Bioluminescence Intensity Research Articles

Protein nanoscaffold enables programmable nanobody-luciferase immunoassembly for sensitive and simultaneous detection of aflatoxin B1 and ochratoxin A

Mycotoxins produced by fungi can contaminate various foods and pose significant health risks. Ensuring food safety demands rapid, highly sensitive analytical techniques. One-step Bioluminescent Enzyme Immunoassays (BLEIAs) employing nanobody-nanoluciferase fusion proteins have recently garnered attention for operational simplicity and heightened sensitivity. Nevertheless, fixed nanobody:nanoluciferase ratios in fusion proteins restrict the customization and sensitivity of traditional BLEIAs. In this study, we present a Scaffold Assembly-based BLEIA (SA-BLEIA) that overcomes these limitations through the programmable conjugation of nanobodies and luciferases onto 60-meric protein nanoscaffolds using SpyTag/SpyCatcher linkages. These nanoscaffolds facilitate the adjustable coupling of anti-aflatoxin B1 and anti-ochratoxin A nanobodies with luciferases, optimizing nanobody/luciferase ratios and diversifying specificities. Compared to conventional methods, SA-BLEIA demonstrates considerably elevated sensitivity for detecting both toxins. The elevated local concentration of luciferase significantly amplifies bioluminescence intensity, permitting reduced substrate consumption and cost-effective detection. The usage of dual-nanobody conjugates facilitates the quantification or simultaneous detection of both mycotoxins in a single test with shared reagents. The assay exhibits exceptional recovery rates in spiked cereal samples, strongly correlating with outcomes from commercial ELISA kits. Overall, this adaptable, highly sensitive, cost-effective, and multiplexed immunoassay underscores the potential of tunable scaffold assembly as a promising avenue for advancing bioanalytical diagnostic tools.

Ecological role of bioluminescence of Black sea ctenophores.

Bioluminescence, which is a manifestation of the vital activity of an organism in the form of electromagnetic radiation in the visible area of the spectrum, is a highly important ecological and optical factor of the marine environment. Until recently, it was believed that microplankton - bacteria and dinoflagellates - exceptionally contribute to the formation of the bioluminescence field in the Black Sea, as well as in other regions of the World Ocean. However, the ctenophoresMnemiopsis leidyiA. Agassiz, 1865, andBeroe ovata Mayer, 1912, which invaded the Black Sea in the 1980s-1990s, are also luminous organisms whose bioluminescence intensity is millions of times greater than that of most microplankton representatives. It is known that the characteristics of bioluminescence can reveal the state of the organism and, consequently, the state of the environment. At present, there is a fairly large number of works devoted to the physiology and ecology of the Black Sea ctenophores. In recent studies, the variability of light emission parameters of ctenophores following their functional state was revealed. Intensity and duration of light emission as parameters of the ctenophore bioluminescent signal as well as the influence of various abiotic and anthropogenic environmental factors on the ctenophore luminescence have been studied. However, the significance of bioluminescence for the living activity of ctenophores remains unclear. In connection with the above, it is extremely important to assess the ecological role of the bioluminescence of the Black Sea ctenophores.

Putative photosensitivity in internal light organs (organs of Pesta) of deep-sea sergestid shrimps

Many marine species can regulate the intensity of bioluminescence from their ventral photophores in order to counterilluminate, a camouflage technique whereby animals closely match the intensity of the downwelling illumination blocked by their bodies, thereby hiding their silhouettes. Recent studies on autogenic cuticular photophores in deep-sea shrimps indicate that the photophores themselves are light sensitive. Here, our results suggest photosensitivity in a second type of autogenic photophore, the internal organs of Pesta, found in deep-sea sergestid shrimps. Experiments were conducted onboard ship on live specimens, exposing the animals to bright light, which resulted in ultrastructural changes that matched those seen in crustacean eyes during the photoreceptor membrane turnover, a process that is crucial for the proper functioning of photosensitive components. In addition, RNA-seq studies demonstrated the expression of visual opsins and phototransduction genes in photophore tissue that are known to play a role in light detection, and electrophysiological measurements indicated that the light organs are responding to light received by the eyes. The long sought after mechanism of counterillumination remains unknown, but evidence of photosensitivity in photophores may indicate a dual functionality of light detection and emission.

Kaempferol promotes non-small cell lung cancer cell autophagy via restricting Met pathway

BackgroundKaempferol is extracted from Hedyotis diffusa, exerting an obvious anti-cancer effect. Here in the present study, we explored the anti-cancer effects and mechanism of kaempferol in non-small cell lung cancer cell (NSCLC). PurposeOur objective is to figure out the molecular mechanism by which kaempferol promotes autophagy in NSCLC cells. Study designA549 and H1299 NSCLC cell lines were used for in vitro experiments. And BALB/c nude mice of NSCLC were used to perform in vivo experiments. MethodsFor in vitro experiments, CCK-8 and EdU assay was used to observe the effect of kaempferol on NSCLC cell proliferation. Confocal microscopy of mCherry-EGFR-LC3 assay and electron microscopy assay were used to detect NSCLC cell autophagy. Protein expression was determined using Western blot, and mRNA expression was determined using qRT-PCR. Flow cytometry was performed to detect the cell apoptosis. For in vivo experiments, a subcutaneously implanted tumor model in BALB/C nude mice was performed using human NSCLC cell line A549-Luc. The kaempferol effect on NSCLC mice model was detected by measuring the tumor weight and bioluminescence intensity. Immunohistochemistry was done to measure the key protein expression from mice tumor tissues. ResultsOur results confirmed that kaempferol inhibited NSCLC cell proliferation significantly. And it promoted NSCLC cell autophagy, leading to NSCLC cell death. Interestingly, Met-was greatly inhibited at both protein and mRNA levels. Meanwhile, PI3K/AKT/mTOR signaling pathway was inhibited accordingly. Furthermore, overexpressing Met-reversed the effect of kaempferol on NSCLC cell viability and cell autophagy with significance. Finally, the above effect and pathway were validated using the xenograft model. ConclusionKaempferol may exert its anti-NSCLC effect by promoting NSCLC cell autophagy. Mechanistically, Met-and its downstream PI3K/AKT/mTOR signaling pathway were involved in the process, which provides a novel mechanism how kaempferol functions in inhibiting NSCLC.

Bioluminescence Imaging with Functional Amyloid Reservoirs in Alzheimer's Disease Models.

Bioluminescence imaging has changed the daily practice of preclinical research on cancer and other diseases over the last few decades; however, it has rarely been applied in preclinical research on Alzheimer's disease (AD). In this Article, we demonstrated that bioluminescence imaging could be used to report the levels of amyloid beta (Aβ) species in vivo. We hypothesized that AkaLumine, a newly discovered substrate for luciferase, could bind to Aβ aggregates and plaques. We further speculated that the Aβ aggregates/fibrils/plaques could be considered as "functional amyloids", which have a reservoir function to sequester and release AkaLumine to control the bioluminescence intensity, which could be used to report the levels of Aβs. Our hypotheses have been validated via in vitro solution tests, mimic studies with brain tissues and mice, two-photon imaging with AD mice, and in vivo bioluminescence imaging using transgenic AD mice that were virally transduced with AkaLuciferase (AkaLuc), a new luciferase that generates bioluminescence in the near-infrared window. As expected, compared to the control group, we observed that the Aβ group showed lower bioluminescence intensity due to AkaLumine sequestering at early time points, while higher intensity was due to AkaLumine releasing at later time points. Lastly, we demonstrated that this method could be used to monitor AD progression and the therapeutic effectiveness of avagacestat, a well-studied gamma-secretase inhibitor. Importantly, a good correlation (R2 = 0.81) was established between in vivo bioluminescence signals and Aβ burdens of the tested AD mice. We believe that our approach can be easily implemented into daily imaging experiments and has tremendous potential to change the daily practice of preclinical AD research.

Mature human induced pluripotent stem cell-derived cardiomyocytes promote angiogenesis through alpha-B crystallin

BackgroundHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be used to treat heart diseases; however, the optimal maturity of hiPSC-CMs for effective regenerative medicine remains unclear. We aimed to investigate the benefits of long-term cultured mature hiPSC-CMs in injured rat hearts.MethodsCardiomyocytes were differentiated from hiPSCs via monolayer culturing, and the cells were harvested on day 28 or 56 (D28-CMs or D56-CMs, respectively) after differentiation. We transplanted D28-CMs or D56-CMs into the hearts of rat myocardial infarction models and examined cell retention and engraftment via in vivo bioluminescence imaging and histological analysis. We performed transcriptomic sequencing analysis to elucidate the genetic profiles before and after hiPSC-CM transplantation.ResultsUpregulated expression of mature sarcomere genes in vitro was observed in D56-CMs compared with D28-CMs. In vivo bioluminescence imaging studies revealed increased bioluminescence intensity of D56-CMs at 8 and 12 weeks post-transplantation. Histological and immunohistochemical analyses showed that D56-CMs promoted engraftment and maturation in the graft area at 12 weeks post-transplantation. Notably, D56-CMs consistently promoted microvessel formation in the graft area from 1 to 12 weeks post-transplantation. Transcriptomic sequencing analysis revealed that compared with the engrafted D28-CMs, the engrafted D56-CMs enriched genes related to blood vessel regulation at 12 weeks post-transplantation. As shown by transcriptomic and western blot analyses, the expression of a small heat shock protein, alpha-B crystallin (CRYAB), was significantly upregulated in D56-CMs compared with D28-CMs. Endothelial cell migration was inhibited by small interfering RNA-mediated knockdown of CRYAB when co-cultured with D56-CMs in vitro. Furthermore, CRYAB overexpression enhanced angiogenesis in the D28-CM grafts at 4 weeks post-transplantation.ConclusionsLong-term cultured mature hiPSC-CMs promoted engraftment, maturation and angiogenesis post-transplantation in infarcted rat hearts. CRYAB, which was highly expressed in D56-CMs, was identified as an angiogenic factor from mature hiPSC-CMs. This study revealed the benefits of long-term culture, which may enhance the therapeutic potential of hiPSC-CMs.

A novel strategy for bioaerosol rapid detection based on broad-spectrum high-efficiency magnetic enrichment and separation combined with ATP bioluminescence

Bioaerosol detection technology represented by laser-induced fluorescence (LIF) cannot effectively detect bioaerosols in the presence of interferents such as plant-derived smoke, industrial waste gas, pollen and pollen debris which can produce strong non-biological fluorescence interference. To overcome this drawback, in this study, a novel method based on broad-spectrum high-efficiency magnetic enrichment and separation combined with adenosine triphosphate (ATP) bioluminescence was proposed for Escherichia coli (E. coli) bioaerosols rapid detection. First, E. coli bioaerosols mixed with interferents were collected. Core-shell Fe3O4@Polydopamine@Polyethyleneimine magnetic particles were used as bioaerosol enrichment materials to enrich E. coli bioaerosol sampling solutions. Subsequently, an ATP bioluminescence assay was performed to determine the concentration of E. coli. A linear relationship was observed between ATP bioluminescence intensity after enrichment and the E. coli bioaerosol concentration in the range of 870–49,098 particles per liter; the bioluminescence intensity measured after enrichment was significantly higher than that before enrichment, and this enrichment method provide a 6-fold better sensitivity in bioaerosol detection. More importantly, this method efficiently enriched and detected bioaerosols in plant-derived smoke. This method can effectively improve the sensitivity of ATP bioluminescence detection, and possesses the advantages of convenient operation and strong anti-interference ability. It also provides a foundation for the effective detection of bioaerosols mixed with interfering substances, and a reference for evaluating the sensitivity and anti-interference of LIF-based instruments.

Creation of Artificial Luciferase 60s from Sequential Insights and Their Applications to Bioassays.

In this study, a series of new artificial luciferases (ALucs) was created using sequential insights on missing peptide blocks, which were revealed using the alignment of existing ALuc sequences. Through compensating for the missing peptide blocks in the alignment, 10 sibling sequences were artificially fabricated and named from ALuc55 to ALuc68. The phylogenetic analysis showed that the new ALucs formed an independent branch that was genetically isolated from other natural marine luciferases. The new ALucs successfully survived and luminesced with native coelenterazine (nCTZ) and its analogs in living mammalian cells. The results showed that the bioluminescence (BL) intensities of the ALucs were interestingly proportional to the length of the appended peptide blocks. The computational modeling revealed that the appended peptide blocks created a flexible region near the active site, potentially modulating the enzymatic activities. The new ALucs generated various colors with maximally approximately 90 nm redshifted BL spectra in orange upon reaction with the authors' previously reported 1- and 2-series coelenterazine analogs. The utilities of the new ALucs in bioassays were demonstrated through the construction of single-chain molecular strain probes and protein fragment complementation assay (PCA) probes. The success of this study can guide new insights into how we can engineer and functionalize marine luciferases to expand the toolbox of optical readouts for bioassays and molecular imaging.

Development of a bioluminescence immunoassay for detecting ochratoxin A based on luciferin oxidation catalyzed by luciferase

In this work, a novel bioluminescence immunoassay inspired by sparkling fireflies at night was established to detect ochratoxin A (OTA). This platform relied on the mechanism by which firefly luciferase hydrolyze ATP to catalyze the transformation of luciferin to oxidized oxyluciferin, which emits bioluminescence. Since the concentration of ATP can be controlled by alkaline phosphatase (ALP), the bioluminescence intensity was related to the concentration of ALP. Thus, the correlation between target OTA and bioluminescence intensity was realized by ALP-mediated enzyme-linked immunosorbent assay (ELISA). This bioluminescence immunoassay exhibited a relatively wide linear range (1.0–316 ng/mL), which was 30 times wider than that of classical HRP-mediated ELISA (0.8–12.8 ng/mL). The limit of detection (LOD) of the proposed bioluminescence immunoassay (0.316 ng/mL) was lower than the maximum OTA residue limits (2 ng/mL to 10 ng/mL) specified by the European Union (EU) and China Government. In addition, the bioluminescence immunoassay showed good selectivity for OTA and satisfactory recoveries in corn samples ranging from 83.39 % to 111.60 %.

The dynamics of biophysical characteristics of the northern Black Sea pelagic ecosystem in the first decades of the XXI century

Wind speed, sea surface temperature chlorophyll-a concentration (satellite data such MODIS-Aqua/Terra), bioluminescence intensity and zooplankton biomass (data from the research vessel), the value of fish traded, the indices of lower-than-normal atmospheric pressure and water mass transfer (model calculations) are the factors used to estimate a modern state of the pelagic ecosystem and its functional characteristics. It has been shown that, in the last two decades (2000-2020), after natural cataclysms associated with eutrophication and trophic stress of invasive plankton species in the 1990s, the ecosystem of the open areas in the northern portion of the Black sea has reached a state of relative stability in its structure and functioning. This state is characterized by the absence of changes in the interannual variability of the factors particular for it. Concepts are discussed which concern regional differences in physical factors and biological interactions over many years.

Intratumoral Restoration of miR-137 Plus Cholesterol Favors Homeostasis of the miR-137/Coactivator p160/AR Axis and Negatively Modulates Tumor Progression in Advanced Prostate Cancer.

MicroRNAs (miRNAs) have gained a prominent role as biomarkers in prostate cancer (PCa). Our study aimed to evaluate the potential suppressive effect of miR-137 in a model of advanced PCa with and without diet-induced hypercholesterolemia. In vitro, PC-3 cells were treated with 50 pmol of mimic miR-137 for 24 h, and gene and protein expression levels of SRC-1, SRC-2, SRC-3, and AR were evaluated by qPCR and immunofluorescence. We also assessed migration rate, invasion, colony-forming ability, and flow cytometry assays (apoptosis and cell cycle) after 24 h of miRNA treatment. For in vivo experiments, 16 male NOD/SCID mice were used to evaluate the effect of restoring miR-137 expression together with cholesterol. The animals were fed a standard (SD) or hypercholesterolemic (HCOL) diet for 21 days. After this, we xenografted PC-3 LUC-MC6 cells into their subcutaneous tissue. Tumor volume and bioluminescence intensity were measured weekly. After the tumors reached 50 mm3, we started intratumor treatments with a miR-137 mimic, at a dose of 6 μg weekly for four weeks. Ultimately, the animals were killed, and the xenografts were resected and analyzed for gene and protein expression. The animals' serum was collected to evaluate the lipid profile. The in vitro results showed that miR-137 could inhibit the transcription and translation of the p160 family, SRC-1, SRC-2, and SRC-3, and indirectly reduce the expression of AR. After these analyses, it was determined that increased miR-137 inhibits cell migration and invasion and impacts reduced proliferation and increased apoptosis rates. The in vivo results demonstrated that tumor growth was arrested after the intratumoral restoration of miR-137, and proliferation levels were reduced in the SD and HCOL groups. Interestingly, the tumor growth retention response was more significant in the HCOL group. We conclude that miR-137 is a potential therapeutic miRNA that, in association with androgen precursors, can restore and reinstate the AR-mediated axis of transcription and transactivation of androgenic pathway homeostasis. Further studies involving the miR-137/coregulator/AR/cholesterol axis should be conducted to evaluate this miR in a clinical context.

Analysis of target organs of Houttuynia cordata: A study on the anti-inflammatory effect of upper respiratory system

Ethnopharmacological relevanceHouttuynia cordata Thunb. (HC) is a traditional anti-pyretic herb that is classified as the lung meridian in traditional Chinese medicine. However, no articles have explored the main organs responsible for the anti-inflammatory activities of HC.Aim of the study: The aim of the study was to investigate the meridian tropism theory of HC in lipopolysaccharide (LPS)-induced pyretic mice, as well as to identify the underlying mechanisms. Materials and methodsTransgenic mice carrying the luciferase gene driven by nuclear factor-κB (NF-κB) were intraperitoneally injected with LPS and orally administered standardized concentrated HC aqueous extract. The phytochemicals present in the HC extract were analyzed using high-performance liquid chromatography. In vivo and ex vivo luminescent imaging from transgenic mice was used to investigate the meridian tropism theory and anti-inflammatory effects of HC. Microarray analysis of gene expression patterns was used to elucidate the therapeutic mechanisms of HC. ResultsHC extract was found to contain phenolic acids, such as protocatechuic acid (4.52%) and chlorogenic acid (8.12%), as well as flavonoids like rutin (2.05%) and quercitrin (7.73%). The bioluminescent intensities induced by LPS in the heart, liver, respiratory system, and kidney were significantly suppressed by HC, while the maximal decrease (about 90% reduction) of induced luminescent intensity was observed in the upper respiratory tract. These data suggested that upper respiratory system might be the target for HC anti-inflammatory abilities. HC affected the processes involved in innate immunity, such as chemokine-mediated signaling pathway, inflammatory response, chemotaxis, neutrophil chemotaxis, and cellular response to interleukin-1 (IL-1). Moreover, HC significantly reduced the proportions of p65-stained cells and the amount of IL-1β in trachea tissues. ConclusionBioluminescent imaging coupled with gene expression profile was used to demonstrate the organ selectivity, anti-inflammatory effects, and therapeutic mechanisms of HC. Our data demonstrated for the first time that HC displayed lung meridian-guiding effects and exhibited great anti-inflammatory potential in the upper respiratory tract. The NF-κB and IL-1β pathways were associated with the anti-inflammatory mechanism of HC against LPS-provoked airway inflammation. Moreover, chlorogenic acid and quercitrin might be involved in the anti-inflammatory properties of HC.

Tibetan medicine Jiuwei Qingpeng San could inhibit influenza A virus induced lung injury via regulating the expression of IFN-γ and its signaling pathway

IntroductionThe Tibetan formula Jiuwei Qingpeng San (JWQPS) has been applied for severe respiratory infection with a centuries-old history and contemporarily applied for influenza induced pneumonia. Although it is empirically effective, its antiviral efficacy and the underlying mechanisms against influenza A (IAV) virus induced lung injury have not been elucidated yet. MethodsThe in vitro anti-viral activity of JWQPS against H1N1 was reflected by the detection of cell viability of PR8 infected BEAS-2B cells. A replication competent PR8 carrying Gaussia luciferase (PR8-Luci) was further utilized to infect BEAS-2B cells to determine the inhibitory activity of virus replication by detection of the intensity of bioluminescence that correlates with viral load. Transcriptome was utilized to analyze the differences of the gene expression profiles in BEAS-2B cell line between the groups with or without JWQPS treatment after PR8 infection. BALB/c mice were intranasally inoculated with 60 or 150 p.f.u. of PR8 and intragastric administered with JWQPS (400 mg/kg/day, 200 mg/kg/day, 100 mg/kg/day). The mice infected with lethal dosages of 60 p.f.u. were monitored and weighed daily for 14 days and the survival rates were calculated. Concentrations of Nucleoprotein (NP) of IAV in BALF that stand for viral load were determined at 3 or 6 d.p.i. by enzyme linked immunosorbent assay (ELISA). Interferons and lymphotactin in the BALF collected at 3 d.p.i. were detected with the corresponding ELISA kits. The level of pro-inflammatory cytokines in BALF collected at 6 d.p.i. were detected using Th1/Th2 Cytokine 11-Plex Mouse ProcartaPlex Panel. The numbers of leukocyte in BALF were counted by an automated blood cell counter. The lung tissues of mice that were not subjected to BALF collection were harvested at 6 d.p.i. to check lung lesions by hematoxylin-eosin staining and lung coefficient calculation. ResultsJWQPS inhibited IAV replication in vitro, down regulated NP in BALF of H1N1 infected BALB/c mice and protected the mice from IAV caused death. The elevated level of lymphotactin, IFN-γ and lymphocytes in BALF were observed 3 days post infection after JWQPS administration. The improved lung lesions, the alleviated leukocytes infiltration, and the decreased pro-inflammatory levels were observed 6 days post infection. Transcriptomic analysis of H1N1 infected lung epithelial cell line revealed JWQPS treatment could dramatically regulate the genes involved in defense response to virus and interferon associated signaling pathways. DiscussionOur study indicated that JWQPS could promote the chemotactic effect of lymphocytes towards site of infection to eliminate the pathogen at early phase of infection and alleviate the development of inflammation thereafter by reducing the occurrence of neutrophils and lymphocytes into the pulmonary alveoli by a rather complicated regulatory network in immune responses.

Activatable NIR‐II Photothermal Lipid Nanoparticles for Improved Messenger RNA Delivery

AbstractEndosomal escape remains a central issue limiting the high protein expression of mRNA therapeutics. Here, we present second near‐infrared (NIR‐II) lipid nanoparticles (LNPs) containing pH activatable NIR‐II dye conjugated lipid (Cy‐lipid) for potentiating mRNA delivery efficiency via a stimulus‐responsive photothermal‐promoted endosomal escape delivery (SPEED) strategy. In acidic endosomal microenvironment, Cy‐lipid is protonated and turns on NIR‐II absorption for light‐to‐heat transduction mediated by 1064 nm laser irradiation. Then, the heat‐promoted LNPs morphology change triggers rapid escape of NIR‐II LNPs from the endosome, allowing about 3‐fold enhancement of enhanced green fluorescent protein (eGFP) encoding mRNA translation capacity compared to the NIR‐II light free group. In addition, the bioluminescence intensity induced by delivered luciferase encoding mRNA in the mouse liver region shows positive correlation with incremental radiation dose, indicating the validity of the SPEED strategy.

Activatable NIR-II Photothermal Lipid Nanoparticles for Improved Messenger RNA Delivery.

Endosomal escape remains a central issue limiting the high protein expression of mRNA therapeutics. Here, we present second near-infrared (NIR-II) lipid nanoparticles (LNPs) containing pH activatable NIR-II dye conjugated lipid (Cy-lipid) for potentiating mRNA delivery efficiencyvia stimulus-responsive photothermal-promoted endosomal escape delivery (SPEED) strategy. In acidic endosomal microenvironment, Cy-lipid is protonated and turns on NIR-II absorption for light-to-heat transduction mediated by 1064 nm laser irradiation. Then, the heat-promoted LNPs morphology change triggers rapid escape of NIR-II LNPs from the endosome, allowing about 3-fold enhancement of enhanced green fluorescent protein (eGFP) encoding mRNA translation capacity compared to the NIR-II light free group. And the bioluminescence intensity induced by delivered luciferase encoding mRNA in mouse liver region shows positive correlation with incremental radiation dose, indicating the validity of the SPEED strategy.

Alteration of enzyme-substrate interactions by ultra-high dilutions of the substrate

The effects of different ultra-high dilutions of luciferin ranged from 1010 to 10102 on luciferin-luciferase reaction of fireflies were investigated. Luciferin substrate concentration varied from 0.0025 nM to 2.5 nM and ATP concentration remained unchanged. This study evaluated the bioluminescence intensity after addition of ultra-high dilutions of luciferin or ultra-high dilutions of water to the bioluminescent system and some multidirectional and significant differences were found. The number of ultra-high dilutions from studied starting materials, for which significant differences were found, increased sharply with decreasing substrate concentration to 0.0025 nM. No differences were found when the effects of ultra-high dilutions of a non-specific control imidazole and ultra-high dilutions of water on the signal intensity were compared. Our results suggest that ultra-high dilutions of luciferin can specifically inhibit the luciferin-luciferase reaction.

Water-Soluble Polymer Polyethylene Glycol: Effect on the Bioluminescent Reaction of the Marine Coelenterate Obelia and Coelenteramide-Containing Fluorescent Protein.

The current paper considers the effects of a water-soluble polymer (polyethylene glycol (PEG)) on the bioluminescent reaction of the photoprotein obelin from the marine coelenterate Obelia longissima and the product of this bioluminescent reaction: a coelenteramide-containing fluorescent protein (CCFP). We varied PEG concentrations (0-1.44 mg/mL) and molecular weights (1000, 8000, and 35,000 a.u.). The presence of PEG significantly increased the bioluminescent intensity of obelin but decreased the photoluminescence intensity of CCFP; the effects did not depend on the PEG concentration or the molecular weight. The photoluminescence spectra of CCFP did not change, while the bioluminescence spectra changed in the course of the bioluminescent reaction. The changes can be explained by different rigidity of the media in the polymer solutions affecting the stability of the photoprotein complex and the efficiency of the proton transfer in the bioluminescent reaction. The results predict and explain the change in the luminescence intensity and color of the marine coelenterates in the presence of water-soluble polymers. The CCFP appeared to be a proper tool for the toxicity monitoring of water-soluble polymers (e.g., PEGs).

Effects of typical marine environmental factors on the bioluminescence intensity of individual Noctiluca scintillans.

Red Noctiluca scintillans (RNS) is one of the major red tide species and dominant bioluminescent plankton in the global offshore. Bioluminescence offers a number of applications for ocean environment assessments such as interval waves study, fish stocks evaluation and underwater target detection making it of significant interest in forecasting bioluminescence occurrence and intensity. RNS is susceptible to changes in marine environmental factors. However, the effects of marine environmental factors on the bioluminescent intensity (BLI, photon s-1) of individual RNS cells (IRNSC) is poorly known. In this study, the effects of temperature, salinity and nutrients on the BLI were studied by field and laboratory culture experiments. In the field experiments, bulk BLI was measured by an underwater bioluminescence assessment tool at various temperature, salinity and nutrient concentrations. To exclude the contribution by other bioluminescent planktons, an identification method of IRNSC was first developed using the features of the bioluminescence flash kinetics (BFK) curve of RNS to identify and extract BLI emitted by an individual RNS cell. To decouple the effects of each environmental factor, laboratory culture experiments were conducted to examine the effects of a single factor on the BLI of IRNSC. The field experiments showed that BLI of IRNSC negatively correlated with temperature (3-27°C) and salinity (30-35‰). The logarithmic BLI can be well fitted using a linear equation with temperature or salinity with Pearson correlation coefficients of -0.95 and -0.80, respectively. The fitting function with salinity was verified by the laboratory culture experiment. On the other hand, no significant correlation was observed between BLI of IRNSC and nutrients. These relationships could be used in the RNS bioluminescence prediction model to improve the prediction accuracy of bioluminescent intensity and spatial distribution.

Bioluminescent eddies of the World Ocean.

Mesoscale eddies of the ocean (with a characteristic diameter of about one hundred km and a life time span of about several weeks) are habitats of plankton organisms, many of which are bioluminescent. The spatial heterogeneity of bioluminescence of the upper mixed layer associated with the impact of mesoscale eddies is poorly studied. The 45-year historical data set was retrieved, in order to select the bathy-photometric surveys carried out in the form of station grids and transects across eddies. Data from 71 expeditions deployed in 1966-2022 to the Atlantic Ocean, Indian Ocean and Mediterranean Sea basin were analyzed, in order for the spatial heterogeneity of bioluminescent fields to be elucidated across eddy fields. The stimulated bioluminescence intensity was characterized by the bioluminescent potential, which represented the maximal amount of radiant energy emitted in a given volume of water by bioluminescent organisms. The normalized bioluminescent potential over oceanographic station grids exhibited correlation with the eddy kinetic energy and zooplankton biomass (r =0.8, at p=0.001 and r =0.7, at p=0.05, respectively), in a broad range of energy and bioluminescence units (0.02-0.2 m2 s-2 ; 0.4-92.0*10-8 W cm-2 L-1 , respectively). Overall, estimates of bioluminescent potential variability on the mesoscale contribute to the assessment of the multiple-scale variation of the bioluminescent field of the World Ocean.

Novel NF-κB reporter mouse for the non-invasive monitoring of inflammatory diseases

Bioluminescence imaging is useful for non-invasively monitoring inflammatory reactions associated with disease progression, and since NF-κB is a pivotal transcription factor that alters expressions of inflammatory genes, we generated novel NF-κB luciferase reporter (NF-κB-Luc) mice to understand the dynamics of inflammatory responses in whole body, and also in various type of cells by crossing NF-κB-Luc mice with cell-type specific Cre expressing mice (NF-κB-Luc:[Cre]). Bioluminescence intensity was significantly increased in NF-κB-Luc (NKL) mice exposed to inflammatory stimuli (PMA or LPS). Crossing NF-κB-Luc mice with Alb-cre mice or Lyz-cre mice generated NF-κB-Luc:Alb (NKLA) and NF-κB-Luc:Lyz2 (NKLL) mice, respectively. NKLA and NKLL mice showed enhanced bioluminescence in liver and macrophages, respectively. To confirm that our reporter mice could be utilized for the non-invasive monitoring of inflammation in preclinical models, we conducted a DSS-induced colitis model and a CDAHFD-induced NASH model in our reporter mice. In both models, our reporter mice reflected the development of these diseases over time. In conclusion, we believe that our novel reporter mouse can be utilized as a non-invasive monitoring platform for inflammatory diseases.