Amorpha-4,11-diene synthase (ADS) and Cyt P450 monooxygenase (CYP71AV1) in Artemisia annua L. are two key enzymes involved in the biosynthesis of artemisinin. The promoters of ADS and CYP71AV1 contain E-box elements, which are putative binding sites for basic helix-loop-helix (bHLH) transcription factors. This study successfully isolated a bHLH transcription factor gene from A. annua, designated as AabHLH1, from a cDNA library of the glandular secretory trichomes (GSTs) in which artemisinin is synthesized and sequestered. AabHLH1 encodes a protein of 650 amino acids containing one putative bHLH domain. AabHLH1 and ADS genes were strongly induced by ABA and the fungal elicitor, chitosan. The transient expression analysis of the AabHLH1-green fluorescent protein (GFP) reporter gene revealed that AabHLH1 was targeted to nuclei. Biochemical analysis demonstrated that the AabHLH1 protein was capable of binding to the E-box cis-elements, present in both ADS and CYP71AV1 promoters, and possessed transactivation activity in yeast. In addition, transient co-transformation of AabHLH1 and CYP71AV1Pro::GUS in A. annua leaves showed a significant activation of the expression of the GUS (β-glucuronidase) gene in transformed A. annua, but mutation of the E-boxes resulted in abolition of activation, suggesting that the E-box is important for the CYP71AV1 promoter activity. Furthermore, transient expression of AabHLH1 in A. annua leaves increased transcript levels of the genes involved in artemisinin biosynthesis, such as ADS, CYP71AV1 and HMGR. These results suggest that AabHLH1 can positively regulate the biosynthesis of artemisinin.

A Rh(III) complex generated in situ from [Cp*RhCl2]2 and (1R,2R)-N-(p-toluenesulfonyl)-1,2-cyclohexanediamine (TsCYDN) serves as a remarkably effective, robust catalyst for the asymmetric transfer hydrogenation of aromatic ketones by HCOONa in water in air, affording alcohols in up to 99% ee.

Actomyosin-mediated cellular contractility is highly conserved for mechanotransduction and signalling. While this phenomenon has been observed in adherent cell models, whether/how contractile forces regulate the function of suspension cells like natural killer (NK) cells during cancer surveillance, is unknown. Here, we demonstrated in coculture settings that the evolutionarily conserved NK cell transcription factor, Eomes, undergoes nuclear shuttling during lung cancer cell surveillance. Biophysical and biochemical analyses revealed mechanistic enhancement of NK cell actomyosin-mediated contractility, which is associated with nuclear flattening, thus enabling nuclear entry of Eomes associated with enhanced NK cytotoxicity. We found that NK cells responded to the presumed immunosuppressive TGFβ in the NK-lung cancer coculture medium to sustain its intracellular contractility through myosin light chain phosphorylation, thereby promoting Eomes nuclear localization. Therefore, our results demonstrate that lung cancer cells provoke NK cell contractility as an early phase activation mechanism and that Eomes is a plausible mechano-responsive protein for increased NK cytotoxicity. There is scope for strategic application of actomyosin-mediated contractility modulating drugs ex vivo, to reinvigorate NK cells prior to adoptive cancer immunotherapy in vivo (177 words).

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