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In utero repair of open neural tube defect (ONTD) using an open hysterotomy approach (hereafter referred to as "Open") has been shown to reduce the need for ventriculo-peritoneal shunting and to improve motor outcomes for affected infants. Laparotomy-assisted fetoscopic repair (hereafter referred to as "Hybrid") is an alternate approach that may confer similar neurological benefits, while reducing the incidence of hysterotomy related complications. In this study we aimed to analyze procedure related maternal and fetal complications of in-utero repair using the Clavien-Dindo classification, and to compare the outcomes of the Hybrid and Open approaches.This was a retrospective cohort study conducted in a single center between September 2011 and July 2021. All patients who met the MOMS criteria and who underwent either Hybrid or Open fetal surgery were included, Maternal complications were classified using a unique adapatation of the Clavien-Dindo scoring system allowing the development of a comprehensive complication index score specific to fetal surgery. Primary fetal outcome was defined as gestational age at delivery and summarized according to the World Health Organization definitions of preterm delivery.There were 146 fetuses with ONTD who were eligible for, and underwent, in utero repair during the study period. Of these, 102 underwent Hybrid fetoscopic repair and 44 underwent Open hysterotomy repair. Gestational age at the time of surgery was higher in the Hybrid group when compared with the Open group (25.1 vs. 24.8 weeks, p=0.004). Maternal body mass index was lower in the Hybrid compared to the Open group (25.4 vs 27.1 kg\mUtilization of our adaptation of the standardized Clavien-Dindo classification to assess the maternal complications associated with in utero ONTD repair provides a new method to objectively assess different fetal surgical approaches. It also provides a much-needed standardized tool to allow objective comparisons between methods, which can be used when counseling patients. The Hybrid ONTD repair was associated with a lower rate of maternal adverse events than the Open approach, and was associated with later gestational age at delivery and less preterm delivery.

The dazzling yellow-green light emission of the common North American firefly Photinus pyralis and other bioluminescent organisms has provided a wide variety of prominent research applications like...

Luciferases catalyze light-emitting reactions that produce a rainbow of colors from their substrates (luciferins), molecular oxygen, and often additional cofactors. These bioluminescence (BL) systems have afforded an incredible variety of basic research and medical applications. Driven by the importance of BL-based non-invasive animal imaging (BLI) applications, especially in support of cancer research, new BL systems have been developed by engineering beetle luciferase (Luc) variants and synthetic substrate combinations to produce red to near-infrared (nIR) light to improve imaging sensitivity and resolution. To stimulate the application of BLI research and advance the development of improved reagents for BLI, we undertook a systematic comparison of the spectroscopic and BL properties of seven beetle Lucs with LH2 and nine substrates, which included two new quinoline ring-containing analogs. The results of these experiments with purified Luc enzymes in vitro and in live HEK293T cells transfected with luc genes have enabled us to identify Luc/analog combinations with improved properties compared to those previously reported and to provide live cell BL data that may be relevant to in vivo imaging applications. Additionally, we found strong candidate enzyme/substrate pairs for in vitro biomarker applications requiring nIR sources with minimal visible light components. Notably, one of our new substrates paired with a previously developed Luc variant was demonstrated to be an excellent in vitro source of nIR and a potentially useful BL system for improved resolution in BLI.

Bioluminescence imaging (BLI) is ubiquitous in scientific research for the sensitive tracking of biological processes in small animal models. However, due to the attenuation of visible light by tissue, and the limited set of near-infrared bioluminescent enzymes, BLI is largely restricted to monitoring single processes in vivo. Here we show, that by combining stabilised colour mutants of firefly luciferase (FLuc) with the luciferin (LH2) analogue infraluciferin (iLH2), near-infrared dual BLI can be achieved in vivo. The X-ray crystal structure of FLuc with a high-energy intermediate analogue, 5’-O-[N-(dehydroinfraluciferyl)sulfamoyl] adenosine (iDLSA) provides insight into the FLuc-iLH2 reaction leading to near-infrared light emission. The spectral characterisation and unmixing validation studies reported here established that iLH2 is superior to LH2 for the spectral unmixing of bioluminescent signals in vivo; which led to this novel near-infrared dual BLI system being applied to monitor both tumour burden and CAR T cell therapy within a systemically induced mouse tumour model., eLife digest Fireflies and some other insects glow to attract mates or prey. This so-called bioluminescence occurs when an enzyme called luciferase modifies the molecule luciferin, which can then emit bright yellow-green light. The gene that encodes the luciferase enzyme has been introduced into cells from mammals, including cancer cells. In the presence of luciferin, these cells begin to glow. The brightness of the bioluminescence depends on how many cancer cells are growing and dividing. The light is bright enough for the cancer cells making luciferase to be transplanted into mice so their behaviour can be examined. However, blood and other tissues absorb the yellow-green light, making it hard to see the cancer cells deep within a mouse. To circumvent this problem, researchers designed a new type of luciferin, called infraluciferin, which emits red light that shines through blood and tissues. There are now also different variants of the luciferase enzyme, which act on infraluciferin to make different shades of red light. Stowe et al. wanted to test if two different biological events happening at the same time could be observed using two shades of bioluminescent red in a single live mouse. First, a mixture of cancer cells containing two versions of luciferase were transplanted into mice. When the mice were then given infraluciferin, the two types of cancer cells could be distinguished based on the different shades of red bioluminescence. In a second experiment, Stowe et al. tracked the treatment of cancer cells with immune cells, by introducing a different version of luciferase into each of the two groups of cells. Over time, the red light produced by the immune cells grew stronger than that of the cancer cells, indicating that the number of cancer cells had decreased and that the treatment was effective. Together, this work shows that it can be simple, cheap and efficient to observe more than one cell type, or even disease, in a living system. This technique may be used by scientists to study different diseases and treatment options in mice. Importantly, it will also reduce the number of animals used to do this research.

Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O2, and added luciferin. Previous Photinus pyralis red-emitting variants with high Kmvalues for ATP have performed disappointingly in live cells despite having much higher relative specific activities than enzymes like Click Beetle Red (CBR). We engineered a luciferase variant PLR3 having a Kmvalue for ATP similar to CBR and ∼2.6-fold higher specific activity. The red-emitting PLR3 was ∼2.5-fold brighter than CBR in living HEK293T and HeLa cells, an improvement consistent with the importance of the Kmvalue in low ATP environments.

In the southern Appalachian area of the United States, the Phausis reticulata firefly, commonly known as the “Blue Ghost,” performs a unique display of bioluminescence. Adult male organisms are observed darting rapidly along paths and riverbeds in dark forests producing long-lasting and mesmerizing bluish-white luminous streaks. Starting with eighteen adult male firefly lanterns, we used a reverse transcriptase and rapid amplification of cDNA ends (RACE) approach to clone the 1635 base pair open reading frame of the P. reticulata luc gene corresponding to a 545 residue protein. Expression of the recombinant luciferase protein in E. coli and characterization studies revealed the true color of the light emission to be green (λmax = 552 nm), strongly suggesting that the field observations result from a Purkinje shift. While the P. reticulata luciferase amino acid sequence is 74.3% identical to the North American P. pyralis luciferase, we were surprised to find that it was 88.4% and 87.7% identical to luciferases from C. ruficollis and D. axillaris both native to mainland Japan. Phylogenetic analysis confirmed the close relationship of the three enzymes that is surprising given the great distance between their natural habitats and the inability of the Japanese fireflies to produce bright bioluminescence. This article is protected by copyright. All rights reserved.

The dazzling yellow-green light emission of the common North American firefly Photinus pyralis and other bioluminescent organisms has provided a wide variety of prominent research applications like reporter gene assays and in vivo imaging methods. While the P. pyralis enzyme has been extensively studied, only recently has a second Photinus luciferase been cloned from the species scintillans. Even though the enzymes share very high sequence identity (89.8%), the color of the light they emit, their specific activity, and their stability to heat, pH, and chemical denaturation are quite different with the scintillans luciferase being generally more resistant. Through the construction and evaluation of the properties of chimeric domain swapped, single point, and various combined variants, we have determined that only 6 amino acid changes are necessary to confer all of the properties of the scintillans enzyme to wild-type P. pyralis luciferase. Altered stability properties were attributed to four of the amino acid changes (T214N/S276T/H332N/E354N) and single mutations each predominantly changed emission color (Y255F) and specific activity (A222C). Results of a crystallographic study of the P. pyralis enzyme containing the 6 changes (Pps6) provide some insight into the structural basis for some of the documented property differences.

Cell-based assays utilizing reporter gene technology have been widely exploited for biosensing, as they provide useful information about the bioavailability and cell toxicity of target analytes. The long assay time due to gene transcription and translation is one of the main drawbacks of cell biosensors. We report the development of two yeast biosensors stably expressing human estrogen receptors α and β and employing NanoLuc as the reporter protein to upgrade the widely used yeast estrogen screening (YES) assays. A viability control strain was also developed based on a chimeric green-emitting luciferase, PLG2, expressed for the first time in Saccharomycescerevisiae. Thanks to their brightness, NanoLuc and PLG2 provided excellent sensitivity, enabling the implementation of these biosensors into low-cost smartphone-based devices. The developed biosensors had a rapid (1h) response and reported on (anti)estrogenic activity via human estrogen receptors α and β as well as general sample toxicity. Under optimized conditions, we obtained LODs of 7.1 ± 0.4nM and 0.38 ± 0.08nM for E2 with nanoYESα and nanoYESβ, respectively. As a proof of concept, we analyzed real samples from plants showing significant estrogenic activity or known to contain significant amounts of phytoestrogens. Graphical abstract.

Whole-cell biosensors present many advantages, including being able to monitor the toxicity and bioavailability of chemicals; cells grown in traditional 2D cultures, however, do not reproduce the complexity of in vivo physiology. In the last years, 3D cell-culture models have garnered great attention due to their capability to better mimic in vivo cellular responses to external stimuli, providing excellent model living organisms. In order to obtain a predictive, sensitive, and robust yet low-cost 3D cell biosensor, we developed a smartphone-based bioluminescent 3D cell biosensor platform for effect-based analysis. We exploited the Nuclear Factor-kappa B (NF-kB) signal transduction pathway, which is induced by several types of stressors and is involved in the regulation of cell-cycle/growth, inflammation, apoptosis, and immunity. The smartphone-based biosensor relies on immobilized HEK293 spheroids genetically engineered with powerful red- and green-emitting luciferases utilized as inflammation and viability reporters. It provides a limit of detection for Tumor Necrosis Factor (TNFα) of 0.15 ± 0.05 ng/mL and could be a useful tool to initially screen environmental samples or other compounds on-site, especially for additional more accurate chemical analyses.

Effective methods for monitoring eukaryotic gene expression and regulation based on bioluminescence - the emission of light by living organisms - are well established. Typically, the expression of a gene of interest is reported on with high sensitivity and over a wide dynamic range by the emission of light from a variety of engineered luciferase genes from beetles and marine organisms. The luciferase reporter genes are expressed downstream of the target gene or promoter and detected after exogenous addition of luciferin substrates. We describe a novel bioluminescence reporter method for the simultaneous monitoring of two genes expressing engineered firefly luciferase variants that emit readily distinguishable green and red light signals. The key feature is the selectivity of the enzymes for two luciferin substrates that determine each emission color. To validate our method, we performed a complex promoter transactivation experiment side-by-side with the Dual-Luciferase Reporter protocol and obtained essentially identical results. Additional comparative experiments demonstrated that our assay system provided improvements in background, cell normalization, and detectability compared to representative available methods. With access to a luminometer equipped with two optical filters, this method is an excellent choice for genetic reporter assays that can be performed with a single reagent solution.

Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications based on the detection of the enzymes or using the proteins to measure ATP levels. Based on studies of chimeric luciferases, we engineered a novel luciferase called PLG2 that has enhanced specific activity, and thermal and pH stability compared to the commonly used Photinus pyralis luciferase. We present here protocols for preparing a single assay mixture containing PLG2 that can be used to readily detect femtomole levels of ATP. Our methodology can be used with a variety of samples, including human and bacterial cells, where measurements of ATP can be used as a biosensor for the detection of viable cells.

Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications based on the detection of the enzymes or using the proteins to measure ATP levels. Based on studies of chimeric luciferases, we engineered a novel luciferase called PLG2 that has enhanced specific activity, and thermal and pH stability compared to the commonly used Photinus pyralis luciferase. We present here protocols for preparing a single assay mixture containing PLG2 that can be used to readily detect femtomole levels of ATP. Our methodology can be used with a variety of samples, including human and bacterial cells, where measurements of ATP can be used as a biosensor for the detection of viable cells.

Bioluminescent (BL) cell-based assays based on two-dimensional (2D) monolayer cell cultures represent well-established bioanalytical tools for preclinical screening of drugs. However, cells in 2D cultures do not often reflect the morphology and functionality of living organisms, thus limiting the predictive value of 2D cell-based assays. Conversely, 3D cell models have the capability to generate the extracellular matrix and restore cell-to-cell communications; thus, they are the most suitable model to mimic in vivo physiology. In this work, we developed a nondestructive real-time BL imaging assay of spheroids for longitudinal studies on 3D cell models. A high-throughput BL 3D cell-based assay in micropatterned 96-well plate format is reported. The assay performance was assessed using the transcriptional regulation of nuclear factor K beta response element in human embryonic kidney (HEK293) cells. We compared concentration-response curves for tumor necrosis factor-α with those obtained using conventional 2D cell cultures. One of the main advantages of this approach is the nonlysing nature of the assay, which allows for repetitive measurements on the same sample. The assay can be implemented in any laboratory equipped with basic cell culture facilities and paves the way to the development of new 3D bioluminescent cell-based assays.

Unlike the enchanting yellow-green flashes of light produced on warm summer evenings by Photinus pyralis, the most common firefly species in North America, the orange lights of Photinus scintillans are infrequently observed. These Photinus species, and likely all bioluminescent beetles, use the same substrates beetle luciferin, ATP and oxygen to produce light. It is the structure of the particular luciferase enzyme that is the key to determining the color of the emitted light. We report here the molecular cloning of the P. scintillans luc gene and the expression and characterization of the corresponding novel recombinant luciferase enzyme. A comparison of the amino acid sequence with that of the highly similar P. pyralis enzyme and subsequent mutagenesis studies revealed that the single conservative amino acid change tyrosine to phenylalanine at position 255 accounted for the entire emission color difference. Additional mutagenesis and crystallographic studies were performed on a H-bond network, which includes the position 255 residue and five other stringently conserved beetle luciferase residues, that is proximal to the substrate/emitter binding site. The results are interpreted in the context of a speculative proposal that this network is key to the understanding of bioluminescence color determination.

A process change with the goal of decreasing and ultimately eliminating bloodstream infections associated with central catheters in critical care patients was implemented at Riverside Methodist Hospital. This process of implementation resulted in a culture change in the hospital’s medical surgical intensive care unit. Keys to success included a multidisciplinary team approach, support from persons with a stake in the process, and provision of continuous feedback. The lessons learned in this journey at Riverside Methodist Hospital can help caregivers at other hospitals decrease the occurrence of these life-threatening infections.

North American firefly Photinus pyralis luciferase, which emits yellow-green light (557nm), has been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. Luciferase variants with red-shifted bioluminescence and high specific activity can be paired with green-emitting counterparts for use in dual-color reporter assays or can be used alone for in vivo imaging. Beginning with a previously reported red-emitting thermostable mutant and using mutagenesis techniques, we engineered two luciferases with redder emission maxima while maintaining satisfactory specific activities and thermostability. The novel enzymes were expressed in HEK293 cells, where they performed similarly to Promega's codon-optimized click beetle red luciferase in model reporter assays. When the firefly luciferase variants were codon-optimized and retested using optimized substrate concentrations, they provided 50- to 100-fold greater integrated light intensities than the click beetle enzyme. These results suggest that the novel enzymes should provide superior performance in dual-color reporter and in vivo imaging applications, and they illustrate the importance of codon optimization for assays in mammalian cells.

The availability of new bioluminescent proteins, obtained by cDNA cloning and mutagenesis of wild-type genes, expanded the applicability of these reporters from the perspective of using more proteins emitting at different wavelengths in the same cell-based assay. By spectrally resolving the light emitted by different reporter proteins, it is in fact possible to simultaneously monitor multiple targets. A new luciferase isolated from Luciola italica has been recently cloned, and thermostable red- and green-emitting mutants were obtained by random and site-directed mutagenesis. Different combinations of luciferases were used in vitro as purified proteins and expressed in bacterial and mammalian cells to test their suitability for multicolor assays. A mammalian triple-color reporter model system was then developed using a green-emitting wild-type Photinus pyralis luciferase, a red thermostable mutant of L. italica luciferase, and a secreted Gaussia princeps luciferase (GLuc) to monitor the two main pathways of bile acid biosynthesis. The two firefly luciferases were used to monitor cholesterol 7-alpha hydroxylase and sterol 27-hydroxylase, while secreted constitutively expressed GLuc was used as an internal vitality control. By treating the cells with chenodeoxycholic acid, it was possible to obtain dose-dependent inhibitions of the two specific signals together with a constant production of GLuc, which allowed for a dynamic evaluation of the metabolic activity of the cells. This is the first triple-color mammalian reporter assay that combines secreted and nonsecreted luciferases requiring different substrates, thus avoiding reciprocal interference between different BL signals. This approach is suitable for high content analysis of gene transcription in living cells to shorten the time for screening assays, increasing throughput and cost-effectiveness.

Firefly luciferase produces light by converting substrate beetle luciferin into the corresponding adenylate that it subsequently oxidizes to oxyluciferin, the emitter of bioluminescence. We have confirmed the generally held notions that the oxidation step is initiated by formation of a carbanion intermediate and that a hydroperoxide (anion) is involved. Additionally, structural evidence is presented that accounts for the delivery of oxygen to the substrate reaction site. Herein, we report key convincing spectroscopic evidence of the participation of superoxide anion in a related chemical model reaction that supports a single electron-transfer pathway for the critical oxidative process. This mechanism may be a common feature of bioluminescence processes in which light is produced by an enzyme in the absence of cofactors.

Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. We previously reported the approximately 2-fold enhanced activity and 1.4-fold greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomole levels of ATP. In addition, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and living cells with 4.4-fold and approximately 3.0-fold greater sensitivity, respectively. PLG2 could be an improved alternative to Promega's luc2 for reporter and imaging applications.

We report the enhanced bioluminescence properties of a chimeric enzyme (PpyLit) that contains the N-domain of recombinant Photinus pyralis luciferase joined to the C-domain of recombinant Luciola italica luciferase. Compared to the P. pyralis enzyme, the novel PpyLit chimera exhibited 1.8-fold enhanced flash-height specific activity, 2.0-fold enhanced integration-based specific activity, 2.9-fold enhanced catalytic efficiency (kcat/Km), and a 1.4-fold greater bioluminescence quantum yield. The results of this study provide an underlying basis of this unusual example of a chimeric enzyme with enhanced catalytic properties that are not simply the sum of the contributions of the two luciferases.

As followers of Spanish-American Modernism are aware, the focus of "Modernista" criticism has sharply shifted in recent years, moving away from an emphasis on form and technical innovations towards a greater concentration on metaphysical themes, in some cases connecting the literary movement with the so-called "Generation of 1898." What has been lacking, however, is a serious study of some of the most important Modernist imagery and how this aspect of poetic technique is actually inseparable from the philosophical themes. This paper examines the use of the image of the abyss in the poetry of Ruben Dario and Amado Nervo, tracing the development of the image into a signifier for the existential anguish suffered by both poets and its eventual shift in meaning accompanying Nervo's renewal of religious faith. In Dario's earliest poetry the image tends to be rather straightforward, associated in some cases with erotic love and in others with the seas and the heavens. However, as the poet matures, the abyss comes to represent an internal void created by a loss of faith, as one sees in such poems as "La fuente," "Yo soy aquel... ," and "Venus." In Nervo's poetry the "abismo" is used to represent the same doubt and anguish, as is the case in "A Felipe II," "Yo no naci para reir," and "La sombra del ala," though it eventually becomes a metaphor for death or what may await us after death, especially in the collection La amada inmovil. Later in life, after Nervo regains his faith, the image comes to represent God, as exemplified by the poems "Inaccesible" and "La leccion," thus bringing the cycle of an image that was originally associated with doubt to a tidy close.

The marine annelid Chaetopterus variopedatus produces bioluminescence by an unknown and potentially novel mechanism. We have advanced the study of this fascinating phenomenon, which has not been investigated for nearly 60 years after initial studies were first reported for this species. Here, we show that the luminous slime produced by the worm exhibits blue fluorescence that matches the bioluminescence emission. This result suggests that the oxyluciferin emitter is present. However, while the blue fluorescence decays over time green fluorescence is increasingly revealed that is likely associated with products of the luminescence reaction. LC/MS and fluorescence analysis of harvested luminescent material revealed riboflavin as the major green fluorescent component. Riboflavin is usually associated with the mechanism of light production in bacteria, yet luminous bacteria were not found in the worm mucus, and accordingly were not reported to be directly responsible for the light emission, which is under nervous control in the worm. We therefore propose a hypothesis in which riboflavin or a structurally related derivative serves as the emitter in the worm's light producing reaction.

Five novel firefly luciferin analogues in which the benzothiazole ring system of the natural substrate was replaced with benzimidazole, benzofuran, benzothiophene, benzoxazole, and indole were synthesized. The fluorescence, bioluminescence, and kinetic properties of the compounds were evaluated with recombinant Photinus pyralis wild type luciferase. With the exception of indole, all of the substrates containing heterocycle substitutions produced readily measurable flashes of light with luciferase. Compared to that of luciferin, the intensities ranged from 0.3 to 4.4% in reactions with varying pH optima and times to reach maximal intensity. The heteroatom changes influenced both the fluorescence and bioluminescence emission spectra, which displayed maxima of 479-528 and 518-574 nm, respectively. While there were some interesting trends in the spectroscopic and bioluminescence properties of this group of structurally similar substrate analogues, the most significant findings were associated with the benzothiophene-containing compound. This synthetic substrate produced slow decay glow kinetics that increased the total light-based specific activity of luciferase more than 4-fold versus the luciferin value. Moreover, over the pH range of 6.2-9.4, the emission maximum is 523 nm, an unusual 37 nm blue shift compared to that of the natural substrate. The extraordinary bioluminescence properties of the benzothiophene luciferin should translate into greater sensitivity for analyte detection in a wide variety of luciferase-based applications.

Beetle luciferases catalyze a two-step reaction that includes the initial adenylation of the luciferin substrate, followed by an oxidative decarboxylation that ultimately produces light. Evidence for homologous acyl-CoA synthetases supports a domain alternation catalytic mechanism in which these enzymes’ C-terminal domain rotates by ∼140° to adopt two conformations that are used to catalyze the two partial reactions. While many structures exist of acyl-CoA synthetases in both conformations, to date only biochemical evidence supports domain alternation with luciferase. We have determined the structure of a cross-linked luciferase enzyme that is trapped in the second conformation. This new structure supports the role of the second catalytic conformation and provides insights into the biochemical mechanism of the luciferase oxidative step.

THE term "frostbite" designates the lesions produced by exposure to cold. Short exposure to extremely low temperatures or longer exposure to moderately low temperatures results in similar conditions, although damage appears to be more localized when caused by low-temperature exposures of short duration.1 During World War II many cases have resulted from prolonged soaking of the feet in cold water, hence the term "immersion foot." In this connection White2 draws a distinction between the lesions of immersion foot and those caused by prolonged exposure in southern latitudes coupled with nutritional deficiencies. Many patients with frostbite or immersion foot have late . . .

Bioluminescent (BL) proteins are a promising tool for diverse applications based on reporter gene technology thanks to their high sensitivity and range of linear response. Due to their widespread use in the environmental, medical and agro-food fields, there is a great need for new BL reporter proteins with improved characteristics to provide researches a wide range of suitable reporters. Few efforts have been made in this direction and further improvement of BL reporter features (e.g., thermostability, narrower emission bandwidth, emission at different wavelengths) tailored for specific applications would be a remarkable progress toward the development of ultrasensitive multiplexed assays either in vitro or in vivo. The suitability of using red- and green-emitting thermostable mutants of Photinus pyralis firefly luciferase and two click beetle luciferases in combination with a secreted luciferase from Gaussia princeps was evaluated to develop a triple-color mammalian assay. Two triple-reporter model mammalian systems were developed in a human hepatoblastoma cell line to monitor the transcriptional regulation of cholesterol 7-alpha hydroxylase (cyp7a1), the enzyme that catalyzes the first and rate-limiting step of the main pathway responsible for cholesterol degradation in humans. These model systems allowed us to evaluate the feasibility of using two intracellular BL reporters and a secreted one in the same cell-based assay. The selection of reporter proteins characterized by similar expression levels was identified as a critical point for the development of a multicolor assay.

Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557 nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. In a previous study designed to produce luciferases for simultaneously monitoring two gene expression events, we identified a very promising blue-shifted emitter (548 nm) that contained the mutations Val241Ile, Gly246Ala, and Phe250Ser [Branchini, B. R., Southworth, T. L., Khattak, N. F., Michelini, E., and Roda, A. (2005) Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications, Anal. Biochem. 345, 140-148]. To establish the basis of the unusual blue-shifted emission, we determined that a simple additive effect of the three individual mutations did not account for the spectral properties of the triple mutant. Instead, the bioluminescence emission spectra of two double mutants containing Phe250Ser and either Val241Ile or Gly246Ala very closely resembled that of the triple mutant. Additional mutagenesis results confirmed that the blue-shifted emission of the double mutants was determined by the synergistic behavior of active site residues. Molecular modeling studies of the Gly246Ala and Phe250Ser double mutant supported the notion that the blue-shifted emission was due to localized changes that increased the hydrophobicity at the emitter site as a result of the addition of a single methyl group at position 246. Moreover, the modeling data suggested that the Ala246 side chain remained close to the emitter through an additional H-bond between Ala246 and the hydroxyl group of Phe250, providing a possible structural basis for the synergistic behavior.

Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. We present studies on the production of a set of thermostable red- and green-emitting luciferase mutants with bioluminescent properties suitable for dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Starting with the luciferase variant Ser284Thr, we introduced the mutations Thr214Ala, Ala215Leu, Ile232Ala, Phe295Leu, and Glu354Lys to produce a new red-emitting enzyme with a bioluminescence maximum of 610 nm, narrow emission bandwidth, favorable kinetic properties, and excellent thermostability at 37 degrees C. By adding the same five changes to luciferase mutant Val241Ile/Gly246Ala/Phe250Ser, we produced a protein with an emission maximum of 546 nm, providing a set of thermostable enzymes whose bioluminescence maxima were separated by 64 nm. Model studies established that the luciferases could be detected at the attomole level and six orders of magnitude higher. In microplate luminometer format, mixtures containing 1.0 fmol total luciferase were quantified from measurements of simultaneously emitted red and green light. The results presented here provide evidence that it is feasible to monitor two distinct activities at 37 degrees C with these novel thermostable proteins.

The cDNA encoding the luciferase from the Italian firefly Luciola italica was cloned using reverse transcriptase-PCR and a gene-specific primer set based on the DNA sequence of Luciola mingrelica. The cDNA sequence of L. italica luciferase was determined to be 1647 base pairs in length with an open reading frame of 548 amino acids. Phylogenetic analysis of the protein sequence demonstrated that this luciferase is closely related to that of other fireflies of the Lampyridae family, particularly within the Luciolinae subfamily, showing 96% homology to luciferases from the fireflies Hotaria unmunsana and Hotaria parvula. The specific activity of the L. italica luciferase was 78% of the North American enzyme, after correction for emission color differences. The bioluminescence emission of the Italian firefly is pH sensitive with maxima at 566 nm and 614 nm at pH 7.8 and 6.0, respectively. Interestingly, the total bioluminescence output was approximately 2-fold greater than that of P. pyralis luciferase due to differences in turnover characteristics evidenced by extended light emission decay kinetics. We expect that this newly discovered luciferase will be suitable for a wide range of bioluminescence applications including in vivo imaging and multiplex assays.

In the first of two half-reactions resulting in the emission of visible light, firefly luciferase forms luciferyl-adenylate from its natural substrates beetle luciferin and Mg-ATP. The acyl-adenylate is subsequently oxidized producing the light emitter oxyluciferin in an electronically excited state. In vitro, under mild conditions of temperature and pH, the acyl-adenylate intermediate is readily hydrolyzed and susceptible to oxidation. We report here the multi-step synthesis and physical and enzymatic characterization of an N-acyl sulfamate analog of luciferyl-adenylate, 5′-O-[(N-dehydroluciferyl)-sulfamoyl]-adenosine (compound 5). This represents the first example of a stable and potent (Ki = 340 nM) reversible inhibitor of firefly luciferase activity based on the structure of the natural acyl-adenylate intermediate. Additionally, we present the results of limited proteolysis studies that demonstrate that the binding of the novel acyl-adenylate analog protects luciferase from proteolysis. The findings presented here are interpreted in the context of the hypothesis that luciferase and the other enzymes in a large superfamily of adenylate-forming proteins adopt two conformations to catalyze two different partial reactions. We anticipate that the novel N-acyl sulfamate analog will be a valuable reagent in future studies designed to elucidate the role of conformational changes in firefly luciferase catalyzed bioluminescence.

Firefly luciferase catalyzes two sequential partial reactions resulting in the emission of light. The enzyme first catalyzes the adenylation of substrate luciferin with Mg-ATP followed by the multistep oxidation of the adenylate to form the light emitter oxyluciferin in an electronically excited state. The beetle luciferases are members of a large superfamily, mainly comprised of nonbioluminescent enzymes that activate carboxylic acid substrates to form acyl-adenylate intermediates. Recently, the crystal structure of a member of this adenylate-forming family, acetyl-coenzyme A (CoA) synthetase, was determined in complex with an unreactive analogue of its acyl-adenylate and CoA [Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M., and Escalante-Semerena, J. C. (2003) Biochemistry 42, 2866-2873]. This structure presented a new conformation for this enzyme family, in which a significant rotation of the C-terminal domain brings residues of a conserved beta-hairpin motif to interact with the active site. We have undertaken a mutagenesis approach to study the roles of key residues of the equivalent beta-hairpin motif in Photinus pyralis luciferase (442IleLysTyrLysGlyTyrGlnVal449) in the overall production of light and the individual adenylation and oxidation partial reactions. Our results strongly suggest that Lys443 is critical for efficient catalysis of the oxidative half-reaction. Additionally, we provide evidence that Lys443 and Lys529, located on opposite sides of the C-terminal domain and conserved in all firefly luciferases, are each essential for only one of the partial reactions of firefly bioluminescence, supporting the proposal that the superfamily enzymes may adopt two different conformations to catalyze the two half-reactions.

Firefly luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a bioluminescence reporter of gene expression. One particularly exciting application is in the area of in vivo bioluminescence imaging. Our interest is in developing improved reagents by identifying Photinus pyralis luciferase mutants that efficiently emit red bioluminescence. In this way, the proven advantages of the P. pyralis protein can be combined with the potential advantages of a red-shifted emitter. Using site-directed mutagenesis techniques, we have identified many mutants emitting red bioluminescence. Unfortunately, these enzymes generally have significantly decreased bioluminescence activity. Interestingly, we discovered a mutation, Ile351Ala, that produced a moderate 16 nm red-shift, while maintaining excellent bioluminescence activity. We then undertook a random mutagenesis approach to identify luciferase mutants that emit further red-shifted bioluminescence with minimal loss of activity. Libraries of mutants were created using an error-prone PCR method and the Ile351Ala luciferase mutant as the template DNA. The libraries were screened by in vivo bacterial assays and the promising mutants were purified to enable accurate determination of bioluminescence emission spectra and total bioluminescence activity. We will report the characterization results, including the identification of the randomly altered amino acids, of several mutants that catalyze bioluminescence with emission maxima of approximately 600 nm.

Beetle luciferases (including those of the firefly) use the same luciferin substrate to naturally display light ranging in color from green (lambda(max) approximately 530 nm) to red (lambda(max) approximately 635 nm). In a recent communication, we reported (Branchini, B. R., Murtiashaw, M. H., Magyar, R. A., Portier, N. C., Ruggiero, M. C., and Stroh, J. G. (2002) J. Am. Chem. Soc. 124, 2112-2113) that the synthetic adenylate of firefly luciferin analogue D-5,5-dimethylluciferin was transformed into the emitter 5,5-dimethyloxyluciferin in bioluminescence reactions catalyzed by luciferases from Photinus pyralis and the click beetle Pyrophorus plagiophthalamus. 5,5-Dimethyloxyluciferin is constrained to exist in the keto form and fluoresces mainly in the red. However, bioluminescence spectra revealed that green light emission was produced by the firefly enzyme, and red light was observed with the click beetle protein. These results, augmented with steady-state kinetic studies, were taken as experimental support for mechanisms of firefly bioluminescence color that require only a single keto form of oxyluciferin. We report here the results of mutagenesis studies designed to determine the basis of the observed differences in bioluminescence color with the analogue adenylate. Mutants of P. pyralis luciferase putative active site residues Gly246 and Phe250, as well as corresponding click beetle residues Ala243 and Ser247 were constructed and characterized using bioluminescence emission spectroscopy and steady state kinetics with adenylate substrates. Based on an analysis of these and recently reported (Branchini, B. R., Southworth, T. L., Murtiashaw, M. H., Boije, H., and Fleet, S. E. (2003) Biochemistry 42, 10429-10436) data, we have developed an alternative mechanism of bioluminescence color. The basis of the mechanism is that luciferase modulates emission color by controlling the resonance-based charge delocalization of the anionic keto form of the oxyluciferin excited state.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate firefly luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We had previously developed [Branchini, B. R., Magyar, R. A., Murtiashaw, M. H., Anderson, S. M., and Zimmer, M. (1998) Biochemistry 37, 15311-15319] a molecular graphics-based working model of the luciferase active site starting with the first X-ray structure [Conti, E., Franks, N. P., and Brick, P. (1996) Structure 4, 287-298] of the enzyme without bound substrates. In our model, the luciferin binding site contains 15 residues that are within 5 A of the substrate. Using site-directed mutagenesis, we made changes at all of these residues and report here the characterization of the corresponding expressed and purified proteins. Of the 15 residues studied, 12 had a significantly (>or=4-fold K(m) difference) altered binding affinity for luciferin and seven residues, spanning the primary sequence region Arg218-Ala348, had substantially (>or=30 nm) red-shifted bioluminescence emission maxima when mutated. We report here an interpretation of the roles of the mutated residues in substrate binding and bioluminescence color determination. The results of this study generally substantiate the accuracy of our model and provide the foundation for future experiments designed to alter the substrate specificity of firefly luciferase.

Stem cuttings from a prostrate clone of Cephalotaxus harringtonia (Forbes) K. Koch (Japanese plum yew) were taken monthly from Sept. 1994 through Aug. 1995, treated with K-IBA at 0 or 10,000 mgliter -1 , placed in a greenhouse under intermittent mist, and evaluated after 16 weeks. Cuttings taken from December to February and treated with K-IBA averaged 85% rooting, 10 roots per cutting, and a total root length of 35 cm. The next highest rooting percentages were for cuttings taken from March to May; poorest rooting occurred for cuttings taken from June to August and September to November, regardless of K-IBA application. Chemical name used: K-indole-3-butyric acid (K-IBA). ported that stem cuttings can be rooted year- round, except during the spring growth flush period, with or without auxin treatment. Root- ing percentages of mid-March cuttings treated with indole-3-butyric acid (IBA) as a talc formulation and nontreated cuttings were simi- lar (Dirr and Heuser, 1987). According to commercial growers, rooting may take ≥6 months, and plants often develop slowly be- cause of sparse root systems. Producing a salable plant often takes 2 to 3 years (Tripp, 1994). This study was initiated to determine the effects of timing and auxin treatment on the rooting of C. harringtonia stem cuttings. Materials and Methods