Cytochrome P450 (P450) enzymes are important in the metabolism of steroids, vitamins, carcinogens, drugs and other compounds. Two of the commonly used assays in this field are the measurements of total P450 and NADPH–P450 reductase in biological preparations. A detailed protocol is presented for the measurement of P450 by its spectral properties, along with a protocol for measuring NADPH–P450 reductase by its NADPH–cytochrome c reduction activity. Each assay can be completed in 5–10 min. Detailed explanations for the rationale of particular sequences in the protocols are provided, along with potential confounding problems.

Dopamine (DA) plays a critical role in the brain, and the ability to directly measure dopaminergic activity is essential for understanding its physiological functions. We therefore developed red fluorescent GPCR-activation–based DA (GRAB DA ) sensors and optimized versions of green fluorescent GRAB DA sensors. In response to extracellular DA, both the red and green GRAB DA sensors exhibit a large increase in fluorescence, with subcellular resolution, subsecond kinetics, and nanomolar to submicromolar affinity. Moreover, the GRAB DA sensors resolve evoked DA release in mouse brain slices, detect evoked compartmental DA release from a single neuron in live flies, and report optogenetically elicited nigrostriatal DA release as well as mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. Co-expressing red GRAB DA with either green GRAB DA or the calcium indicator GCaMP6s allows simultaneously tracking neuronal activity and dopaminergic signaling in distinct circuits in vivo .

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The monoamine neuromodulator dopamine (DA) plays a critical role in the brain, and the 21 ability to directly measure dopaminergic activity is essential for understanding its 22 physiological functions. We therefore developed the first red fluorescent GPCR-activation-23 based DA (GRABDA) sensors and optimized versions of green fluorescent GRABDA sensors 24 following our previous studies. In response to extracellular DA, both the red and green 25 GRABDA sensors have a large increase in fluorescence (ΔF/F0 values of 150% and 340%, 26 respectively), with subcellular resolution, subsecond kinetics, and nanomolar to 27 submicromolar affinity. Moreover, both the red and green GRABDA sensors readily resolve 28 evoked DA release in mouse brain slices, detect compartmental DA release in live flies with 29 single-cell resolution, and report optogenetically elicited nigrostriatal DA release as well as 30 mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. 31 Importantly, co-expressing red GRABDA with either green GRABDA or the calcium indicator 32GCaMP6s provides a robust tool for simultaneously tracking neuronal activity and 33 dopaminergic signaling in distinct circuits in vivo. 35Dopamine (DA) is an essential monoamine neuromodulator produced primarily in the midbrain and 36 released throughout the central nervous system. A multitude of brain functions are regulated by DA, 37including motor control, motivation, learning and memory, and emotional control 1-9 . Consistent with 38these key physiological roles, altered DA signaling has been implicated in a variety of brain 39 disorders, including Parkinson's disease, addiction, schizophrenia, attention-deficit/hyperactivity 40 disorder, and posttraumatic stress disorder 10-20 . Thus, tools that can sense changes in DA 41 concentration with high spatiotemporal resolution, high specificity, and high sensitivity will greatly 42 facilitate our study of the diverse functions that the dopaminergic system plays under both 43 physiological and pathological conditions. 44Previous techniques for measuring DA dynamics, including microdialysis, electrochemical 45 probes, reporter cells, and gene expression-based assays, lack sufficient spatiotemporal resolution 46 and/or molecular specificity [21][22][23][24][25][26][27][28][29][30] . Recently, our group 31 and Patriarchi et al. 32 independently 47 developed two series of genetically encoded, G-protein-coupled receptor (GPCR)-based DA 48 sensors called GRABDA and dLight, respectively. Taking advantage of naturally occurring DA 49 receptors, these sensors convert a ligand-stabilized conformational change in the DA receptor into 50 an optical response via a conformation-sensitive fluorescent protein inserted in the receptor's third 51 intracellular loop. Our first-generation DA receptor-based sensors called GRABDA1m and 52 GRABDA1h were used to detect cell type-specific DA dynamics in several organisms, including 53 Drosophila, zebrafish, mice, and zebra finches 31,33-35 . Here, we employed semi-rational en...

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BACKGROUND Although decoding the molecular mechanisms underlying insecticide resistance has often proven difficult, recent progress has revealed that specific mutations in the ryanodine receptor (RyR) of the diamondback moth, Plutella xylostella, can confer resistance to diamide insecticides. The extent to which specific RyR mutations contribute to the diamide resistance phenotype, the associated genetic traits and fitness costs remain limited. RESULTS Three field‐evolved PxRyR mutations (G4946E, I4790 M, and I4790 K) were respectively introgressed into a common susceptible background strain (IPP‐S) of P. xylostella with marker‐assisted backcrossing. The mutations alone can result in moderate to high levels of resistance to five commercial diamides (flubendiamide, chlorantraniliprole, cyantraniliprole, tetraniliprole, and cyclaniliprole), and the resistance intensity mediated by the three mutations was hierarchical in order of I4790 K (1199‐ to >2778‐fold) > G4946E (39‐ to 739‐fold) > I4790 M (16‐ to 57‐fold). Flubendiamide resistance was autosomal and incompletely recessive, and was significantly linked with the introgressed mutations in the three constructed strains. In addition, the resistance levels to flubendiamide of hybrid progeny from any two resistant strains fell in between the status of their parents. Furthermore, by comparing the net replacement rate, the fitness of 4946E, 4790 M and 4790 K strains were 0.77, 0.93 and 0.92 relative to the IPP‐S strain, respectively. CONCLUSION Three independent PxRyR mutations confer varying degrees of resistance to diamides in P. xylostella. Among the three mutations, I4790 K confers highest levels of resistance (> 1000‐fold) to all five commercial diamides. The findings can guide resistance management practices for diamides in P. xylostella and other arthropods.

We report a comprehensive genetic, metabolomic, and biochemical study on the catalytic properties of Streptomyces coelicolor cytochrome P450 (P450) 154A1, known to have a unique heme orientation in its crystal structure. Deletion of the P450 154A1 gene compromised the long-term stability of the bacterial spores. A novel dipentaenone (1) with a high degree of conjugation was identified as an endogenous substrate of P450 154A1 using a metabolomics approach. The biotransformation of 1 by P450 154A1 was shown to be an unexpected intramolecular cyclization to a Paternò–Büchi-like product, without oxidation/reduction.