Utilization of multiple-dilution fluorescence fingerprint facilitates prediction of chemical attributes in spice extracts.

Fluorescence Fingerprint (FF) is a powerful tool for rapid quality assessment of various foods and plant-derived products. However, the conventional utilization of FFs measured at a single dilution level (DL) to substitute chemical analyses is extremely challenging, especially for multicomponent materials like spice extracts because fluorescence intensity and concentration widely differ between components, with complex phenomena like inner filter effects. Here, we proposed a new strategy to use the meta-data comprised of FFs measured at multiple DLs with machine learning to estimate common chemical attributes including total polyphenol and flavonoid contents, and antioxidant abilities. This strategy achieved more consistently satisfactory performance in estimation of all chemical attributes of spice extracts compared to using a single DL. Hence, the workflow employed in this study is expected to serve as an alternative method to quickly evaluate the chemical quality of spice extracts, as well as other plant products and food materials.

Brassinosteroid-induced gene repression requires specific and tight promoter binding of BIL1/BZR1 via DNA shape readout.

BRZ-INSENSITIVE-LONG 1 (BIL1)/BRASSINAZOLE-RESISTANT 1 (BZR1) and its homologues are plant-specific transcription factors that convert the signalling of the phytohormones brassinosteroids (BRs) to transcriptional responses, thus controlling various physiological processes in plants. Although BIL1/BZR1 upregulates some BR-responsive genes and downregulates others, the molecular mechanism underlying the dual roles of BIL1/BZR1 is still poorly understood. Here we show that BR-responsive transcriptional repression by BIL1/BZR1 requires the tight binding of BIL1/BZR1 alone to the 10 bp elements of DNA fragments containing the known 6 bp core-binding motifs at the centre. Furthermore, biochemical and structural evidence demonstrates that the selectivity for two nucleobases flanking the core motifs is realized by the DNA shape readout of BIL1/BZR1 without direct recognition of the nucleobases. These results elucidate the molecular and structural basis of transcriptional repression by BIL1/BZR1 and contribute to further understanding of the dual roles of BIL1/BZR1 in BR-responsive gene regulation.

Highlighting the potential utility of MBP crystallization chaperone for Arabidopsis BIL1/BZR1 transcription factor-DNA complex.

The maltose-binding protein (MBP) fusion tag is one of the most commonly utilized crystallization chaperones for proteins of interest. Recently, this MBP-mediated crystallization technique was adapted to Arabidopsis thaliana (At) BRZ-INSENSITIVE-LONG (BIL1)/BRASSINAZOLE-RESISTANT (BZR1), a member of the plant-specific BZR TFs, and revealed the first structure of AtBIL1/BZR1 in complex with target DNA. However, it is unclear how the fused MBP affects the structural features of the AtBIL1/BZR1-DNA complex. In the present study, we highlight the potential utility of the MBP crystallization chaperone by comparing it with the crystallization of unfused AtBIL1/BZR1 in complex with DNA. Furthermore, we assessed the validity of the MBP-fused AtBIL1/BZR1-DNA structure by performing detailed dissection of crystal packings and molecular dynamics (MD) simulations with the removal of the MBP chaperone. Our MD simulations define the structural basis underlying the AtBIL1/BZR1-DNA assembly and DNA binding specificity by AtBIL1/BZR1. The methodology employed in this study, the combination of MBP-mediated crystallization and MD simulation, demonstrates promising capabilities in deciphering the protein-DNA recognition code.

Evaluation of spice and herb as phyto-derived selective modulators of human retinaldehyde dehydrogenases using a simple in vitro method.

Selective modulation of retinaldehyde dehydrogenases (RALDHs)-the main aldehyde dehydrogenase (ALDH) enzymes converting retinal into retinoic acid (RA), is very important not only in the RA signaling pathway but also for the potential regulatory effects on RALDH isozyme-specific processes and RALDH-related cancers. However, very few selective modulators for RALDHs have been identified, partly due to variable overexpression protocols of RALDHs and insensitive activity assay that needs to be addressed. In the present study, deletion of the N-terminal disordered regions is found to enable simple preparation of all RALDHs and their closest paralog ALDH2 using a single protocol. Fluorescence-based activity assay was employed for enzymatic activity investigation and screening for RALDH-specific modulators from extracts of various spices and herbs that are well-known for containing many phyto-derived anti-cancer constituents. Under the established conditions, spice and herb extracts exhibited differential regulatory effects on RALDHs/ALDH2 with several extracts showing potential selective inhibition of the activity of RALDHs. In addition, the presence of magnesium ions was shown to significantly increase the activity for the natural substrate retinal of RALDH3 but not the others, while His-tag cleavage considerably increased the activity of ALDH2 for the non-specific substrate retinal. Altogether we propose a readily reproducible workflow to find selective modulators for RALDHs and suggest potential sources of selective modulators from spices and herbs.