Fractional deletion of Compound Kushen Injection indicates cytokine signaling pathways are critical for its perturbation of the cell cycle

Published online: 02 October 2019We used computational and experimental biology approaches to identify candidate mechanisms of action of aTraditional Chinese Medicine, Compound Kushen Injection (CKI), in a breast cancer cell line (MDA-MB-231). Because CKI is a complex mixture of plant secondary metabolites, we used a high-performance liquid chromatography (HPLC) fractionation and reconstitution approach to define chemical fractions required for CKI to induce apoptosis. The initial fractionation separated major from minor compounds, and it showed that major compounds accounted for little of the activity of CKI. Furthermore, removal of no single major compound altered the effect of CKI on cell viability and apoptosis. However, simultaneous removal of two major compounds identified oxymatrine and oxysophocarpine as critical with respect to CKI activity. Transcriptome analysis was used to correlate compound removal with gene expression and phenotype data. Many compounds in CKI are required to trigger apoptosis but significant modulation of its activity is conferred by a small number of compounds. In conclusion, CKI may be typical of many plant based extracts that contain many compounds in that no single compound is responsible for all of the bioactivity of the mixture and that many compounds interact in a complex fashion to influence a network containing many targets.T. N . Aung, S. Nourmohammadi, Z. Qu, Y. Harata-Lee, J. Cui, H. Y. Shen, A. J. Yool, T. Pukala, Hong Du, R. D. Kortschak, W. Wei, D. L. Adelso

Arabidopsis thaliana Pol IV subunit mutant nrpd1a-3 is associated with a deletion in RHD6

Published December 12, 2019In eukaryotes, RNA and chromatin-based pathways control transposable elements (TE) to minimize the deleterious consequences of genetic invasion, transposition, mutation and chromosome instability (Matzke and Mosher, 2014). In higher plants, the multi-subunit nuclear RNA polymerase IV (Pol IV) specializes in transcribing the 24 nucleotide class of small RNAs that target TE’s for DNA cytosine methylation and silencing in the RNA-directed DNA methylation (RdDM) pathway (Herr et al., 2005; Onodera et al., 2005). In Arabidopsis, the Pol IV has two alternative subunits encoded by the NRPD1a and NRPD1b and a common subunit encoded by NRPD2A (Herr et al., 2005; Kanno et al., 2005; Onodera et al., 2005; Pontier et al., 2005). The null mutant for NRPD1a is defective in the RdDM pathway and also displays a late flowering phenotype under short day conditions (Pontier et al., 2005; Eamens et al., 2008). Interestingly, we observed a root hair defective phenotype in the nrpd1a-3 mutant allele which has not been previously reported (Fig. 1A). When grown on vertically oriented agar medium, nrpd1a-3 seedlings lacked root hairs or defective root hair elongation when compared to the Col-0 wild type control which showed normal root hair distribution and length in the root maturation zone (Fig. 1A). Further investigation of other NRPD1A mutant alleles, nrpd1a-1, nrpd1a-2, in the C-24 accession, and nrpd1a-4 in the Col-0 accession revealed that these alleles had a similar root hair number to the wild type controls (Fig. 1B). To investigate if the root hair defective phenotype in nrpd1a-3 was a spurious event in our laboratory’s seed stock or an earlier event, we re-ordered the same mutant from the ABRC stock Centre and tested the root hair phenotype. We observed the same root hair defective phenotype in both nrpd1a-3 seed stocks. We next crossed the nrpd1a-3 mutant (Col-0) to wild type accession C-24, self-fertilized the F1to create a F2mapping population and mapped polymorphic DNA markers across the 5 chromosomes revealed the gene for the root hair defective phenotype was located between DNA markers ciw3 and F26B6 on chromosome 1 (Berendzen et al., 2005). Next we whole-genome sequenced DNA from both Col-0 and nrpd1a-3 using Illumina short read technology, and after GATK (McKenna et al., 2010) and DELLY (Rausch et al., 2012) analysis of the annotated gene models in the genetic window, we identified only one nucleotide mutation, a 1,310 bp deletion in Root Hair Defective 6 (RHD6), in nrpd1a-3 (Fig. 1C). RHD6 is a bHLH transcription factor that positively regulates root hair initiation (Masucci and Schiefelbein, 1994) and loss of function mutations cause a root hair defective phenotype. Semi-quantitative RT-PCR showed RHD6 mRNA was undetectable in the nrpd1a-3 roots when compared to Col-0 wild-type and nrpd1a-4 suggesting that the loss of RHD6 was the likely candidate for the root hair defective phenotype observed in the nrpd1a-3 mutant (Fig. 1D). We confirmed the 1,310 bp deletion that deleted part of exon 2, all of exons 3-5 and part of the 3’ UTR by PCR and Sanger sequencing. Together the genetic mapping and the undetectable RHD6 mRNA transcript strongly suggests that the root hair defective phenotype only observed in the nrpd1a-3 mutant background is caused by the deletion in RHD6. In the Arabidopsis research community, sometimes phenotypes caused by an unlinked mutation to a gene of interest have been incorrectly associated in the research field for many years (Enders et al., 2015; Habets and Offringa, 2015), and so our discovery of a deletion in RHD6 in nrpd1a-3 will allow the community to not incorrectly associate the defective root hair phenotype with POLIV function.Rakesh David, R. Daniel Kortschak, and Iain Searl

A new strategy for identifying mechanisms of drug-drug interaction using transcriptome analysis: Compound Kushen Injection as a proof of principle

Published online: 04 November 2019Drug-drug interactions (DDIs), especially with herbal medicines, are complex, making it difficult to identify potential molecular mechanisms and targets. We introduce a workflow to carry out DDI research using transcriptome analysis and interactions of a complex herbal mixture, Compound Kushen Injection (CKI), with cancer chemotherapy drugs, as a proof of principle. Using CKI combined with doxorubicin or 5-Fu on cancer cells as a model, we found that CKI enhanced the cytotoxic effects of doxorubicin on A431 cells while protecting MDA-MB-231 cells treated with 5-Fu. We generated and analysed transcriptome data from cells treated with single treatments or combined treatments and our analysis showed that opposite directions of regulation for pathways related to DNA synthesis and metabolism which appeared to be the main reason for different effects of CKI when used in combination with chemotherapy drugs. We also found that pathways related to organic biosynthetic and metabolic processes might be potential targets for CKI when interacting with doxorubicin and 5-Fu. Through co-expression analysis correlated with phenotype results, we selected the MYD88 gene as a candidate major regulator for validation as a proof of concept for our approach. Inhibition of MYD88 reduced antagonistic cytotoxic effects between CKI and 5-Fu, indicating that MYD88 is an important gene in the DDI mechanism between CKI and chemotherapy drugs. These findings demonstrate that our pipeline is effective for the application of transcriptome analysis to the study of DDIs in order to identify candidate mechanisms and potential targets.Hanyuan Shen, Zhipeng Qu, Yuka Harata-Lee, Jian Cui, Thazin N we Aung, Wei Wang, R. Daniel Kortschak & David L. Adelso

Cnidarian gene expression patterns and the origins of bilaterality – are cnidarians reading the same game plan as "higher" animals?

[Extract] The past few years have seen a dramatic increase in the available data on gene sequence and gene expression for cnidarians and other "lower" Metazoa, and a flurry of recent papers has drawn on these to address the origins of bilaterality. Cnidarianhomologs of many genes that play key roles in the specification of both the A/P and D/V axes of bilaterians have been characterized, and their patterns of expression determined. Some of these expression patterns are consistent with the conservation of function between Cnidaria and Bilateria, but others clearly differ. Moreover, in some cases very different interpretations have been made on the basis of the same, or similar,\ud data. In part, these differences reflect the inevitable uncertainties associated with the depth of the divergence between cnidarians and bilaterians. In this paper we briefly summarize the cnidarian data on gene expression\ud and organization relevant to axis formation, the varying interpretations of these data, and where they conflict. Our conclusion is that the oral-aboral axis probably does correspond to the anterior-posterior axis of bilaterians,\ud but that its polarity remains uncertain, and that many of the same genes are involved in determining the directive axis of cnidarians and the dorsal-ventral axis of bilaterians, but with sufficient differences in expression that exact homologies are uncertain