Bioluminescence Measurements Research Articles

Convection-delivered adenoviral gene therapy reprograms the immunosuppressive glioblastoma microenvironment.

2039 Background: Immune checkpoint inhibition has not improved outcomes for glioblastoma patients, and single- cell approaches reveal the glioblastoma microenvironment is largely comprised of immunosuppressive cells. We hypothesized intratumor convection enhanced delivery (CED) of adenoviral gene therapy could recruit and activate anti-tumor immune cells in the glioblastoma microenvironment. Methods: Syngeneic GL261 (3x105 cells/mouse) or SB28 glioblastoma (3 x 104) cells were implanted into the frontal lobe of immunocompetent C57BL/6J mice (18 mice/arm). Intracranial bioluminescence (BLI) and body weight (BW) measurements were used to assess glioblastoma growth and treatment toxicity, respectively. After tumor engraftment, glioblastomas were treated with conformal ionizing radiation mimicking stereotactic radiosurgery (SRS) in human patients as a positive control for tumor inhibition (18Gy/1Fx). Attenuated adeno-associated virus 9 (AAV9) vectors encoding Gfp as a negative control, or encoding experimental cytokines driving recruitment and activation of anti-tumor immune cells in other intracranial tumors ( Ccl4, Il1b, or Apoa1) were delivered using CED (2x1011 vg/mouse). Glioblastomas were collected for histologic, single-cell, and molecular analyses 5 days after treatment (6 mice/arm) and at endpoints after monitoring for survival (12 mice/arm). CED targeting was validated using AAV9-GFP and confocal microscopy. Treatment responses were assessed using H&E, IHC, multiplexed cytokine assays, and single cell mass cytometry (CyTOF) to define immune cell types in the glioblastoma microenvironment. Results: Histologic analyses revealed AAV9-CCL4, AAV9-IL1B, or SRS induced glioblastoma macrophage infiltration, and AAV9-IL1B, AAV9-APOA1, or SRS induced glioblastoma T cell infiltration. AAV9-APOA1 (18.5 days versus 15 days, p < 0.001) or AAV9-IL1B (16.5 days versus 15 days, p = 0.001) CED treatments prolonged median survival from SB28 allografts. Glioblastoma cytokine analysis revealed inhibition of pro-tumor cytokines (IL6, LIF) after experimental CED treatments. Systemic cytokines were minimally changed by CED treatments. CyTOF showed decreased immunosuppressive macrophage infiltration and increased CD8+ T cell or microglia infiltration of the glioblastoma microenvironment after experimental CED treatments. There was no evidence of systemic or central toxicity in any treatment condition. Conclusions: Convection-enhanced delivered of adenoviral gene therapy reprograms the glioblastoma immune microenvironment and improves survival in an immunologically “cold” syngeneic glioblastoma model.

Endothelial Pannexin1‐purinergic signaling pulmonary hypertension

Endothelial cell (EC) Ca2+ signals dilate small pulmonary arteries (PAs) and maintain a low resting pulmonary arterial pressure (PAP). Impairment of endothelial Ca2+ signaling mechanisms is a crucial contributor to elevated PAP in pulmonary hypertension (PH). Studies in systemic arteries show that flow/shear stress activates endothelial Ca2+ signals to cause vasodilation. Although PAs are a “high‐flow” vascular bed, flow‐induced endothelial Ca2+ signaling mechanisms have not been investigated in PH. In this regard, we recently showed that Ca2+ influx through endothelial transient receptor potential vanilloid 4 (TRPV4EC) ion channels dilates PAs and lowers resting PAP (Daneva et al., PNAS, 2021). Moreover, flow/shear stress activates the efflux of adenosine triphosphate (ATP) through endothelial Pannexin 1 (Panx1EC) in PAs. Panx1EC‐effluxed ATP, in turn, stimulates endothelial purinergic P2Y2 receptor (P2Y2REC) to increase TRPV4EC channels activity (Daneva et al., eLife, 2021). I hypothesized that impaired Panx1EC—P2Y2REC—TRPV4EC signaling axis reduces flow‐induced dilation of PAs and elevates PAP in PH. Confocal Ca2+ imaging studies in fluo‐4‐loaded en face PAs (~ 50 mm) showed that the baseline and agonist (GSK1016790A or GSK101)‐induced activity of TRPV4EC sparklets (individual Ca2+ influx signals through TRPV4EC channels) is reduced in the mice exposed to Sugen 5416 + chronic hypoxia (Su+CH; 3 weeks; 10% O2) compared to the normoxic control mice, indicating a decrease in TRPV4EC channel activity in PH. The expression of TRPV4 channels at the mRNA level, however, was not altered in Su+CH mice. The activation of TRPV4EC sparklets by exogenous ATP (1 mM) was also reduced in PAs from Su+CH mice, suggesting an impairment of ATP‐P2Y2REC‐TRPV4EC channel signaling axis in PH. Accordingly, ATP‐induced, endothelium‐dependent dilation was absent in PAs from Su+CH mice. We previously showed that basal ATP efflux in PAs predominantly occurs through endothelial Panx1, and flow/shear stress increases ATP efflux entirely through the activation of endothelial Panx1. Bioluminescence measurements of extracellular ATP concentration showed lower baseline ATP levels in PAs from Su+CH mice than control mice, suggesting a decrease in ATP efflux in PH. Importantly, flow/shear stress (4 – 15 dynes/cm2) increased the efflux of ATP from normal mice, but not in PAs from Su+CH mice, indicating that flow/shear stress‐Panx1EC signaling is also impaired in PH. Together, these data support the idea that flow‐Panx1EC and P2Y2REC‐TRPV4EC signaling are impaired in PH. Targeting individual elements of this pathway may provide novel therapeutic options in PH and other pulmonary vascular disorders characterized by endothelial dysfunction. Future studies will assess the individual signaling linkages of the flow‐Panx1EC‐P2Y2REC‐TRPV4EC signaling axis in PH.

Laboratory experiment of ATP measurement using Mars soil simulant: as a method for extraterrestrial life detection

We present a laboratory experiment of ATP measurement using Mars soil simulant and Escherichia coli (E. coli) with a point of view for future application to searching extraterrestrial life. We used Mars Global Simulant MGS-1 (Exolith Lab) as soil simulant, added E. coli suspension to it, then the soil simulant with E. coli was dried. Various configurations of samples with different E. coli density, 1.75 × 102, 1.75 × 103, 1.75 × 104, 1.75 × 105, and 1.75 × 106 cells (g soil)−1, were prepared together with controls. ATP extraction reagent and luminescence reagent were added to the sample, and bioluminescence measurement was performed. The result suggests significant detection of ATP for samples with E. coli density used in this work. Similar experiments but without the soil simulant were carried out, and results with and without the soil simulant are compared. Based on the ATP measurement studied in this work, we discussed extraterrestrial life search, planetary protection, relation with the panspermia hypothesis, and also other applications.Graphical abstract

A Dual-Reporter Platform for Screening Tumor-Targeted Extracellular Vesicles.

Extracellular vesicle (EV)-mediated transfer of biomolecules plays an essential role in intercellular communication and may improve targeted drug delivery. In the past decade, various approaches to EV surface modification for targeting specific cells or tissues have been proposed, including genetic engineering of parental cells or postproduction EV engineering. However, due to technical limitations, targeting moieties of engineered EVs have not been thoroughly characterized. Here, we report the bioluminescence resonance energy transfer (BRET) EV reporter, PalmReNL-based dual-reporter platform for characterizing the cellular uptake of tumor-homing peptide (THP)-engineered EVs, targeting PDL1, uPAR, or EGFR proteins expressed in MDA-MB-231 breast cancer cells, simultaneously by bioluminescence measurement and fluorescence microscopy. Bioluminescence analysis of cellular EV uptake revealed the highest binding efficiency of uPAR-targeted EVs, whereas PDL1-targeted EVs showed slower cellular uptake. EVs engineered with two known EGFR-binding peptides via lipid nanoprobes did not increase cellular uptake, indicating that designs of EGFR-binding peptide conjugation to the EV surface are critical for functional EV engineering. Fluorescence analysis of cellular EV uptake allowed us to track individual PalmReNL-EVs bearing THPs in recipient cells. These results demonstrate that the PalmReNL-based EV assay platform can be a foundation for high-throughput screening of tumor-targeted EVs.

Agreement of Bioluminescence Measurements and Visual Assessment in Monitoring Occlusal Surfaces of Permanent Teeth

Background: Caries lesion activity is typically assessed by visual–tactile criteria. Regular monitoring is required to detect the transition of lesions and to ensure that the initial assessment was valid. This clinical study aimed to evaluate the agreement of bioluminescence measurements (Calcivis imaging system, Cis) with visual examination to assess caries lesion activity and to monitor occlusal surfaces. Methods: The occlusal surfaces of ninety-one permanent posterior teeth were assessed for the presence or absence of active caries lesions with ICCMS criteria and Cis measurements at three visit times: baseline (t1) and six months (t2) and 12 months (t3) after baseline. Results: At the baseline visit, 70% of the included occlusal sites were assessed visually as active lesions (ICCMS codes 1 and 2). At t3, 64.8% of the sites showed signs of an active lesion. The percentage agreements between the visual and Cis methods were 87.8% (t1), 89.9% (t2) and 88.6% (t3). The corresponding κ-values were 0.71 (95% CI 0.52;0.87), 0.75 (95% CI 0.59;0.89) and 0.77 (95% CI 0.61;0.90), respectively. No significant difference between the visual and bioluminescence systems was found at any visit (p > 0.05). The results based on cluster randomization (generalized estimation equations) showed no significant differences between the visual and Cis findings for all visits (p = 0.108, Wald Χ2 with 1 df = 2.587). Conclusion: The bioluminescence system demonstrated substantial agreement for the activity assessment of occlusal lesions compared to the findings obtained by visual assessment over twelve months.

A Novel In Vitro Method to Assess the Microbial Barrier Function of Tissue Adhesives Using Bioluminescence Imaging Technique.

Background. Tissue glues can minimize treatment invasiveness, mitigate the risk of infection, and reduce surgery time; ergo, they have been developed and used in surgical procedures as wound closure devices beside sutures, staples, and metallic grafts. Regardless of their structure or function, tissue glues should show an acceptable microbial barrier function before being used in humans. This study proposes a novel in vitro method using Escherichia coli Lux and bioluminescence imaging technique to assess the microbial barrier function of tissue glues. Different volumes and concentrations of E. coli Lux were applied to precured or cured polyurethane-based tissue glue placed on agar plates. Plates were cultured for 1 h, 24 h, 48 h, and 72 h with bioluminescence signal measurement subsequently. Herein, protocol established a volume of 5 μL of a 1 : 100 dilution of E. coli Lux containing around 2 × 107 CFU/mL as optimal for testing polyurethane-based tissue glue. Measurement of OD600nm, determination of CFU/mL, and correlation with the bioluminescence measurement in p/s unit resulted in a good correlation between CFU/mL and p/s and demonstrated good reproducibility of our method. In addition, this in vitro method could show that the tested polyurethane-based tissue glue can provide a reasonable barrier against the microbial penetration and act as a bacterial barrier for up to 48 h with no penetration and up to 72 h with a low level of penetration through the material. Overall, we have established a novel, sensitive, and reproducible in vitro method using the bioluminescence imaging technique for testing the microbial barrier function of new tissue glues.

The effect of spin exchange interaction on protein structural stability.

Partially charged chiral molecules act as spin filters, with preference for electron transport toward one type of spin ("up" or "down"), depending on their handedness. This effect is named the chiral induced spin selectivity (CISS) effect. A consequence of this phenomenon is spin polarization concomitant with electric polarization in chiral molecules. These findings were shown by adsorbing chiral molecules on magnetic surfaces and investigating the spin-exchange interaction between the surface and the chiral molecule. This field of study was developed using artificial chiral molecules. Here we used such magnetic surfaces to explore the importance of the intrinsic chiral properties of proteins in determining their stability. First, proteins were adsorbed on paramagnetic and ferromagnetic nanoparticles in a solution, and subsequently urea was gradually added to induce unfolding. The structural stability of proteins was assessed using two methods: bioluminescence measurements used to monitor the activity of the Luciferase enzyme, and fast spectroscopy detecting the distance between two chromophores implanted at the termini of a Barnase core. We found that interactions with magnetic materials altered the structural and functional resilience of the natively folded proteins, affecting their behavior under varying mild denaturing conditions. Minor structural disturbances at low urea concentrations were impeded in association with paramagnetic nanoparticles, whereas at higher urea concentrations, major structural deformation was hindered in association with ferromagnetic nanoparticles. These effects were attributed to spin exchange interactions due to differences in the magnetic imprinting properties of each type of nanoparticle. Additional measurements of proteins on macroscopic magnetic surfaces support this conclusion. The results imply a link between internal spin exchange interactions in a folded protein and its structural and functional integrity on magnetic surfaces. Together with the accumulating knowledge on CISS, our findings suggest that chirality and spin exchange interactions should be considered as additional factors governing protein structures.

Investigating Mitochondrial Ca2+ Dynamics in Isolated Mitochondria and Intact Cells: Application of Fluorescent Dyes and Genetic Reporters.

Mitochondrial Ca2+ buffering is a hallmark of eukaryotic cellular physiology, contributing to the spatiotemporal shaping of the cytosolic Ca2+ signals and regulation of mitochondrial bioenergetics. Often, this process is altered in a pathological context; therefore, it can be scrutinized experimentally for therapeutic intervention. In this chapter, we describe fluorescence and bioluminescence measurement of mitochondrial Ca2+ in both isolated mitochondria and intact cells.

Methods for Detecting PER2:LUCIFERASE Bioluminescence Rhythms in Freely Moving Mice.

Circadian rhythms are driven by daily oscillations of gene expression. An important tool for studying cellular and tissue circadian rhythms is the use of a gene reporter, such as bioluminescence from the reporter gene luciferase controlled by a rhythmically expressed gene of interest. Here we describe methods that allow measurement of circadian bioluminescence from a freely moving mouse housed in a standard cage. Using a LumiCycle In Vivo (Actimetrics), we determined conditions that allow detection of circadian rhythms of bioluminescence from the PER2 reporter, PER2::LUC, in freely behaving mice. The LumiCycle In Vivo applies a background subtraction that corrects for effects of room temperature on photomultiplier tube (PMT) output. We tested delivery of d-luciferin via a subcutaneous minipump and in the drinking water. We demonstrate spikes in bioluminescence associated with drinking bouts. Further, we demonstrate that a synthetic luciferase substrate, CycLuc1, can support circadian rhythms of bioluminescence, even when delivered at a lower concentration than d-luciferin, and can support longer-term studies. A small difference in phase of the PER2::LUC bioluminescence rhythms, with females phase leading males, can be detected with this technique. We share our analysis scripts and suggestions for further improvements in this method. This approach will be straightforward to apply to mice with tissue-specific reporters, allowing insights into responses of specific peripheral clocks to perturbations such as environmental or pharmacological manipulations.

Influence of the Smoking and Spitting Behaviors in Public Smoking Areas on the Microbial Contamination and Spread

Objectives : The purpose of this study was to investigate the effect of spitting behavior on microbial public health in smoking areas. Through this investigation, bacterial concentration and spread of bacteria from the smoking areas were evaluated for the prospective public health and environment.Methods : The number of spitting and the spitting location in the smoking areas were measured visually by observers. For the microbial analysis, filter papers (5 × 5 cm) as sample collectors had been attached to the surface of the smoking areas for a given time. Using the collected filter paper, ATP bioluminescence measurement (RLU), colony forming unit (CFU) were measured. For the estimation of spitting behaviors in the smoking area on the spread microorganisms, saliva containing filters were selected from the areas, and fluorescent powder (0.01 g/cm2) was added to the papers. After 4 hours, the papers around the smoking areas were collected and the fluorescence activity was measured.Results and Discussion : The results showed that there was a clear relationship between the spitting behavior and the microbial activity in the smoking areas. In the case of sampling area A, the highest number of spitting was observed at the lunch hour (102 ± 16 times), and the RLU from the sample collected near the smoking area showed about 5,139 ± 1,267 RLU. When the RLU around the smoking area was measured, the lowest values were found at distance of 11 and 17 m from the cigarette bin (1,329 ± 148 and 1,204 ± 203 RLU), and it was confirmed that smoking and spitting did not occur at this sampling points. In the case of sampling area B, the highest number of spitting (45 ± 6 times) was observed at evening hour, and the RLU from the sample collected near the smoking area showed about 5,274 ± 1,297 RLU. The lowest value was in the sampling area B shown at 11 and 18 m (1,181 ± 243 RLU and 1,148 ± 168 RLU) from the cigarette bin, and it was also confirmed that smoking and spitting did not occur at this sampling points. The results indicate that the spitting during the smoking induced the increases of the microbial contamination of the public environment. The fluorescence powder diffusion analysis indicated that the saliva on the ground surface would enhance the spread of the microorganisms from the smoking area.Conclusions : Spitting in smoking areas can pose a high risk of microbial infection for smokers and non-smokers in smoking areas. Thus extensive and systematic attention related to the smoking manner must be paid to protect our public health.

Engineered Interleukin-15 Autocrine Signaling Invigorates Anti-CD123 CAR-NK Cells

Introduction: Acute Myeloid Leukemia (AML) is an aggressive neoplastic disorder with poor outcomes in children and adults. NK cell adoptive transfer is an anti-cancer immunotherapy that has promise for AML treatment. We aimed to improve NK cell anti-tumor efficacy with expression of a Chimeric Antigen Receptor (CAR) on the cell surface. Our CAR consists of an extracellular single-chain variable fragment targeting the AML-associated antigen CD123 (IL3Rα) and intracellular domains derived from 2B4 and TCRζ. We sought to improve the persistence and long-term functionality of our CAR-NKs by introducing transgenic interleukin-15 (IL15).Methods: CD3-depleted PBMCs were first activated with lethally irradiated feeder cells, then transduced with transiently produced replication incompetent γ-retrovirus (αCD123.2B4.ζ, αCD123.2B4.ζ-IRES-sIL15, sIL15-IRES-mOrange) on day 4 of culture. CAR expression was measured on day 8 using FACS. Secretion of IL15 was verified with ELISA. Cytotoxicity was measured using ffLuc expressing target cells and bioluminescence (BL) measurement. In serial stimulation assays, target cells were repleted daily to maintain a 1:1 effector:target ratio. Immunophenotype and cell counts were assessed by FACS. Transcriptomic analysis (RNAseq) was performed on RNA derived from NK cells purified on D10. Xenograft modeling was performed using NSG mice engrafted with MV-4-11.ffLuc or MOLM-13.ffLuc AML cell lines. Mice were treated with NK cells on D4 or D4-7-10. Untreated mice served as controls. Tumor growth was serially tracked in vivo using BL imaging. NK cell persistence and expansion were measured in peripheral blood.Results: The 2B4.ζ CAR was well expressed on the surface of transduced NK cells (median transduction efficiency 95%, range 85-97%, n=3). 2B4.ζ CAR-NK treatment prolonged survival of AML engrafted mice when compared to treatment with unmodified NKs (median survival: 63 vs 55 days; n=8 mice; p=0.014). Serial peripheral blood analysis revealed a steady decline in circulating NK cells, which were undetectable in all cohorts within 21 days. NK cells were then engineered for constitutive secretion of IL15, with and without CAR expression. 2B4.ζ/sIL15 CAR-NKs had the most potent 24h-cytotoxicity against CD123+ targets (Fig. 1). After a 10-day chronic stimulation with MV-4-11, 2B4.ζ/sIL15- and sIL15-NKs expanded (x1.2 and x5.9 respectively), while NK cells without sIL15 decreased in number. In this assay, only 2B4.ζ/sIL15 CAR-NKs exhibited sustained tumor killing. Transcriptomic analysis after 10 days of serial stimulation showed sample clustering dependent on IL15 secretion. Differential gene expression analysis (DESeq2) identified upregulation of genes associated with cell cycle progression, apoptosis regulation, chemokine signaling, and NK cell mediated cytotoxicity in NK cells secreting IL15 compared to those without. In multiparameter flow cytometric analysis, 2B4.ζ/sIL15 CAR-NKs had a higher percentage of NK cells populating clusters defined by higher surface expression of NK cell activating receptors (NKp30, NKG2D, LFA-1) compared to 2B4.ζ and unmodified NK cells. In our MV-4-11 xenograft model, NKs armed with secreted IL15 expanded in vivo and had improved persistence. A single dose (D4) of 2B4.ζ/sIL15 CAR-NKs demonstrated an initial antitumor response, equivalent to that seen following 3 doses (D4-7-10) of 2B4.ζ CAR-NKs. However, mice treated with IL15-secreting NKs had short survival (Fig. 2). Compared to control mice, peripheral blood analysis showed increasing systemic hIL15 and higher levels of hTNFα. In our more aggressive MOLM-13 xenograft model, both single dose 2B4.ζ/sIL15 CAR-NK and multiple dose 2B4.ζ CAR-NK treatment prolonged survival compared to treatment with unmodified NKs. (27 and 26 vs 20 days; n=5 mice; p<0.01; Fig. 2).Conclusion: 2B4.ζ CAR-NKs have limited antitumor efficacy and short persistence in vivo. NK cells armored with secreted IL15 have enhanced anti-AML cytotoxicity and in vitro persistence. Introduction of IL15 secretion confers a distinctly activated phenotype that is maintained during chronic antigen stimulation. Constitutive local IL15 secretion improves in vivo NK cell persistence but may cause lethal toxicity when employed against AML. These results warrant further study and should impact the development of CAR-NK clinical products for patients with AML. [Display omitted] DisclosuresHo: Rodeo Therapeutics/Amgen: Patents & Royalties; Exelixis: Consultancy; Sanofi: Research Funding. Bonifant: Kiadis Pharma: Research Funding; BMS: Research Funding; Merck, Sharpe, Dohme: Research Funding.

Efficacy of Removing Bacteria and Organic Dirt from Hands-A Study Based on Bioluminescence Measurements for Evaluation of Hand Hygiene When Cooking.

The objective of this study was to evaluate the efficacy of dirt removal (bacteria and organic matter) of several hand-cleaning procedures. The results from the hand hygiene experiment indicated that washing hands with warm water and soap for 20 s is the most effective method investigated when hands are either dirty or greasy. Even if not proper washing, rinsing under running water for 5 s is a cleaning procedure that may significantly reduce the probability of cross-contamination, as it removes 90% of the hands’ dirt. Although less effective than water and soap, the usage of antibacterial wipes was significantly more effective than wet wipes, indicating that they are a better choice when water and soap are not available. The results of this study enable us to inform consumers about the effectiveness of hand-cleaning procedures applied in their homes when cooking. Moreover, it can make consumers understand why, during the COVID-19 pandemic, authorities recommended washing hands as a preventive measure of infection and using an anti-bacterial hand gel or wiping hands with an antimicrobial wipe if water and soap are not available.

Quantifying dose-, strain-, and tissue-specific kinetics of parainfluenza virus infection.

Human parainfluenza viruses (HPIVs) are a leading cause of acute respiratory infection hospitalization in children, yet little is known about how dose, strain, tissue tropism, and individual heterogeneity affects the processes driving growth and clearance kinetics. Longitudinal measurements are possible by using reporter Sendai viruses, the murine counterpart of HPIV 1, that express luciferase, where the insertion location yields a wild-type (rSeV-luc(M-F*)) or attenuated (rSeV-luc(P-M)) phenotype. Bioluminescence from individual animals suggests that there is a rapid increase in expression followed by a peak, biphasic clearance, and resolution. However, these kinetics vary between individuals and with dose, strain, and whether the infection was initiated in the upper and/or lower respiratory tract. To quantify the differences, we translated the bioluminescence measurements from the nasopharynx, trachea, and lung into viral loads and used a mathematical model together a nonlinear mixed effects approach to define the mechanisms distinguishing each scenario. The results confirmed a higher rate of virus production with the rSeV-luc(M-F*) virus compared to its attenuated counterpart, and suggested that low doses result in disproportionately fewer infected cells. The analyses indicated faster infectivity and infected cell clearance rates in the lung and that higher viral doses, and concomitantly higher infected cell numbers, resulted in more rapid clearance. This parameter was also highly variable amongst individuals, which was particularly evident during infection in the lung. These critical differences provide important insight into distinct HPIV dynamics, and show how bioluminescence data can be combined with quantitative analyses to dissect host-, virus-, and dose-dependent effects.

Use of bioluminescence measurements for detection of artificial demineralization adjacent to orthodontic brackets.

Enamel demineralization can occur as aside effect during orthodontic treatment with fixed appliances and should be detected as early as possible. Anew approach to assess demineralization is asystem consisting of aphotosensitive protein that binds to free calcium ions at the enamel surface. Acamera is then used to visualize the bioluminescence spots. This invitro study aimed to evaluate the ability of the bioluminescence technology to assess artificially demineralized enamel adjacent to various orthodontic brackets. In all, 108 human enamel samples were allocated randomly to groups with different orthodontic bracket material: stainless steel, titanium, ceramic. Initial lesions were created adjacent to the brackets. The samples were assessed by bioluminescence before and after demineralization. Images were assessed for presence of bioluminescence spots (yes/no). To quantify the bioluminescence measurements, the images' pixel values(P) were calculated within adefined area(F) adjacent to each bracket before and after demineralization. Quantitative light-induced fluorescence measurements (ΔF, ΔQ) were performed as the reference standard for demineralization. After demineralization, bioluminescence spots were visible (yes/no decision) in 87% of the samples. The pixel analysis of the bioluminescence spots showed significantly higher pixel values after demineralization compared to baseline (p < 0.0001). The bracket material had no influence on the bioluminescence measurements. All samples showed fluorescence loss with amedian ΔF of -9.52% (±3.15) and amedian ΔQ of -1.01% × mm2 (±3.34), respectively. The bioluminescence technology is apromising tool to demonstrate demineralization adjacent to different orthodontic brackets invitro.

Abstract 2929: Comparative analysis of circulating human tumor DNA and bioluminescent imaging in monitoring tumor burden and therapeutic response of ionizing radiation in intratibial human prostate tumor xenografts

Abstract Purpose/Objective: Pre-clinical models of prostate cancer (PCa) metastases provide critical insight into tumor biology and therapeutic response. Current models require sophisticated reporter transgene models and/or expensive tools to noninvasively monitor tumors in metastatic sites, such as bone. Recent developments in the measurement of circulating tumor DNA (ctDNA) provide an alternative method to noninvasively assess tumor burden at a low cost. Here we present a comparative analysis of human PCa xenograft tumor growth and therapeutic response by simultaneous measurement of ctDNA and bioluminescence. Materials and methods: Male athymic nude mice were used for intra-tibial (n=10) modeling. LNCaP-CMV-Luc cells were implanted in the tibial medullary canal. Tumor burden was evaluated at pre and post-IR (focal exposure 6 Gy on day 28 post-implantation) time-points by in vivo bioluminescent imaging and quantitative real-time polymerase chain reaction (qPCR) for human LINE1/Alu DNA. qPCR was performed on plasma-derived DNA consecutively on the same day as bioluminescence. Results: Human genomic DNA quantification was sensitive to 0.1 pg and linear by hAlu (R2=0.98, for 0.1pg-150ng) and LINE DNA (R2=0.99, for 0.1pg-150ng) qPCR. hAlu DNA qPCR produces a much lower Ct value when compared to LINE1; possibly due to its higher copies in the human genome (Ct value 28 versus 22/0.1 pg). Following tumor inoculation, ctDNA level and tumor bioluminescence were strongly and significantly correlated over time (LINE1, r=0.65 and Alu, r=0.59, p&amp;lt;0.002). Following radiation, we observed an immediate (day 32) but transient decrease in the levels of ctDNA and bioluminescence, followed by a gradual increase due to tumor relapse. The correlation between ctDNA level and bioluminescence was not maintained, but responses followed a similar trend. Conclusions: In the present study, we report that the volume of human PCa xenografts can be accurately measured in the intra-tibial compartments of athymic nude mice by qPCR of circulating human Alu and LINE1 elements. Plasma-derived ctDNA levels highly correlated with tumor bioluminescence over time. Tumor directed radiation therapy exhibited slightly differential responses in ctDNA level and tumor bioluminescence, but overall both measurements followed a similar trend. Citation Format: Alok Mishra, Kenji Zennami, Esteban Velarde, Jonathan B. Coulter, Srinivasan Yegnasubramanian, Shawn E. Lupold, Theodore L. DeWeese. Comparative analysis of circulating human tumor DNA and bioluminescent imaging in monitoring tumor burden and therapeutic response of ionizing radiation in intratibial human prostate tumor xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2929.

Use of Bioluminescence for Monitoring Brown Coal Mine Waters from Deep and Surface Drainage

Open-pit mines can cause environmental changes, such as alterations of landscape structure, hydrology, air quality, and river sediments; they can also generate cones of depression. We propose a new method for surveys of mine waters using the example of an open-pit mine in central Poland. This study examines the correlations between bioluminescence and the color of brown coal mine waters and tests whether values of the three-color coordinate system reflected the physicochemical quality of mine waters measured in real-time and in the field. Our results show that alkalinity, pH reaction, and conductivity are higher in surface drainage, while values of trophic parameters (soluble reactive phosphates, total phosphorus, nitrates) are greater in samples representing subsurface drainage. Correlation analysis of bioluminescence with mine water quality parameters showed that only water color had a strong association with bioluminescence. This correlation is stronger for surface drainage, than for mine waters from subsurface drainage. Direct measurement of bioluminescence, resulting from adenosine 5`-triphosphate (ATP) using a luminometer, is a fast and reliable method for evaluation of the characteristics of mine waters in real-time.

Development of an Experimental Ex Vivo Wound Model to Evaluate Antimicrobial Efficacy of Topical Formulations.

Wound infections are considered a major cause for wound-associated morbidity. There is a high demand for alternative, robust, and affordable methods that can provide relatable and reproducible results when testing topical treatments, both in research and in the pharmaceutical industry. Here we present an ex vivo wound infection model using porcine skin and a burn wounding method, allowing for the efficacy evaluation of topical antimicrobial formulations. Utilizing this model, we demonstrate the potential of topical treatments after infecting the wounds with clinically significant bacteria, P. aeruginosa and S. aureus. We show that the method is compatible with several analytical tools used to analyze infection and antimicrobial effects. Both bacterial strains successfully infected the wound surface, as well as deeper regions of the tissue. Quantification of viable bacteria on the wound surface and in the tissue, longitudinal measurements of bioluminescence, fluorescence microscopy, and scanning electron microscopy were used to confirm the effects of antibacterial treatments. Furthermore, we show that biofilms are formed on the wound surface, indicating that the demonstrated method mirrors typical in vivo infections.

Bioluminescence Measurement of Time-Dependent Dynamic Changes of CYP-Mediated Cytotoxicity in CYP-Expressing Luminescent HepG2 Cells.

We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC50) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC50 value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC50 values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement.

Intracellular Ionic Strength Sensing Using NanoLuc.

Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.

Generation of Mouse Primary Hypothalamic Neuronal Cultures for Circadian Bioluminescence Assays.

An endogenous circadian clock system enables organisms to adapt to time-of-day dependent environmental changes. In consequence, most physiological processes exhibit daily rhythms of, e.g., energy metabolism, immune function, sleep, or hormone production. Hypothalamic circadian clocks have been identified to play a particular role in coordinating many of these processes. Primary neuronal cultures are widely used as a physiologically relevant model to study molecular events within neurons. However, as circadian rhythms include dynamic molecular changes over longer timescales that vary between individual cells, longitudinal measurement methods are essential to investigate the regulation of circadian clocks of hypothalamic neurons. Here we provide a protocol for generating primary hypothalamic neuronal cultures expressing a circadian luciferase reporter. Such reporter cells can be used to longitudinally monitor cellular circadian rhythms at high temporal resolution by performing bioluminescence measurements.