Hu, C.-M. | bioRxiv Science

bioRxiv2022

Selection and validation of internal control genes for quantitative real-time RT-qPCR normalization of Phlebopus portentosus gene expression under different conditions

Phlebopus portentosus (Berk. and Broome) Boedijin is an attractive edible mushroom and considered as the unique bolete to have achieved artificial cultivation in vitro. Gene expression analysis has become widely used in researches of edible fungi and is important to uncover the functions of genes involved in complex biological processes. Selecting appropriate reference genes is crucial to ensuring reliable results of gene expression analysis by RT-qPCR. In our study, a total of 12 candidate control genes were selected from 25 traditional housekeeping genes based on the expression stability in 9 transcriptomes of 3 developmental stages. These genes were further evaluated using geNorm, NormFinder and RefFinder under different conditions and developmental stages. The results revealed that MSF1-domain-containing protein (MSF1), synaptobrevin (SYB), mitogen-activated protein kinase genes (MAPK), TATA binding protein 1 (TBP1) and SPRY domain protein (SPRY) were the most stable reference genes in all sample treatments, while elongation factor 1-alpha (EF1), Actin and ubiquitin-conjugating enzyme (UBCE) were the most unstably expressed. The gene SYB was selected based on the transcriptome results and was identified as a novel reference gene in P. portentosus. This is the first detailed study for identification reference genes in this fungus and may provide new insights into selecting genes and quantifying gene expression.

microbiology↗

bioRxiv2022

Targeting Reductive Metabolic Shifts by T315I Mutation in BCR-ABL Myeloid Leukemia for Therapy

T315I mutation of Bcr-Abl in chronic myeloid leukemia (CML) leads to therapeutic resistance. It is known that Bcr-Abl transformation causes ROS-induced DNA damages and replication stress, which can be exploited for anti-nucleotide therapy. We developed a small compound, JMF4073, which inhibited pyrimidylate kinases and selectively eliminated Bcr-Abl-transformed, but not untransformed myeloid cells, due to dTTP exhaustion and ROS-induced replication stress. However, T315I-Bcr-Abl-transformed cells were less vulnerable to JMF4073 because of higher dTTP pool and low replication stress. Unlike WT-Bcr-Abl-transformed cells, T315I-Bcr-Abl cells lacked Sirt1- regulated OXPHOS with increased glutamine flux to reductive carboxylation in TCA cycle and glutathione synthesis. Blocking mitochondrial pyruvate carrier (MPC) by UK-5099 reduced NADH and glutathione levels with replication stress induction, thereby converting T315I-Bcr-Abl cells sensitive to JMF4073 with dTTP and dCTP depletion. The combination of JMF4073 with UK-5099 showed in vivo eradication of T315I-Bcr-Abl-CML. These data reveal that T315I mutation causes reductive metabolic shifts in Bcr-Abl-CML, and demonstrate the therapeutic option by co-targeting MPC and pyrimidylate kinases.

cancer biology↗