Project description:We used single-cell sequencing data and imaging to investigate Eukaryotic plankton from environmental marine samples collected from Coogee, NSW, Australia.
Project description:In this study we applied MASC-seq (massive and parallel microarray sequencing, https://doi.org/10.1038/ncomms13182), a scRNA-seq method that facilitates sequencing of thousands of cells in parallel, and that couples microscope images with the single cell transcriptome data. For this method, fixed cells are spread over a microarray with 100 μm-sized spots of DNA capture probes with spot-specific indices. The cells are first imaged using a scanning microscope and then permeabilized, releasing their RNA out of the cells and bind to the probes on the array. cDNA is synthesized, harvested and sequenced, and, using the spot-specific barcode-sequences, cDNA sequences stemming from a specific spot (i.e., cell) can be linked to the microscope image of the corresponding cell. However, until now, the MASC-seq method has only been applied to mammalian cells. The aim of this study was to test and adapt the MASC-seq method for application on unicellular eukaryotic plankton. We applied and optimized the method on three cultured plankton representatives, abundant in communities of aquatic environments, Phaeodactylum tricornutum (a diatom, silica and polysaccharide cell walls 23), Heterocapsa sp. (a dinoflagellate, cellulose thecal plates 24) and Tetrahymena thermophila (a ciliate, lipid membrane 25) which all have different size and diverse cell surface structures common to plankton. We optimized several steps in the protocol to make it more suitable for planktonic cells and compared the results from MASC-seq generated single cell transcriptomes to bulk RNA sequencing.
Project description:Roux-en-Y gastric bypass (RYGB) is highly effective in reversing obesity and associated diabetes. Recent observations in humans suggest a contributing role of increased circulating bile acids in mediating such effects. Here we use a diet-induced obesity mouse model and compared metabolic remission when bile flow was diverted through a gallbladder anastomosis to jejunum, ileum or duodenum (sham control). We found that only bile diversion to the ileum results in physiologic changes similar to RYGB including sustained improvements in weight, glucose tolerance and hepatic steatosis despite differential effects on hepatic gene expression. Circulating free fatty acids and triglycerides decrease while bile acids increase, particularly conjugated tauro-b-muricholic acid, an FXR antagonist. Activity of the hepatic FXR/FGF15 axis was reduced and associated with altered gut microbiota. Thus bile diversion, independent of surgical rearrangement of the gastrointestinal tract, imparts significant weight loss accompanied by improved glucose and lipid homeostasis that are hallmarks of RYGB. Total RNA from n = 5 DIO, n = 4 GB-IL, n = 5 RYGB mice livers was extracted of total RNA and submitted fro RNAseq
Project description:Microeukaryotic plankton communities are keystone components for keeping aquatic primary productivity. Currently, variations in microeukaryotic plankton diversity have often been explained by local ecological factors but not by evolutionary constraints. We used amplicon sequencing of 100 water samples across five years to investigate the ecological preferences of the microeukaryotic plankton community in a subtropical riverine ecosystem. We found that microeukaryotic plankton diversity was less associated with bacterial abundance (16S rRNA gene copy number) than bacterial diversity. Further, environmental effects exhibited a larger influence on microeukaryotic plankton community composition than bacterial community composition, especially at fine taxonomic levels. The evolutionary constraints of microeukaryotic plankton community increased with decreasing taxonomic resolution (from 97% to 91% similarity levels), but not significant change from 85% to 70% similarity levels. However, compared with the bacterial community, the evolutionary constraints were shown to be more affected by environmental variables. This study illustrated possible controlling environmental and bacterial drivers of microeukaryotic diversity and community assembly in a subtropical river, thereby indirectly reflecting on the quality status of the water environment by providing new clues on the microeukaryotic community assembly.