KPC-luciferase-expressing cells elicit an anti-tumor immune response in a mouse model of pancreatic cancer.

Mouse models for the study of pancreatic ductal adenocarcinoma (PDAC) are well-established and representative of many key features observed in human PDAC. To monitor tumor growth, cancer cells that are implanted in mice are often transfected with reporter genes, such as firefly luciferase (Luc), enabling in vivo optical imaging over time. Since Luc can induce an immune response, we aimed to evaluate whether the expression of Luc could affect the growth of KPC tumors in mice by inducing immunogenicity. Although both cell lines, KPC and Luc transduced KPC (KPC-Luc), had the same proliferation rate, KPC-Luc tumors had significantly smaller sizes or were absent 13 days after orthotopic cell implantation, compared to KPC tumors. This coincided with the loss of bioluminescence signal over the tumor region. Immunophenotyping of blood and spleen from KPC-Luc tumor-bearing mice showed a decreased number of macrophages and CD4+ T cells, and an increased accumulation of natural killer (NK) cells in comparison to KPC tumor mice. Higher infiltration of CD8+ T cells was found in KPC-Luc tumors than in their controls. Moreover, the immune response against Luc peptide was stronger in splenocytes from mice implanted with KPC-Luc cells compared to those isolated from KPC wild-type mice, indicating increased immunogenicity elicited by the presence of Luc in the PDAC tumor cells. These results must be considered when evaluating the efficacy of anti-cancer therapies including immunotherapies in immunocompetent PDAC or other cancer mouse models that use Luc as a reporter for bioluminescence imaging.

Establishment of a Cleavage-Based Single-Plasmid Dual-Luciferase Surrogate Reporter for the Cleavage Efficiency Evaluation of CRISPR-Cas12a Systems and Its Primary Application.

CRISPR-Cas technology is a widely utilized gene-editing tool that involves gRNA-guided sequence recognition and Cas nuclease-mediated cleavage. The design and evaluation of gRNA are essential for enhancing CRISPR/Cas editing efficiency. Various assays such as single-strand annealing, in vitro cleavage, and T7 endonuclease I (T7EI) are commonly used to assess gRNA-mediated Cas protein cleavage activity. In this study, a firefly luciferase and Renilla luciferase co-expressed and a cleavage-based single-plasmid dual-luciferase surrogate reporter was built to evaluate the gRNA-mediated Cas12a cleavage efficiency. The cleavage activities of CRISPR-Cas12a can be quantitatively determined by the recovery degree of firefly luciferase activity. The cleavage efficiency of CRISPR-Cas12a can be quantitatively measured by the recovery of firefly luciferase activity. By using this system, the cleavage efficiency of CRISPR-Cas12a on hepatitis B virus (HBV)/D expression plasmid was evaluated, revealing a negative correlation between gRNA cleavage efficiency and HBV gene expression measured using an enzyme-linked immunosorbent assay. This simple, efficient, and quantifiable system only requires the dual-luciferase vector and CRISPR-Cas12a vector, making it a valuable tool for selecting effective gRNAs for gene editing.

Temperature-dependent intracellular crystallization of firefly luciferase in mammalian cells is suppressed by D-luciferin and stabilizing inhibitors.

Firefly luciferase (Fluc) from Photinus pyralis is one of the most widely used reporter proteins in biomedical research. Despite its widespread use, Fluc's protein phase transition behaviors and phase separation characteristics have not received much attention. Current research uncovers Fluc's intrinsic property to phase separate in mammalian cells upon a simple cell culture temperature change. Specifically, Fluc spontaneously produced needle-shaped crystal-like inclusion bodies upon temperature shift to the hypothermic temperatures ranging from 25 °C to 31 °C. The crystal-like inclusion bodies were not associated with or surrounded by membranous organelles and were likely built from the cytosolic pool of Fluc. Furthermore, the crystal-like inclusion formation was suppressed when cells were cultured in the presence of D-luciferin and its synthetic analog, as well as the benzothiazole family of so-called stabilizing inhibitors. These two classes of compounds inhibited intracellular Fluc crystallization by different modes of action as they had contrasting effects on steady-state luciferase protein accumulation levels. This study suggests that, under substrate insufficient conditions, the excess Fluc phase separates into a crystal-like state that can modulate intracellular soluble enzyme availability and protein turnover rate.

A BSL-2 compliant mouse model of SARS-CoV-2 infection for efficient and convenient antiviral evaluation.

Animal models of authentic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require operation in biosafety level 3 (BSL-3) containment. In the present study, we established a mouse model employing a single-cycle infectious virus replicon particle (VRP) system of SARS-CoV-2 that can be safely handled in BSL-2 laboratories. The VRP [ΔS-VRP(G)-Luc] contains a SARS-CoV-2 genome in which the spike gene was replaced by a firefly luciferase (Fluc) reporter gene (Rep-Luci), and incorporates the vesicular stomatitis virus glycoprotein on the surface. Intranasal inoculation of ΔS-VRP(G)-Luc can successfully transduce the Rep-Luci genome into mouse lungs, initiating self-replication of Rep-Luci and, accordingly, inducing acute lung injury mimicking the authentic SARS-CoV-2 pathology. In addition, the reporter Fluc expression can be monitored using a bioluminescence imaging approach, allowing a rapid and convenient determination of viral replication in ΔS-VRP(G)-Luc-infected mouse lungs. Upon treatment with an approved anti-SARS-CoV-2 drug, VV116, the viral replication in infected mouse lungs was significantly reduced, suggesting that the animal model is feasible for antiviral evaluation. In summary, we have developed a BSL-2-compliant mouse model of SARS-CoV-2 infection, providing an advanced approach to study aspects of the viral pathogenesis, viral-host interactions, as well as the efficacy of antiviral therapeutics in the future.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious and pathogenic in humans; thus, research on authentic SARS-CoV-2 has been restricted to biosafety level 3 (BSL-3) laboratories. However, due to the scarcity of BSL-3 facilities and trained personnel, the participation of a broad scientific community in SARS-CoV-2 research had been greatly limited, hindering the advancement of our understanding on the basic virology as well as the urgently necessitated drug development. Previously, our colleagues Jin et al. had generated a SARS-CoV-2 replicon by replacing the essential spike gene in the viral genome with a Fluc reporter (Rep-Luci), which can be safely operated under BSL-2 conditions. By incorporating the Rep-Luci into viral replicon particles carrying vesicular stomatitis virus glycoprotein on their surface, and via intranasal inoculation, we successfully transduced the Rep-Luci into mouse lungs, developing a mouse model mimicking SARS-CoV-2 infection. Our model can serve as a useful platform for SARS-CoV-2 pathological studies and antiviral evaluation under BSL2 containment.

Role of Histidine 310 in Amydetes vivianii firefly luciferase pH and metal sensitivities and improvement of its color tuning properties.

Firefly luciferases emit yellow-green light and are pH-sensitive, changing the bioluminescence color to red in the presence of heavy metals, acidic pH and high temperatures. These pH and metal-sensitivities have been recently harnessed for intracellular pH indication and toxic metal biosensing. However, whereas the structure of the pH sensor and the metal binding site, which consists mainly of two salt bridges that close the active site (E311/R337 and H310/E354), has been identified, the specific role of residue H310 in pH and metal sensing is still under debate. The Amydetes vivianii firefly luciferase has one of the lowest pH sensitivities among the group of pH-sensitive firefly luciferases, displaying high bioluminescent activity and special spectral selectivity for cadmium and mercury, which makes it a promising analytical reagent. Using site-directed mutagenesis, we have investigated in detail the role of residue H310 on pH and metal sensitivity in this luciferase. Negatively charged residues at position 310 increase the pH sensitivity and metal sensitivity; H310G considerably increases the size of the cavity, severely impacting the activity, H310R closes the cavity, and H310F considerably decreases both pH and metal sensitivities. However, no substitution completely abolished pH and metal sensitivities. The results indicate that the presence of negatively charged and basic side chains at position 310 is important for pH sensitivity and metals coordination, but not essential, indicating that the remaining side chains of E311 and E354 may still coordinate some metals in this site. Furthermore, a metal binding site search predicted that H310 mutations decrease the affinity mainly for Zn, Ni and Hg but less for Cd, and revealed the possible existence of additional binding sites for Zn, Ni and Hg.

Diagnosis of Nonalcoholic Fatty Liver Disease via a H2S-Responsive Bioluminescent Probe Combined with Firefly Luciferase mRNA Delivery.

The early detection of nonalcoholic fatty liver disease (NAFLD) through bioluminescent probes is of great significance. However, there remains a challenge to apply them in nontransgenic natural animals due to the lack of exogenous luciferase. To address this issue, we herein report a new strategy for in situ monitoring of endogenous hydrogen sulfide (H2S) in the liver of NAFLD mice by leveraging a H2S-responsive bioluminescent probe (H-Luc) combined with firefly luciferase (fLuc) mRNA delivery. The probe H-Luc was created by installing a H2S recognition moiety, 2,4-dinitrophenol, onto the luciferase substrate (d-luciferin), which is allowed to release cage-free d-luciferin in the presence of H2S via a nucleophilic aromatic substitution reaction. In the meantime, the intracellular luciferase was introduced by lipid nanoparticle (LNP)-mediated fLuc mRNA delivery, rendering it suitable for bioluminescence (BL) imaging in vitro and in vivo. Based on this luciferase-luciferin system, the endogenous H2S could be sensitively and selectively detected in living cells, showing a low limit of detection (LOD) value of 0.72 µM. More importantly, after systematic administration of fLuc mRNA-loaded LNPs in vivo, H-Luc was able to successfully monitor the endogenous H2S levels in the NAFLD mouse model for the first time, displaying a 28-fold higher bioluminescence intensity than that in the liver of normal mice. We believe that this strategy may shed new light on the diagnosis of inflammatory liver disease, further elucidating the roles of H2S.

Analysis of the impact of pluronic acid on the thermal stability and infectivity of AAV6.2FF.

BACKGROUND: The advancement of AAV vectors into clinical testing has accelerated rapidly over the past two decades. While many of the AAV vectors being utilized in clinical trials are derived from natural serotypes, engineered serotypes are progressing toward clinical translation due to their enhanced tissue tropism and immune evasive properties. However, novel AAV vectors require formulation and stability testing to determine optimal storage conditions prior to their use in a clinical setting. RESULTS: Here, we evaluated the thermal stability of AAV6.2FF, a rationally engineered capsid with strong tropism for lung and muscle, in two different buffer formulations; phosphate buffered saline (PBS), or PBS supplemented with 0.001% non-ionic surfactant Pluronic F68 (PF-68). Aliquots of AAV6.2FF vector encoding the firefly luciferase reporter gene (AAV6.2FF-ffLuc) were incubated at temperatures ranging from -20°C to 55°C for varying periods of time and the impact on infectivity and particle integrity evaluated. Additionally, the impact of several rounds of freeze-thaw treatments on the infectivity of AAV6.2FF was investigated. Vector infectivity was measured by quantifying firefly luciferase expression in HEK 293 cells and AAV particle integrity was measured by qPCR quantification of encapsidated viral DNA. CONCLUSIONS: Our data demonstrate that formulating AAV6.2FF in PBS containing 0.001% PF-68 leads to increased stability and particle integrity at temperatures between -20℃ to 21℃ and protection against the destructive effects of freeze-thaw. Finally, AAV6.2FF-GFP formulated in PBS supplemented with 0.001% PF-68 displayed higher transduction efficiency in vivo in murine lung epithelial cells following intranasal administration than vector buffered in PBS alone further demonstrating the beneficial properties of PF-68.

The Biodistribution of Replication-Defective Simian Adenovirus 1 Vector in a Mouse Model.

The administration route affects the biodistribution of a gene transfer vector and the expression of a transgene. A simian adenovirus 1 vector carrying firefly luciferase and GFP reporter genes (SAdV1-GFluc) were constructed, and its biodistribution was investigated in a mouse model by bioluminescence imaging and virus DNA tracking with real-time PCR. Luciferase activity and virus DNA were mainly found in the liver and spleen after the intravenous administration of SAdV1-GFluc. The results of flow cytometry illustrated that macrophages in the liver and spleen as well as hepatocytes were the target cells. Repeated inoculation was noneffective because of the stimulated serum neutralizing antibodies (NAbs) against SAdV-1. A transient, local expression of low-level luciferase was detected after intragastric administration, and the administration could be repeated without compromising the expression of the reporter gene. Intranasal administration led to a moderate, constant expression of a transgene in the whole respiratory tract and could be repeated one more time without a significant increase in the NAb titer. An immunohistochemistry assay showed that respiratory epithelial cells and macrophages in the lungs were transduced. High luciferase activity was restricted at the injection site and sustained for a week after intramuscular administration. A compromised transgene expression was observed after a repeated injection. When these mice were intramuscularly injected for a third time with the human adenovirus 5 (HAdV-5) vector carrying a luciferase gene, the luciferase activity recovered and reached the initial level, suggesting that the sequential use of SAdV-1 and HAdV-5 vectors was practicable. In short, the intranasal inoculation or intramuscular injection may be the preferred administration routes for the novel SAdV-1 vector in vaccine development.

Visualization of Endogenous Hypochlorite in Drug-Induced Liver Injury Mice via a Bioluminescent Probe Combined with Firefly Luciferase mRNA-Loaded Lipid Nanoparticles.

Drug-induced liver injury (DILI) is a common liver disease with a high rate of morbidity, and its pathogenesis is closely associated with the overproduction of highly reactive hypochlorite (ClO-) in the liver. However, bioluminescence imaging of endogenous hypochlorite in nontransgenic natural mice remains challenging. Herein, to address this issue, we report a strategy for imaging ClO- in living cells and DILI mice by harnessing a bioluminescent probe formylhydrazine luciferin (ClO-Luc) combined with firefly luciferase (fLuc) mRNA-loaded lipid nanoparticles (LNPs). LNPs could efficiently deliver fLuc mRNA into living cells and in vivo, expressing abundant luciferase in the cytoplasm in situ. In the presence of ClO-, probe ClO-Luc locked by formylhydrazine could release cage-free d-luciferin through oxidation and follow-up hydrolysis reactions, further allowing for bioluminescence imaging. Moreover, based on the luciferase-luciferin system, it was able to sensitively and selectively detect ClO- in vitro with a limit of detection of 0.59 µM and successfully monitor the endogenous hypochlorite generation in the DILI mouse model for the first time. We postulate that this work provides a new method to elucidate the roles of ClO- in related diseases via bioluminescence imaging.

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.

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.

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.

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.