| 1. |
- Brandt, Christian, et al.
(author)
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Abundance Tracking by Long-Read Nanopore Sequencing of Complex Microbial Communities in Samples from 20 Different Biogas/Wastewater Plants
- 2020
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In: Applied Sciences. - : MDPI AG. - 2076-3417. ; 10
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Journal article (peer-reviewed)abstract
- Anaerobic digestion (AD) has long been critical technology for green energy, but the majority of the microorganisms involved are unknown and are currently not cultivable, which makes abundance tracking difficult. Developments in nanopore long-read sequencing make it a promising approach for monitoring microbial communities via metagenomic sequencing. For reliable monitoring of AD via long reads, we established a robust protocol for obtaining less fragmented, high-quality DNA, while preserving bacteria and archaea composition, for a broad range of different biogas reactors. Samples from 20 different biogas/wastewater reactors were investigated, and a median of 20.5 Gb sequencing data per nanopore flow cell was retrieved for each reactor using the developed DNA isolation protocol. The nanopore sequencing data were compared against Illumina sequencing data while using different taxonomic indices for read classifications. The Genome Taxonomy Database (GTDB) index allowed sufficient characterisation of the abundance of bacteria and archaea in biogas reactors with a dramatic improvement (1.8- to 13-fold increase) in taxonomic classification compared to the RefSeq index. Both technologies performed similarly in taxonomic read classification with a slight advantage for Illumina in regard to the total proportion of classified reads. However, nanopore sequencing data revealed a higher genus richness after classification. Metagenomic read classification via nanopore provides a promising approach to monitor the abundance of taxa present in a microbial AD community as an alternative to 16S ribosomal RNA studies or Illumina Sequencing.
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| 2. |
- Cunningham, Janet, et al.
(author)
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Impact of time and temperature on gut microbiota and SCFA composition in stool samples
- 2020
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In: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 15:8
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Journal article (peer-reviewed)abstract
- Gut dysbiosis has been implicated in the pathophysiology of a growing number of non-communicable diseases. High through-put sequencing technologies and short chain fatty acid (SCFA) profiling enables surveying of the composition and function of the gut microbiota and provide key insights into host-microbiome interactions. However, a methodological problem with analyzing stool samples is that samples are treated and stored differently prior to submission for analysis potentially influencing the composition of the microbiota and its metabolites. In the present study, we simulated the sample acquisition of a large-scale study, in which stool samples were stored for up to two days in the fridge or at room temperature before being handed over to the hospital. To assess the influence of time and temperature on the microbial community and on SCFA composition in a controlled experimental setting, the stool samples of 10 individuals were exposed to room and fridge temperatures for 24 and 48 hours, respectively, and analyzed using 16S rRNA gene amplicon sequencing, qPCR and nuclear magnetic resonance spectroscopy. To best of our knowledge, this is the first study to investigate the influence of storage time and temperature on the absolute abundance of methanogens, and ofLactobacillus reuteri. The results indicate that values obtained for methanogens,L.reuteriand total bacteria are still representative even after storage for up to 48 hours at RT (20 degrees C) or 4 degrees C. The overall microbial composition and structure appeared to be influenced more by laboratory errors introduced during sample processing than by the actual effects of temperature and time. Although microbial activity was demonstrated by elevated SCFA at both 4 degrees C and RT, SCFAs ratios were more stable over the different conditions and may be considered as long as samples are come from similar storage conditions.
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| 3. |
- Hippe, Andreas, et al.
(author)
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EGFR/Ras-induced CCL20 production modulates the tumour microenvironment.
- 2020
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In: British Journal of Cancer. - : Springer Science and Business Media LLC. - 0007-0920 .- 1532-1827. ; 123:6, s. 942-954
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Journal article (peer-reviewed)abstract
- BACKGROUND: The activation of the EGFR/Ras-signalling pathway in tumour cells induces a distinct chemokine repertoire, which in turn modulates the tumour microenvironment.METHODS: The effects of EGFR/Ras on the expression and translation of CCL20 were analysed in a large set of epithelial cancer cell lines and tumour tissues by RT-qPCR and ELISA in vitro. CCL20 production was verified by immunohistochemistry in different tumour tissues and correlated with clinical data. The effects of CCL20 on endothelial cell migration and tumour-associated vascularisation were comprehensively analysed with chemotaxis assays in vitro and in CCR6-deficient mice in vivo.RESULTS: Tumours facilitate progression by the EGFR/Ras-induced production of CCL20. Expression of the chemokine CCL20 in tumours correlates with advanced tumour stage, increased lymph node metastasis and decreased survival in patients. Microvascular endothelial cells abundantly express the specific CCL20 receptor CCR6. CCR6 signalling in endothelial cells induces angiogenesis. CCR6-deficient mice show significantly decreased tumour growth and tumour-associated vascularisation. The observed phenotype is dependent on CCR6 deficiency in stromal cells but not within the immune system.CONCLUSION: We propose that the chemokine axis CCL20-CCR6 represents a novel and promising target to interfere with the tumour microenvironment, and opens an innovative multimodal strategy for cancer therapy.
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| 4. |
- Moestedt, Jan, et al.
(author)
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Acetate and Lactate Production During Two-Stage Anaerobic Digestion of Food Waste Driven by Lactobacillus and Aeriscardovia
- 2020
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In: Frontiers in Energy Research. - : FRONTIERS MEDIA SA. - 2296-598X. ; 8
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Journal article (peer-reviewed)abstract
- Background: In a previous study, single-stage processes were compared with two-stage processes, using either food waste alone or mixed with thin stillage as substrate. Overall methane yield increased (by 12%) in two-stage compared with single-stage digestion when using food waste, but decreased when food waste was co-digested with thin stillage (50:50 on VS basis). The obtained difference in methane yield was likely caused by a higher acetate level in the first stage reactor operating with food waste alone (around 20 g/L) compared to the reactor also treating thin stillage (around 8 g /L). The present study sought to shed additional light on possible causes of the large difference in methane yield by scrutinizing the microbial community in the first- and second-stage reactors, using a combined Illumina sequencing and qPCR approach. Results: In the first-stage process, acid-tolerant Aeriscardovia and Lactobacillus formed a highly efficient consortium. For food waste with high levels of acetate (20 g/L, equal to 0.14 g acetate/g VS) was produced but when thin stillage was added the pH was lower (<4), resulting in lactate production exceeding acetate production. This difference in hydrolysate composition between the reactors resulted in development of slightly different communities in the second-stage, for both hydrolysis, fermentation, and acetogenesis. High acetate concentration appeared to promote proliferation of different syntrophic consortia, such as various syntrophic acetate oxidizers, members of the genus Syntrophomonas and candidate phylum Cloacimonetes, likely explaining the higher methane yields with two-step compared with single-stage digestion of food waste. Conclusion: Using food waste as sole substrate resulted in enrichment of Lactobacillus and Aeriscardovia and high acetate yields in the first-stage reactor. This was beneficial for biogas yield in two-stage digestion, where efficient acid-degrading syntrophic consortia developed. Addition of thin stillage contributed to low pH and higher lactate production, which resulted in decreased methane yield in the two-stage process compared with using food waste as sole substrate.
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| 5. |
- Singh, Abhijeet, et al.
(author)
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High-Throughput Sequencing and Unsupervised Analysis of Formyltetrahydrofolate Synthetase (FTHFS) Gene Amplicons to Estimate Acetogenic Community Structure.
- 2020
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In: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 11
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Journal article (peer-reviewed)abstract
- The formyltetrahydrofolate synthetase (FTHFS) gene is a molecular marker of choice to study the diversity of acetogenic communities. However, current analyses are limited due to lack of a high-throughput sequencing approach for FTHFS gene amplicons and a dedicated bioinformatics pipeline for data analysis, including taxonomic annotation and visualization of the sequence data. In the present study, we combined the barcode approach for multiplexed sequencing with unsupervised data analysis to visualize acetogenic community structure. We used samples from a biogas digester to develop proof-of-principle for our combined approach. We successfully generated high-throughput sequence data for the partial FTHFS gene and performed unsupervised data analysis using the novel bioinformatics pipeline "AcetoScan" presented in this study, which resulted in taxonomically annotated OTUs, phylogenetic tree, abundance plots and diversity indices. The results demonstrated that high-throughput sequencing can be used to sequence the FTHFS amplicons from a pool of samples, while the analysis pipeline AcetoScan can be reliably used to process the raw sequence data and visualize acetogenic community structure. The method and analysis pipeline described in this paper can assist in the identification and quantification of known or potentially new acetogens. The AcetoScan pipeline is freely available at https://github.com/abhijeetsingh1704/AcetoScan.
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