Taxonomy of anaerobic digestion microbiome reveals biases associated with the applied high throughput sequencing strategies

In the past few years, many studies investigated the anaerobic digestion microbiome by means of 16S rRNA amplicon sequencing. Results obtained from these studies were compared to each other without taking into consideration the followed procedure for amplicons preparation and data analysis. This negligence was mainly due to the lack of knowledge regarding the biases influencing specific steps of the microbiome investigation process. In the present study, the main technical aspects of the 16S rRNA analysis were checked giving special attention to the approach used for high throughput sequencing. More specifically, the microbial compositions of three laboratory scale biogas reactors were analyzed before and after addition of sodium oleate by sequencing the microbiome with three different approaches: 16S rRNA amplicon sequencing, shotgun DNA and shotgun RNA. This comparative analysis revealed that, in amplicon sequencing, abundance of some taxa (Euryarchaeota and Spirochaetes) was biased by the inefficiency of universal primers to hybridize all the templates. Reliability of the results obtained was also influenced by the number of hypervariable regions under investigation. Finally, amplicon sequencing and shotgun DNA underestimated the Methanoculleus genus, probably due to the low 16S rRNA gene copy number encoded in this taxon

Deciphering the molecular mechanisms of amino acid sensing by mTOR

The mechanistic target of Rapamycin (mTOR) is a serine-threonine kinase and the master regulator of cell growth and metabolism. The mTOR complex 1 (mTORC1) integrates nutrient availability signals to promote anabolic and inhibit catabolic processes in the cell by phosphorylating multiple substrates. Dysregulation of the mTORC1 pathway is involved in ageing and disease. mTORC1 is recruited to the lysosomal surface by the Rag GTPases upon re-stimulation with amino acids after starvation, where it is activated by the small GTPase Rheb. Although the lysosome-centric model of mTORC1 activation is well-described, growing evidence suggests that amino acids do not activate mTORC1 only on the lysosomes. With this work, I sought to expand the model of mTORC1 activation, by showing that mTORC1 can be activated by amino acids towards specific substrates in a lysosome- and Rag-independent manner. Moreover, I sought to examine mTORC1 activity in unchallenged cells and explain its lysosomal localization when nutrients are abundant. The findings of this work shed light on a largely unexplored part of mTORC1 regulation and lead to a better understanding of the mechanism behind mTORC1 activation by amino acids