| 1. |
- Bergman, Petra, et al.
(author)
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Next-generation sequencing identifies microRNAs that associate with pathogenic autoimmune neuroinflammation in rats.
- 2013
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In: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 190:8, s. 4066-75
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Journal article (peer-reviewed)abstract
- MicroRNAs (miRNAs) are known to regulate most biological processes and have been found dysregulated in a variety of diseases, including multiple sclerosis (MS). In this study, we characterized miRNAs that associate with susceptibility to develop experimental autoimmune encephalomyelitis (EAE) in rats, a well-established animal model of MS. Using Illumina next-generation sequencing, we detected 544 miRNAs in the lymph nodes of EAE-susceptible Dark Agouti and EAE-resistant Piebald Virol Glaxo rats during immune activation. Forty-three miRNAs were found differentially expressed between the two strains, with 81% (35 out of 43) showing higher expression in the susceptible strain. Only 33% of tested miRNAs displayed differential expression in naive lymph nodes, suggesting that a majority of regulated miRNAs are EAE dependent. Further investigation of a selected six miRNAs indicates differences in cellular source and kinetics of expression. Several of the miRNAs, including miR-146a, miR-21, miR-181a, miR-223, and let-7, have previously been implicated in immune system regulation. Moreover, 77% (33 out of 43) of the miRNAs were associated with MS and other autoimmune diseases. Target genes likely regulated by the miRNAs were identified using computational predictions combined with whole-genome expression data. Differentially expressed miRNAs and their targets involve functions important for MS and EAE, such as immune cell migration through targeting genes like Cxcr3 and cellular maintenance and signaling by regulation of Prkcd and Stat1. In addition, we demonstrated that these three genes are direct targets of miR-181a. Our study highlights the impact of multiple miRNAs, displaying diverse kinetics and cellular sources, on development of pathogenic autoimmune inflammation.
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| 2. |
- Blagodatskikh, Konstantin A., et al.
(author)
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Improved DOP-PCR (iDOP-PCR) : A robust and simple WGA method for efficient amplification of low copy number genomic DNA
- 2017
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:9
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Journal article (peer-reviewed)abstract
- Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high-and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.
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| 3. |
- Ignatov, Konstantin B., et al.
(author)
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A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification
- 2014
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In: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 57:2, s. 81-87
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Journal article (peer-reviewed)abstract
- The sensitivity and robustness of various DNA detection and amplification techniques are to a large extent determined by the properties of the DNA polymerase used. We have compared the performance of conventional Taq and Bst DNA polymerases to a novel Tag DNA polymerase mutant (SD DNA polymerase), which has a strong strand displacement activity, in PCR (including amplification of GC-rich and complex secondary structure templates), long-range PCR (LR PCR), loop-mediated amplification (LAMP), and polymerase chain displacement reaction (PCDR). Our results demonstrate that the strand displacement activity of SD DNA polymerase, in combination with the robust polymerase activity, provides a notable improvement in the sensitivity and efficiency of all these methods.
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| 4. |
- Ignatov, Konstantin B., et al.
(author)
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Fragmentation Through Polymerization (FTP) : A new method to fragment DNA for next-generation sequencing
- 2019
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 14:4
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Journal article (peer-reviewed)abstract
- Fragmentation of DNA is the very important first step in preparing nucleic acids for next-generation sequencing. Here we report a novel Fragmentation Through Polymerization (FTP) technique, which is a simple, robust, and low-cost enzymatic method of fragmentation. This method generates double-stranded DNA fragments that are suitable for direct use in NGS library construction and allows the elimination of the additional step of reparation of DNA ends.
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| 5. |
- Kramarova, Tatiana, 1976-
(author)
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Limiting factors in ATP synthesis
- 2006
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Doctoral thesis (other academic/artistic)abstract
- The aim of the present study was to investigate the biosynthesis of the ATP synthase in various tissues, and to test hypotheses about possible models of activation of several mitochondrial proteins, the ATP/ADP translocase and UCPs, that could utilize the proton gradient, thus bypassing the ATP synthase. We have examined the role of the expression of the P1 isoform of the c-Fo subunit in the biogenesis of ATP synthase in brown adipose tissue. Our findings point to a role for the c-Fo subunit in defining the final content of the ATP synthase in brown adipose tissue.We have analyzed sequences in the 3’UTR of the β subunit F1-ATPase mRNA that are important for formation of RNA-protein complexes. We could detect protein complexes that bind to two different sequence regions of the 3’UTR, one being the poly(A) tail and an adjacent region), and the other being a sequence stretch at the 3’ end of the 3’UTR able to form a stem-loop structure, which is evolutionarily conserved throughout mammalian species. We investigated a role of the ATP/ADP carrier (ANT) in fatty acid-induced uncoupling in brown-fat mitochondria. We conclude that the ANT cannot substitute for UCP1 in fatty acid uncoupling in brown-fat mitochondria from mice lacking UCP1. We propose that the two ANT isoforms mediate proton translocation under different conditions.We have investigated a role of UCP1 in defence against oxidative stress. We found that products of oxidative stress (4-HNE) could neither reactivate purine nucleotide-inhibited UCP1, nor induce additional activation of innately active UCP1 in brown-fat mitochondria from UCP1(+/+) and UCP1(-/-) mice. We conclude that UCP1 is not involved in defence against oxidative stress. We evaluated possible uncoupling activity of UCP3 in skeletal muscle from warm- and cold-acclimated UCP1(+/+) and UCP1(-/-) mice. We conclude that no evidence exists for a higher UCP3-mediated uncoupling activity; a high UCP3 content in cold-acclimated UCP1(-/-) mice could possibly be linked to improved fatty acid oxidative capacity.
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| 6. |
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| 7. |
- Kramarova, Tatiana V., et al.
(author)
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Mitochondrial ATP synthase levels in brown adipose tissue are governed by the c-Fo subunit P1 isoform
- 2008
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In: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 22:1, s. 55-63
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Journal article (peer-reviewed)abstract
- Despite the significance of mitochondrial ATP synthase for mammalian metabolism, the regulation of the amount of ATP synthase in mammalian systems is not understood. As brown adipose tissue mitochondria contain very low amounts of ATP synthase, relative to respiratory chain components, they constitute a physiological system that allows for examination of the control of ATP synthase assembly. To examine the role of the expression of the P1-isoform of the c-F-o subunit in the biogenesis of ATP synthase, we made transgenic mice that express the P1-c subunit isoform under the promoter of the brown adipose tissue-specific protein UCP1. In the resulting UCP1p1 transgenic mice, total P1-c subunit mRNA levels were increased; mRNA levels of other F1F(o)-ATPase subunits were unchanged. In isolated brown-fat mitochondria, protein levels of the total c-Fo subunit were increased. Remarkably, protein levels of ATP synthase subunits that are part of the F-1-ATPase complex were also increased, as was the entire Complex V. Increased ATPase and ATP synthase activities demonstrated an increased functional activity of the F1Fo-ATPase. Thus, the levels of the c-F-o subunit P1-isoform are crucial for defining the final content of the ATP synthase in brown adipose tissue. The level of c-F-o subunit may be a determining factor for F1Fo-ATPase assembly in all higher eukaryotes.-Kramarova, T. V., Shabalina, I. G., Andersson, U., Westerberg, R., Carlberg, I., Houstek, J., Nedergaard, J., Cannon, B. Mitochondrial ATP synthase levels in brown adipose tissue are governed by the c-F-o subunit P1 isoform.
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| 8. |
- Shabalina, Irina G, et al.
(author)
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Cold tolerance of UCP1-ablated mice : A skeletal muscle mitochondria switch toward lipid oxidation with marked UCP3 up-regulation not associated with increased basal, fatty acid- or ROS-induced uncoupling or enhanced GDP effects.
- 2010
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In: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0005-2728. ; 1797:6-7, s. 968-80
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Journal article (peer-reviewed)abstract
- Mice lacking the thermogenic mitochondrial membrane protein UCP1 (uncoupling protein 1) - and thus all heat production from brown adipose tissue - can still adapt to a cold environment (4 degrees C) if successively transferred to the cold. The mechanism behind this adaptation has not been clarified. To examine possible adaptive processes in the skeletal muscle, we isolated mitochondria from the hind limb muscles of cold-acclimated wild-type and UCP1(-/-) mice and examined their bioenergetic chracteristics. We observed a switch in metabolism, from carbohydrate towards lipid catabolism, and an increased total mitochondrial complement, with an increased total ATP production capacity. The UCP1(-/-) muscle mitochondria did not display a changed state-4 respiration rate (no uncoupling) and were less sensitive to the uncoupling effect of fatty acids than the wild-type mitochondria. The content of UCP3 was increased 3-4 fold, but despite this, endogenous superoxide could not invoke a higher proton leak, and the small inhibitory effect of GDP was unaltered, indicating that it was not mediated by UCP3. Double mutant mice (UCP1(-/-) plus superoxide dismutase 2-overexpression) were not more cold sensitive than UCP1(-/-), bringing into question an involvement of reactive oxygen species (ROS) in activation of any alternative thermogenic mechanism. We conclude that there is no evidence for an involvement of UCP3 in basal, fatty-acid- or superoxide-stimulated oxygen consumption or in GDP sensitivity. The adaptations observed did not imply any direct alternative process for nonshivering thermogenesis but the adaptations observed would be congruent with adaptation to chronically enhanced muscle activity caused by incessant shivering in these mice.
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| 9. |
- Shabalina, Irina G., et al.
(author)
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The Environmental Pollutants Perfluorooctane Sulfonate and Perfluorooctanoic Acid Upregulate Uncoupling Protein 1 (UCP1) in Brown-Fat Mitochondria Through a UCP1-Dependent Reduction in Food Intake
- 2015
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In: Toxicological Sciences. - : Oxford University Press (OUP). - 1096-6080 .- 1096-0929. ; 146:2, s. 334-343
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Journal article (peer-reviewed)abstract
- The environmental pollutants perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) cause a dramatic reduction in the size of the major adipose tissue depots and a general body weight decrease when they are added to the food of mice. We demonstrate here that this is mainly due to a reduction in food intake; this reduction was not due to food aversion. Remarkably and unexpectedly, a large part of the effect of PFOA/PFOS on food intake was dependent on the presence of the uncoupling protein 1 (UCP1) in the mice. Correspondingly, PFOA/PFOS treatment induced recruitment of brown adipose tissue mitochondria: increased oxidative capacity and increased UCP1-mediated oxygen consumption (thermogenesis). In mice pair-fed to the food intake during PFOA/PFOS treatment in wildtype mice, brown-fat mitochondrial recruitment was also induced. We conclude that we have uncovered the existence of a regulatory component of food intake that is dependent upon brown adipose tissue thermogenic activity. The possible environmental consequences of this novel PFOA/PFOS effect (a possible decreased fitness) are noted, as well as the perspectives of this finding on the general understanding of control of food intake control and its possible extension to combatting obesity.
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