Discovery of Red-Shifting Mutations in Firefly Luciferase Using High-Throughput Biochemistry.

Photinus pyralis luciferase (FLuc) has proven a valuable tool for bioluminescence imaging, but much of the light emitted from the native enzyme is absorbed by endogenous biomolecules. Thus, luciferases displaying red-shifted emission enable higher resolution during deep-tissue imaging. A robust model of how protein structure determines emission color would greatly aid the engineering of red-shifted mutants, but no consensus has been reached to date. In this work, we applied deep mutational scanning to systematically assess 20 functionally important amino acid positions on FLuc for red-shifting mutations, predicting that an unbiased approach would enable novel contributions to this debate. We report dozens of red-shifting mutations as a result, a large majority of which have not been previously identified. Further characterization revealed that mutations N229T and T352M, in particular, bring about unimodal emission with the majority of photons being >600 nm. The red-shifting mutations identified by this high-throughput approach provide strong biochemical evidence for the multiple-emitter mechanism of color determination and point to the importance of a water network in the enzyme binding pocket for altering the emitter ratio. This work provides a broadly applicable mutational data set tying FLuc structure to emission color that contributes to our mechanistic understanding of emission color determination and should facilitate further engineering of improved probes for deep-tissue imaging.

Cloning and characterization of luciferase from an Asian firefly Pygoluciola qingyu and its comparison with other beetle luciferases.

The bioluminescence system of luminescent beetles has extensive applications in biological imaging, protein labeling and drug screening. To explore wild luciferases with excellent catalytic activity and thermal stability, we cloned the luciferase of Pygoluciola qingyu, one species living in areas of high temperature and with strong bioluminescence, by combining transcriptomic sequencing and reverse transcription polymerase chain reaction (RT-PCR). The total length of luciferase gene is 1638 bp and the luciferase consists 544 amino acids. The recombinant P. qingyu luciferase was produced in vitro and its characteristics were compared with those of eight luciferases from China firefly species and two commercial luciferases. Compared with these luciferases, the P. qingyu luciferase shows the highest luminescence activity at room temperature (about 25-28 â„ƒ) with similar KM value for D-luciferin and ATP to the Photinus pyralis luciferase. The P. qingyu luciferase activity was highest at 35 â„ƒ and can keep high activity at 30-40 â„ƒ, which suggests the potential of P. qingyu luciferase for in vivo and cell application. Our results provide new insights into P. qingyu luciferase and give a new resource for the application of luciferases.

[Construction and identification of a stable CT26 cell line expressing CD19-FLUC-GFP].

Solid tumors lack well-defined targets for chimeric antigen receptor T-cell (CAR-T) therapy. Therefore, introducing a known target molecule, CD19, into solid tumor cell lines via lentiviral transduction to investigate the cytotoxicity of CD19 CAR-T cells can potentially support CAR-T cell therapy against solid tumors. In this study, a stable colon cancer CT26 cell line, CT26-CD19-FLUC-GFP, expressing CD19, firefly luciferase (FLUC), and green fluorescent protein (GFP), was constructed using a triple-plasmid lentiviral system. The growth characteristics of this cell line were consistent with those of the CT26 cell line. Subsequent flow cytometry analysis confirmed stable expression of CD19 and GFP in CT26-CD19-FLUC-GFP cells after serial passaging up to the 5th, 10th, and 22nd generations. Further validation revealed significantly higher levels of CD19 mRNA and FLUC expression in CT26-CD19-FLUC-GFP cells continuously passaged up to the 22nd generation compared to the control CT26 cells. In comparison to T cells, CD19 CAR-T cells demonstrated substantial cytotoxicity against CT26-CD19-FLUC-GFP cells and MC38-CD19 cells. One week after intraperitoneal implantation of CT26-CD19-FLUC-GFP cells into mice, FLUC expression in the peritoneal region could be detected. These results indicate the successful establishment of a stable CT26 cell line expressing CD19-FLUC-GFP, which can be specifically targeted by CD19 CAR-T cells.

A comparative study of structural and catalytic activity alterations in firefly luciferase induced by carbon quantum dots containing amine and carboxyl functional groups.

Despite of growing interest in use of carbon-based nanomaterials as carriers of functional proteins, less attention has been paid to the effects of these nanomaterials on the structure and function of the proteins. In this study, with the aim of shedding light on the mechanisms of interaction between carbon-based nanomaterials and proteins, the interactions of carbon quantum dots (CQDs) containing amine (CQD-NH2) or carboxyl groups (CQD-COOH) with Photinus pyralis firefly luciferase enzyme were investigated by experimental and computational approaches. The structural changes and reduction in activity of the luciferase upon treatment with CQDs were experimentally proved. CQD-NH2 induced more reduction in enzyme activity (15 %) compared to CQD-COOH (7.4 %). The interactions CQD-NH2 with luciferase led to higher affinity of the enzyme for its substrate. It was found by molecular dynamic simulations that CQD-NH2 binds to multiple regions on the surface of luciferase. Secondary structure analysis showed that CQD-NH2 had more profound effects on the active site amino acids, the adjacent amino acids to the active site and the residues involved in ATP binding site. In addition, CQD-NH2 interactions with luciferase were suggested to be stronger than CQD-COOH based on the number of hydrogen bonds and the binding energies.

Firefly Rats: Illuminating the Scientific Community in Transplantation Research.

Fireflies produce light through luciferase-catalyzed reactions involving luciferin, oxygen, and adenosine triphosphate, distinct from other luminescent organisms. This unique feature has revolutionized molecular biology and physiology, serving as a valuable tool for cellular research. Luciferase-based bioluminescent imaging enabled the creation of transgenic animals, such as Firefly Rats. Firefly Rats, created in 2006, ubiquitously express luciferase and have become a critical asset in scientific investigations. These rats have significantly contributed to transplantation and tissue engineering studies. Their low immunogenicity reduces graft rejection risk, making them ideal for long-term tracking of organ/tissue/cellular engraftments. Importantly, in the islet transplantation setting, the ubiquitous luciferase expression in these rats does not alter islet morphology or function, ensuring accurate assessments of engrafted islets. Firefly Rats have illuminated the path of transplantation research worldwide for over a decade and continue accelerating scientific advancements in many fields.

Quantification of Xylem-Specific Thermospermine-Dependent Translation of SACL Transcripts with Dual Luciferase Reporter System.

Thermospermine (Tspm) is a polyamine found to play a crucial role in xylem development in Arabidopsis thaliana. Tspm promotes the translation of the SACL genes by counteracting the activity of a cis element in their 5'-leader region that suppresses the translation of the main ORF. Here we describe a method to test the Tspm-dependent translational regulation of the 5'-leader of the SACL mRNAs in Nicotiana benthamiana leaves and A. thaliana mesophyll protoplasts with a dual luciferase assay. The dual luciferase reporter system is used to assess gene expression and is based on the detection of the Firefly luciferase luminescence driven by a specific promoter. However, it can also be used to evaluate the cis elements found in 5'-leader that influence the translation of the main ORF in a transcript. We have used a modified version of the pGreenII 0800 LUC plasmid carrying a double 35S promoter, followed by a poly-linker sequence in phase with the Firefly luciferase gene (pGreen2x35SLUC) where the full 5'-leader sequence of SACL3 was cloned. This construct was used for Agrobacterium tumefaciens infiltration of N. benthamiana leaves and for transfection of A. thaliana mesophyll protoplasts, followed by mock or Tspm treatments. The resulting translation of the Firefly luciferase in these organisms and conditions was then tested by measuring luminescence with the dual luciferase assay and a luminometer. These experiments have allowed us to quantify the positive effect of Tspm in the translation of SACL3 transcripts.

The orange light emitting luciferase from the rare Euryopa clarindae adult railroadworm (Coleoptera:Phengodidae): structural/functional and evolutionary relationship with green and red emitting luciferases.

Railroadworms luciferases emit the widest range of bioluminescence colors among beetles, ranging from green to red, being model enzymes to investigate the structure and bioluminescence colors relationships. Only three active railroadworms luciferases from the larval stage have been cloned and investigated: the Phrixothrix hirtus head lanterns red-emitting luciferase (PhRE); the Phrixothrix vivianii lateral lanterns green emitting luciferases (PvGR) and the Phengodes sp. dorsal lanterns yellow-green emitting luciferase (Ph). No active luciferase emitting in the yellow-orange region, however, has been cloned yet. Here we report the cloning and characterization of the orange emitting luciferase from the adult males of a rare Brazilian Cerrado railroadworm, Euryopa clarindae, and the transcriptional identification of two isozymes from the Amazon forest Mastinomorphus sp. railroadworm. The luciferase of E. clarindae has 548 residues, emits orange bioluminescence (600 nm), and displays intermediate kinetic values [KM(luciferin) = 50 µM, KM(ATP) ~ 170 µM] between those reported for green-emitting lateral lanterns and red emitting head lanterns luciferases. It displays 74-78% identity with the lateral lanterns luciferases of other railroadworms and 70% with the head lantern PhRE luciferase, and 96% with the larval Mastinomorphus sp. Mast-1, suggesting that this larva could be from the Euryopa genus. The phylogenetic analysis and kinetic/functional properties, place this orange-emitting enzyme as an intermediate form between the green-emitting lateral lanterns and red-emitting head lanterns luciferases. Major structural differences that could be associated with bioluminescence color determination are a relatively larger cavity size, and substitutions in the loops 223-235 and 311-316, especially N/C/T311, and their interactions which may help to close the bottom of LBS.

New synthetic red- and orange-emitting luciferases to upgrade in vitro and 3D cell biosensing.

Bioluminescence (BL), i.e., the emission of light in living organisms, has become an indispensable tool for a plethora of applications including bioassays, biosensors, and in vivo imaging. Current efforts are focused on the obtainment of new luciferases having optimized properties, such as improved thermostability at 37 °C, pH-insensitive emission, high quantum yield, extended kinetics and red-shifted emission. To address these issues we have obtained two new synthetic luciferases, an orange and a red-emitting luciferase, which were designed to achieve high sensitivity (BoLuc) and multiplexing capability (BrLuc) for in vitro and in vivo biosensing using as a starting template a recently developed thermostable synthetic luciferase (BgLuc). Both luciferases were characterized in terms of emission behaviour and thermal and pH stability showing promising features as reporter proteins and BL probes. As proof-of-principle application, an inflammation assay based on Human Embryonic Kidney (HEK293T) 3D cell cultures was developed using either the orange or the red-emitting mutant. The assay provided good analytical performance, with limits of detection for Tumor Necrosis Factor (TNFα) of 0.06 and 0.12 ng mL-1 for BoLuc and BrLuc, respectively. Moreover, since these luciferases require the same substrate, D-luciferin, they can be easily implemented in dual-color assays with a significant reduction of total cost per assay.

A Low-Cost, Sensitive Reporter System Using Membrane-Tethered Horseradish Peroxidase for Efficient Gene Expression Analysis.

Reporter gene assays are essential for high-throughput analysis, such as drug screening or determining downstream signaling activation/inhibition. However, use of this technology has been hampered by the high cost of the substrate (e.g., d-Luciferin (d-Luc)) in the most common firefly luciferase (FLuc) reporter gene assay. Although alternate luciferase is available worldwide, its substrate has remained expensive, and a more affordable option is still in demand. Here, we present a membrane-tethered horseradish peroxidase (mHRP), a new reporter system composed of a cell membrane expressing HRP that can preserve its enzymatic function on the cell surface, facilitates contact with HRP substrates (e.g., ABTS and TMB), and avoids the cell lysis process and the use of the high-priced luciferase substrate. An evaluation of the light signal sensitivity of mHRP compared to FLuc showed that both had comparable signal sensitivity. We also identified an extended substrate half-life of more than 5-fold that of d-Luc. Of note, this strategy provided a more stable detection signal, and the cell lysis process is not mandatory. Furthermore, with this strategy, we decreased the total amount of time taken for analysis and increased the time of detection limit of the reporter assay. Pricing analysis showed a one-third to one twenty-eighth price drop per single test of reporter assay. Given the convenience and stability of the mHRP reporter system, we believe that our strategy is suitable for use as an alternative to the luciferase reporter assay.

Synthesis and Bioluminescence of 'V'-Shaped Firefly Luciferin Analogues Based on A Novel Benzobisthiazole Core.

The design of π-extended conjugation 'V'-shaped red shifted bioluminescent D-luciferin analogues based on a novel benzobisthiazole core is described. The divergent synthetic route allowed access to a range of amine donor substituents through an SN Ar reaction. In spectroscopic studies, the 'V'-shaped luciferins exhibited narrower optical band gaps, more red-shifted absorption and emission spectra than D-luciferin. Their bioluminescence characteristics were recorded against four different luciferases (PpyLuc, FlucRed, CBR2 and PLR3). With native luciferase PpyLuc, the 'V'-shaped luciferins demonstrated more red-shifted emissions than D-luciferin (λbl =561 nm) by 60 to 80 nm. In addition, the benzobisthiazole luciferins showed a wide range of bioluminescence spectra from the visible light region (λbl =500 nm) to the nIR window (>650 nm). The computational results validate the design concept which can be used as a guide for further novel D-luciferin analogues based upon other 'V'-shaped heterocyclic cores.

Development of a Novel ATP Bioluminescence Assay Based on Engineered Probiotic Saccharomyces boulardii Expressing Firefly Luciferase.

Quantitative analysis of adenosine triphosphate (ATP) has been widely used as a diagnostic tool in the food and medical industries. Particularly, the pathogenesis of a few diseases including inflammatory bowel disease (IBD) is closely related to high ATP concentrations. A bioluminescent D-luciferin/luciferase system, which includes a luciferase (FLuc) from the firefly Photinus pyralis as a key component, is the most commonly used method for the detection and quantification of ATP. Here, instead of isolating FLuc produced in recombinant Escherichia coli, we aimed to develop a whole-cell biocatalyst system that does not require extraction and purification of FLuc. To this end, the gene coding for FLuc was introduced into the genome of probiotic Saccharomyces boulardii using the CRISPR/Cas9-based genome editing system. The linear relationship (r2 = 0.9561) between ATP levels and bioluminescence generated from the engineered S. boulardii expressing FLuc was observed in vitro. To explore the feasibility of using the engineered S. boulardii expressing FLuc as a whole-cell biosensor to detect inflammation biomarker (i.e., ATP) in the gut, a colitis mouse model was established using dextran sodium sulfate as a colitogenic compound. Our findings demonstrated that the whole-cell biosensor can detect elevated ATP levels during gut inflammation in mice. Therefore, the simple and powerful method developed herein could be applied for non-invasive IBD diagnosis.

Optimization of Constitutive Promoters Using a Promoter-Trapping Vector in Burkholderia pyrrocinia JK-SH007.

Selecting suitable promoters to drive gene overexpression can provide significant insight into the development of engineered bacteria. In this study, we analyzed the transcriptome data of Burkholderia pyrrocinia JK-SH007 and identified 54 highly expressed genes. The promoter sequences were located using genome-wide data and scored using the prokaryotic promoter prediction software BPROM to further screen out 18 promoter sequences. We also developed a promoter trap system based on two reporter proteins adapted for promoter optimization in B. pyrrocinia JK-SH007: firefly luciferase encoded by the luciferase gene set (Luc) and trimethoprim (TP)-resistant dihydrofolate reductase (TPr). Ultimately, eight constitutive promoters were successfully inserted into the probe vector and transformed into B. pyrrocinia JK-SH007. The transformants were successfully grown on Tp antibiotic plates, and firefly luciferase expression was determined by measuring the relative light unit (RLU). Five of the promoters (P4, P9, P10, P14, and P19) showed 1.01-2.51-fold higher activity than the control promoter λ phage transcriptional promoter (PRPL). The promoter activity was further validated via qPCR analysis, indicating that promoters P14 and P19 showed stable high transcription levels at all time points. Then, GFP and RFP proteins were overexpressed in JK-SH007. In addition, promoters P14 and P19 were successfully used to drive gene expression in Burkholderia multivorans WS-FJ9 and Escherichia coli S17-1. The two constitutive promoters can be used not only in B. pyrrocinia JK-SH007 itself to gene overexpression but also to expand the scope of application.

Effects of synonymous mutations on kinetic properties and structure of firefly luciferase: Molecular dynamics simulation, molecular docking, RNA folding, and experimental study.

Although synonymous mutations have long been thought to lack striking results, a growing body of research shows these mutations have highly variable effects. In this study, the impact of synonymous mutations in the development of thermostable luciferase was investigated using a combination of experimental and theoretical approaches. Using bioinformatics analysis, the codon usage features in the Lampyridae family's luciferases were studied and four synonymous mutations of Arg in luciferase were created. An exciting result was that the analysis of kinetic parameters showed a slight increase in the thermal stability of the mutant luciferase. AutoDock Vina, %MinMax algorithm, and UNAFold Server were used to perform molecular docking, folding rate, and RNA folding, respectively. Here, it was assumed that in the region (Arg337) with a moderate propensity for coil, synonymous mutation altered the rate of translation, which in turn may lead to a slight change in the structure of the enzyme. According to the molecular dynamics simulation data, local minor global flexibility is observed in the context of the protein conformation. A plausible explanation is that this flexibility may strengthen hydrophobic interactions due to its sensitivity to a molecular collision. Accordingly, thermostability originated mainly from hydrophobic interaction.

Using bioluminescence to image gene expression and spontaneous behavior in freely moving mice.

Bioluminescence imaging (BLI) of gene expression in live animals is a powerful method for monitoring development, tumor growth, infections, healing, and other progressive, long-term biological processes. BLI remains an effective approach for reducing the number of animals needed to monitor dynamic changes in gene activity because images can be captured repeatedly from the same animals. When examining these ongoing changes, it is sometimes necessary to remove rhythmic effects on the bioluminescence signal caused by the circadian clock's daily modulation of gene expression. Furthermore, BLI using freely moving animals remains limited because the standard procedures can alter normal behaviors. Another obstacle with conventional BLI of animals is that luciferin, the firefly luciferase substrate, is usually injected into mice that are then imaged while anesthetized. Unfortunately, the luciferase signal declines rapidly during imaging as luciferin is cleared from the body. Alternatively, mice are imaged after they are surgically implanted with a pump or connected to a tether to deliver luciferin, but stressors such as this surgery and anesthesia can alter physiology, behavior, and the actual gene expression being imaged. Consequently, we developed a strategy that minimizes animal exposure to stressors before and during sustained BLI of freely moving unanesthetized mice. This technique was effective when monitoring expression of the Per1 gene that serves in the circadian clock timing mechanism and was previously shown to produce circadian bioluminescence rhythms in live mice. We used hairless albino mice expressing luciferase that were allowed to drink luciferin and engage in normal behaviors during imaging with cooled electron-multiplying-CCD cameras. Computer-aided image selection was developed to measure signal intensity of individual mice each time they were in the same posture, thereby providing comparable measurements over long intervals. This imaging procedure, performed primarily during the animal's night, is compatible with entrainment of the mouse circadian timing system to the light cycle while allowing sampling at multi-day intervals to monitor long-term changes. When the circadian expression of a gene is known, this approach provides an effective alternative to imaging immobile anesthetized animals and can removing noise caused by circadian oscillations and body movements that can degrade data collected during long-term imaging studies.

Effect of pH on the secondary structure and thermostability of beetle luciferases: structural origin of pH-insensitivity.

Beetle luciferases were classified into three functional groups: (1) pH-sensitive yellow-green-emitting (fireflies) which change the bioluminescence color to red at acidic pH, high temperatures and presence of heavy metals; (2) the pH-insensitive green-yellow-emitting (click beetles, railroad worms and firefly isozymes) which are not affected by these factors, and (3) pH-insensitive red-emitting. Although the pH-sensing site in firefly luciferases was recently identified, it is unclear why some luciferases are pH-insensitive despite the presence of some conserved pH-sensing residues. Through circular dichroism, we compared the secondary structural changes and unfolding temperature of luciferases of representatives of these three groups: (1) pH-sensitive green-yellow-emitting Macrolampis sp2 (Mac) and Amydetes vivianii (Amy) firefly luciferases; (2) the pH-insensitive green-emitting Pyrearinus termitilluminans larval click beetle (Pte) and Aspisoma lineatum (Al2) larval firefly luciferases, and (3) the pH-insensitive red-emitting Phrixotrix hirtus railroadworm (PxRE) luciferase. The most blue-shifted luciferases, independently of pH sensitivity, are thermally more stable at different pHs than the red-shifted ones. The pH-sensitive luciferases undergo increases of α-helices and thermal stability above pH 6. The pH-insensitive Pte luciferase secondary structure remains stable between pH 6 and 8, whereas the Al2 luciferase displays an increase of the ß-sheet at pH 8. The PxRE luciferase also displays an increase of α-helices at pH 8. The results indicate that green-yellow emission in beetle luciferases can be attained by: (1) a structurally rigid scaffold which stabilizes a single closed active site conformation in the pH-insensitive luciferases, and (2) active site compaction above pH 7.0 in the more flexible pH-sensitive luciferases.

Copper-mediated oxidation of imidazopyrazinones inhibits marine luciferase activity.

The development of bioluminescence-based tools has seen steady growth in the field of chemical biology over the past few decades ranging in uses from reporter genes to assay development and targeted imaging. More recently, coelenterazine-utilizing luciferases such as Gaussia, Renilla, and the engineered nano-luciferases have been utilized due to their intense luminescence relative to firefly luciferin/luciferase. The emerging importance of these systems warrants investigations into the components that affect their light production. Previous work has reported that one marine luciferase, Gaussia, is potently inhibited by copper salt. The mechanism for inhibition was not elucidated but was hypothesized to occur via binding to the enzyme. In this study, we provide the first report of a group of nonhomologous marine luciferases also exhibiting marked decreases in light emission in the presence of copper (II). We investigate the mechanism of action behind this inhibition and demonstrate that the observed copper inhibition does not stem from a luciferase interaction but rather the chemical oxidation of imidazopyrazinone luciferins generating inert, dehydrated luciferins.

A High-Throughput Amenable Dual Luciferase System for Measuring Toxoplasma gondii Bradyzoite Viability after Drug Treatment.

It is estimated that more than 2 billion people are chronically infected with the intracellular protozoan parasite Toxoplasma gondii (T. gondii). Despite this, there is currently no vaccine to prevent infection in humans, and there is no recognized curative treatment to clear tissue cysts. A major hurdle for identifying effective drug candidates against chronic-stage cysts has been the low throughput of existing in vitro assays for testing the survival of bradyzoites. We have developed a luciferase-based platform for specifically determining bradyzoite survival within in vitro cysts in a 96-well plate format. We engineered a cystogenic type II T. gondii PruΔku80Δhxgpr strain for stage-specific expression of firefly luciferase in the cytosol of bradyzoites and nanoluciferase for secretion into the lumen of the cyst (DuaLuc strain). Using this DuaLuc strain, we found that the ratio of firefly luciferase to nanoluciferase decreased upon treatment with atovaquone or LHVS, two compounds that are known to compromise bradyzoite viability. The 96-well format allowed us to test several additional compounds and generate dose-response curves for calculation of EC50 values indicating relative effectiveness of a compound. Accordingly, this DuaLuc system should be suitable for screening libraries of diverse compounds and defining the potency of hits or other compounds with a putative antibradyzoite activity.

Development of nanoluciferase-based sensing system that can specifically detect 1α,25-dihydroxyvitamin D in living cells.

Previously, we reported a FLucN-LXXLL+LBD-FLucC system that detects VDR ligands using split firefly luciferase techniques, ligand binding domain (LBD) of VDR, and LXXLL sequences that interact with LBD after VDR ligand binding. In vivo, 25-hydroxyvitamin D3 (25(OH)D3) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) act as VDR ligands that bind to VDR, and regulate bone-related gene expression. Therefore, the amount of 25(OH)D3 and 1α,25(OH)2D3 are indicators of bone-related diseases such as rickets and osteoporosis. In this study, we have developed a novel LgBiT-LXXLL+LBD-SmBiT system using NanoLuc Binary Technology (NanoBiT), which has an emission intensity several times higher than that of the split-type firefly luciferase. Furthermore, by using genetic engineering techniques, we attempted to construct a novel system that can specifically detect 1α,25(OH)2D3. Because histidine residues at positions 305 and 397 play important roles in forming a hydrogen bond with a hydroxyl group at position C25 of 25(OH)D3 and 1α,25(OH)2D3, His305 and His397 were each substituted by other amino acids. Consequently, the three mutant VDRs, H305D, H397N, and H397E were equally useful to detect 1α,25(OH)2D3 specifically. In addition, among the 58 variants of the LXXLL sequences, LPYEGSLLLKLLRAPVEE showed the greatest increase in luminescence upon the addition of 25(OH)D3 or 1α,25(OH)2D3. Thus, our novel system using NanoBiT appear to be useful for detecting native vitamin D or its derivatives.

Realization of firefly bioluminescence cycle in vitro and in cells.

The quantum yield (Q) of the "cold light" of firefly bioluminescence (BL) is remarkably high due to its nonradiative decay is extremely minimized. Thus, an artificial firefly represents the new generation of biomimetic "cold light" source with highest energy utilization. However, to manufacture a firefly-biomimetic "cold light" in vitro, one has to overcome several challenges including realization of the firefly BL cycle by incorporating the two important enzymes (i.e., firefly luciferase (Fluc) and luciferin-regenerating enzyme (LRE)) in one system. Here in this work, using self-prepared Fluc, LRE, and the main substrates, we realized the firefly BL cycle both in vitro and in cells. Moreover, using combinational analyses of HPLC and nESI-CID-MS/MS, we identified the main chemicals in the metabolic pathways underlying the firefly BL cycle. Using theoretical simulations, we revealed an optimum chemical route which balances the reaction cycle to achieve the highest BL intensity with the least chemical supplies. We anticipate that this pioneering study of the firefly cycle would provide industry with the opportunity to design tunable, economical, biomimetic "cold light" device in near future.

A new enzymatic assay to quantify inorganic pyrophosphate in plasma.

Inorganic pyrophosphate (PPi) is a crucial extracellular mineralization regulator. Low plasma PPi concentrations underlie the soft tissue calcification present in several rare hereditary mineralization disorders as well as in more common conditions like chronic kidney disease and diabetes. Even though deregulated plasma PPi homeostasis is known to be linked to multiple human diseases, there is currently no reliable assay for its quantification. We here describe a PPi assay that employs the enzyme ATP sulfurylase to convert PPi into ATP. Generated ATP is subsequently quantified by firefly luciferase-based bioluminescence. An internal ATP standard was used to correct for sample-specific interference by matrix compounds on firefly luciferase activity. The assay was validated and shows excellent precision (< 3.5%) and accuracy (93-106%) of PPi spiked into human plasma samples. We found that of several anticoagulants tested only EDTA effectively blocked conversion of ATP into PPi in plasma after blood collection. Moreover, filtration over a 300,000-Da molecular weight cut-off membrane reduced variability of plasma PPi and removed ATP present in a membrane-enclosed compartment, possibly platelets. Applied to plasma samples of wild-type and Abcc6-/- rats, an animal model with established low circulating levels of PPi, the new assay showed lower variability than the assay that was previously in routine use in our laboratory. In conclusion, we here report a new and robust assay to determine PPi concentrations in plasma, which outperforms currently available assays because of its high sensitivity, precision, and accuracy.