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Calcium-dependent molecular fMRI using a magnetic nanosensor.

ABSTRACT: Calcium ions are ubiquitous signalling molecules in all multicellular organisms, where they mediate diverse aspects of intracellular and extracellular communication over widely varying temporal and spatial scales 1 . Though techniques to map calcium-related activity at a high resolution by optical means are well established, there is currently no reliable method to measure calcium dynamics over large volumes in intact tissue 2 . Here, we address this need by introducing a family of magnetic calcium-responsive nanoparticles (MaCaReNas) that can be detected by magnetic resonance imaging (MRI). MaCaReNas respond within seconds to [Ca2+] changes in the 0.1-1.0?mM range, suitable for monitoring extracellular calcium signalling processes in the brain. We show that the probes permit the repeated detection of brain activation in response to diverse stimuli in vivo. MaCaReNas thus provide a tool for calcium-activity mapping in deep tissue and offer a precedent for the development of further nanoparticle-based sensors for dynamic molecular imaging with MRI.

SUBMITTER: Okada S

PROVIDER: S-EPMC6086382 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Calcium-dependent molecular fMRI using a magnetic nanosensor.

Okada Satoshi S Bartelle Benjamin B BB Li Nan N Breton-Provencher Vincent V Lee Jiyoung J JJ Rodriguez Elisenda E Melican James J Sur Mriganka M Jasanoff Alan A

Nature nanotechnology 20180430 6

Calcium ions are ubiquitous signalling molecules in all multicellular organisms, where they mediate diverse aspects of intracellular and extracellular communication over widely varying temporal and spatial scales <sup>1</sup> . Though techniques to map calcium-related activity at a high resolution by optical means are well established, there is currently no reliable method to measure calcium dynamics over large volumes in intact tissue <sup>2</sup> . Here, we address this need by introducing a f ...[more]

PMID: 29713073

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Project description:(+)-Catechin is an important antioxidant of green tea (Camelia sinensis (L.) O. Kuntze). Catechin is known for its positive role in anticancerous activity, extracellular matrix degradation, cell death regulation, diabetes, and other related disorders. As a result of enormous interest in and great demand for catechin, its biosynthesis using metabolic engineering has become the subject of concentrated research with the aim of enhancing (+)-catechin production. Metabolic flux is an essential concept in the practice of metabolic engineering as it helps in the identification of the regulatory element of a biosynthetic pathway. In the present study, an attempt was made to analyze the metabolic flux of the (+)-catechin biosynthesis pathway in order to decipher the regulatory element of this pathway. Firstly, a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor (FLIP-Cat, fluorescence indicator protein for (+)-catechin) was developed for real-time monitoring of (+)-catechin flux. In vitro characterization of the purified protein of the nanosensor showed that the nanosensor was pH stable and (+)-catechin specific. Its calculated Kd was 139 µM. The nanosensor also performed real-time monitoring of (+)-catechin in bacterial cells. In the second step of this study, an entire (+)-catechin biosynthesis pathway was constructed and expressed in E. coli in two sets of plasmid constructs: pET26b-PT7-rbs-PAL-PT7-rbs-4CL-PT7-rbs-CHS-PT7-rbs-CHI and pET26b-T7-rbs-F3H-PT7-rbs- DFR-PT7-rbs-LCR. The E. coli harboring the FLIP-Cat was transformed with these plasmid constructs. The metabolic flux analysis of (+)-catechin was carried out using the FLIP-Cat. The FLIP-Cat successfully monitored the flux of catechin after adding tyrosine, 4-coumaric acid, 4-coumaroyl CoA, naringenin chalcone, naringenin, dihydroquercetin, and leucocyanidin, individually, with the bacterial cells expressing the nanosensor as well as the genes of the (+)-catechin biosynthesis pathway. Dihydroflavonol reductase (DFR) was identified as the main regulatory element of the (+)-catechin biosynthesis pathway. Information about this regulatory element of the (+)-catechin biosynthesis pathway can be used for manipulating the (+)-catechin biosynthesis pathway using a metabolic engineering approach to enhance production of (+)-catechin.

Dataset Information

Calcium-dependent molecular fMRI using a magnetic nanosensor.

Publications

Calcium-dependent molecular fMRI using a magnetic nanosensor.

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