| 1. |
- Bood, Mattias, et al.
(författare)
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Pentacyclic adenine: a versatile and exceptionally bright fluorescent DNA base analogue
- 2018
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Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 9:14, s. 3494-3502
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Tidskriftsartikel (refereegranskat)abstract
- Emissive base analogs are powerful tools for probing nucleic acids at the molecular level. Herein we describe the development and thorough characterization of pentacyclic adenine (pA), a versatile base analog with exceptional fluorescence properties. When incorporated into DNA, pA pairs selectively with thymine without perturbing the B-form structure and is among the brightest nucleobase analogs reported so far. Together with the recently established base analog acceptor qAnitro, pA allows accurate distance and orientation determination via Forster resonance energy transfer (FRET) measurements. The high brightness at emission wavelengths above 400 nm also makes it suitable for fluorescence microscopy, as demonstrated by imaging of single liposomal constructs coated with cholesterolanchored pA-dsDNA, using total internal reflection fluorescence microscopy. Finally, pA is also highly promising for two-photon excitation at 780 nm, with a brightness (5.3 GM) that is unprecedented for a base analog.
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| 2. |
- Füchtbauer, Anders Foller, 1984, et al.
(författare)
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Fluorescent RNA cytosine analogue - an internal probe for detailed structure and dynamics investigations
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- The bright fluorescent cytosine analogue tCO stands out among fluorescent bases due to its virtually unquenched fluorescence emission in duplex DNA. However, like most reported base analogues, it has not been thoroughly characterized in RNA. We here report on the first synthesis and RNA-incorporation of tCO, and characterize its base-mimicking and fluorescence properties in RNA. As in DNA, we find a high quantum yield inside RNA duplexes (< Phi(F)> = 0.22) that is virtually unaffected by the neighbouring bases (Phi(F) = 0.20-=0.25), resulting in an average brightness of 1900 M-1 cm(-1). The average fluorescence lifetime in RNA duplexes is 4.3 ns and generally two lifetimes are required to fit the exponential decays. Fluorescence properties in ssRNA are defined by a small increase in average quantum yield (< Phi(F) > = 0.24) compared to dsRNA, with a broader distribution (Phi(F) = 0.17-=0.34) and slightly shorter average lifetimes. Using circular dichroism, we find that the tCO-modified RNA duplexes form regular A-form helices and in UV-melting experiments the stability of the duplexes is only slightly higher than that of the corresponding natural RNA ( = + 2.3 degrees C). These properties make tC(0) a highly interesting fluorescent RNA base analogue for detailed FRET-based structural measurements, as a bright internal label in microscopy, and for fluorescence anisotropy measurements of RNA dynamics.
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| 3. |
- Aase, Audun, et al.
(författare)
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Validate or falsify: Lessons learned from a microscopy method claimed to be useful for detecting Borrelia and Babesia organisms in human blood
- 2016
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Ingår i: INFECTIOUS DISEASES. - : TAYLOR & FRANCIS LTD. - 2374-4235 .- 2374-4243. ; 48:6, s. 411-419
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Tidskriftsartikel (refereegranskat)abstract
- Background A modified microscopy protocol (the LM-method) was used to demonstrate what was interpreted as Borrelia spirochetes and later also Babesia sp., in peripheral blood from patients. The method gained much publicity, but was not validated prior to publication, which became the purpose of this study using appropriate scientific methodology, including a control group. Methods Blood from 21 patients previously interpreted as positive for Borrelia and/or Babesia infection by the LM-method and 41 healthy controls without known history of tick bite were collected, blinded and analysed for these pathogens by microscopy in two laboratories by the LM-method and conventional method, respectively, by PCR methods in five laboratories and by serology in one laboratory. Results Microscopy by the LM-method identified structures claimed to be Borrelia- and/or Babesia in 66% of the blood samples of the patient group and in 85% in the healthy control group. Microscopy by the conventional method for Babesia only did not identify Babesia in any samples. PCR analysis detected Borrelia DNA in one sample of the patient group and in eight samples of the control group; whereas Babesia DNA was not detected in any of the blood samples using molecular methods. Conclusions The structures interpreted as Borrelia and Babesia by the LM-method could not be verified by PCR. The method was, thus, falsified. This study underlines the importance of doing proper test validation before new or modified assays are introduced.
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| 4. |
- Astvaldsdottir, A., et al.
(författare)
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Oral health and dental care of older persons-A systematic map of systematic reviews
- 2018
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Ingår i: Gerodontology. - : Wiley. - 0734-0664 .- 1741-2358. ; 35:4, s. 290-304
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Forskningsöversikt (refereegranskat)abstract
- Objectives: To examine the current knowledge on oral health status and dental care of older persons through a systematic mapping of systematic reviews of low or moderate risk of bias. Background: Geriatric dentistry covers all aspects of oral health and oral care of older persons. Oral health is part of general health and contributes to a person's physical, psychological and social wellbeing. Methods: A literature search was performed in three different databases (PubMed, The Cochrane Library and Cinahl) within 12 domains: Dental caries, periodontitis, Orofacial pain and temporomandibular joint (TMJ) pain, mucosal lesions, oral motor function, dry mouth, halitosis, interaction between oral status and other medical conditions, ability to interrelate and communicate, quality of life, ethics and organisation of dental care for older persons. Systematic reviews were identified and scrutinised, highlighting scientific knowledge and knowledge gaps. Results: We included 32 systematic reviews of which 14 were judged to be of low/moderate risk of bias. Most of the domains lack systematic reviews with low or moderate risk of bias. In two of the domains evidence was identified; in institutionalised people aged 65 or older, effective oral hygiene can prevent pneumonia. Furthermore, there is an evidence of a relationship between malnutrition (protein energy-related malnutrition, PEM) and poor appetite and edentulousness. Conclusions: There is an urgent need for further research and evidence-based knowledge within most domains in geriatric dentistry and in other fields related to oral health and dental care for older persons striving for multi-disciplinary research programmes.
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| 5. |
- Dumat, Blaise, 1984, et al.
(författare)
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Second-Generation Fluorescent Quadracyclic Adenine Analogues: Environment-Responsive Probes with Enhanced Brightness
- 2015
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Ingår i: Chemistry - A European Journal. - : Wiley. - 0947-6539 .- 1521-3765. ; 21:10, s. 4039-4048
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Tidskriftsartikel (refereegranskat)abstract
- Fluorescent base analogues comprise a group of increasingly important molecules for the investigation of nucleic acid structure, dynamics, and interactions with other molecules. Herein, we report on the quantum chemical calculation aided design, synthesis, and characterization of four new putative quadracyclic adenine analogues. The compounds were efficiently synthesized from a common intermediate through a two-step pathway with the Suzuki-Miyaura coupling as the key step. Two of the compounds, qAN1 and qAN4, display brightnesses (εΦF) of 1700 and 2300, respectively, in water and behave as wavelength-ratiometric pH probes under acidic conditions. The other two, qAN2 and qAN3, display lower brightnesses but exhibit polarity-sensitive dual-band emissions that could prove useful to investigate DNA structural changes induced by DNA-protein or -drug interactions. The four qANs are very promising microenvironment-sensitive fluorescent adenine analogues that display considerable brightness for such compounds. Polarity and pH probes: 2-Aminopurine has long been the standard for fluorescent base analogues. Four new fluorescent probes suitable for the replacement of adenine in nucleic acids are presented. Based on their high structural similarity to their parent compound, quadracyclic adenine, they have the potential to be excellent A analogues. Their improved photophysical properties also suggest that they could be significantly brighter than 2-aminopurine inside nucleic acid systems (see figure; ΦF: fluorescence quantum yield).
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| 6. |
- Eskeland, Sveinung, et al.
(författare)
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Assessing the clinical and bacteriological outcomes of vaccination with recombinant Asp14 and OmpA against A. phagocytophilum in sheep
- 2019
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Ingår i: Veterinary Immunology and Immunopathology. - : ELSEVIER. - 0165-2427 .- 1873-2534. ; 218
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Tidskriftsartikel (refereegranskat)abstract
- Anaplasma phagocytophilum is a tick borne bacterium, causing disease in sheep and other mammals, including humans. The bacterium has great economic and animal welfare implications for sheep husbandry in Northern Europe. With the prospect of a warmer and more humid climate, the vector availability will likely increase, resulting in a higher prevalence of A. phagocytophilum. The current preventive measures, as pyrethroids acting on ticks or long acting antibiotics controlling bacterial infection, are suboptimal for prevention of the disease in sheep. Recently, the increased awareness on antibiotic- and pyrethorid resistance, is driving the search for a new prophylactic approach in sheep against A. phagocytophilum. Previous studies have used an attenuated vaccine, which gave insufficient protection from challenge with live bacteria. Other studies have focused on bacterial membrane surface proteins like Asp14 and OmpA. An animal study using homologous proteins to Asp14 and OmpA of A. marginate, showed no protective effect in heifers. In the current study, recombinant proteins of Asp14 (rAsp14) and OmpA (rOmpA) of A. phagocytophilum were produced and prepared as a vaccine for sheep. Ten lambs were vaccinated twice with an adjuvant emulsified with rAsp14 or rOmpA, three weeks apart and challenged with a live strain of A. phagocytophilum (GenBank acc.nr M73220) on day 42. The control group consisted of five lambs injected twice with PBS and adjuvant. Hematology, real time qPCR, immunodiagnostics and flow cytometric analyses of peripheral blood mononuclear cells were performed. Vaccinated lambs responded with clinical signs of A.phagocytophilum infection after challenge and bacterial load in the vaccinated group was not reduced compared to the control group. rAsp14 vaccinated lambs generated an antibody response against the vaccine, but a clear specificity for rAsp14 could not be established. rOmpA-vaccinated lambs developed a strong specific antibody response on days 28 after vaccination and 14 days post-challenge. Immunofluorescent staining and flow cytometric analysis of peripheral blood mononuclear monocytes revealed no difference between the three groups, but the percentage of CD4, CD8(+), gamma delta TcR+, lambda-Light chain(+), CD11b(+), CD14(+) and MHC II+ cells, within the groups changed during the study, most likely due to the adjuvant or challenge with the bacterium. Although an antigen specific antibody response could be detected against rOmpA and possibly rAsp14, the vaccines seemed to be ineffective in reducing clinical signs and bacterial load caused by A. phagocytophilum. This is the first animal study with recombinant Asp14 and OmpA aimed at obtaining clinical protection against A. phagocytophilum in sheep.
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| 7. |
- Fisher, Rachel S., et al.
(författare)
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Pulse-shaped two-photon excitation of a fluorescent base analogue approaches single-molecule sensitivity
- 2018
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Ingår i: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9084 .- 1463-9076. ; 20:45, s. 28487-28498
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Tidskriftsartikel (refereegranskat)abstract
- Fluorescent nucleobase analogues (FBAs) have many desirable features in comparison to extrinsic fluorescent labels, but they are yet to find application in ultrasensitive detection. Many of the disadvantages of FBAs arise from their short excitation wavelengths (often in the ultraviolet), making two-photon excitation a potentially attractive approach. Pentacyclic adenine (pA) is a recently developed FBA that has an exceptionally high two-photon brightness. We have studied the two-photon-excited fluorescence properties of pA and how they are affected by incorporation in DNA. We find that pA is more photostable under two-photon excitation than via resonant absorption. When incorporated in an oligonucleotide, pA has a high two-photon cross section and emission quantum yield, varying with sequence context, resulting in the highest reported brightness for such a probe. The use of a two-photon microscope with ultrafast excitation and pulse shaping has allowed the detection of pA-containing oligonucleotides in solution with a limit of detection of ∼5 molecules, demonstrating that practical single-molecule detection of FBAs is now within reach.
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| 8. |
- Freeman, N.S., et al.
(författare)
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Chromophoric Nucleoside Analogues: Synthesis and Characterization of 6-Aminouracil-Based Nucleodyes
- 2016
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Ingår i: Journal of Organic Chemistry. - : American Chemical Society (ACS). - 0022-3263 .- 1520-6904. ; 81:11, s. 4530-4539
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Tidskriftsartikel (refereegranskat)abstract
- Nucleodyes, visibly colored chromophoric nucleoside analogues, are reported. Design criteria are outlined and the syntheses of cytidine and uridine azo dye analogues derived from 6-aminouracil are described. Structural analysis shows that the nucleodyes are sound structural analogues of their native nucleoside counterparts, and photophysical studies demonstrate that the nucleodyes are sensitive to microenvironmental changes. Quantum chemical calculations are presented as a valuable complementary tool for the design of strongly absorbing nucleodyes, which overlap with the emission of known fluorophores. Förster critical distance (R0) calculations determine that the nucleodyes make good FRET pairs with both 2-aminopurine (2AP) and pyrrolocytosine (PyC). Additionally, unique tautomerization features exhibited by 5-(4-nitrophenylazo)-6-oxocytidine (8) are visualized by an extraordinary crystal structure.
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| 9. |
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| 10. |
- Larsen, Anders Foller, 1984, et al.
(författare)
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Development of bright fluorescent quadracyclic adenine analogues: TDDFT-calculation supported rational design
- 2015
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (epsilon Phi(F) = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs.
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