QuantSeq 3’ mRNA-Seq for Ion Torrent

Metabolic responses of Aspergillus terreus under low dissolved oxygen and pH levels

Pajareeya Songserm, Aphichart Karnchanatat, Sitanan Thitiprasert, Somboon Tanasupawat, Suttichai Assabumrungrat, Shang-Tian Yang, Nuttha Thongchul

Annals of Microbiology, doi:10.1007/s13213-018-1330-6

The metabolic responses of Aspergillus terreus NRRL1960 to stress conditions (low dissolved oxygen and pH with limited nitrogen and phosphate) in the two-phase fermentation were investigated in this study. The fermentation kinetics suggested that itaconate production was suppressed under low dissolved oxygen (DO) concentrations. A slight change in pH caused a significant change in itaconate production. The transcriptomic data revealed that under low DO concentration, the glycolytic pathway was uncoupled from the oxidative phosphorylation, resulting in the activation of substrate-level phosphorylation as an alternative route for ATP regeneration. The downregulation of pdh genes, the genes encoding ATP synthase and succinate dehydrogenase, confirmed the observation of the uncoupling of the oxidative TCA cycle from glycolysis. It was found that the upregulation of pyc resulted in a large pool of oxaloacetate in the cytosol. This induced the conversion of oxaloacetate to malate. The upregulation of the gene encoding fumarate hydratase with the subsequent formation of fumarate was found to be responsible for the regeneration of NADPH and ATP under the condition of a low dissolved oxygen level. The large pool of oxaloacetate drove itaconic acid production also via the oxidative TCA cycle. Nevertheless, the downregulation of ATP synthase genes resulted in the deficiency of the proton-pumping H+ ATPase and the subsequent stress due to the failure to maintain the physiological pH. This resulted in itaconate production at a low titer. The fermentation kinetics and the transcriptomic data provided in this study can be used for further process optimization and control to improve itaconate production performance.

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Investigating the cellular processes anti-ageing compounds interact with can identify genes and pathways involved in ageing. The macrolide lactone FK506 was identified in a phenotypic screen as extending lifespan in yeast and C. elegans through an unknown mechanism. FK506 also ameliorates neurodegeneration and age-related weight gain in rodents. Here, the mechanism of action of FK506 has been investigated in two experimental systems: C. elegans and 3T3-L1 mouse adipocytes.

As the general mechanisms of ageing are well conserved between C. elegans and mammals, C. eleganshas been used to understand how FK506 acts at an organismal level. Firstly, the result of the phenotypic screen was confirmed. FK506 treatment induced lifespan extension in C. elegans in the presence of population crowding stress, but not in the absence of crowding. FK506 treatment inhibited neither E. coli OP50 growth nor C. elegans pharyngeal pumping, demonstrating that FK506 did not induce dietary restriction to extend lifespan. FK506 treatment increased C. elegans thrashing and pharynx pumping rates in early adulthood and delayed accumulation of gut bacteria, showing that FK506 extended healthspan. A transcriptome analysis of FK506-treated C. elegans allowed the identification of transcripts whose levels change and potential pathways by which FK506 manifests its effect. To explore this and to identify potential targets of FK506, the cellular functions required for FK506 to extend C. elegans lifespan and healthspan were investigated using RNA-seq, RNAi, genetic mutation and co-treatment with small molecule inhibitors and inducers. Interestingly, FK506 was found to have different mechanisms of action on lifespan and healthspan. The mechanism of FK506 on C. elegans thrashing rate was DAF-16 dependent, did not require population crowding stress, had a partial interaction with FUdR and autophagy, and may involve Ca2+ flux. The mechanism of FK506-induced C. elegans lifespan extension overlapped with dietary restriction and was dependent on calcineurin, TOR-independent regulation of autophagy and the presence of population crowding stress.

FK506 may modulate body weight by influencing metabolism and/or acting on adipocytes directly. FK506-treated aged 3T3-L1 adipocytes accumulated significantly less lipid, indicating that FK506 acts directly on adipocytes. RNA-seq of FK506-treated adipocytes found that translation-associated RNAs were upregulated whilst RNAs associated with lipid metabolism were downregulated. An ER-localised FK506-binding protein was up regulated in both C. elegans and 3T3-L1 adipocytes, fkb-4 and Fkbp2 respectively.

In conclusion, FK506 has been confirmed as a potential anti-ageing treatment, through its ability to extend lifespan and healthspan in C. C. elegans. In addition, FK506 has also been shown to act directly on mouse adipocytes, resulting in a reduction in lipid accumulation. This action could explain how FK506 caused weight loss in obese aged rats, restoring body mass to a healthy adult weight.

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Targeting the PD-1/PD-L1 pathway potentiates immunoediting to counterbalance neutral evolution in a mouse model of colorectal cancer

Zlatko Trajanoski, Mirjana Efremova, Victoria Klepsch, Pornpimol Charoentong, Francesca Finotello, Dietmar Rieder, Hubert Hackl, Natasch Hermann-Kleiter, Gottfried Baier, Anne Krogsdam

BioRxiv, doi:10.1101/099747

Background: The cancer immunoediting hypothesis postulates a dual role of the immune system: protecting the host by eliminating tumor cells, and shaping the developing tumor by editing the cancer genome. However, to what extent immunoediting is shaping the cancer genome in common malignancies is still a matter of debate. Moreover, the impact of cancer immunotherapy with checkpoint blockers on modulating immunoediting remains largely unexplored. Results: Here we employed a mouse model of colorectal cancer (CRC), next-generation sequencing, and computational analyses to elucidate the impact of evolutionary and immune-related forces on editing the tumor. We first carried out genomic and transcriptomic analyses of a widely-used model, MC38 cell line and show that this is a valid model for hypermutated and microsatellite-unstable CRC. Analyses of the data from longitudinal samples of wild type and immunodeficient RAG1 knockout mice transplanted with MC38 cells revealed that upregulation of checkpoint molecules and infiltration of Tregs are the major tumor escape mechanisms. Strikingly, the impact of neutral evolution on sculpting the tumor outweighed immunoediting by T cell dependent and T cell independent mechanisms in the progressing tumors. We also show that targeting the PD-1/PD-L1 pathway potentiated immunoediting and rendered tumors more homogeneous. Conclusions: In summary, our study demonstrates that neutral evolution is the major force that sculpts the tumor during progression, and that checkpoint blockade effectively enforces T cell dependent immunoselective pressure in this model. The results have important implication for basic research studies on the mechanisms of resistance to checkpoint blockade and for clinical translation.

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