Genome assembly of Genji firefly (Nipponoluciola cruciata) reveals novel luciferase-like luminescent proteins without peroxisome targeting signal.

The Genji firefly, Nipponoluciola cruciata, is an aquatic firefly endemic to Japan, inhabiting a wide area of the Japanese archipelago. The luminescence of fireflies is a scientifically interesting phenomenon, and many studies have evaluated this species in Japan. In this study, we sequenced the whole genome of male N. cruciata and constructed a high-quality genome assembly of 662 Mb with a BUSCO completeness of 99.1% in the genome mode. Using the detected set of 15,169 protein-coding genes, the genomic structures and genetic background of luminescence-related genes were also investigated. We found four new firefly luciferase-like genes in the genome. The highest bioluminescent activity was observed for LLa2, which originated from ancestral PDGY, a mitochondrial acyl-CoA synthetase. A thioesterase candidate, NcruACOT1, which is involved in d-luciferin biosynthesis, was expressed in the lantern. Two opsins were also detected and the absorption wavelength of the UV-type opsin candidate shifted from UV to blue. These findings provide an important resource for unravelling the adaptive evolution of fireflies in terms of luminescence and vision.

Quantitative Analysis of Bioluminescence Optical Signal.

Bioluminescence is light emission based on the luciferin-luciferase enzymatic reaction in living organisms. Optical signals from bioluminescence (BL) reactions are available for bioanalysis and bioreporters for gene expression, in vitro, in vivo, and ex vivo bioimaging, immunoassay, and other applications. Although there are numerous bioanalysis methods based on BL signal measurements, the BL signal is measured as a relative value, and not as an absolute value. Recently, some approaches have been established to completely quantify the BL signal, resulting in, for instance, the redetermination of the quantum yield of the BL reaction and counting the photon number of the BL signal at the single-cell level. Reliable and reproducible understanding of biological events in the bioanalysis and bioreporter fields can be achieved by means of standardized absolute optical signal measurements, which is described in an International Organization for Standardization (ISO) document.

Quantum yield enhancement of firefly bioluminescence with biomolecular condensates.

The enzymatic luminescence reactions of fireflies are accelerated in the presence of biomolecular condensates comprising a positively charged peptide and ATP. We revealed that this acceleration is caused by the enrichment of reaction elements, local pH changes, and promotion of inhibitory intermediate dissociation, improving the bioluminescence quantum yield by approximately 10%.

Biosynthesis-Inspired Deracemizative Production of D-Luciferin In Vitro by Combining Luciferase and Thioesterase.

Due to the strict enantioselectivity of firefly luciferase (FLuc), only D-luciferin can be used as a substrate for the bioluminescence (BL) reaction. Unfortunately, luciferin racemizes easily and accumulation of nonluminous L-luciferin has negative influences on the light-emitting reaction. By a detailed analysis of luciferin chirality, however, it becomes clarified that L-luciferin is the biosynthetic precursor of D-luciferin in fireflies and undergoes the enzymatic chiral inversion. By the chiral inversion reaction, the enantiopurity of luciferin can be maintained in the reaction mixture for applications using FLuc. Thus, chirality is crucial for the BL reaction and essential for investigating and applying the biosynthesis of D-luciferin. Here, we describe the methods for the analysis of chiral inversion reaction using high-performance liquid chromatography (HPLC) with a chiral column. We also introduce an example of an in vitro deracemizative BL reaction system using a combination of FLuc and fatty acyl-CoA thioesterase, which is inspired by the chiral inversion mechanism in the biosynthetic pathway of D-luciferin.

Few-Photon Spectral Confocal Microscopy for Cell Imaging Using Superconducting Transition Edge Sensor.

A superconducting transition edge sensor (TES) is an energy-dispersive single-photon detector that distinguishes the wavelength of each incident photon from visible to near-infrared (NIR) without using spectral dispersive elements. Here, we introduce an application of the TES technique for confocal laser scanning microscopy (CLSM) as proof of our concept of ultra-sensitive and wide-band wavelength range color imaging for biological samples. As a reference sample for wide-band observation, a fixed fluorescence-labeled cell sample stained with three different color dyes was observed using our TES-based CLSM method. The three different dyes were simultaneously excited by irradiating 405 and 488 nm lasers, which were coupled using an optical fiber combiner. Even when irradiated at low powers of 80 and 120 nW with the 405 and 488 nm lasers respectively, emission signals were spectrally detected by the TES and categorized into four wavelength bands: up to 500 nm (blue), from 500 to 600 nm (green), from 600 to 800 nm (red), and from 800 to 1,200 nm (NIR). Using a single scan, an RGB color image and an NIR image of the fluorescent cell sample were successfully captured with tens of photon signals in a 40 ms exposure time for each pixel. This result demonstrates that TES is a useful wide-band spectral photon detector in the field of life sciences.

Coelenterazine Analogue with Human Serum Albumin-Specific Bioluminescence.

A synthetic luciferin comprising an imidazopyrazinone core, named HuLumino1, was designed to generate specific bioluminescence with human serum albumin (HSA) in real serum samples. HuLumino1 was developed by attaching a methoxy-terminated alkyl chain to C-6 of coelenterazine and by eliminating a benzyl group at C-8. HSA levels were quantified within 5% error margins of an enzyme-linked immunosorbent assay without the need for any sample pretreatments because of the high specificity of HuLumino1.

Evidence-based Care Bundles for Preventing Surgical Site Infections in Spinal Instrumentation Surgery.

STUDY DESIGN: A retrospective study, using prospectively collected data. OBJECTIVE: The aim of this study was to evaluate the impact of evidence-based care bundles for preventing surgical site infections (SSIs) in spinal instrumentation surgery. SUMMARY OF BACKGROUND DATA: About half of all SSIs are preventable via evidence-based methods. For successful SSI prevention, the bacterial load must be minimized, and methicillin-resistant Staphylococcus aureus (MRSA) protection must be maximized. However, it is difficult to cover all of these requirements by single preventative method. METHODS: We screened consecutive patients scheduled for spinal instrumentation surgeries at a single tertiary referral hospital for high surgical, SSI, and MRSA colonization risks. Evidence-based care bundles were implemented for high-risk patients and included 1) additional vancomycin prophylaxis, 2) diluted povidone-iodine irrigation, and 3) nasal and body decontamination. Patient demographics, comorbidities, operative features, and SSIs reported to the Japanese Nosocomial Infections Surveillance system were prospectively obtained in the same method by the same assessor and were used for the analyses. The results were compared before and after the application of the bundle. RESULTS: There were 1042 spinal instrumentation surgeries (741 before and 301 after care bundles) performed from November 2010 to December 2015. Of 301 surgeries, 57 cases (18.9%) received care bundles. There were no significant differences in patient backgrounds before and after the intervention. The SSI rate decreased significantly from 3.8% to 0.7% (P < 0.01) after the intervention, with an overall 82% relative risk reduction. A significant protective effect was observed in the multivariate analysis (adjusted odds ratio 0.18, 95% confidence interval: 0.04-0.77, P = 0.02). There were no MRSA-related SSIs among those that received care bundles, even though MRSA was the predominant pathogen in the study population. CONCLUSION: Evidence-based care bundles, applied in selected high-risk spinal instrumentation cases, minimized bacterial load, maximized MRSA protection, and significantly reduced SSI rates without topical vancomycin powder. LEVEL OF EVIDENCE: 4.

An allylated firefly luciferin analogue with luciferase specific response in living cells.

An allylated firefly luciferin was successfully synthesized and its bioluminescence properties were evaluated. When applied to cellular imaging in combination with Eluc, which is one of the commercially available luciferases, this analogue displayed a luciferase-specific bioluminescence signal with prolonged emission (>100 min).

Biosynthesis-inspired deracemizative production of d-luciferin by combining luciferase and thioesterase.

Due to the strict enantioselectivity of firefly luciferase, only d-luciferin can be used as a substrate for bioluminescence reactions. Unfortunately, luciferin racemizes easily and accumulation of nonluminous l-luciferin has negative influences on the light emitting reaction. Thus, maintaining the enantiopurity of luciferin in the reaction mixture is one of the most important demands in bioluminescence applications using firefly luciferase. In fireflies, however, l-luciferin is the biosynthetic precursor of d-luciferin, which is produced from the L-form undergoing deracemization. This deracemization consists of three successive reactions: l-enantioselective thioesterification by luciferase, in situ epimerization, and hydrolysis by thioesterase. In this work, we introduce a deracemizative luminescence system inspired by the biosynthetic pathway of d-luciferin using a combination of firefly luciferase from Luciola cruciata (LUC-G) and fatty acyl-CoA thioesterase II from Escherichia coli (TESB). The enzymatic reaction property analysis indicated the importance of the concentration balance between LUC-G and TESB for efficient d-luciferin production and light emission. Using this deracemizative luminescence system, a highly sensitive quantitative analysis method for l-cysteine was constructed. This LUC-G-TESB combination system can improve bioanalysis applications using the firefly bioluminescence reaction by efficient deracemization of D-luciferin.

Few-photon color imaging using energy-dispersive superconducting transition-edge sensor spectrometry.

Highly sensitive spectral imaging is increasingly being demanded in bioanalysis research and industry to obtain the maximum information possible from molecules of different colors. We introduce an application of the superconducting transition-edge sensor (TES) technique to highly sensitive spectral imaging. A TES is an energy-dispersive photodetector that can distinguish the wavelength of each incident photon. Its effective spectral range is from the visible to the infrared (IR), up to 2800 nm, which is beyond the capabilities of other photodetectors. TES was employed in this study in a fiber-coupled optical scanning microscopy system, and a test sample of a three-color ink pattern was observed. A red-green-blue (RGB) image and a near-IR image were successfully obtained in the few-incident-photon regime, whereas only a black and white image could be obtained using a photomultiplier tube. Spectral data were also obtained from a selected focal area out of the entire image. The results of this study show that TES is feasible for use as an energy-dispersive photon-counting detector in spectral imaging applications.

[Investigation of the Pathophysiology and Diagnostic Methods Through a Case of Acute HIV Infection in which the Western Blot Method Remained Negative Over the Long Term and the 4th Generation Screening Assay Became Negative for a Certain Period of Time after Initiating Antiretroviral Therapy].

Confirmatory tests using Western blot (WB) and HIV-1 nucleic acid testing (HIV-1 RNA) following a positive screening test are required for the diagnosis of HIV-1 infection according to the current Japanese guidelines for HIV-1/2 diagnosis. We report herein on a rare case in a patient who remained negative for WB over 10 months in spite of being positive by fourth-generation immunoassays (4thGIA) and who subsequently seroreverted by 4thGIA for three months after initiating antiretroviral therapy. Case: A man in his early twenties previously visited a hospital because of fever in October 2012. Laboratory data revealed leukocytopenia, thrombocytopenia and increased serum ferritin, suggesting hemophagocytic syndrome (HPS). During that visit, he tested positive for a 4thGIA, but negative for HIV-1 WB and his result of HIV-1 RNA result was detected invalid because of the presence of some inhibitory material in his RNA preparation. Thereafter, he was diagnosed as having cytomegalovirus-associated HPS treatment was for which initiated. In January 2013, he developed Pneumocystis jirovecii pneumonia, and his HIV-1 RNA viral load was 7.7 × 105 copies/mL in February 2013. Acute HIV infection was suspected, because the HIV-1 WB remained negative. He was started on antiretroviral therapy in April 2013. His 4thGIA was converted to negative in May 2013 and was reconverted to positive in August 2013. HIV-1 WB, however, continued to be indeterminant until February 2014, in which it turned positive for the first time according to the CDC criteria. Methods and Results: The genetic analyses of HIV-1 were done on the gag, env, nef and pol region of the HIV-1 gene from the patient. There was no clear element to delay antibody production on the virus side. Preserved specimens of the patient were measured with eight kinds of HIV screening assay. It was thought that the fourth generation assay was positive only by the presence of the antigen until March 2013 because the antibody had not been detected. Discussion: We encountered a case of acute HIV infection in which the WB result was negative for 10 months after the first positive response of the 4thGIA. The 4thGIA is essential for the early diagnosis and early treatment of HIV infection; therefore, the 4thGIA should be strictly recommended to avoid the use of older generations of immunoassay in the diagnostic guidelines. The role of the WB test should be examined closely from various aspects for use as a confirmatory test under recent laboratory situations in which highly sensitive and specific methods, e.g. the 4th GIA, have become available. In addition, unnecessary confusion due to the diversities of antibody formation should be avoided. The antibody detection tests for HIV are still necessary and indispensable for the confirmation of the disease or the diagnosis of the acute infection stage. Therefore development of a newer antibody measuring method which could achieve an easier operation and should have a higher sensitivity and specificity for HIV confirmation is strongly expected.

Pathological background of subcortical hyperintensities on diffusion-weighted images in a case of neuronal intranuclear inclusion disease.

AIMS: Neuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disorder with eosinophilic intranuclear inclusion bodies. The variable symptoms of NIID increase the difficulty in an antemortem diagnosis. NIID shows leukoencephalopathy on brain magnetic resonance imaging MRI, but the significance of the radiological findings have not been clarified. METHODS: We examined an autopsied case of NIID with subcortical linear hyperintensities on diffusion weighted imaging (DWI) and leukoencephalopathy on fluid attenuation inversion recovery (FLAIR) imaging. Semiquantitative analysis was performed by merging coronal sections of DWI and identical hematoxylin-eosin (H & E) stained brain specimens. The severity of spongiotic changes, the common pathological findings of NIID, were quantified and compared with MRI lesions classified by DWI signals. RESULTS: The white matter showed diffuse myelin pallor, and multiple focal spongiotic changes were present in the subcortical white matter proximal to the U-fibers. Spongiotic changes were restricted in the lesions with subcortical linear DWI high signals. CONCLUSION: Subcortical DWI high signals in NIID strongly correlate with pathological spongiotic changes of NIID. Subcortical spongiotic changes may be a characteristic finding of NIID.
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Quantum Yield Determination Based on Photon Number Measurement, Protocols for Firefly Bioluminescence Reactions.

Quantum yield (QY), which is defined as the probability of photon production by a single bio/chemiluminescence reaction, is an important factor to characterize luminescence light intensity emitted diffusively from the reaction solution mixture. Here, methods to measure number of photons to determine QY according to the techniques of national radiometry standards are described. As an example, experiments using firefly bioluminescence reactions are introduced.

Highly sensitive luciferase reporter assay using a potent destabilization sequence of calpain 3.

Reporter assays that use luciferases are widely employed for monitoring cellular events associated with gene expression in vitro and in vivo. To improve the response of the luciferase reporter to acute changes of gene expression, a destabilization sequence is frequently used to reduce the stability of luciferase protein in the cells, which results in an increase of sensitivity of the luciferase reporter assay. In this study, we identified a potent destabilization sequence (referred to as the C9 fragment) consisting of 42 amino acid residues from human calpain 3 (CAPN3). Whereas the half-life of Emerald Luc (ELuc) from the Brazilian click beetle Pyrearinus termitilluminans was reduced by fusing PEST (t1/2=9.8 to 2.8h), the half-life of C9-fused ELuc was significantly shorter (t1/2=1.0h) than that of PEST-fused ELuc when measurements were conducted at 37°C. In addition, firefly luciferase (luc2) was also markedly destabilized by the C9 fragment compared with the humanized PEST sequence. These results indicate that the C9 fragment from CAPN3 is a much more potent destabilization sequence than the PEST sequence. Furthermore, real-time bioluminescence recording of the activation kinetics of nuclear factor-κB after transient treatment with tumor necrosis factor α revealed that the response of C9-fused ELuc is significantly greater than that of PEST-fused ELuc, demonstrating that the use of the C9 fragment realizes a luciferase reporter assay that has faster response speed compared with that provided by the PEST sequence.

A firefly inspired one-pot chemiluminescence system using n-propylphosphonic anhydride (T3P).

A simple reaction procedure for chemiluminescence of firefly luciferin (D-luc) using n-propylphosphonic anhydride (T3P) is reported. A luminescent photon is produced as a result of one-pot reaction, only requiring mixing with the substrate carboxylic acid and T3P in the presence of a mild organic base.

ABCG2 dysfunction causes hyperuricemia due to both renal urate underexcretion and renal urate overload.

Gout is a common disease which results from hyperuricemia. We have reported that the dysfunction of urate exporter ABCG2 is the major cause of renal overload (ROL) hyperuricemia, but its involvement in renal underexcretion (RUE) hyperuricemia, the most prevalent subtype, is not clearly explained so far. In this study, the association analysis with 644 hyperuricemia patients and 1,623 controls in male Japanese revealed that ABCG2 dysfunction significantly increased the risk of RUE hyperuricemia as well as overall and ROL hyperuricemia, according to the severity of impairment. ABCG2 dysfunction caused renal urate underexcretion and induced hyperuricemia even if the renal urate overload was not remarkable. These results show that ABCG2 plays physiologically important roles in both renal and extra-renal urate excretion mechanisms. Our findings indicate the importance of ABCG2 as a promising therapeutic and screening target of hyperuricemia and gout.

Common dysfunctional variants in ABCG2 are a major cause of early-onset gout.

Gout is a common disease which mostly occurs after middle age, but more people nowadays develop it before the age of thirty. We investigated whether common dysfunction of ABCG2, a high-capacity urate transporter which regulates serum uric acid levels, causes early-onset gout. 705 Japanese male gout cases with onset age data and 1,887 male controls were genotyped, and the ABCG2 functions which are estimated by its genotype combination were determined. The onset age was 6.5 years earlier with severe ABCG2 dysfunction than with normal ABCG2 function (P = 6.14 × 10(-3)). Patients with mild to severe ABCG2 dysfunction accounted for 88.2% of early-onset cases (twenties or younger). Severe ABCG2 dysfunction particularly increased the risk of early-onset gout (odds ratio 22.2, P = 4.66 × 10(-6)). Our finding that common dysfunction of ABCG2 is a major cause of early-onset gout will serve to improve earlier prevention and therapy for high-risk individuals.

Interconversion of ketoprofen recognition in firefly luciferase-catalyzed enantioselective thioesterification reaction using from Pylocoeria miyako (PmL) and Hotaria parvura (HpL) just by mutating two amino acid residues.

We identified the critical amino acid residues for substrate recognition using two firefly luciferases from Pylocoeria miyako (PmL) and Hotaria parvura (HpL), as these two luciferase enzymes exhibit different activities toward ketoprofen. Specifically, PmL can catalyze the apparent enantioselective thioesterification reaction, while HpL cannot. By comparing the amino acid sequences around the active site, we identified two residues (I350 and M397 in PmL and F351 and S398 in HpL) that were different between the two enzymes, and the replacement of these amino acids resulted in changing the ketoprofen recognition pattern. The inactive HpL was converted to the active enzyme toward ketoprofen and vice versa for PmL. These residues also affected the enantioselectivity toward ketoprofen; however, the bioluminescent color was not affected. In addition, using molecular dynamics calculations, the replacement of these two amino acids induced changes in the state of hydrogen bonding between ketoprofen and the S349 side chain through the active site water. As S349 is not considered to influence color tuning, these changes specifically caused the differences in ketoprofen recognition in the enzyme.

Cloning and characterization of luciferase from a Fijian luminous click beetle.

Luminous click beetle is distributed almost exclusively in Central and South America with a single genus in Melanesia. Among these click beetles, the description of Melanesian species has been fragmentary, and its luciferase gene and phylogenetic relation to other click beetles still remain uncertain. We collected a living luminous click beetle, Photophorus jansonii in Fiji. It emits green-yellow light from two spots on the pronotum and has no ventral luminous organ. Here, we cloned a luciferase gene from this insect by RT-PCR. The deduced amino acid sequence showed high identity of ~85% to the luciferases derived from other click beetle species. The luciferase of the Fijian click beetle was produced as a recombinant protein to characterize its biochemical properties. The Km for D-luciferin and ATP were 173 and 270 µm, respectively. The luciferase was pH-insensitive and the spectrum measured at pH 8.0 showed a peak at 559 nm, which was in the range of green-yellow light as seen in the luminous spot of the living Fijian click beetle. The Fijian click beetle luciferase was assigned to the Elateridae clade by a phylogenetic analysis, but it made a clearly different branch from Pyrophorus group examined in this study.