Bioluminescence Intensity Research Articles

Decoding wheat contamination through self-assembled whole-cell biosensor combined with linear and non-linear machine learning algorithms

The contamination of mycotoxins is a serious problem around the world. It has detrimental effects on human beings and leads to tremendous economic loss. It is essential to develop a rapid and non-destructive method for contamination recognition particularly for early alarm. In this study, the whole-cell biosensor array was constructed and employed for rapid recognition of wheat contamination by combining with machine learning algorithms. Seven key VOCs were explored through univariate coupling to multivariate analysis of orthogonal partial least squares-discrimination analysis (OPLS-DA) models. The promoters of dnaK, katG, oxyR, soxS obtained from the stress-responsive of key VOCs were fused to the bacterial operon and fabricated on the whole-cell biosensor. The constructed whole-cell biosensor array was consisted with four kinds of sensors and 18 sensor unit. The bioluminescent intensity combined with linear machine learning algorithm of partial least squares discriminant analysis (PLS-DA) and non-linear algorithms of back propagating artificial neural network (BP-ANN) and least square support vector machine (LS-SVM) were employed to establish discrimination models for mold contamination especially for early warning. The Monte-Carlo strategy was performed to generate thirty subsets for modeling to give more reliable results. As a result, the whole-cell biosensor combined with non-linear algorithm of LS-SVM was practicable for detecting mold identification for wheat early-warning with the accuracy of 97.24%. Additionally, this study provides practical and effective methods not only for wheat quality guarantee and supervision but also for other foodstuffs.

Quantifying Bioluminescent Light Intensity in Breaking Waves Using Numerical Simulations

AbstractBreaking‐wave induced bioluminescence is a critical component of the biogeochemical process in the ocean. Understanding bioluminescence is important for monitoring red tides caused by bioluminescent microorganisms. In this study, we present the first numerical effort to quantify bioluminescent light intensity based on high‐fidelity direct numerical simulations of breaking waves and a quantitative bioluminescent model. The dynamics of breaking waves are extensively validated through comparison with existing studies. We find that the time‐averaged and Lagrangian‐averaged shear stress saturates as surface tension effects decrease and wave steepness increases. The spatial distribution of light intensity correlates with the wave crest overturning and air bubbles generated in plunging breakers. Furthermore, we observe that the maximum light intensity asymptotically approaches the emission of single cells, suggesting the potential for cost‐effective prediction models in future studies.

Superluminescent variants of Gaussia luciferase in living animal deep-tissue imaging

Gaussia luciferase (Gluc) is widely recognized as a powerful bioluminescent tool for biosensing and molecular imaging. However, its utility in mammalian deep tissue imaging and high-throughput applications is hampered by two primary limitations: the emission of blue light and the transient nature of its bioluminescence reaction. In this study, we have engineered two novel luciferases, BeM and SuM, by incorporating known amino acid mutations that have demonstrated the ability to stabilize, enhance, or shift the wavelength of Gluc-catalyzed luminescence towards the red spectrum. The BeM and SuM-coelenterazine systems exhibited a more than 100-fold increase in bioluminescence intensity in vitro, and over a 10-fold enhancement in vivo, compared to the conventional firefly luciferase-D-luciferin system. This significant improvement enables the noninvasive deep tissue imaging of a small number of cells within living mice. BeM and SuM emerge as the most efficient Gaussia luciferase variants to date, showcasing their immense potential as bioengineered light sources. Their applications could span various fields, ranging from fundamental scientific research to clinical diagnostics and therapeutic interventions, marking a significant leap forward in bioluminescence technology.

Enhancing Bioluminescence Imaging of Cultured Tissue Explants Using Optical Telecompression.

Long-term observation of single-cell oscillations within tissue networks is now possible by combining bioluminescence reporters with stable tissue explant culture techniques. This method is particularly effective in revealing the network dynamics in systems with slow oscillations, such as circadian clocks. However, the low intensity of luciferase-based bioluminescence requires signal amplification using specialized cameras (e.g., I-CCDs and EM-CCDs) and prolonged exposure times, increasing baseline noise and reducing temporal resolution. To address this limitation, we implemented a cost-effective optical enhancement technique called telecompression, first used in astrophotography and now commonly used in digital photography. By combining a high numerical aperture objective lens with a magnification-reducing relay lens, we significantly increased the collection efficiency of the bioluminescence signal without raising the baseline CCD noise. This method allows for shorter exposure times in time-lapse imaging, enhancing temporal resolution and enabling more precise period estimations. Our implementation demonstrates the feasibility of telecompression for enhancing bioluminescence imaging for the tissue-level network observation of circadian clocks.

Features of Bioluminescence Dynamics of Photobacterium phosphoreum IMV B-7071

The problem of the prolonged and stable intensity of bioluminescent signals is relevant in the development of any test systems that use biological objects. The aim of this work was to study the features of bioluminescence dynamics of Photobacterium phosphoreum IMV B-7071 in a liquid and on different stationary media. Methods. Bioluminescence studies were performed in liquid, agarose, and cellulose-cotton media. Bacterial suspensions were cultivated at 21°C in the mediums with standard composition. We studied both the background glow and its dynamics under conditions of mixing a liquid medium. Bioluminescence was recorded using digital photography with subsequent image processing of the samples. The measurements of luminescence were made by digital photo or video recording using Olympus digital camera SP560UZ, CANON 700D, and mobile device camera Samsung Galaxy 9 Note with specialized applications for mobile devices "Colorimeter (Lab Tools Apps)" and Camera Color Counter (Keuwsoft) at maximum light sensitivity in the automatic white balance mode at a fixed distance from the sample. Image processing was carried out using ImageJ and Origin Pro. Spectra of bacterial luminescence and its dynamics over time were measured using an LOMO MDR-23 spectrometer in the range of 200–750 nm. Results. The results of the study prove that in aqueous or solid agar and also on cellulose cotton medium, the intensity of bioluminescence of P. phosphoreum gradually increases, reaching a maximum within approximately 2 days, after which it slowly fades. It was established that the bioluminescence of photobacterium P. phosphoreum is a non-stationary process and has characteristic features of temporal dynamics associated with both the dynamics of the oxygen concentration in the environment of bacterial suspensions and the dynamics of the bacterial population density. Analysis of the luminescence spectra of bacteria shows that luminescence occurs mainly in the blue and green regions of the spectrum with luminescence maxima in the range of 460–520 nm, but the ultra-weak glow is also registered in the UV and red spectral ranges. The variability of photobacterial luminescence spectra over time in the spectral ranges of the main luminophores causes color fluctuations between the blue and green ranges. Conclusions. The key parameters of multi-day background and short-term induced bioluminescence dynamics of photobacteria in different environments were clarified, and the certain variability of the spectral characteristics of luminescent radiation over time was shown. The revealed features of the dynamics of the bioluminescence of P. phosphoreum must be taken into account in practical application to assess the toxicity of substances of various nature, as well as in environmental monitoring.

Application of the luminous bacterium Photobacterium phosphoreum for toxicity monitoring of selenite and its reduction to selenium(0) nanoparticles

Luminous marine bacteria are traditionally used as a bioassay due to the convenience and high rate of registering the intensity of their physiological function – luminescence. This study aimed to develop the application of Photobacterium phosphoreum in traditional and novel fields – toxicity monitoring and biotechnology. We demonstrated (1) effects of selenite ions on bioluminescence, and (2) biotransformation of selenite to selenium(0) in the form of nanoparticles. The effects of selenite (SeO32−) on the intensity of bacterial bioluminescence were studied, and its dependencies on exposure time and concentration of Na2SeO3 were analyzed. Bioluminescence activation and inhibition were revealed; dose–effect dependencies corresponded to the hormesis model. The toxicity of SeO32− was characterized by an effective concentration of 10−3 M. Effects of SeO32− on reactive oxygen species (ROS) in bacterial suspensions were studied. High positive correlations were found between the bioluminescence intensity and ROS content, which indicates the decisive role of ROS and associated redox processes in the bioeffects of selenite ions. Scanning and transmission electron microscopy revealed the presence of nano-structures in the bacteria exposed to selenite. The energy dispersion spectrum detected a high content of selenium in the nanoparticles. The particle size distribution depended on Na2SeO3 concentration; maxima of the distribution varied within 45–55 nm.

Cytotoxic and radical activities of metal-organic framework modified with iron oxide: Biological and physico-chemical analyses

Metal-organic framework (MOF) modified with iron oxide, Fe3O4-MOF, is a perspective drug delivery agent, enabling magnetic control and production of active hydroxyl radicals, •OH, via the Fenton reaction. This paper studies cytotoxic and radical activities of Fe-containing nanoparticles (NPs): Fe3O4-MOF and its components – bare Fe3O4 and MOF (MIL-88B). Luminous marine bacteria Photobacteriumphosphoreum were used as a model cellular system to monitor bioeffects of the NPs. Neither the NPs of Fe3O4-MOF nor MOF showed cytotoxic effects in a wide range of concentrations (<10 mg/L); while Fe3O4 was toxic at >3·10−3 mg/L. The NPs of Fe3O4 did not affect the bacterial bioluminescence enzymatic system; their toxic effect was attributed to cellular membrane processes. The integral content of reactive oxygen species (ROS) was determined using a chemiluminescence luminol assay. Bacteria mitigated excess of ROS in water suspensions of Fe3O4-MOF and MOF, maintaining bioluminescence intensity closer to the control; this resulted in low toxicity of these NPs. We estimated the activity of •OH radicals in the NPs samples with physical and chemical methods – spin capture technology (using electron paramagnetic resonance spectroscopy) and methylene blue degradation. Physico-chemical interpretation of cellular responses is provided in terms of iron content, iron ions release and •OH radical production.

Photographic monitoring of glowworm Arachnocampa luminosa (Diptera: Keroplatidae) bioluminescence in a tourist cave reveals diurnal and annual cycles

AbstractGlowworms are the bioluminescent larvae of a group of dipteran insects related to fungus gnats. They require sheltered, consistently moist conditions and are found in aggregations on the walls and ceilings of caves and near streams in wet forests where they attract flying insects as prey. The Waitomo Glowworm Cave in New Zealand receives many thousands of visitors each year to see the colony of the glowworm, Arachnocampa luminosa. The cave climate is managed to ensure the glowworms are not harmed by influxes of dry air, as happened in the 1970s. To monitor the population and warn of catastrophic population declines, time‐lapse photographic monitoring of the glowworm population began in 2011 using a permanent, fixed camera. Photographs are taken 30 min apart. The population exhibits synchronised diurnal cycles of bioluminescence intensity. The time of the acrophase (the peak) of the diurnal cycle varied seasonally between 5 pm in early southern spring and 8 pm in summer. Cross‐correlation analyses with cave and water temperatures incorporating time lags suggest that this annual cycle could be related to changes in the composition or density of prey insects. Annual cycles also occur in the number of glowing larvae and their overall intensity. In most years, the numbers are lowest in winter and increase in spring to produce the brightest display through summer. The summer peak is not seen every year and autocorrelation of the 13‐year time series of count shows signs of a 3‐ to 4‐year cycle beyond the annual periodicity. The availability of prey in the cave chamber could influence the annual cycles in glowworm density, underscoring the need for a deeper knowledge of the bionomics of prey species, mainly Chironomidae (non‐biting midges). The photographic monitoring has proven to be a useful component of the management of the glowworm population.

Long-Chain Alkylthio Cyclodextrin Derivatives for Modulation of Quorum-Sensing-Based Bioluminescence in Aliivibrio fischeri Model System.

Quorum sensing (QS) allows bacteria to coordinate their activities by producing and detecting low-molecular-weight signal molecules based on population density, thereby controlling the infectivity of bacteria through various virulence factors. Quorum-sensing inhibition is a promising approach to tackle bacterial communication. Cyclodextrins (CDs) are a class of cyclic oligosaccharides that reversibly encapsulate the acyl chain of the signal molecules, thereby preventing their binding to receptors and interrupting bacterial communication. This results in the inhibition of the expression of various properties, including different virulence factors. To examine the potential quorum-quenching (QQ) ability of newly prepared cyclodextrin derivatives, we conducted short-term tests using Aliivibrio fischeri, a heterotrophic marine bacterium capable of bioluminescence controlled by quorum sensing. α- and β-cyclodextrins monosubstituted with alkylthio moieties and further derivatized with quaternary ammonium groups were used as the test agents. The effect of these cyclodextrins on the quorum-sensing system of A. fischeri was investigated by adding them to an exponential growth phase of the culture and then measuring bioluminescence intensity, population growth, and cell viability. Our results demonstrate that the tested cyclodextrins have an inhibitory effect on the quorum-sensing system of A. fischeri. The inhibitory effect varies based on the length of the alkyl chain, with alkylthio substitution enhancing it and the presence of quaternary ammonium groups decreasing it. Our findings suggest that cyclodextrins can be a promising therapeutic agent for the treatment of bacterial infections.

Diagnosis of Nonalcoholic Fatty Liver Disease via a H2S-Responsive Bioluminescent Probe Combined with Firefly Luciferase mRNA Delivery.

The early detection of nonalcoholic fatty liver disease (NAFLD) through bioluminescent probes is of great significance. However, there remains a challenge to apply them in nontransgenic natural animals due to the lack of exogenous luciferase. To address this issue, we herein report a new strategy for in situ monitoring of endogenous hydrogen sulfide (H2S) in the liver of NAFLD mice by leveraging a H2S-responsive bioluminescent probe (H-Luc) combined with firefly luciferase (fLuc) mRNA delivery. The probe H-Luc was created by installing a H2S recognition moiety, 2,4-dinitrophenol, onto the luciferase substrate (d-luciferin), which is allowed to release cage-free d-luciferin in the presence of H2S via a nucleophilic aromatic substitution reaction. In the meantime, the intracellular luciferase was introduced by lipid nanoparticle (LNP)-mediated fLuc mRNA delivery, rendering it suitable for bioluminescence (BL) imaging in vitro and in vivo. Based on this luciferase-luciferin system, the endogenous H2S could be sensitively and selectively detected in living cells, showing a low limit of detection (LOD) value of 0.72 μM. More importantly, after systematic administration of fLuc mRNA-loaded LNPs in vivo, H-Luc was able to successfully monitor the endogenous H2S levels in the NAFLD mouse model for the first time, displaying a 28-fold higher bioluminescence intensity than that in the liver of normal mice. We believe that this strategy may shed new light on the diagnosis of inflammatory liver disease, further elucidating the roles of H2S.

CAFFEIC ACID STIMULATES IN VIVO LUMINESCENCE OF THE MYCELIA OF THE HIGHER FUNGI NEONOTHOPANUS NAMBI AND ARMILLARIA BOREALIS

CAFFEIC ACID STIMULATES IN VIVO LUMINESCENCE OF THE MYCELIA OF THE HIGHER FUNGI NEONOTHOPANUS NAMBI AND ARMILLARIA BOREALIS

Effect of salinity on the bioluminescence intensity of the heterotrophic dinoflagellates Noctiluca scintillans and Polykrikos kofoidii and the autotrophic dinoflagellate Alexandrium mediterraneum

Many dinoflagellate species are bioluminescent, which is one of the anti-predation mechanisms in these species. In addition, dinoflagellate species experience a wide range of salinities in the ocean. However, the effects of salinity on their bioluminescence intensity has only been investigated for one species. Here, we explored the effect of salinity on the bioluminescence intensity of the heterotrophic dinoflagellate Noctiluca scintillans NSDJ2010 feeding on the chlorophyte Dunaliella salina, the heterotrophic dinoflagellate Polykrikos kofoidii PKJH1607 feeding on the dinoflagellate Alexadrium minutum, and the autotrophic dinoflagellate Alexandrium mediterraneum AMYS1807. Moreover, to determine the cell volume and growth effects on bioluminescence intensity, the cell volume and growth rate of three bioluminescent dinoflagellates were simultaneously investigated. The mean 200-s-integrated bioluminescence intensity (BL) per cell, equivalent to the total bioluminescence, of N. scintillans, P. kofoidii, and A. mediterraneum was significantly affected by salinity and increased with increasing salinity from 10 to 40. The results of the present study suggest that the total bioluminescence of N. scintillans, P. kofoidii, and A. mediterraneum in offshore and oceanic waters is greater than that in estuarine waters.

Abstract 75: Luciferase reporter cell lines allow simultaneous incorporation of tumor cells, innate immune cells, and adaptive immune cells for in-depth immune checkpoint studies

Abstract Despite the considerable success of immune checkpoint therapies targeting T cells, a sizable proportion of patients experience resistance or relapse due to the immunosuppressive nature of the tumor microenvironment. Myeloid cells, a major component that suppresses effector lymphocytes, have emerged as an alternative and promising therapeutic target. However, there is a deep lack of widely accessible immunological models capable of representing the intricate three-way interaction between tumor cells, T cells, and myeloid cells. To address this need, we conducted a comprehensive protein profiling of human tumor and immune cell lines available at ATCC for various established and novel immune checkpoint molecules. Cell lines with high endogenous expression of the immune checkpoint proteins, such as programmed death-ligand 1 and 2 (PD-L1 and PD-L2), cluster of differentiation 155 (CD155), B7 homolog 3 (B7-H3), sialic acid-binding Ig-like lectin 10 (Siglec-10), or signal-regulatory protein alpha (SIRPα), were selected and constructed into luciferase reporter cell lines. For tumor reporter cell lines, a gamma interferon activation site (GAS) response element was placed upstream of the luciferase gene in the lentiviral vector, enabling the activation of the JAK-STAT signaling pathway within tumor cells to induce luciferase expression. In myeloid reporter cell lines, a nuclear factor kappa B (NF-κB) response element replaced GAS to monitor the activation of the NF-κB signaling pathway. In the presence of corresponding immune checkpoint inhibitors that enhance T cell-mediated anti-tumor activity, these reporter cell lines produce a bioluminescent signal based on luciferase expression. This signal can be easily detected and quantified to assess the efficacy of the inhibitor. Our data revealed that bioluminescence intensity in the tumor and myeloid reporter cell lines increased by &amp;gt;100-fold in a dose-dependent manner in response to interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) stimulation, respectively, and by &amp;gt;50-fold in response to the conditioned media collected from activated primary T cells. Furthermore, in co-culture assays involving various combinations of immune cell and tumor cell types with corresponding immune checkpoint inhibitors, these reporter cell lines demonstrated a significant increase in bioluminescence intensity. In conclusion, these newly established luciferase reporter cell lines offer an excellent ex vivo model for cancer immunotherapy. These cell lines naturally express immune checkpoint proteins, enabling the sensitive and reproducible monitoring of combinatorial responses from various immune cell types. Citation Format: Hyeyoun Chang, Alicia C. Walker, John G. Foulke, Luping Chen, Fang Tian, Zhizhan Gu. Luciferase reporter cell lines allow simultaneous incorporation of tumor cells, innate immune cells, and adaptive immune cells for in-depth immune checkpoint studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 75.

Abstract 2841: Development and validation of a systemic human multiple myeloma model utilizing luciferase expressing MM1.S tumor cells

Abstract Multiple Myeloma (MM) is a complex hematologic malignancy characterized by clonal proliferation of transformed plasma cells leading to overproduction of monoclonal immunoglobulins and subsequently end-stage organ damage. Despite therapeutic advances, MM is an incurable disease with poor prognosis in high-risk patients, and thus relevant preclinical models are necessary to develop novel treatment strategies. We developed a Multiple Myeloma in vivo model by systemic implantation of the luciferase-expressing glucocorticoid sensitive tumor cell line MM1.S (MM1.S-Luc) into immunodeficient NCG mice. First, we generated a MM1.S-Luc monoclonal cell line from a polyclonal pool of stable luciferase-expressing cells by limiting dilution. Single cells from the stable pool were expanded under selective pressure and four MM1.S-Luc monoclonal lines were selected based on in vitro growth properties and bioluminescence intensity. Next, we determined the in vivo tumor growth profile by testing the monoclonal lines at two different cell inoculums, and disease progression was monitored by in vivo serial bioimaging. MM1.S-Luc tumor growth kinetics revealed a long latency and based on our in vivo imaging results, the tumor preferentially localized to the long bones, lungs, and mandible. For validation of the MM1.S-Luc in vivo model, we determined the efficacy and survival benefit in response to various clinically relevant standard of care agents including Dexamethasone, Daratumumab, Cyclophosphamide, Vincristine, Panobinostat, and Bortezomib. The quantification of tumor burden by bioluminescent imaging showed reduced tumor burden and prolonged survival with Panobinostat and Cyclophosphamide. In summary, we have developed a systemic MM1.S-Luc model that recapitulates the disease dissemination and invasiveness of multiple myeloma for evaluating novel therapeutic approaches to treatment. Citation Format: Bincy John, Christopher Currie, Nicole Westrick, Tyler Rowe, Andrew Wong, Elizabeth Rainbolt, Zachary Ward, Chassidy Hall, David Harris. Development and validation of a systemic human multiple myeloma model utilizing luciferase expressing MM1.S tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2841.

Breaking the Mold: Towards Rapid and Cost-Effective Microbial Contamination Detection in Paints and Cosmetics Using ATP-Bioluminescence

Traditional culture-based methods, though a “gold standard” for bacterial detection in various industrial sectors, do often not fulfill today’s high requirements regarding rapidity, on-site applicability, and cost-efficiency both during operation and evaluation. Here, the feasibility of using an adenosine triphosphate (ATP)-based assay for determining microbial contaminations in paints and cosmetics was investigated and compared with standard plate count techniques and dipslides. Therefore, we initially determined the level of sensitivity and assessed the accuracy and concordance among the different methods via spiking tests using a mix of frequently abundant bacterial species to simulate microbial contamination. Bioluminescence intensity was linearly proportional to log colony counts over five orders of magnitude (R2 = 0.99), indicating a high level of sensitivity. Overall, the accuracy varied depending on the test specimen, most probably due to matrix-related quenching effects. Although the degree of conformity was consistently higher at target concentrations ≥ 105 CFU·mL−1, microbial contaminations were detectable down to 103 CFU·mL−1, thus meeting the high requirements of various industries. ATP-based results tended to be within an order of magnitude lower than the reference. However, bearing that in mind, the developed assay serves as a rapid, real-time alternative for routine quality control and hygiene monitoring.

Estimation of bioluminescence intensity of the dinoflagellates Noctiluca scintillans, Polykrikos kofoidii, and Alexandrium mediterraneum populations in Korean waters using cell abundance and water temperature

Many dinoflagellates produce bioluminescence. To estimate the intensity of bioluminescence produced by populations of the heterotrophic dinoflagellates Noctiluca scintillans and Polykrikos kofoidii and autotrophic dinoflagellate Alexandrium mediterraneum in Korean waters, we measured cellular bioluminescence intensity as a function of water temperature and calculated population bioluminescence intensity with cell abundances and water temperature. The mean 200-second-integrated bioluminescence intensity per cell (BLcell) of N. scintillans satiated with the chlorophyte Dunaliella salina decreased continuously with increasing water temperature from 5 to 25°C. However, the BLcell of P. kofoidii satiated with the mixotrophic dinoflagellate Alexandrium minutum continuously increased from 5 to 15°C but decreased at temperatures exceeding this (to 30°C). Similarly, the BLcell of A. mediterraneum continuously increased from 10 to 20°C but decreased between 20 and 30°C. The difference between highest and lowest BLcell of N. scintillans, P. kofoidii, and A. mediterraneum at the tested water temperatures was 3.5, 11.8, and 21.0 times, respectively, indicating that water temperature clearly affected BLcell. The highest estimated population bioluminescence intensity (BLpopul) of N. scintillans in Korean waters in 1998–2022 was 4.22 × 1013 relative light unit per liter (RLU L-1), which was 1,850 and 554,000 times greater than that of P. kofoidii and A. mediterraneum, respectively. This indicates that N. scintillans populations produced much brighter bioluminescence in Korean waters than the populations of P. kofoidii or A. mediterraneum.

Regional delivery of mesothelin-targeted chimeric antigen receptor T-cell effectively and safely targets colorectal cancer liver metastases in mice.

Liver metastasis is the major cause of colorectal cancer related death. Mesothelin (MSLN)-targeted chimeric antigen receptor (CAR) T-cell therapy has been illustrated effective and safe through regional delivery of breast cancer, ovarian cancer and malignant mesothelioma tumors. Herein, we investigated the safety, efficacy, and immune microenvironment of regional delivery of MSLN (CAR) T-cell in the treatment of colorectal carcinoma liver metastases (CRLM). Second-generation MSLN CAR T-cells were administered by portal vein (PV) or caudal vein (CV, systemic administration) delivery in an orthotopic MSLN+ CRLM nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/γc-/- (NSG) mouse model. A total of 20 mice were randomly divided into control group, non-transduced T cell (NT)-CV group, NT-PV group, MSLN CAR T-cell CV (MSLN-CV) group, and MSLN CAR T-cell PV (MSLN-PV) group, with each group containing four mice to examine the safety and efficacy. The bioluminescence intensity (BLI) of tumor burden, tumor tissue macroscopic and microscopic observation were used to evaluate treatment efficacy. The safety was examined by body weight, survival time, and vital organ damage of mice. CAR T-cell infiltration and cytokine concentration were analyzed by flow cytometry, and immunostaining. The change of immune microenvironment between regional delivery and systemic delivery was investigated on an immune reconstructed CRLM patient-derived xenograft (PDX) model. Additionally, T cell subsets and immunosuppressive markers were examined. PV administration of 1×107/100 μL MSLN CAR T-cells in 20 NSG mice was well tolerated, and no overt toxicity was observed. The tumor burden in the PV group was obviously alleviated. The BLI was (0.73±0.52)×109 in PV group and (1.97±0.11)×109 in CV group (P<0.05), CD8+ granzyme B (GB)+ T cell percentage (MSLN-CV 4.42%±0.47% vs. MSLN-PV 13.5%±4.67%, P<0.01) and cytokine concentration were obviously increased in the MSLN-PV group. In the immune reconstituted CRLM PDX model, intratumor (IT) delivery of MSLN CAR T-cells exhibited much more infiltration of CD4+ and CD8+ T cells accompanied with elevated expression levels of PD-1, LAG-3, and TIM-3. Regional delivery of MSLN-targeted CAR T-cell therapy has encouraging results in the orthotopic CRLM NSG mouse model and PDX model, and converts the tumor microenvironment from cold to hot. This study may provide a new therapeutic approach for CRLM. Further clinical study is needed.

Fall-winter sea surface temperature anomalies affect subsequent spring-summer phytoplankton succession and bioluminescence patterns in the Black Sea coastal waters near Crimea

Seasonal and interannual dynamics of bioluminescence intensity and succession of the major phytoplankton taxonomic groups were analyzed using the six-year monitoring of Crimean coastal waters (the northern Black Sea) in 2009–2014. Monitoring program included regular CTD and bioluminescence intensity casts in the upper 60 m layer by means of “Salpa-M” sonde accompanied with phytoplankton sampling from subsurface (∼0.2 m) and from the layer of maximal bioluminescence intensity. Years with anomalous warm and cold preceding fall-winter periods were defined on the basis of remotely sensed monthly sea surface temperature (SST) and long-term records on coastal SST measuring station. It was shown that succession of phytoplankton and net vertical bioluminescence patterns in subsequent spring-summer seasons depend upon the SST anomaly during preceding fall-winter period. In “cold” years a minimal bioluminescence intensity associated with a diminished biomass and species abundance of luminous dinoflagellates as well as diatoms and dinoflagellates groups as a whole were observed. A number of luminous dinoflagellate species dropped out of spring phytoplankton community (e.g., Scrippsiella cf. acuminata). As a result, a typical spring peak of bioluminescence in the upper mixed layer has not been observed during these years. In contrast maximal bioluminescence intensity and phytoplankton abundance in spring-summer was observed in “warm” years; biomass and bioluminescence intensity peaks shift to earlier month (April) in comparison to “moderate” years; period of dinoflagellate dominance was more extended during the spring-summer succession phase. Overall, group of dinoflagellates including luminous species, seems to be the most sensitive group of algae to climatic SST anomalies in coastal waters of the northern Black Sea. Hence, the bioluminescence in the upper sea layer can act as an indicator of climate-induced phytoplankton community restructuring.

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.