Simultaneously quantifying intracellular FAD and FMN using a novel strategy of intrinsic fluorescence four-way calibration

Kang, Chao; Xu, Minli; Yan, Xiu-Fang; Liu, Yajuan; Yu, Ru‐Qin

doi:10.1016/j.talanta.2018.12.076

Cited by 18 publications

(7 citation statements)

References 40 publications

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“…2A, the flavins secreted by Lp B2 mainly consist of RF (and/or FMN) (~80%), which is then the compound in charge of the vitamin B2 biofortification of foods by this bacterium. In contrast, the major dietary source of vitamin B 2 is FAD, as this is the prevalent flavin compound within both eukaryotic and bacterial cells (Kimata et al, 2018; Kang et al, 2019; Fig. 2).…”

Section: Discussionmentioning

confidence: 99%

High vitamin B₂ production by the Lactiplantibacillus plantarum B2 strain carrying a mutation in the aptamer P1 helix of the FMN-riboswitch that regulates expression of the bacterial rib operon

Ripa

Já

et al. 2021

Preprint

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Manufacturing of probiotics and functional foods using lactic acid bacteria that overproduce vitamin B2 has gained growing interest due to ariboflavinosis problems affecting populations of both developing and affluent countries. Two isogenic Lactiplantibacillus plantarum strains, namely a riboflavin-producing parental strain (UFG9) and a roseoflavin-resistant strain (B2) that carries a mutation in the FMN-aptamer of the potential rib operon riboswitch, were analyzed for production and intra- and extracellular accumulation of flavins, as well as for regulation of the rib operon expression. Strain B2 accumulated in the medium one of the highest levels of riboflavin+FMN ever reported for lactic acid bacteria, exceeding by ~25 times those accumulated by UFG9. Inside the cells, concentration of FAD was similar in both strains, while that of riboflavin+FMN was ~6-fold higher in B2. Mutation B2 could decrease the stability of the aptamer's regulatory P1 helix even in the presence of the effector, thus promoting the antiterminator structure of the riboswitch ON state. Although the B2-mutant riboswitch showed an impaired regulatory activity, it retained partial functionality being still sensitive to the effector. The extraordinary capacity of strain B2 to produce riboflavine, together with its metabolic versatility and probiotic properties, can be exploited for manufacturing multifunctional foods.

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Section: Discussionmentioning

confidence: 99%

High vitamin B₂ production by the Lactiplantibacillus plantarum B2 strain carrying a mutation in the aptamer P1 helix of the FMN-riboswitch that regulates expression of the bacterial rib operon

Ripa

Já

et al. 2021

Preprint

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“…Fluorescence emission was isolated with

bandpass filter for NAD(P)H and a

bandpass filter for FAD and/or FMN. 3 , 6 , 7 Fluorescence emission was detected with a H7422PA-40 GaAsP photomultiplier tube (Hamamatsu Corporation, Bridgewater, NJ, USA). Powers on the sample ranged from 3.0 to 5.0 mW (not including the SNARF measurements, which were measured at an average power of 0.3 mW).…”

Section: Methodsmentioning

confidence: 99%

“…FAD and FMN are fluorescent flavins, in which FAD is more prevalent in the cell. 2 , 3 FAD and FMN are primarily localized in the mitochondria and bound to enzymes as a cofactor, although some proteins that use FAD or FMN as a cofactor are localized to other parts of the cell. 4 , 5 Fluorescence spectra of NAD(P)H, FAD, and FMN have been previously published.…”

Section: Introductionmentioning

confidence: 99%

“… 4 , 5 Fluorescence spectra of NAD(P)H, FAD, and FMN have been previously published. 3 , 6 , 7 The fluorescence spectra of FAD and FMN show substantial overlap, so FMN may contribute to FAD lifetime in FLIM measurements. 3 NAD(P)H and FAD each have two distinct lifetimes due to free- and protein-bound states.…”

Section: Introductionmentioning

confidence: 99%

“… 3 , 6 , 7 The fluorescence spectra of FAD and FMN show substantial overlap, so FMN may contribute to FAD lifetime in FLIM measurements. 3 NAD(P)H and FAD each have two distinct lifetimes due to free- and protein-bound states. For NAD(P)H, the short lifetime (

) corresponds to free, unbound NAD(P)H, and the long lifetime (

) corresponds to protein-bound NAD(P)H. 8 Conversely, for FAD, free FAD has a long lifetime, and protein-bound FAD has a short lifetime.…”

Section: Introductionmentioning

confidence: 99%

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Extracellular pH affects the fluorescence lifetimes of metabolic co-factors

et al. 2021

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Significance: Autofluorescence measurements of the metabolic cofactors NADH and flavin adenine dinucleotide (FAD) provide a label-free method to quantify cellular metabolism. However, the effect of extracellular pH on flavin lifetimes is currently unknown.Aim: To quantify the relationship between extracellular pH and the fluorescence lifetimes of FAD, flavin mononucleotide (FMN), and reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H].Approach: Human breast cancer (BT474) and HeLa cells were placed in pH-adjusted media. Images of an intracellular pH indicator or endogenous fluorescence were acquired using twophoton fluorescence lifetime imaging. Fluorescence lifetimes of FAD and FMN in solutions were quantified over the same pH range. Results:The relationship between intracellular and extracellular pH was linear in both cell lines. Between extracellular pH 4 to 9, FAD mean lifetimes increased with increasing pH. NAD(P)H mean lifetimes decreased with increasing pH between extracellular pH 5 to 9. The relationship between NAD(P)H lifetime and extracellular pH differed between the two cell lines. Fluorescence lifetimes of FAD, FAD-cholesterol oxidase, and FMN solutions decreased, showed no trend, and showed no trend, respectively, with increasing pH.Conclusions: Changes in endogenous fluorescence lifetimes with extracellular pH are mostly due to indirect changes within the cell rather than direct pH quenching of the endogenous molecules.

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Label‐Free Assessment of Key Biological Autofluorophores: Material Characteristics and Opportunities for Clinical Applications

Campbell,

Gosnell,

Agha

et al. 2024

Advanced Materials

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Autofluorophores are endogenous fluorescent compounds that naturally occur in the intra and extracellular spaces of all tissues and organs. Most have vital biological functions – like the metabolic cofactors NAD(P)H and FAD+, as well as the structural protein collagen. Others have been considered to be waste products – like lipofuscin and advanced glycation end products – which accumulate with age and are associated with cellular dysfunction. Due to their natural fluorescence these materials have great utility for enabling non‐invasive, label‐free assays with direct ties to biological function. Numerous technologies, with different advantages and drawbacks, have been applied to their assessment, including fluorescence lifetime imaging microscopy, hyperspectral microscopy, and flow cytometry. Here, we review the applications of label‐free autofluorophore assessment for clinical and health‐research applications, with specific attention to biomaterials, disease detection, surgical guidance, treatment monitoring and tissue assessment – fields which greatly benefit from non‐invasive methodologies capable of continuous, in vivo characterisation.This article is protected by copyright. All rights reserved

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Simultaneously quantifying intracellular FAD and FMN using a novel strategy of intrinsic fluorescence four-way calibration

Cited by 18 publications

References 40 publications

High vitamin B₂ production by the Lactiplantibacillus plantarum B2 strain carrying a mutation in the aptamer P1 helix of the FMN-riboswitch that regulates expression of the bacterial rib operon

High vitamin B₂ production by the Lactiplantibacillus plantarum B2 strain carrying a mutation in the aptamer P1 helix of the FMN-riboswitch that regulates expression of the bacterial rib operon

Extracellular pH affects the fluorescence lifetimes of metabolic co-factors

Label‐Free Assessment of Key Biological Autofluorophores: Material Characteristics and Opportunities for Clinical Applications

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Simultaneously quantifying intracellular FAD and FMN using a novel strategy of intrinsic fluorescence four-way calibration

Cited by 18 publications

References 40 publications

High vitamin B2 production by the Lactiplantibacillus plantarum B2 strain carrying a mutation in the aptamer P1 helix of the FMN-riboswitch that regulates expression of the bacterial rib operon

High vitamin B2 production by the Lactiplantibacillus plantarum B2 strain carrying a mutation in the aptamer P1 helix of the FMN-riboswitch that regulates expression of the bacterial rib operon

Extracellular pH affects the fluorescence lifetimes of metabolic co-factors

Label‐Free Assessment of Key Biological Autofluorophores: Material Characteristics and Opportunities for Clinical Applications

Contact Info

Product

Resources

About

High vitamin B₂ production by the Lactiplantibacillus plantarum B2 strain carrying a mutation in the aptamer P1 helix of the FMN-riboswitch that regulates expression of the bacterial rib operon

High vitamin B₂ production by the Lactiplantibacillus plantarum B2 strain carrying a mutation in the aptamer P1 helix of the FMN-riboswitch that regulates expression of the bacterial rib operon