Quorum sensing inhibitors (QSIs), as a kind of ideal antibiotic substitutes, have been recommended to be used in combination with traditional antibiotics in medical and aquaculture fields. Due to the co-existence of QSIs and antibiotics in environmental media, it is necessary to evaluate their joint risk. However, there is little information about the acute toxicity of mixtures for QSIs and antibiotics. In this study, 10 QSIs and 3 sulfonamides (SAs, as the representatives for traditional antibiotics) were selected as the test chemicals, and their acute toxic effects were determined using the bioluminescence of Aliivibrio fischeri (A. fischeri) as the endpoint. The results indicated that SAs and QSIs all induced S-shaped dose-responses in A. fischeri bioluminescence. Furthermore, SAs possessed greater acute toxicity than QSIs, and luciferase (Luc) might be the target protein of test chemicals. Based on the median effective concentration (EC50) for each test chemical, QSI-SA mixtures were designed according to equitoxic (EC50(QSI):EC50(SA) = 1:1) and non-equitoxic ratios (EC50(QSI):EC50(SA) = 1:10, 1:5, 1:0.2, and 1:0.1). It could be observed that with the increase of QSI proportion, the acute toxicity of QSI-SA mixtures enhanced while the corresponding TU values decreased. Furthermore, QSIs contributed more to the acute toxicity of test binary mixtures. The joint toxic actions of QSIs and SAs were synergism for 23 mixtures, antagonism for 12 mixtures, and addition for 1 mixture. Quantitative structure-activity relationship (QSAR) models for the acute toxicity QSIs, SAs, and their binary mixtures were then constructed based on the lowest CDOCKER interaction energy (Ebind-Luc) between Luc and each chemical and the component proportion in the mixture. These models exhibited good robustness and predictive ability in evaluating the toxicity data and joint toxic actions of QSIs and SAs. This study provides reference data and applicable QSAR models for the environmental risk assessment of QSIs, and gives a new perspective for exploring the joint effects of QSI-antibiotic mixtures.

Bioluminescence imaging (BLI) is a powerful method for visualizing biological processes and tracking cells. Engineered bioluminescent bacteria that utilize luciferase-catalyzed biochemical reactions to generate luminescence have become useful analytical tools for in vitro and in vivo bacterial imaging. Accordingly, this review initially introduces the development of engineered bioluminescent bacteria that use different luciferase-luciferin pairs as analytical tools and their applications for in vivo BLI, including real-time bacterial tracking of infection, probiotic investigation, tumor-targeted therapy, and drug screening. Applications of engineered bioluminescent bacteria as whole-cell biosensors for sensing biological changes in vitro and in vivo are then discussed. Finally, we review the optimizations and future directions of bioluminescent bacteria for imaging. This review aims to provide fundamental insights into bacterial BLI and highlight the potential development of this technique in the future.

The current luminescent bacteria test for acute toxicity with short contact time was invalid for antibiotics, and the non-uniformed contact times reported in the literature for long-term toxicity assessment led to incomparable results. Herein, a representative long-term toxicity assessment method was established which unified the contact time of antibiotics and Vibrio fischeri within the bioluminescence increasing period (i.e. 10-100% maximum luminescence) of control samples. The effects of excitation and detoxification of antibiotics such as β-lactams were discovered. Half maximal inhibitory concentration (IC50) of toxic antibiotics (0.00069-0.061 mmol/L) obtained by this method was 2-3 orders of magnitude lower than acute test, quantifying the underestimated toxicity. As antibiotics exist in natural water as mixtures, an equivalent concentration addition (ECA) model was built to predict mixture toxicity based on physical mechanism rather than mathematical method, which showed great fitting results (R2 = 0.94). Furthermore, interaction among antibiotics was investigated. Antibiotics acting during bacterial breeding period had strong synergistic inhibition (IC50 relative deviation from 0.1 to 0.6) such as macrolides and quinolones. Some antibiotics produced increasing synergistic inhibition during concentration accumulation, such as macrolides. The discharge of antibiotics with severe long-term toxicity and strong synergistic inhibition effect should be seriously restricted.

Zeolitic imidazolate framework (ZIF) is of wide interest in biomedical applications due to its extraordinary properties such as high storage capacity, functionality and favorable biocompatibility. However, more comprehensive safety assessments are still essential before ZIF is broadly used in biomedicine. Using the characteristic that aldehyde groups on the surface of ZIF-90 can be modified with other functional groups, a series of ZIF-90s modified with different functional groups (oxime group, carboxyl group, amino group and sulfhydryl group) were synthesized to investigate the effect of functionalization on the toxicity of ZIF-90. ZIF-90 series showed concentration-dependent toxic effects on Photobacterium phosphoreum T3 and the functionalized ZIF-90s are more toxic than pristine ZIF-90, with the ZIF-90 modified with amino group (ZIF-90-NH2) showing the strongest toxicity (IC50 = 23.06 mg/L). Based on the results of the cellular assay and stability exploration, we concluded that corresponding imidazole-ligand release and the property of positively charged are responsible for the elevated toxicity of ZIF-90-NH2. Cell membrane damage, oxidative damage and luminescence damage are the main contributors to the toxic effects of ZIF-90 series. This study explored the effect of surface functionalization on the toxicity of ZIF and proposed mechanistic clues for the safety application of ZIF.

Bioluminescence in beetles has long fascinated biologists, with diverse applications in biotechnology. To date, however, our understanding of its evolutionary origin and functional variation mechanisms remains poor. To address these questions, we obtained high-quality reference genomes of luminous and nonluminous beetles in 6 Elateroidea families. We then reconstructed a robust phylogenetic relationship for all luminous families and related nonluminous families. Comparative genomic analyses and biochemical functional experiments suggested that gene evolution within Elateroidea played a crucial role in the origin of bioluminescence, with multiple parallel origins observed in the luminous beetle families. While most luciferase-like proteins exhibited a conserved nonluminous amino acid pattern (TLA346 to 348) in the luciferin-binding sites, luciferases in the different luminous beetle families showed divergent luminous patterns at these sites (TSA/CCA/CSA/LVA). Comparisons of the structural and enzymatic properties of ancestral, extant, and site-directed mutant luciferases further reinforced the important role of these sites in the trade-off between acyl-CoA synthetase and luciferase activities. Furthermore, the evolution of bioluminescent color demonstrated a tendency toward hypsochromic shifts and variations among the luminous families. Taken together, our results revealed multiple parallel origins of bioluminescence and functional divergence within the beetle bioluminescent system.

External light sources, such as lasers, light emitting diodes (LEDs) and lamps, are widely applied in photodynamic therapy (PDT); however, their use is severely limited by the nature of shallow tissue penetration depth. The recent exploration of light delivery or local generation on tumor sites has attracted much attention, owing to the fact that these systems are significantly endowed with high tissue penetration. In this review, we briefly introduced the principle of \"on-spot light generation or delivery systems\" in PDT. These systems are divided into different categories: (1) implantable luminescence, (2) mechanoluminescence, (3) electrochemiluminescence, (4) Cerenkov luminescence, (5) chemiluminescence, and (6) bioluminescence. Finally, their applications, advantages, and disadvantages in PDT will be appropriately summarized and further discussed in detail. We believe that this review will provide general guidance for the further design of light generation or delivery systems and clinical studies for PDT-mediated cancer treatments with unparalleled merits.

Self-luminous probes with near-infrared (NIR) emission are powerful tools for deep-penetration and autofluorescence-free imaging, owing to the joint optimization of both excitation and emission. However, the limited emission wavelength and requirement for multistep energy transfer limit its potency. In this study, the concept of direct wavelength conversion is established from visible light (vis) to NIR-IIb using an exquisitely designed sensitizer-activator ion pair. The manipulation of the doping hosts enables a pair of energy levels between the sensitizer and activator. Based on this a class of broadband vis-responsive nanocrystals with intense NIR-II emission is prepared. The stability and quantum yield (up to 7.4%) of the nanocrystals are further enhanced by ZnS passivation via coherent epitaxial growth. By coupling luciferase, the self-luminous probe can convert bioluminescence to NIR-IIb luminescence (>1500 nm) through a one-step energy transfer. A maximum penetrable thickness of 6 mm is achieved in the porcine tissue model. Collectively, the distinctive photon-conversion performance of this probe offers the prospect of high-resolution labeling of deep-seated lesions.

Assessing heavy metal bioavailability and toxicity during bioprocess is critical for advancing green biotechnology. The capability of whole-cell bioreporters to measure heavy metal bioavailability has been increasingly recognized. The advantages of this technology being applied to bioprocess monitoring are less studied. Here we investigate the potential of a cadmium- and lead-sensitive bioreporter to be used for heavy metals as a class, which holds great interest for bioprocess applications. We evaluated the bioavailability of eight individual heavy metals with bioreporter zntA, as well as the bioavailability and toxicity of mixed metals. The bioavailability and toxicity of heavy metals in bioprocess samples were also evaluated. We have demonstrated for the first time that the zntA bioreporter can effectively detect the bioavailability of zinc, nickel, and cobalt with limit of detection lower than 0.01, 0.08 and 0.5 mg·L-1, respectively. The detection limits meet the requirements of the WHO, the U.S. Environmental Protection Agency, and the China drinking water quality standards, which makes this approach reasonable for monitoring heavy metal bioavailability in bioprocess. LIVE/DEAD toxicity experiments have been conducted for the detection of mixed metal solution toxicity to zntA bioreporter which shows an EC50 (as EC50, concentration for 50% of maximal effect) value of mixed metal solution is 3.84 mg·L-1. Samples from wastewater treatment plants, sludge treatment plants and kitchen waste fermentation processes were analyzed to extend upon the laboratory results. The results of this study confirm the potential for practical applications of bioreporter technology in bioprocess monitoring. In turn, development for such practical applications is key to achieve the necessary level of commercialization to further make the routine use of bioreporters in bioprocess monitoring feasible.

DNA-based probes have gained significant attention as versatile tools for biochemical analysis, benefiting from their programmability and biocompatibility. However, most existing DNA-based probes rely on fluorescence as the signal output, which can be problematic due to issues like autofluorescence and scattering when applied in complex biological materials such as living cells or tissues. Herein, we report the development of bioluminescent nucleic acid (bioLUNA) sensors that offer laser excitation-independent and ratiometric imaging of the target in vivo. The system is based on computational modelling and mutagenesis investigations of a genetic fusion between circular permutated Nano-luciferase (NLuc) and HaloTag, enabling the conjugation of the protein with a DNAzyme. In the presence of Zn2+ , the DNAzyme sensor releases the fluorophore-labelled strand, leading to a reduction in bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. Consequently, this process induces ratiometric changes in the bioluminescent signal. We demonstrated that this bioLUNA sensor enabled imaging of both exogenous Zn2+ in vivo and endogenous Zn2+ efflux in normal epithelial prostate and prostate tumors. This work expands the DNAzyme sensors to using bioluminescence and thus has enriched the toolbox of nucleic acid sensors for a broad range of biomedical applications.

Cell-free biosensors have inspired low-cost and field-applicable methods to detect antibiotic contaminants. However, the satisfactory sensitivity of current cell-free biosensors is mostly achieved by sacrificing the rapidity, which prolongs turnaround time by hours. Additionally, the software-based result interpretation provides an obstacle for delivering these biosensors to untrained individuals. Here, we present a bioluminescence-based cell-free biosensor, termed enhanced Bioluminescence sensing of Ligand-Unleashed RNA Expression (eBLUE). The eBLUE leveraged antibiotic-responsive transcription factors to regulate the transcription of RNA arrays that can serve as scaffolds for reassembling and activating multiple luciferase fragments. This process converted target recognition into an amplified bioluminescence response, enabling smartphone-based quantification of tetracycline and erythromycin directly in milk within 15 min. Moreover, the detection threshold of eBLUE can be easily tuned according to the maximum residue limits (MRLs) established by government agencies. Owing to this tunable nature, the eBLUE was further repurposed as an on-demand semi-quantification platform, allowing for fast (∼20 min) and software-free identification of safe and MRL-exceeding milk samples only by glancing over the smartphone photographs. Overall, the sensitivity, rapidity and user-friendliness of eBLUE demonstrate its potentials for practical applications, especially in resource-limited and household settings.