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Cyanogenic glycosides are ancient biomolecules found in more than 2,650 higher plant species as well as in a few arthropod species. Cyanogenic glycosides are amino acid-derived b-glycosides of a-hydroxynitriles. In analogy to cyanogenic plants, cyanogenic arthropods may use cyanogenic glycosides as defence compounds. Many of these arthropod species have been shown to de novo synthesize cyanogenic glycosides by biochemical pathways that involve identical intermediates to those known from plants, while the ability to sequester cyanogenic glycosides appears to be restricted to Lepidopteran species. In plants, two atypical multifunctional cytochromes P450 and a soluble family 1 glycosyltransferase form a metabolon to facilitate channelling of the otherwise toxic and reactive intermediates to the end product in the pathway, the cyanogenic glycoside. The glucosinolate pathway present in Brassicales and the pathway for cyanoalk(en)yl glucoside synthesis such as rhodiocyanosides A and D in Lotus japonicus exemplify how cytochromes P450 in the course of evolution may be recruited for novel pathways. The use of metabolic engineering using cytochromes P450 involved in biosynthesis of cyanogenic glycosides allows for the generation of acyanogenic cassava plants or cyanogenic Arabidopsis thaliana plants as well as L. japonicus and A. thaliana plants with altered cyanogenic, cyanoalkenyl or glucosinolate profiles.Keywords Metabolic engineering AE Metabolons AE CYP79 AE Systems biology AE Plant-insect interactions Cyanogenic glycosides are ancient biomoleculesCyanogenic glycosides (A1) are amino acidderived b-glycosides of a-hydroxynitriles. They have been found in more than 2,650 higher plant

Cassava (Manihot esculenta) is a eudicotyledonous plant that produces the valine-and isoleucine-derived cyanogenic glucosides linamarin and lotaustralin with the corresponding oximes and cyanohydrins as key intermediates. CYP79 enzymes catalyzing amino acid-to-oxime conversion in cyanogenic glucoside biosynthesis are known from several plants including cassava. The enzyme system converting oxime into cyanohydrin has previously only been identified in the monocotyledonous plant great millet (Sorghum bicolor). Using this great millet CYP71E1 sequence as a query in a Basic Local Alignment Search Tool-p search, a putative functional homolog that exhibited an approximately 50% amino acid sequence identity was found in cassava. The corresponding full-length cDNA clone was obtained from a plasmid library prepared from cassava shoot tips and was assigned CYP71E7. Heterologous expression of CYP71E7 in yeast afforded microsomes converting 2-methylpropanal oxime (valine-derived oxime) and 2-methylbutanal oxime (isoleucine-derived oxime) to the corresponding cyanohydrins, which dissociate into acetone and 2-butanone, respectively, and hydrogen cyanide. The volatile ketones were detected as 2.4-dinitrophenylhydrazone derivatives by liquid chromatography-mass spectrometry. A K S of approximately 0.9 mM was determined for 2-methylbutanal oxime based on substrate-binding spectra. CYP71E7 exhibits low specificity for the side chain of the substrate and catalyzes the conversion of aliphatic and aromatic oximes with turnovers of approximately 21, 17, 8, and 1 min 21 for the oximes derived from valine, isoleucine, tyrosine, and phenylalanine, respectively. A second paralog of CYP71E7 was identified by database searches and showed approximately 90% amino acid sequence identity. In tube in situ polymerase chain reaction showed that in nearly unfolded leaves, the CYP71E7 paralogs are preferentially expressed in specific cells in the endodermis and in most cells in the first cortex cell layer. In fully unfolded leaves, the expression is pronounced in the cortex cell layer just beside the epidermis and in specific cells in the vascular tissue cortex cells. Thus, the transcripts of the CYP71E7 paralogs colocalize with CYP79D1 and CYP79D2. We conclude that CYP71E7 is the oxime-metabolizing enzyme in cyanogenic glucoside biosynthesis in cassava.

Lotus japonicus accumulates the hydroxynitrile glucosides lotaustralin, linamarin, and rhodiocyanosides A and D. Upon tissue disruption, the hydroxynitrile glucosides are bioactivated by hydrolysis by specific b-glucosidases. A mixture of two hydroxynitrile glucoside-cleaving b-glucosidases was isolated from L. japonicus leaves and identified by protein sequencing as LjBGD2 and LjBGD4. The isolated hydroxynitrile glucoside-cleaving b-glucosidases preferentially hydrolyzed rhodiocyanoside A and lotaustralin, whereas linamarin was only slowly hydrolyzed, in agreement with measurements of their rate of degradation upon tissue disruption in L. japonicus leaves. Comparative homology modeling predicted that LjBGD2 and LjBGD4 had nearly identical overall topologies and substrate-binding pockets. Heterologous expression of LjBGD2 and LjBGD4 in Arabidopsis (Arabidopsis thaliana) enabled analysis of their individual substrate specificity profiles and confirmed that both LjBGD2 and LjBGD4 preferentially hydrolyze the hydroxynitrile glucosides present in L. japonicus. Phylogenetic analyses revealed a third L. japonicus putative hydroxynitrile glucoside-cleaving b-glucosidase, LjBGD7. Reverse transcriptionpolymerase chain reaction analysis showed that LjBGD2 and LjBGD4 are expressed in aerial parts of young L. japonicus plants, while LjBGD7 is expressed exclusively in roots. The differential expression pattern of LjBGD2, LjBGD4, and LjBGD7 corresponds to the previously observed expression profile for CYP79D3 and CYP79D4, encoding the two cytochromes P450 that catalyze the first committed step in the biosyntheis of hydroxynitrile glucosides in L. japonicus, with CYP79D3 expression in aerial tissues and CYP79D4 expression in roots.

The maturation and adoption of pervasive computing technologies have dramatically altered the face of healthcare. With the introduction of mobile devices, body area networks, and embedded computing systems, care providers can use continuous, ecologically valid information to overcome geographic and temporal barriers and thus provide more effective and timely health assessments. In this paper, we review recent technological developments that can be harnessed to replicate, enhance, or design new methods for assessment of functional performance. Enabling technologies in wearable sensors, ambient sensors, mobile technologies and virtual reality make it possible to quantify real-time functional performance and changes in cognitive health. These technologies, their uses for functional health assessment, and their challenges for adoption are presented in this paper.