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| 2. |
- Elbasani, E, et al.
(författare)
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Kaposi's Sarcoma-Associated Herpesvirus Reactivation by Targeting of a dCas9-Based Transcription Activator to the ORF50 Promoter
- 2020
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Ingår i: Viruses. - : MDPI AG. - 1999-4915. ; 12:9
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Tidskriftsartikel (refereegranskat)abstract
- CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site. To our surprise, the virus reactivation induced by binding of the stable DD-dCas9-VP192 on the ORF50 promoter was even more efficient than reactivation induced by ectopic expression of ORF50. This suggests that recruitment of additional transcriptional activators to the ORF50 promoter, in addition to ORF50 itself, are needed for the efficient virus production. Further, we show that CRISPRa can be applied to selectively express the early lytic gene, ORF57, without disturbing the viral latency. Therefore, CRISPRa-based systems can be utilized to facilitate virus–host interaction studies by controlling the expression of not only cellular but also of specific KSHV genes.
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| 3. |
- Kuil, Teun, et al.
(författare)
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Pyrophosphate as allosteric regulator of ATP-phosphofructokinase in Clostridium thermocellum and other bacteria with ATP- and PPi-phosphofructokinases
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor, some organisms also encode for PPi-dependent Pfks. Despite this central role, the biochemical characteristics as well as the physiological role of both Pfks is often not known. Clostridium thermocellum is an example of a microorganism that encodes for both Pfks, however, only PPi-Pfk activity has been detected in cell-free extracts and little is known about the regulation and function of both enzymes. In this study, the ATP- and PPi-Pfk of C. thermocellum were purified and biochemically characterized. No allosteric regulators were found for PPi-Pfk amongst common effectors. With fructose-6-P, PPi, fructose-1,6-bisP, and Pi PPi-Pfk showed high specificity (KM < 0.62 mM) and activity (Vmax > 156 U mgprotein-1). In contrast, ATP-Pfk showed much lower affinity (K0.5 of 9.26 mM) and activity (14.5 U mgprotein-1) with fructose-6-P. In addition to ATP, also GTP, UTP and ITP could be used as phosphoryl donors. The catalytic efficiency with GTP was 7-fold higher than with ATP, suggesting that GTP is the preferred substrate. The enzyme was activated by NH4+, and pronounced inhibition was observed with GDP, FBP, PEP, and especially with PPi (Ki of 0.007 mM). Characterization of purified ATP-Pfks originating from eleven different bacteria, encoding for only ATP-Pfk or for both ATP- and PPi-Pfk, identified that PPi inhibition of ATP-Pfks could be a common phenomenon for organisms with a PPi-dependent glycolysis.
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| 4. |
- Kuil, Teun, et al.
(författare)
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Pyrophosphate as allosteric regulator of ATP-phosphofructokinase in Clostridium thermocellum and other bacteria with ATP- and PPi-phosphofructokinases
- 2023
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 743
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Tidskriftsartikel (refereegranskat)abstract
- The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor, some organisms also encode for PPi-dependent Pfks. Despite this central role, the biochemical characteristics as well as the physiological role of both Pfks is often not known. Clostridium thermocellum is an example of a microorganism that encodes for both Pfks, however, only PPi-Pfk activity has been detected in cell-free extracts and little is known about the regulation and function of both enzymes. In this study, the ATP- and PPi-Pfk of C. thermocellum were purified and biochemically characterized. No allosteric regulators were found for PPi-Pfk amongst common effectors. With fructose-6-P, PPi, fructose-1,6-bisP, and Pi PPi-Pfk showed high specificity (KM < 0.62 mM) and maximum activity (Vmax > 156 U mg-1). In contrast, ATP-Pfk showed much lower affinity (K0.5 of 9.26 mM) and maximum activity (14.5 U mg-1) with fructose-6-P. In addition to ATP, also GTP, UTP and ITP could be used as phosphoryl donors. The catalytic efficiency with GTP was 7-fold higher than with ATP, suggesting that GTP is the preferred substrate. The enzyme was activated by NH4+, and pronounced inhibition was observed with GDP, FBP, PEP, and especially with PPi (Ki of 0.007 mM). Characterization of purified ATP-Pfks originating from eleven different bacteria, encoding for only ATP-Pfk or for both ATP- and PPi-Pfk, identified that PPi inhibition of ATP-Pfks could be a common phenomenon for organisms with a PPi-dependent glycolysis.
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| 5. |
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| 6. |
- Pekcan-Hekim, Zeynep, et al.
(författare)
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Bridge under troubled water : Turbulence and niche partitioning in fish foraging
- 2016
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Ingår i: Ecology and Evolution. - : Wiley-Blackwell. - 2045-7758. ; 6:24, s. 8919-8930
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Tidskriftsartikel (refereegranskat)abstract
- The coexistence of competing species relies on niche partitioning. Competitive exclusion is likely inevitable at high niche overlap, but such divide between competitors may be bridged if environmental circumstances displace competitor niches to enhance partitioning. Foraging-niche dimension can be influenced by environmental characteristics, and if competitors react differently to such conditions, coexistence can be facilitated. We here experimentally approach the partitioning effects of environmental conditions by evaluating the influence of water turbulence on foraging-niche responses in two competing fish species, Eurasian perch Perca fluviatilis and roach Rutilus rutilus, selecting from planktonic and benthic prey. In the absence of turbulence, both fish species showed high selectivity for benthic chironomid larvae. R. rutilus fed almost exclusively on zoobenthos, whereas P. fluviatilis complemented the benthic diet with zooplankton (mainly copepods). In turbulent water, on the other hand, the foraging-niche widths of both R. rutilus and P. fluviatilis increased, while their diet overlap simultaneously decreased, caused by 20% of the R. rutilus individuals turning to planktonic (mainly bosminids) prey, and by P. fluviatilis increasing foraging on littoral/benthic food sources. We show that moderate physical disturbance of environments, such as turbulence, can enhance niche partitioning and thereby coexistence of competing foragers. Turbulence affects prey but not fish swimming capacities, with consequences for prey-specific distributions and encounter rates with fish of different foraging strategies (pause-travel P. fluviatilis and cruise R. rutilus). Water turbulence and prey community structure should hereby affect competitive interaction strengths among fish species, with consequences for coexistence probability as well as community and system compositions.
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| 7. |
- Uhlig, E., et al.
(författare)
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The live bacterial load and microbiota composition of prepacked “ready-to-eat” leafy greens during household conditions, with special reference to E. coli
- 2022
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Ingår i: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605. ; 377
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Tidskriftsartikel (refereegranskat)abstract
- Ready-to-eat (RTE) leafy greens are popular products that unfortunately have been associated with numerous foodborne illness outbreaks. Since the influence of consumer practices is essential for their quality and safety, the objective of this study was to analyze the microbiota of RTE products throughout shelf life during simulated household conditions. Products from different companies were analyzed in terms of plate counts, and resealed and unopened packages were compared. High bacterial loads were found, up to a total plate count of 9.6 log10 CFU/g, and Enterobacteriaceae plate counts up to 6.0 CFU/g on the expiration date. The effect of consumer practice varied, thus no conclusions regarding resealed or unopened bags could be drawn. The tested products contained opportunistic pathogens, such as Enterobacter homaechei, Hafnia paralvei and Pantoea agglomerans. Amplicon sequencing revealed that the relative abundance of major taxonomic groups changed during shelf life; Pseudomonadaceae and Xanthomonadaceae decreased, while Flavobacteriaceae and Marinomonadaceae inceased. Inoculation with E. coli CCUG 29300T showed that the relative abundance of Escherichia-Shigella was lower on rocket than on other tested leafy greens. Inoculation with E. coli strain 921 indicate growth at the beginning of shelf-life time, while E. coli 731 increases at the end, seemingly able to adapt to cold storage conditions. The high levels of live microorganisms, the detection of opportunistic pathogens, and the ability of E. coli strains to grow at refrigeration temperature raise concerns and indicate that the shelf life may be shortened to achieve a safer product. Due to variations between products, further studies are needed to define how long the shelf-life of these products should be, to ensure a safe product even at the end of the shelf-life period.
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