Bioluminescence Research Articles

Modified vaginal lactobacilli expressing fluorescent and luminescent proteins for more effective monitoring of their release from nanofibers, safety and cell adhesion

Electrospun nanofibers offer a highly promising platform for the delivery of vaginal lactobacilli, providing an innovative approach to preventing and treating vaginal infections. To advance the application of nanofibers for the delivery of lactobacilli, tools for studying their safety and efficacy in vitro need to be established. In this study, fluorescent (mCherry and GFP) and luminescent (NanoLuc luciferase) proteins were expressed in three vaginal lactobacilli (Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii) and a control Lactiplantibacillus plantarum with the aim to use this technology for close tracking of lactobacilli release from nanofibers and their adhesion on epithelial cells. The recombinant proteins influenced the growth of the bacteria, but not their ability to produce hydrogen peroxide. Survival of lactobacilli in nanofibers immediately after electrospinning varied among species. Bacteria retained fluorescence upon incorporation into PEO nanofibers, which was vital for evaluation of their rapid release. In addition, fluorescent labelling facilitated efficient tracking of bacterial adhesion to Caco-2 epithelial cells, while luminescence provided important quantitative insights into bacterial attachment, which varied from 0.5 to 50% depending on the species. The four lactobacilli in dispersion or in nanofibers were not detrimental for the viability of Caco-2 cells, and did not demonstrate hemolytic activity highlighting the safety profiles of both bacteria and PEO nanofibers. To summarize, this study contributes to the development of a promising delivery system, tailored for local administration of safe vaginal lactobacilli.

Advanced Bioluminescence Reporter with Engineered Gaussia Luciferase via Sequence-Guided Mutagenesis.

Gaussia luciferase (GLuc) is the preeminent secreted luciferase widely used in cell-based reporter assays. By employing sequence-guided mutagenesis informed by alignments of diverse copepod luciferase sequences, we identified key amino acids that significantly enhance bioluminescence (BL) intensity. Among the mutated proteins expressed in bacteria, five individual mutations (M60L, K88Q, F89Y, I90L, or S103T) independently increased BL intensity by 1.8 to 7.5-fold compared to wild-type GLuc in the presence of coelenterazine substrates. Remarkably, the combination of all five mutations in GLuc (designated as GLuc5) resulted in an unexpected 29-fold enhancement in BL intensity. Subsequent evaluation of the GLuc5-secreted reporter in transfected mammalian cells confirmed its superior BL performance across multiple cell lines. These findings suggest that the mutated residues are likely crucial for enhancing BL intensity in GLuc, supporting its potential to serve as a highly sensitive biosensor or reporter for a wide range of biological applications.

Light‐Based Juxtacrine Signaling Between Synthetic Cells

Cell signaling through direct physical cell–cell contacts plays vital roles in biology during development, angiogenesis, and immune response. Intercellular communication mechanisms between synthetic cells constructed from the bottom up are majorly reliant on diffusible chemical signals, thus limiting the range of responses in receiver cells. Engineering contact‐dependent signaling between synthetic cells promises to unlock more complicated signaling schemes with spatial responses. Herein, a light‐activated contact‐dependent communication scheme for synthetic cells is designed and demonstrated. A split luminescent protein is utilized to limit signal generation exclusively to contact interfaces of synthetic cells, driving the recruitment of a photoswitchable protein in receiver cells, akin to juxtacrine signaling in living cells. The modular design not only demonstrates contact‐dependent communication between synthetic cells but also provides a platform for engineering orthogonal contact‐dependent signaling mechanisms.

Ancient Secretory Pathways Contributed to the Evolutionary Origin of an Ecologically Impactful Bioluminescence System.

Evolutionary innovations in chemical secretion-such as the production of secondary metabolites, pheromones, and toxins-profoundly impact ecological interactions across a broad diversity of life. These secretory innovations may involve a "legacy-plus-innovation" mode of evolution, whereby new biochemical pathways are integrated with conserved secretory processes to create novel products. Among secretory innovations, bioluminescence is important because it evolved convergently many times to influence predator-prey interactions, while often producing courtship signals linked to increased rates of speciation. However, whether or not deeply conserved secretory genes are used in secretory bioluminescence remains unexplored. Here, we show that in the ostracod Vargula tsujii, the evolutionary novel c-luciferase gene is co-expressed with many conserved genes, including those related to toxin production and high-output protein secretion. Our results demonstrate that the legacy-plus-innovation mode of secretory evolution, previously applied to sensory modalities of olfaction, gustation, and nociception, also encompasses light-producing signals generated by bioluminescent secretions. This extension broadens the paradigm of secretory diversification to include not only chemical signals but also bioluminescent light as an important medium of ecological interaction and evolutionary innovation.

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.

Three birds with one stone: A nano zeolitic imidazolate framework-luciferase-antimicrobial peptide biocomposite-based biosensing platform for improving enzyme stability, detection sensitivity, and sterilization effect

Adenosine triphosphate-driven bioluminescence (ATP-BL) biosensors were widely employed for on-site screening of bacteria. However, unstable luciferase with strict storage conditions limited its field detection. Moreover, the traditional ATP-BL biosensors lacked the sterilization effect. In this study, a robust and multifunctional zeolitic imidazolate framework-90 decorated with luciferase and polyethylene glycol–antimicrobial peptides (ZIF-90@Luc-AMP) was prepared. It could not only enhance the activity and stability of luciferase at ambient temperatures (≥1 month) but also sensitively detect target bacteria via the bioluminescence (BL) method and kill them (three birds with one stone). During the detection process, the target pathogen Escherichia coli O157:H7 (E. coli O157:H7) was specifically captured on a phage-modified stir bar and formed a sandwich complex with ZIF-90@Luc-AMP. With the synergistic bacteriolysis of ZIF-90@Luc-AMP and the phage, the captured E. coli O157:H7 was decomposed and emitted adenosine triphosphate (ATP), achieving the sterilization effect. Meanwhile, the introduced luciferase catalyzed ATP to produce a BL signal, which was proportional to the concentration of E. coli O157:H7. Combining ZIF-90@Luc-AMP and a phage-labeled stir bar for target recognition and enrichment, the entire analytical process could be easily manipulated within 30 min with a low limit of detection (20 CFU/mL) through a portable ATP bioluminescent meter. The multifunctional ZIF-90@Luc-AMP biosensor with high stability, specificity, and sensitivity was also appropriate for rapid pathogen screening and antibacterial application in the wild.

Strength of activation and temporal dynamics of bioluminescent-optogenetics in response to systemic injections of the luciferin

BioLuminescent OptoGenetics (“BL-OG”) is a chemogenetic method that can evoke optogenetic reactions in the brain non-invasively. In BL-OG, an enzyme that catalyzes a light producing reaction (i.e., a luciferase) is tethered to an optogenetic element that is activated in response to bioluminescent light. Bioluminescence is generated by injecting a chemical substrate (luciferin, e.g., h-Coelenterazine; h-CTZ) that is catalyzed by the luciferase. By directly injecting the luciferin into the brain, we show that bioluminescent light is proportional to spiking activity, and this relationship scales as a function of luciferin dosage. Here, we build on these previous observations by characterizing the temporal dynamics and dose response curves of bioluminescence generated by luminopsins (LMOs), a proxy of BL-OG effects, to intravenous (IV) injections of the luciferin. We imaged bioluminescence through a thinned skull of mice running on a wheel, while delivering h-CTZ via the tail vein with different dosage concentrations and injection rates. The data reveal a systematic relationship between strength of bioluminescence and h-CTZ dosage, with higher concentration generating stronger bioluminescence. We also found that bioluminescent activity occurs rapidly (< 60 s after IV injection) regardless of concentration dosage. However, as expected, the onset time of bioluminescence is delayed as the injection rate decreases. Notably, the strength and time decay of bioluminescence is invariant to the injection rate of h-CTZ. Taken together, these data show that BL-OG effects are highly consistent across injection parameters of h-CTZ, highlighting the reliability of BL-OG as a minimally invasive neuromodulation method.

Establishment of Translational Luciferase-Based Cancer Models to Evaluate Antitumoral Therapies.

Luciferase (luc) bioluminescence (BL) is the most used light-emitting protein that has been engineered to be expressed in multiple cancer cell lines, allowing for the detection of tumor nodules in vivo as it can penetrate most tissues. The goal of this study was to develop an oncolytic adenovirus (OAd)-resistant human triple-negative breast cancer (TNBC) that could express luciferase. Thus, when combining an OAd with chemotherapies or targeted therapies, we would be able to monitor the ability of these compounds to enhance OAd antitumor efficacy using BL in real time. The TNBC cell line HCC1937 was stably transfected with the plasmid pGL4.50[luc2/CMV/Hygro] (HCC1937/luc2). Once established, HCC1937/luc2 was orthotopically implanted in the 4th mammary gland fat pad of NSG (non-obese diabetic severe combined immunodeficiency disease gamma) female mice. Bioluminescence imaging (BLI) revealed that the HCC1937/luc2 cell line developed orthotopic breast tumor and lung metastasis over time. However, the integration of luc plasmid modified the HCC1937 phenotype, making HCC1937/luc2 more sensitive to OAdmCherry compared to the parental cell line and blunting the interferon (IFN) antiviral response. Testing two additional luc cell lines revealed that this was not a universal response; however, proper controls would need to be evaluated, as the integration of luciferase could affect the cells' response to different treatments.

Biosensing strategies using recombinant luminescent proteins and their use for food and environmental analysis.

Progress in synthetic biology and nanotechnology plays at present a major role in the fabrication of sophisticated and miniaturized analytical devices that provide the means to tackle the need for new tools and methods for environmental and food safety. Significant research efforts have led to biosensing experiments experiencing a remarkable growth with the development and application of recombinant luminescent proteins (RLPs) being at the core of this boost. Integrating RLPs into biosensors has resulted in highly versatile detection platforms. These platforms include luminescent enzyme-linked immunosorbent assays (ELISAs), bioluminescence resonance energy transfer (BRET)-based sensors, and genetically encoded luminescent biosensors. Increased signal-to-noise ratios, rapid response times, and the ability to monitor dynamic biological processes in live cells are advantages inherent to the approaches mentioned above. Furthermore, novel fusion proteins and optimized expression systems to improve their stability, brightness, and spectral properties have enhanced the performance and pertinence of luminescent biosensors in diverse fields. This review highlights recent progress in RLP-based biosensing, showcasing their implementation for monitoring different contaminants commonly found in food and environmental samples. Future perspectives and potential challenges in these two areas of interest are also addressed, providing a comprehensive overview of the current state and a forecast of the biosensing strategies using recombinant luminescent proteins to come.

Identification of NanoLuciferase Substrates Transported by Human ABCB1 and ABCG2 and Their Zebrafish Homologs at the Blood-Brain Barrier.

ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier (BBB) impede delivery of therapeutic agents to the brain, including agents to treat neurodegenerative diseases and primary and metastatic brain cancers. Two transporters, ABCB1 and ABCG2, are highly expressed at the BBB and are responsible for the efflux of numerous clinically useful chemotherapeutic agents, including irinotecan, paclitaxel, and doxorubicin. Based on a previous mouse model, we have generated transgenic zebrafish in which expression of NanoLuciferase (NanoLuc) is controlled by the promoter of glial fibrillary acidic protein, leading to expression in zebrafish glia. To identify agents that disrupt the BBB, including inhibitors of ABCB1 and ABCG2, we identified NanoLuc substrates that are also transported by ABCB1, ABCG2, and their zebrafish homologs. These substrates will elevate the amount of bioluminescent light produced in the transgenic zebrafish with BBB disruption. We transfected HEK293 cells with NanoLuc and either human ABCB1, ABCG2, or their zebrafish homologs Abcb4 or Abcg2a, respectively, that are expressed at the zebrafish BBB. We evaluated the luminescence and transporter substrate status of 16 NanoLuc substrates. We identified eight substrates that were efficiently pumped out by ABCB1, six by Abcb4, seven by ABCG2, and seven by Abcg2a. These data will aid in the development of a transgenic zebrafish model of the BBB to identify novel BBB disruptors and should prove useful in the development of other animal models that use NanoLuc as a reporter. SIGNIFICANCE STATEMENT: The ATP-binding cassette (ABC) transporters ABCB1 and ABCG2 at the blood-brain barrier (BBB) hinder pharmacological treatment of brain-related diseases. Consequently, there is a need for tools to identify BBB disruptors. This study screened 16 NanoLuciferase substrates, identifying the brightest and those that were transported by human and zebrafish ABC transporters at the BBB. This work supports and complements development of a transgenic zebrafish model, in which NanoLuciferase is expressed within glial cells, enabling detection of BBB disruption.

Phage LysSA163-CBD mediated specific recognition coupled with ATP bioluminescence for the sensitive detection of viable Staphylococcus aureus in food matrices

BackgroundStaphylococcus aureus is a significant foodborne pathogen, commonly detected in milk and meat products. Conventional detection methods have limited sensitivity and specificity, which are time-consuming and susceptible to background interference from complex samples, and cannot effectively distinguish live and dead bacteria. ResultsHerein, we developed a novel adenosine triphosphate (ATP) bioluminescence method coupled with magnetic separation, which is based on phage-encoded endolysin LysSA163-CBD (CBD 163) for rapid and specific detection of viable Staphylococcus aureus. The expressed protein (CBD 163) was derived from the phage LSA2301 and was successfully expressed in Escherichia coli BL21 following an induction period of 4 h at 37 °C, with a molecular weight approximating 40 kDa. The optimal conditions for CBD-magnetic beads (cMBs) to capture S. aureus cells were determined to be 100 μL/mL cMBs at 25 °C for 30 min. The viable S. aureus cells were disrupted by the Cetyl trimethyl ammonium bromide (CTAB) to release intracellular ATP. Then, the ATP reacted with the firefly luciferase and D-Luciferin-based bioluminescence (BL) reagents solution to generate intensive BL signal. The CBD-magnetic separation-ATP bioluminescence (cMS-BL) assay was able to quickly detect viable S. aureus via ATP bioluminescence in 45 min, with a detection range from 5 × 103 to 5 × 107 CFU/mL and limit of detection (LOD) of 190 CFU/mL. Additionally, the cMS-BL method exhibited high specificity and anti-interference ability, which has been successfully applied to quantify S. aureus cells in crayfish-tail, chicken, and skim milk. Significance and noveltyThese results demonstrate the potential of CBD 163 as a versatile and robust biorecognition element for rapid and specific detection of viable S. aureus in food matrices. The proposed phage-derived bacteria-binding proteins-based protocol for BL detection shows various advantages, including high sensitivity, simple operation, and the capability to distinguish live bacteria, providing a strategy for designing high-quality biorecognition element toward foodborne pathogens.

Light-triggered protease-mediated release of actin-bound cargo from synthetic cells.

Synthetic cells offer a versatile platform for addressing biomedical and environmental challenges, due to their modular design and capability to mimic cellular processes such as biosensing, intercellular communication, and metabolism. Constructing synthetic cells capable of stimuli-responsive secretion is vital for applications in targeted drug delivery and biosensor development. Previous attempts at engineering secretion for synthetic cells have been confined to non-specific cargo release via membrane pores, limiting the spatiotemporal precision and specificity necessary for selective secretion. Here, we designed and constructed a protein-based platform termed TEV Protease-mediated Releasable Actin-binding protein (TRAP) for selective, rapid, and triggerable secretion in synthetic cells. TRAP is designed to bind tightly to reconstituted actin networks and is proteolytically released from bound actin, followed by secretion via cell-penetrating peptide membrane translocation. We demonstrated TRAP's efficacy in facilitating light-activated secretion of both fluorescent and luminescent proteins. By equipping synthetic cells with a controlled secretion mechanism, TRAP paves the way for the development of stimuli-responsive biomaterials, versatile synthetic cell-based biosensing systems, and therapeutic applications through the integration of synthetic cells with living cells for targeted delivery of protein therapeutics.

Regiospecific Coelenterazine Analogs for Bioassays and Molecular Imaging.

Bioluminescence (BL) generated by luciferase-coelenterazine (CTZ) reactions is broadly employed as an optical readout in bioassays and in vivo molecular imaging. In this study, we demonstrate a systematic approach to elucidate the luciferase-CTZ binding chemistry with a full set of regioisomeric CTZ analogs, where all the functional groups were regiochemically modified. When the chemical structures were categorized into Groups 1-6, the even-numbered Groups (2, 4, and 6) of the CTZ analogs are found to be exceptionally bright with NanoLuc enzyme. A CTZ analogue M2 was the brightest with NanoLuc and the reason was deciphered by a computational analysis of the binding modes. We also report that (i) the regioisomeric CTZ analogs collectively create unique intensity patterns according to each marine luciferase, (ii) the quantitative structure-activity relationship analysis revealed the roles of respective functional groups of CTZ analogs, and (iii) the regioisomeric CTZ analogs also exert red shifts of the BL spectra and color variation: that is, the λmax values are near 500 nm with NanoLuc, near 530 nm with ALuc16, and near 570 nm with RLuc86SG. The advantages of the regioisomeric CTZ analogs were finally demonstrated using (i) a dual-luciferase system with M2-specific NanoLuc and native CTZ-specific ALuc16, (ii) an estrogen activatable single-chain BL probe by imaging, and (iii) BL imaging of live mice bearing tumors expressing NanoLuc and RLuc8.6SG. This study is the first systematic approach to elucidate the regiochemistry in BL imaging studies. This study provides new insights into how CTZ analogs regiochemically work in BL reporter systems and guides the specific applications to molecular imaging.

Direct measurements of luminal Ca 2+ with endo-lysosomal GFP-aequorin reveal functional IP 3 receptors.

Endo-lysosomes are considered acidic Ca 2+ stores but direct measurements of luminal Ca 2+ within them are limited. Here we report that the Ca 2+ -sensitive luminescent protein aequorin does not reconstitute with its cofactor at highly acidic pH but that a significant fraction of the probe is functional within a mildly acidic compartment when targeted to the endo-lysosomal system. We leveraged this probe (ELGA) to report Ca 2+ dynamics in this compartment. We show that Ca 2+ uptake is ATP-dependent and sensitive to blockers of endoplasmic reticulum Ca 2+ pumps. We find that the Ca 2+ mobilizing messenger IP 3 which typically targets the endoplasmic reticulum evokes robust luminal responses in wild type cells, but not in IP 3 receptor knock-out cells. Responses were comparable to those evoked by activation of the endo-lysosomal ion channel TRPML1. Stimulation with IP 3 -forming agonists also mobilized the store in intact cells. Super-resolution microscopy analysis confirmed the presence of IP 3 receptors within the endo-lysosomal system, both in live and fixed cells. Our data reveal a physiologically-relevant, IP 3 -sensitive store of Ca 2+ within the endo-lysosomal system.

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.

Fungal burden, dimorphic transition and candidalysin: Role in Candida albicans-induced vaginal cell damage and mitochondrial activation in vitro.

Candida albicans (C. albicans) can behave as a commensal yeast colonizing the vaginal mucosa, and in this condition is tolerated by the epithelium. When the epithelial tolerance breaks down, due to C. albicans overgrowth and hyphae formation, the generated inflammatory response and cell damage lead to vulvovaginal candidiasis (VVC) symptoms. Here, we focused on the induction of mitochondrial reactive oxygen species (mtROS) in vaginal epithelial cells after C. albicans infection and the involvement of fungal burden, morphogenesis and candidalysin (CL) production in such induction. Bioluminescent (BLI) C. albicans, C. albicans PCA-2 and C. albicans 529L strains were employed in an in vitro infection model including reconstituted vaginal epithelium cells (RVE), produced starting from A-431 cell line. The production of mtROS was kinetically measured by using MitoSOX™ Red probe. The potency of C. albicans to induced cell damage to RVE and C. albicans proliferation have also been evaluated. C. albicans induces a rapid mtROS release from vaginal epithelial cells, in parallel with an increase of the fungal load and hyphal formation. Under the same experimental conditions, the 529L C. albicans strain, known to be defective in CL production, induced a minor mtROS release showing the key role of CL in causing epithelial mithocondrial activation. C. albicans PCA-2, unable to form hyphae, induced comparable but slower mtROS production as compared to BLI C. albicans yeasts. By reducing mtROS through a ROS scavenger, an increased fungal burden was observed during RVE infection but not in fungal cultures grown on abiotic surface. Collectively, we conclude that CL, more than fungal load and hyphae formation, seems to play a key role in the rapid activation of mtROS by epithelial cells and in the induction of cell-damage and that mtROS are key elements in the vaginal epithelial cells response to C. albicans.

A bioluminescent probe for imaging human cytochrome P450 2J2 activity in vitro and in vivo

Human Cytochrome P450 2J2 (CYP 2J2) is closely associated with various physiological diseases. However, bioluminescence (BL) tools for monitoring CYP 2J2 activity have not yet been reported. Herein, we rationally designed a BL probe, PJ-Luc, capable of real-time imaging of CYP 2J2. In vitro studies demonstrated that PJ-Luc can effectively respond to exogenous CYP 2J2 with high selectivity and a low detection limit (LOD = 0.11 nM). Further, living cell experiments indicated that PJ-Luc can accurately quantify the ratio of endogenous CYP 2J2 amounts in HepG-2-Luc and A549-Luc cells. Moreover, an ultrahigh BL signal-to-noise ratio (S/N = 2.3×105) was achieved in tumor-bearing mouse models. These results suggest that PJ-Luc has great potential in the diagnosis of tumors and other CYP-2J2-related diseases.

Toward a brighter constellation: multiorgan neuroimaging of neural and vascular dynamics in the spinal cord and brain.

Pain comprises a complex interaction between motor action and somatosensation that is dependent on dynamic interactions between the brain and spinal cord. This makes understanding pain particularly challenging as it involves rich interactions between many circuits (e.g., neural and vascular) and signaling cascades throughout the body. As such, experimentation on a single region may lead to an incomplete and potentially incorrect understanding of crucial underlying mechanisms. We aimed to develop and validate tools to enable detailed and extended observation of neural and vascular activity in the brain and spinal cord. The first key set of innovations was targeted to developing novel imaging hardware that addresses the many challenges of multisite imaging. The second key set of innovations was targeted to enabling bioluminescent (BL) imaging, as this approach can address limitations of fluorescent microscopy including photobleaching, phototoxicity, and decreased resolution due to scattering of excitation signals. We designed 3D-printed brain and spinal cord implants to enable effective surgical implantations and optical access with wearable miniscopes or an open window (e.g., for one- or two-photon microscopy or optogenetic stimulation). We also tested the viability for BL imaging and developed a novel modified miniscope optimized for these signals (BLmini). We describe "universal" implants for acute and chronic simultaneous brain-spinal cord imaging and optical stimulation. We further describe successful imaging of BL signals in both foci and a new miniscope, the "BLmini," which has reduced weight, cost, and form-factor relative to standard wearable miniscopes. The combination of 3D-printed implants, advanced imaging tools, and bioluminescence imaging techniques offers a coalition of methods for understanding spinal cord-brain interactions. Our work has the potential for use in future research into neuropathic pain and other sensory disorders and motor behavior.

A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae.

Modular Cloning (MoClo) is based on libraries of standardized genetic parts that can be directionally assembled via Golden Gate cloning in one-pot reactions into transcription units and multigene constructs. Here, a team of bachelor students established a MoClo toolkit for the protist Leishmania tarentolae in the frame of the international Genetically Engineered Machine (iGEM) competition. Our modular toolkit is based on a domesticated version of a commercial LEXSY expression vector and comprises 34 genetic parts encoding various affinity tags, targeting signals as well as fluorescent and luminescent proteins. We demonstrated the utility of our kit by the successful production of 16 different tagged versions of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in L. tarentolae liquid cultures. While highest yields of secreted recombinant RBD were obtained for GST-tagged fusion proteins 48 h post induction, C-terminal peptide tags were often degraded and resulted in lower yields of secreted RBD. Fusing secreted RBD to a synthetic O-glycosylation SP20 module resulted in an apparent molecular mass shift around 10 kDa. No disadvantage regarding the production of RBD was detected when the three antibiotics of the LEXSY system were omitted during the 48-h induction phase. Furthermore, the successful purification of secreted RBD from the supernatant of L. tarentolae liquid cultures was demonstrated in pilot experiments. In summary, we established a MoClo toolkit and exemplified its application for the production of recombinant proteins in L. tarentolae.

Abstract 7427: Automated, real-time acquisition and quantification of peak, plateau-phase in vivo bioluminescent data

Abstract It is widely recognized that plateau-phase bioluminescent (BL) kinetic curve data should be used when reporting on BLI results from substrate-injection BLI model systems. The basis for this understanding is that plateau-phase BL data is uniquely suited in several ways: (i) it will optimize the detection of target, luciferase-expressing cells (especially when they are low in copy number or deep within the animal model), (ii) it will optimize BLI data reproducibility (with there being no change in signal values across the plateau phase), and (iii) it will also provide BLI data values that correlate consistently with the number of viable target cells in a given BLI model. Given this critical value of plateau-phase BL data, there is, nevertheless, a simple practical issue to be addressed: The overall manual process of generating BL kinetic curves, and then identifying and quantifying plateau-phase data is not insignificant—it takes time!In this poster, we review the performance of “Kinetics,” a new feature in the Aura software platform from Spectral Instruments Imaging, LLC. Kinetics is designed to collect, present, and analyze BL kinetic curve data for up to 10 mice, in a completely automated and real-time fashion. Data outputs include a live graph that simultaneously presents individual mouse, whole body ROI (Total photon/sec) values vs. time post-luciferase injection, and analogous mean BL values per mouse group. Here in, we present an evaluation of the performance of Kinetics under various testing conditions. We initially evaluated Kinetics’ ability to acquire, present and analyze the BL kinetic curves of 5 phantoms. The photon output rates of these phantoms (Total photons/sec) were programmed to mimic the rise, plateau, and fall sequence typically seen BLI studies. In an analogous fashion, we then tested Kinetics’ ability to acquire, present, and analyze the BL kinetic curves of 5 to 10 live mice from several oncology models. Furthermore, we used Kinetics in oncology efficacy studies to monitor for expected changes in BL kinetic curve shapes (i.e., for changes in the onset times and durations of the rise, plateau and fall phases in BL kinetic curves) between different treatment groups (e.g., between positive controls groups and one or more treatment groups). In the same studies, we also used Kinetics to check for changes in BL kinetic curve shapes that might occur for a given treatment group across several time points, after pathogen challenge. Results from this set of experiments consistently and clearly illustrated the capability of Kinetics to acquire, present and analyze individual and mean BL kinetic curve data for up to 10 mice at a time, in an automated and real-time fashion. We believe that Kinetics from Spectral Instruments Imaging, LLC, has the potential to revolutionize the ease with which plateau-phase, BL kinetic curve data can be collected, presented, and used in a wide range of preclinical BLI studies. Citation Format: Andrew Van Praagh, Bo Nelson, Paul Ballieu, Melanie Smith, Mike Rule. Automated, real-time acquisition and quantification of peak, plateau-phase in vivo bioluminescent data [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 7427.