Activation of Protease and Luciferase Using Engineered Nostoc punctiforme PCC73102 DnaE Intein with Altered Split Position

Inteins, self-catalytic enzymes, have been widely used in the field of protein engineering and chemical biology. Here, Nostoc punctiforme PCC73102 (Npu) DnaE intein was engineered to have an altered split position. An 11-residue N-intein of DnaE in which Gly and Asp were substituted for Tyr4 and Glu5, respectively, was designed, and the active C-intein variants were acquired by a GFP fluorescence-based screening. The designed N-intein and the obtained active C-intein variants were used to construct a turn-on system for enzyme activities such as human immunodeficiency 1 protease and NanoLuc luciferase. Based on the NanoLuc-intein fusion, we developed two intein pairs, each of which is capable of reacting preferentially, by interchanging the charged amino acids on N- and C-inteins. The specific splicing reactions were easily monitored and discriminated by bioluminescence resonance energy transfer (BRET).     

A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6′-Amino-Analogs for Bioimaging Purposes

Beetle luciferases produce bioluminescence (BL) colors ranging from green to red, having been extensively used for many bioanalytical purposes, including bioimaging of pathogen infections and metastasis proliferation in living animal models and cell culture. For bioimaging purposes in mammalian tissues, red bioluminescence is preferred, due to the lower self-absorption of light at longer wavelengths by hemoglobin, myoglobin and melanin. Red bioluminescence is naturally produced only by Phrixothrix hirtus railroad worm luciferase (PxRE), and by some engineered beetle luciferases. However, Far-Red (FR) and Near-Infrared (NIR) bioluminescence is best suited for bioimaging in mammalian tissues due to its higher penetrability. Although some FR and NIR emitting luciferin analogs have been already developed, they usually emit much lower bioluminescence activity when compared to the original luciferin-luciferases. Using site-directed mutagenesis of PxRE luciferase in combination with 6′-modified amino-luciferin analogs, we finally selected novel FR combinations displaying BL ranging from 636–655 nm. Among them, the combination of PxRE-R215K mutant with 6′-(1-pyrrolidinyl)luciferin proved to be the best combination, displaying the highest BL activity with a catalytic efficiency ~2.5 times higher than the combination with native firefly luciferin, producing the second most FR-shifted bioluminescence (650 nm), being several orders of magnitude brighter than commercial AkaLumine with firefly luciferase. Such combination also showed higher thermostability, slower BL decay time and better penetrability across bacterial cell membranes, resulting in ~3 times higher in vivo BL activity in bacterial cells than with firefly luciferin. Overall, this is the brightest FR emitting combination ever reported, and is very promising for bioimaging purposes in mammalian tissues.     

Image-Based In Vitro Screening Reveals the Trypanostatic Activity of Hydroxymethylnitrofurazone against Trypanosoma cruzi

Hydroxymethylnitrofurazone (NFOH) is a therapeutic candidate for Chagas disease (CD). It has negligible hepatotoxicity in a murine model compared to the front-line drug benznidazole (BZN). Here, using Trypanosoma cruzi strains that express bioluminescent and/or fluorescent reporter proteins, we further investigated the in vitro and in vivo activity of NFOH to define whether the compound is trypanocidal or trypanostatic. The in vitro activity was assessed by exploiting the fluorescent reporter strain using wash-out assays and real-time microscopy. For animal experimentation, BALB/c mice were inoculated with the bioluminescent reporter strain and assessed by highly sensitive in vivo and ex vivo imaging. Cyclophosphamide treatment was used to promote parasite relapse in the chronic stage of infection. Our data show that NFOH acts by a trypanostatic mechanism, and that it is more active than BZN in vitro against the infectious trypomastigote form of Trypanosoma cruzi. We also found that it is more effective at curing experimental infections in the chronic stage, compared with the acute stage, a feature that it shares with BZN. Therefore, given its reduced toxicity, enhanced anti-trypomastigote activity, and curative properties, NFOH can be considered as a potential therapeutic option for Chagas disease, perhaps in combination with other trypanocidal agents.     

一种新的多光谱光学参数等效方法

通过建立与分析生物发光断层成像的系统方程,提出了一种新的多光谱光学参数等效方法,将多光谱生物发光断层成像问题转换成与之等价的混合谱问题,从而有效降低了系统方程的规模和计算量.研究结果表明:新提出的光源加权法,比现有的算术平均法更加准确、鲁棒,相对误差始终在5%以内.     

Identification of luciferyl adenylate and luciferyl coenzyme a synthesized by firefly luciferase

The firefly luciferase reaction intermediate luciferyl adenylate was detected by RP-HPLC analysis when the luciferase reaction was performed under a nitrogen atmosphere. Although this compound is always specified as an intermediate in the light-production reaction, this is the first report of its identification by HPLC in a luciferase assay medium. Under a low-oxygen atmosphere, luciferase can catalyze the synthesis of luciferyl coenzyme A from luciferin, ATP, and coenzyme A, but in air dehydroluciferyl coenzyme A was produced. The luciferase-catalyzed synthesis of these coenzyme A derivatives may be a consequence of the postulated recent evolutionary origin of firefly luciferases from an ancestral acyl-coenzyme A synthetase.     

发光细菌法在环境污染物监测中的进展与应用

发光细菌法具有快速、灵敏且成本低廉等诸多优点,因此在环境污染物监测领域备受关注。该文主要介绍了发光细菌的发光机理、供试淡水菌种、发光细菌检测仪的开发及检测方法的改进优化,同时对目前国内利用发光细菌法在环境污染物监测中的应用情况进行了详细阐述。     

Bettering Biology?

Is there a danger that in inventing protocells we are turning away too quickly from nature's own ‘beautifully engineered self-assembly system’? Bill Watts , a partner at Max Fordham Consulting Engineers, asks us to take another look at the possibilities of biology for creating a wholly sustainable architecture that takes its aesthetic prompts from natural forms. Copyright © 2011 John Wiley & Sons, Ltd.     

Biochemical Analysis Leads to Improved Orthogonal Bioluminescent Tools

Engineered luciferase-luciferin pairs have expanded the number of cellular targets that can be visualized in tandem. While light production relies on selective processing of synthetic luciferins by mutant luciferases, little is known about the origin of selectivity. The development of new and improved pairs requires a better understanding of the structure−function relationship of bioluminescent probes. In this work, we report a biochemical approach to assessing and optimizing two popular bioluminescent pairs: Cashew/d -luc and Pecan/4′-BrLuc. Single mutants derived from Cashew and Pecan revealed key residues for selectivity and thermal stability. Stability was further improved through a rational addition of beneficial residues. In addition to providing increased stability, the known stabilizing mutations surprisingly also improved selectivity. The resultant improved pair of luciferases are >100-fold selective for their respective substrates and highly thermally stable. Collectively, this work highlights the importance of mechanistic insight for improving bioluminescent pairs and provides significantly improved Cashew and Pecan enzymes which should be immediately suitable for multicomponent imaging applications.     

How to Select Firefly Luciferin Analogues for In Vivo Imaging

Bioluminescence reactions are widely applied in optical in vivo imaging in the life science and medical fields. Such reactions produce light upon the oxidation of a luciferin (substrate) catalyzed by a luciferase (enzyme), and this bioluminescence enables the quantification of tumor cells and gene expression in animal models. Many researchers have developed single-color or multicolor bioluminescence systems based on artificial luciferin analogues and/or luciferase mutants, for application in vivo bioluminescence imaging (BLI). In the current review, we focus on the characteristics of firefly BLI technology and discuss the development of luciferin analogues for high-resolution in vivo BLI. In addition, we discuss the novel luciferin analogues TokeOni and seMpai, which show potential as high-sensitivity in vivo BLI reagents.     

NIR-II Self-Luminous Molecular Probe for In Vivo Inflammation Tracking and Cancer PDT Effect Self-Evaluating

Optical imaging in the second near-infrared (NIR-II, 900–1700 nm) window has been extensively investigated for bioimaging. However, a strong autofluorescence background from real-time excitation light significantly reduces the images’ quality of NIR-II fluorescence (FL) imaging. To resolve this issue, a NIR-II self-luminous small molecule (CLPD) based on bioluminescence (BL) resonance energy transfer (BRET) mechanism is first developed. The reactive oxygen species (ROS) can trigger NIR-II BL and reduce the NIR-II FL signals of the CLPD simultaneously, enabling ROS-correlated ratiometric BL/FL imaging. CLPD is used for high-contrast NIR-II BL imaging of osteoarthritis as well as guiding the treatment process by ratiometric BL/FL imaging. Moreover, CLPD is applied for NIR-II BL imaging of tumor triggered by the generated ROS during PDT. A correlation between the ratiometric NIR-II BL/FL signal and tumor size is constructed, providing a trustworthy tool for early assessment of PDT effect. Overall, this study presents a novel NIR-II self-luminous small molecular probe for in vivo imaging and provides a strategy for design a self-evaluation system of therapeutic effect.