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Aim: To investigate whether DNA from two obligate anaerobes, Fusobacterium nucleatum and Peptostreptococcus anaerobius, is recoverable after loss of cell viability induced by air exposure. Methodology: Harvested cultures of F. nucleatum and P. anaerobius were killed by exposure to air and stored in phosphate-buffered saline. Dead cells were incubated aerobically for up to 6 months. Every month, the presence of detectable DNA in the cell pellet and supernatant was assessed by conventional and quantitative PCR. Cell staining techniques were used to characterize the cell wall permeability of air-killed cells. Scanning electron microscopy was used to examine viable, freshly killed and stored cells. Results: With conventional PCR, amplifiable DNA was detectable over 6 months in all samples. Quantitative PCR showed a progressive fall in DNA concentration in nonviable cell pellets and a concomitant rise in DNA concentration in the supernatant. DNA staining showed that some air-killed cells retained an intact cell wall. After storage, SEM of both air-killed species revealed shrivelling of the cells, but some cells of P. anaerobius retained their initial form. Conclusion: Amplifiable DNA from F. nucleatum and P. anaerobius was detectable 6 months after loss of viability. Air-killed anaerobes initially retained their cell form, but cells gradually shriveled over time. The morphological changes were more pronounced with the gram-negative F. nucleatum than the gram-positive P. anaerobius. Over 6 months, there was a gradual increase in cell wall permeability with progressive leakage of DNA. Bacterial DNA was recoverable long after loss of cell viability.
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| 6. |
- Brundin, Malin, 1972-
(författare)
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Stability of bacterial DNA in relation to microbial detection in teeth
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The fate of DNA from dead cells is an important issue when interpreting results from root canal infections analysed by the PCR technique. DNA from dead bacterial cells is known to be detectable long time after cell death and its stability is dependent on many different factors. This work investigated factors found in the root canal that could affect the recovery of microbial DNA. In an ex vivo experiment, DNA from non-viable gram-positive Enterococcus faecalis was inoculated in instrumented root canals and recovery of DNA was assessed by PCR over a two-year period. DNA was still recoverable two years after cell death in 21/25 teeth. The fate of DNA from the gram-negative bacteria Fusobacterium nucleatum and the gram-positive Peptostreptococcus anaerobius was assessed in vitro. DNA from dead F. nucleatum and P. anaerobius could be detected by PCR six months post cell death even though it was clear that the DNA was released from the cells due to lost of cell wall integrity during the experimental period. The decomposition rate of extracellular DNA was compared to cell-bound and it was evident that DNA still located inside the bacterium was much less prone to decay than extracellular DNA.Free (extracellular) DNA is very prone to decay in a naked form. Binding to minerals is known to protect DNA from degradation. The fate of extracellular DNA was assessed after binding to ceramic hydroxyapatite and dentine. The data showed that free DNA, bound to these materials, was protected from spontaneous decay and from enzymatic decomposition by nucleases.The main conclusions from this thesis were: i) DNA from dead bacteria can be detected by PCR years after cell death ex vivo and in vitro. ii) Cell-bound DNA is less prone to decomposition than extracellular DNA. iii) DNA is released from the bacterium some time after cell death. iv) Extracellular DNA bound to hydroxyapatite or dentine is protected from spontaneous decomposition and enzymatic degradation.
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| 7. |
- Brundin, Malin, et al.
(författare)
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Starvation response and growth in serum of Fusobacterium nucleatum, Peptostreptococcus anaerobius, Prevotella intermedia, and Pseudoramibacter alactolyticus.
- 2009
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Ingår i: Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics. - : Elsevier BV. - 1079-2104 .- 1528-395X. ; 108:1, s. 129-34
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Tidskriftsartikel (refereegranskat)abstract
- The microbiota inhabiting the untreated root canal differ markedly from those found in post-treatment disease, yet there is limited information on the microbial characteristics distinguishing the different infections. We hypothesized that starvation survival is a key microbial property in species selection. This study analyzed starvation-survival behavior over 60 days of species representative of the untreated root canal infection: Fusobacterium nucleatum, Peptostreptococcus anaerobius, Prevotella intermedia and Pseudoramibacter alactolyticus. All species did not survive 1 day in water. In 1% serum, the 4 species could not survive beyond 2-3 weeks. They required a high initial cell density and >or=10% serum to survive the observation period. The results highlight a poor starvation-survival capacity of these 4 species compared with species prevalent in post-treatment infection, which are well equipped to endure starvation and survive in low numbers on minimal serum. These findings point to starvation-survival capacity as a selection factor for microbial participation in post-treatment disease.
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| 8. |
- Figdor, David, et al.
(författare)
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Contamination Controls for Analysis of Root Canal Samples by Molecular Methods : An Overlooked and Unsolved Problem
- 2016
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Ingår i: Journal of Endodontics. - : Elsevier. - 0099-2399 .- 1878-3554. ; 42:7, s. 1003-1008
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Forskningsöversikt (refereegranskat)abstract
- Introduction: It has been almost 20 years since molecular methods were first described for the analysis of root canal microbial flora. Contamination control samples are essential to establish DNA decontamination before taking root canal samples, and this review assessed those studies. Methods: Using PubMed, a search was conducted for studies using molecular microbial analysis for the investigation of endodontic samples. Studies were grouped according to the cleaning protocol, acquisition methods, and processing of control samples taken to check for contamination. Results: Of 136 studies applying molecular analysis to root canal samples, 21 studies performed surface cleaning and checking nucleotide decontamination with contamination control samples processed by polymerase chain reaction. Only 1 study described disinfection, sampling from the access cavity,, and processing by polymerase chain reaction and reported the result; that study reported that all samples contained contaminating bacterial DNA. Conclusions: Cleaning, disinfection, and checking for contamination are basic scientific prerequisites for this type of investigation; yet, this review identifies it as an overlooked issue. On the basis of this review, we call for improved scientific practice in this field.
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| 9. |
- Figdor, David, et al.
(författare)
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[Obituary] Göran Sundqvist (1937-2020)
- 2020
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Ingår i: International Endodontic Journal. - : John Wiley & Sons. - 0143-2885 .- 1365-2591. ; 53:5, s. 726-727
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Tidskriftsartikel (refereegranskat)
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| 10. |
- Figdor, David, et al.
(författare)
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[OBITUARY] Göran Sundqvist (1937-2020)
- 2020
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Ingår i: Australian endodontic journal. - : Australian Society of Endodontology. - 1329-1947 .- 1747-4477. ; 46:1, s. 170-171
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Tidskriftsartikel (refereegranskat)
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