| 1. |
- Alekseenko, Alisa, et al.
(författare)
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Direct detection of SARS-CoV-2 using non-commercial RT-LAMP reagents on heat-inactivated samples
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.
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| 2. |
- Bergman, Birgitta, et al.
(författare)
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Trichodesmium : a widespread marine cyanobacterium with unusual nitrogen fixation properties
- 2013
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Ingår i: FEMS Microbiology Reviews. - : Oxford University Press (OUP). - 0168-6445 .- 1574-6976. ; 37:3, s. 286-302
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Forskningsöversikt (refereegranskat)abstract
- The last several decades have witnessed dramatic advances in unfolding the diversity and commonality of oceanic diazotrophs and their N(2) -fixing potential. More recently substantial progress in diazotrophic cell biology has provided a wealth of information on processes and mechanisms involved. The substantial contribution by the diazotrophic cyanobacterial genus Trichodesmium to the nitrogen influx of the global marine ecosystem is by now undisputable and of paramount ecological importance, while the underlying cellular and molecular regulatory physiology has only recently started to unfold. Here we explore and summarize current knowledge, related to optimization of its diazotrophic capacity, from genomics to ecophysiological processes, via eg. cellular differentiation (diazocytes) and temporal regulations, and suggest cellular research avenues that now ought to be explored. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
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| 3. |
- Cai, Fei, et al.
(författare)
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Bioinformatic Identification and Structural Characterization of a New Carboxysome Shell Protein
- 2012
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Ingår i: Functional Genomics and Evolution of Photosynthetic Systems. - Dordrecht : Springer Netherlands. - 9789400715325 - 9789400715332 ; , s. 345-356
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Bokkapitel (refereegranskat)abstract
- Bacterial Microcompartments (BMCs) are organelles composed of a polyhedral protein shell that encapsulates metabolically related enzymes. The best characterized BMC, the carboxysome, which functions to enhance CO2 fixation by D-ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), is found in all cyanobacteria. It is an essential part of the cyanobacterial CO2 concentrating mechanism. The shell of BMCs is composed of small (similar to 100 amino acids) proteins with a conserved primary structure known as the BMC domain. Proteins that contain BMC domains were shown to form hexamers that assemble in layers to form the facets of BMC shells. Previous structural models of the carboxysome shell were built from proteins which contain a single BMC domain. Recently, a new carboxysome shell protein was detected bioinformatically in Prochlorococcus and Synechococcus species. The crystal structure of this protein, CsoS1D, unexpectedly was the first tandem BMC domain protein structurally characterized. These data, together with transcriptomic evidence suggested that CsoS1D is a novel alpha-carboxysome shell protein with functionally important features. Here we used bioinformatic and comparative structural modeling to show that a hypothetical protein found in all beta cyanobacterial genomes is the ortholog of CsoS1D. We also discuss observations of other tandem BMC domain proteins, and we propose the hypothesis that the carboxysome shell may be a dynamic structure that responds to the environmental conditions within the cell.
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| 4. |
- Ekman, Martin, et al.
(författare)
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Cellular and functional specificity among ferritin-like proteins in the multicellular cyanobacterium Nostoc punctiforme.
- 2014
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Ingår i: Environmental Microbiology. - Oxford : Wiley-Blackwell. - 1462-2912 .- 1462-2920. ; 16:3, s. 829-844
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Tidskriftsartikel (refereegranskat)abstract
- Ferritin-like proteins constitute a remarkably heterogeneous protein family, including ferritins, bacterioferritins and Dps proteins. The genome of the filamentous heterocyst-forming cyanobacterium Nostoc punctiforme encodes five ferritin-like proteins. In the present paper, we report a multidimensional characterization of these proteins. Our phylogenetic and bioinformatics analyses suggest both structural and physiological differences among the ferritin-like proteins. The expression of these five genes responded differently to hydrogen peroxide treatment, with a significantly higher rise in transcript level for Npun_F3730 as compared with the other four genes. A specific role for Npun_F3730 in the cells tolerance against hydrogen peroxide was also supported by the inactivation of Npun_F3730, Npun_R5701 and Npun_R6212; among these, only the ΔNpun_F3730 strain showed an increased sensitivity to hydrogen peroxide compared with wild type. Analysis of promoter-GFP reporter fusions of the ferritin-like genes indicated that Npun_F3730 and Npun_R5701 were expressed in all cell types of a diazotrophic culture, while Npun_F6212 was expressed specifically in heterocysts. Our study provides the first comprehensive analysis combining functional differentiation and cellular specificity within this important group of proteins in a multicellular cyanobacterium.
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| 5. |
- Li, Xin, et al.
(författare)
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Differential transcriptional regulation of orthologous dps genes from two closely related heterocyst-forming cyanobacteria
- 2015
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 362:6
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Tidskriftsartikel (refereegranskat)abstract
- In cyanobacteria, DNA-binding proteins from starved cells (Dps) play an important role in the cellular response to oxidative and nutritional stresses. In this study, we have characterized the cell-type specificity and the promoter regions of two orthologous dps genes, Npun_R5799 in Nostoc punctiforme and alr3808 in Anabaena sp. PCC 7120. A transcriptional start site (TSS), identical in location to the previously identified proximal TSS of alr3808, was identified for Npun_R5799 under both combined nitrogen supplemented and N-2-fixing growth conditions. However, only alr3808 was also transcribed from a second distal TSS. Sequence homologies suggest that the promoter region containing the distal TSS is not conserved upstream of orthologous genes among heterocyst-forming cyanobacteria. The analysis of promoter GFP-reporter strains showed a different role in governing cell-type specificity between the proximal and distal promoter of alr3808. We here confirmed the heterocyst specificity of the distal promoter of alr3808 and described a very early induction of its expression during proheterocyst differentiation. In contrast, the complete promoters of both genes were active in all cells. Even though Npun_R5799 and alr3808 are orthologs, the regulation of their respective expression differs, indicating distinctions in the function of these cyanobacterial Dps proteins depending on the strain and cell type.
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| 6. |
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| 7. |
- Lin, Senjie, et al.
(författare)
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Two flavodoxin genes in Trichodesmium (Oscillatoriales, Cyanophyceae): Remarkable sequence divergence and possible functional diversification
- 2009
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Ingår i: Journal of Experimental Marine Biology and Ecology. - : Elsevier. - 0022-0981 .- 1879-1697. ; 371:1, s. 93-101
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Tidskriftsartikel (refereegranskat)abstract
- We analyzed flavodoxin gene sequences and transcription patterns in the marine nitrogen-fixing cyanobacterium Trichodesmium Ehrenberg ex Gomont 1892. While a typical cyanobacterial flavodoxin ortholog (fld1) was identified from cultured (strain IMS101) and environmental Trichodesmium by PCR and Southern blot hybridization, a second flavodoxin (fld2) was identified in the genome sequence. BLAST and phylogenetic analyses indicated that the two fld genes were highly divergent: fld1 was most closely related to fld common in cyanobacteria, wheras fld2 formed a distinct cluster with a fld so far only found in the unicellular diazotrophic cyanobacteria Cyanothece and Crocosphaera (in which fld2 was the only fld). The fld2 cluster was more closely allied with a fld in non-cyanobacterial diazotrophs such as Azotobacter and Azoarcus than fld1 in other cyanobacteria. This result suggests different evolutionary history of these two genes. Real-Time RT–PCR analysis on iron-replete and iron-deplete Trichodesmium cultures revealed remarkable diel dynamics in transcription of both flds but surprisingly failed to show induction under iron stress. Addition of nitrate to these cultures depressed nifH and fld2 transcription while elevating the transcription of fld1 in iron-stressed cultures. A positive correlation between fld and nifH transcript abundances was found, which was stronger in the case of fld2 than fld1. Our data suggest that these two flavodoxins in Trichodesmium may be functionally diversified and that use of flavodoxin as an iron stress indicator needs to be validated for each organism with consideration of diel dynamics of gene expression.
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| 8. |
- Moparthi, Vamsi, et al.
(författare)
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The two Dps proteins, NpDps2 and NpDps5, are involved in light-induced oxidative stress tolerance in the N-2-fixing cyanobacterium Nostoc punctiforme
- 2016
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier. - 0005-2728 .- 1879-2650. ; 1857:11, s. 1766-1776
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Tidskriftsartikel (refereegranskat)abstract
- Cyanobacteria are photosynthetic prokaryotes that are considered biotechnologically prominent organisms for production of high-value compounds. Cyanobacteria are subject to high-light intensities, which is a challenge that needs to be addressed in design of efficient bio-engineered photosynthetic organisms. Dps proteins are members of the ferritin superfamily and are omnipresent in prokaryotes. They play a major role in oxidative stress protection and iron homeostasis. The filamentous, heterocyst-forming Nostoc punctiforme, has five Dps proteins. In this study we elucidated the role of these Dps proteins in acclimation to high light intensity, the gene loci organization and the transcriptional regulation of all five dps genes in N. punctiforme was revealed, and dps-deletion mutant strains were used in physiological characterization. Two mutants defective in Dps2 and Dps5 activity displayed a reduced fitness under increased illumination, as well as a differential Photosystem (PS) stoichiometry, with an elevated Photosystem II to Photosystem I ratio in the dps5 deletion strain. This work establishes a Dps-mediated link between light tolerance, H2O2 detoxification, and iron homeostasis, and provides further evidence on the non-redundant role of multiple Dps proteins in this multicellular cyanobacterium.
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| 9. |
- Sandh, Gustaf, et al.
(författare)
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Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic filaments
- 2014
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND:In the filamentous cyanobacterium Nostoc punctiforme ATCC 29133, removal of combined nitrogen induces the differentiation of heterocysts, a cell-type specialized in N2 fixation. The differentiation involves genomic, structural and metabolic adaptations. In cyanobacteria, changes in the availability of carbon and nitrogen have also been linked to redox regulated posttranslational modifications of protein bound thiol groups. We have here employed a thiol targeting strategy to relatively quantify the putative redox proteome in heterocysts as compared to N2-fixing filaments, 24 hours after combined nitrogen depletion. The aim of the study was to expand the coverage of the cell-type specific proteome and metabolic landscape of heterocysts.RESULTS:Here we report the first cell-type specific proteome of newly formed heterocysts, compared to N2-fixing filaments, using the cysteine-specific selective ICAT methodology. The data set defined a good quantitative accuracy of the ICAT reagent in complex protein samples. The relative abundance levels of 511 proteins were determined and 74% showed a cell-type specific differential abundance. The majority of the identified proteins have not previously been quantified at the cell-type specific level. We have in addition analyzed the cell-type specific differential abundance of a large section of proteins quantified in both newly formed and steady-state diazotrophic cultures in N. punctiforme. The results describe a wide distribution of members of the putative redox regulated Cys-proteome in the central metabolism of both vegetative cells and heterocysts of N. punctiforme.CONCLUSIONS:The data set broadens our understanding of heterocysts and describes novel proteins involved in heterocyst physiology, including signaling and regulatory proteins as well as a large number of proteins with unknown function. Significant differences in cell-type specific abundance levels were present in the cell-type specific proteomes of newly formed diazotrophic filaments as compared to steady-state cultures. Therefore we conclude that by using our approach we are able to analyze a synchronized fraction of newly formed heterocysts, which enabled a better detection of proteins involved in the heterocyst specific physiology.
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| 10. |
- Sandh, Gustaf, et al.
(författare)
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Comparative proteomic profiles of the marine cyanobacterium Trichodesmium erythraeum IMS101 under different nitrogen regimes
- 2011
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 11:3, s. 406-419
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Tidskriftsartikel (refereegranskat)abstract
- Trichodesmium is a marine filamentous diazotrophic cyanobacterium and an important contributor of "new" nitrogen in the oligotrophic surface waters of the tropical and subtropical oceans. It is unique in that it exclusively fixes N-2 at daytime, although it belongs to the non-heterocystous filamentous segment of the cyanobacterial radiation. Here we present the first quantitative proteomic analysis of Trichodesmium erythraeum IMS101 when grown under different nitrogen regimes using 2-DE/MALDI-TOF-MS. Addition of combined nitrogen (NO3-) prevented development of the morphological characteristics of the N-2-fixing cell type (diazocytes), inhibited expression of the nitrogenase enzyme subunits and consequently N-2 fixation activity. The diazotrophic regime (N-2 versus NO3- cultures) elicited the differential expression of more than 100 proteins, which represented 13.5% of the separated proteins. Besides proteins directly related to N-2 fixation, proteins involved in the synthesis of reducing equivalents and the generation of a micro-oxic environment were strongly up-regulated, as was in particular Dps, a protein related to iron acquisition and potentially other vital cellular processes. In contrast, proteins involved in the S-adenosylmethionine (SAM) cycle, synthesis of amino acids and production of carbon skeletons for storage and synthesis of amino acids were suppressed. The data are discussed in the context of Trichodesmium's unusual N-2-fixing physiology.
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