Decrease In Bioluminescence Research Articles

Assessment of environmental risk related to the polycyclic aromatic hydrocarbons (PAH) in the sediments along the eastern Adriatic coast

Polycyclic aromatic hydrocarbons (PAH) were investigated in the sediments at 24 locations in the coastal area of Adriatic Sea. Total PAH concentrations ranged from 14,98 to 26145,62 µg kg-1, with the highest levels determined in the sediments of Šibenik Bay and the lowest in the sediment near Island of Pag. Unsubstituted PAH were prevailing PAH group in the sediments from the Šibenik Bay and in the Coastal area, while methyl-substituted PAH were dominant group in the sediments of the Kaštela Bay. In order to estimate the possible toxicological significance of PAH concentrations, Sediment Quality Assessment was done according to three different sets of sediment quality guidelines. A high environmental risk was calculated for each station in Šibenik Bay; for the station in vicinity of the Industrial port of Split and for the two stations in Coastal zone. Low environmental risk was determined in less urbanised areas such as Island of Pag. Sediment extracts were further analysed for acute toxicity by measurements of decrease in bioluminescence upon exposure to sediment extracts. Obtained results show disagreement between sediment toxicity predicted upon PAH levels in sediment and experimentally obtained sediment toxicity. Sediment extracts in which low PAH levels were measured showed high acute toxicity indicating that concentrations of PAH alone are a poor indicator of sediment toxicity. Correlation analysis between concentrations of specific PAH compounds and toxicity revealed the strongest association between acute sediment toxicity and sulphur-containing PAH suggesting the S-PAH should be considered in sediment toxicity assessment.

Abstract 3831: Utilizing decellularized triple-negative breast cancer patient-derived tumor models as drug testing platforms

Abstract Triple-negative breast cancer (TNBC) is an aggressive, heterogenous disease that lacks approved targeted therapeutics. Due to the complexity of tumor microenvironment (TME) interactions in TNBC, it is crucial to identify therapeutic targets that regulate the extracellular matrix (ECM). Traditional 2D TNBC models do not accurately mimic the cell-matrix interactions and structural matrix proteins that are key in maintaining the unique TME in individual patient tumors. Here, we introduce our decellularized TNBC patient-derived xenograft (PDX) models to address this gap in knowledge. Utilizing our decellularized models, we can investigate the unique ECM composition and cell-matrix interactions in PDX models from understudied patients with diverse clinical presentations. We can also use our decellularized tumor scaffolds as drug testing models. We utilize our tissue decellularization method on PDX tumor scaffolds derived from two of our established TNBC PDX models (TU-BcX-56S and TU-BcX-4QX). Decellularization of tumor scaffolds was confirmed by DNA quantification and histological analysis (H&E, Masson’s Trichrome, and Movat’s staining). MDA-MB-231 cells expressing GFP and luciferase were seeded onto scaffolds and treated with 100nM of Paclitaxel, a microtubule-stabilizing cytotoxic chemotherapeutic, or DMSO for 48 hours. Concordantly, cells in 2D culture were treated with 100nM of Paclitaxel or DMSO for 48 hours. Using the IVIS imaging system, we measured the bioluminescence of cells on our seeded scaffolds before treatment and at 24 and 48 hours after treatment. Cells in 2D culture showed a significant decrease in bioluminescent signal (p-value < 0.05) in the Paclitaxel group compared to the DMSO group at 48 hours. Cells seeded on TU-BcX-56S scaffold also showed a significant decrease in bioluminescent signal (p-value < 0.05) in the Paclitaxel group compared to the DMSO group at 48 hours. Interestingly, cells seeded on TU-BcX-4QX scaffold showed a decrease in bioluminescence in both the treatment and control groups at 24 and 48 hours, albeit there was no significant difference. Using qPCR, we investigated scaffold regulation of ECM gene composition in our treatment and control groups. These results demonstrate that the unique ECM composition and architecture of these scaffolds are important drivers of cell behavior. Here, we demonstrate that our decellularized PDX tumor scaffold models can be used as drug testing platforms. Furthermore, our models recapitulate key structural matrix proteins in the TNBC TME, can be used to investigate ECM onco-architecture and composition, and can be used in therapeutic discovery research. Citation Format: Maryl Wright, Khoa Nguyen, Elizabeth Martin, Melyssa Bratton, Bridgette Collins-Burow, Matthew Burow. Utilizing decellularized triple-negative breast cancer patient-derived tumor models as drug testing platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3831.

Abstract 4558: Antitumor activity of AMG757, a half-life extended (HLE) bispecific T-cell engager (BiTE®) immune therapy targeting DLL3, in human PDX and orthotopic mouse models of small cell lung cancer (SCLC)

Abstract SCLC is a highly aggressive neuroendocrine tumor with poor prognosis and limited therapeutic options. Delta-like ligand 3 (DLL3) is a tumor-associated antigen that is highly specific for SCLC; expression is elevated in tumors but minimal and mainly cytoplasmic in normal tissues. AMG 757 is an HLE BiTE® immune therapy that is designed to redirect T-cell cytotoxicity to cancer cells by binding to DLL3 on the surface of cancer cells and CD3 on T cells. AMG 757 is being evaluated in a phase 1 clinical trial in patients with relapsed/refractory SCLC (NCT03319940). We evaluated the efficacy and pharmacodynamic (PD) effects of AMG 757 in three preclinical models of SCLC that express DLL3. We used the LXFS 1129 patient-derived xenograft (PDX) model to assess AMG 757 efficacy in established, subcutaneously implanted tumors similar to human SCLC tumors with their associated stroma. We also developed two orthotopic models of SCLC (SHP-77 and H82) to assess AMG 757 efficacy and PD at biologically relevant sites. In the SHP-77 orthotopic model, cells injected intravenously trafficked to the lungs where they formed diffuse tumors similar to primary SCLC tumors. In the H82 orthotopic model, cells injected intravenously formed discrete metastatic lesions in the liver, mimicking a major site for SCLC metastasis. In each model, mice bearing established tumors received a single infusion of human T cells and were then dosed with AMG 757 or a control HLE BiTE molecule. In the LXFS 1129 PDX model, treatment with AMG 757 induced complete tumor regression in 8 out of 10 mice, whereas treatment with the control HLE BiTE molecule showed no tumor growth inhibition. In the orthotopic SHP-77 model, treatment with AMG 757 led to significant tumor growth inhibition in the lungs relative to treatment with a control HLE BiTE molecule; the bioluminescence (BLI) signal decreased to near the limit of detection 22 days after start of treatment. AMG 757 antitumor activity was associated with increased T-cell trafficking and expansion in tumors; a single dose of AMG 757 significantly increased the number of human CD4+ and CD8+ cells in lungs as assessed by flow cytometry. In the orthotopic H82 model, AMG 757 treatment completely cleared visible lesions from the liver, as assessed by a decrease in BLI to near-background levels and macroscopic analysis of the liver tissue. AMG 757 antitumor activity was associated with increased CD8+ T-cell infiltration as assessed by flow cytometry and IHC and upregulation of the T cell surface activation markers CD25, CD69, PD-1, and 4-1BB as assessed by flow cytometry. Together, these preclinical data demonstrate that AMG 757 can recruit and engage T cells to tumors, consistent with the BiTE® mechanism of action, and that AMG 757-mediated redirected T-cell lysis can drive significant antitumor activity in established SCLC tumor models. Citation Format: Keegan Cooke, Juan Estrada, Jinghui Zhan, Jonathan Werner, Sean Caenepeel, Mike Giffin, Julie M. Bailis, Angela Coxon, Paul E. Hughes, Jude Canon. Antitumor activity of AMG757, a half-life extended (HLE) bispecific T-cell engager (BiTE®) immune therapy targeting DLL3, in human PDX and orthotopic mouse models of small cell lung cancer (SCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4558.

Influence of invader ctenophores on bioluminescence variability off the coast of Western Crimea

In the second half of the XX century, Black Sea ecosystem has undergone significant changes: a number of storm winds and upwellings decreased, precipitation abundance increased, coastal waters salinity decreased, temperature increased; moreover, ctenophores invaded. As a result, in the late 1980s, Black Sea pelagic ecosystem abruptly got restructured. This research is based on the studies performed in 1965–1966 and 2007–2012 near Sevastopol (Western Crimea) using the remote sensing data. Analysis of satellite data over the past 20 years showed the presence of positive dynamics in surface water temperature in Sevastopol water area. In the mid-1960s, the annual bioluminescence was characterized by seasonal peaks of dinophytes luminescence. In recent years, this rhythm has changed due to ctenophores invasion. The increase in Mnemiopsis leidyi abundance leads to a decrease in bioluminescence of luminous microalgae being consumed by these ctenophores. Due to Beroe ovata invasion and reproduction, M. leidyi biomass decreased; as a result, bioluminescence increased.

Repetitive Detection of Aromatic Hydrocarbon Contaminants with Bioluminescent Bioreporters Attached on Tapered Optical Fiber Elements.

In this study, we show the repetitive detection of toluene on a tapered optical fiber element (OFE) with an attached layer of Pseudomonas putida TVA8 bioluminescent bioreporters. The bioluminescent cell layer was attached on polished quartz modified with (3-aminopropyl)triethoxysilane (APTES). The repeatability of the preparation of the optical probe and its use was demonstrated with five differently shaped OFEs. The intensity of measured bioluminescence was minimally influenced by the OFE shape, possessing transmittances between 1.41% and 5.00%. OFE probes layered with P. putida TVA8 were used to monitor liquid toluene over a two-week period. It was demonstrated that OFE probes layered with positively induced P. putida TVA8 bioreporters were reliable detectors of toluene. A toluene concentration of 26.5 mg/L was detected after <30 min after immersion of the probe in the toluene solution. Additional experiments also immobilized constitutively bioluminescent cells of E. coli 652T7, on OFEs with polyethyleneimine (PEI). These OFEs were repetitively induced with Lauria-Bertani (LB) nutrient medium. Bioluminescence appeared 15 minutes after immersion of the OFE in LB. A change in pH from 7 to 6 resulted in a decrease in bioluminescence that was not restored following additional nutrient inductions at pH 7. The E. coli 652T7 OFE probe was therefore sensitive to negative influences but could not be repetitively used.

Bioluminescence Detection of Superoxide Anion Using Aequorin.

Although the superoxide anion (O2-·) is generated during normal cellular respiration and has fundamental roles in a wide range of cellular processes, such as cell proliferation, migration, apoptosis, and homeostasis, its dysregulation is associated with a variety of diseases. Regarding these prominent roles in biological systems, the development of accurate methods for quantification of superoxide anion has attracted tremendous research attention. Here, we evaluated aequorin, a calcium-dependent photoprotein, as a potential bioluminescent reporter protein of superoxide anion. The mechanism is based on the measurement of aequorin bioluminescence, where the lower the concentration of coelenterazine under the oxidation of superoxide anion, the lower the amount aequorin regeneration, leading to a decrease in bioluminescence. The bioluminescence intensity of aequorin was proportional to the concentration of superoxide anion in the range from 4 to 40 000 pM with a detection limit (S/N = 3) of 1.2 pM, which was 5000-fold lower than those of the chemiluminescence methods. The proposed method exhibited high sensitivity and has been successfully applied to the determination of superoxide anion in the plant cell samples. The results could suggest a photoprotein-based bioluminescence system as a highly sensitive, specific, and simple bioluminescent probe for in vitro detection of superoxide anion.

Iso-propyl stilbene: a life cycle signal?

Members of the Gram-negative bacterial genus Photorhabdus are all highly insect pathogenic and exist in an obligate symbiosis with the entomopathogenic nematode worm Heterorhabditis. All members of the genus produce the small-molecule 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) as part of their secondary metabolism. IPS is a multi-potent compound that has antimicrobial, antifungal, immunomodulatory and anti-cancer activities and also plays an important role in symbiosis with the nematode. In this study we have examined the response of Photorhabdus itself to exogenous ectopic addition of IPS at physiologically relevant concentrations. We observed that the bacteria had a measureable phenotypic response, which included a decrease in bioluminescence and pigment production. This was reflected in changes in its transcriptomic response, in which we reveal a reduction in transcript levels of genes relating to many fundamental cellular processes, such as translation and oxidative phosphorylation. Our observations suggest that IPS plays an important role in the biology of Photorhabdus bacteria, fulfilling roles in quorum sensing, antibiotic-competition advantage and maintenance of the symbiotic developmental cycle.

Rapid in situ assessment of Cu-ion mediated effects and antibacterial efficacy of copper surfaces

Release of metal ions from metal-based surfaces has been considered one of the main drivers of their antimicrobial activity. Here we describe a method that enables parallel assessment of metal ion release from solid metallic surfaces and antimicrobial efficacy of these surfaces in a short time period. The protocol involves placement of a small volume of bioluminescent bacteria onto the tested surface and direct measurement of bioluminescence at various time points. In this study, two recombinant Escherichia coli strains, one expressing bioluminescence constitutively and applicable for general antimicrobial testing, and the other induced by Cu ions, were selected. Decrease in bioluminescence of constitutive E. coli on the surfaces showed a good correlation with the decrease in bacterial viability. Response of Cu-inducible E. coli showed a correlation with Cu content in the tested surfaces but not with Cu dissolution suggesting the role of direct bacteria-surface contact in Cu ion-driven antibacterial effects. In summary, the presented protocol enables the analysis of microbial toxicity and bioavailability of surface-released metal ions directly on solid surfaces within 30–60 min. Although optimized for copper and copper alloy surfaces and E. coli, the method can be extended to other types of metallic surfaces and bacterial strains.

Mechanism characterization of bacterial inactivation of atmospheric air plasma gas and activated water using bioluminescence technology

Abstract We assessed the efficacy of bacterial inactivation using a dielectric barrier discharge in three different plasma setups: plasma gas (PG), and direct and indirect plasma activated water (PAW), where deionized water was placed either between or away from the electrodes, respectively. We used bioluminescent Escherichia coli K12 lux as a model bacteria in a biosensor format to study the inactivation kinetics and mechanism of action of produced PG and PAW. The results showed that uninterrupted application of PG decreased bioluminescence rapidly by 1-log within the first minute and 3.6-log after 10 min of treatment. Exposing the bacterial culture with a sublethal dose of PAW (1 mL) rapidly decreased the bioluminescence; however, luminescence slowly recovered after exposure. Subsequent treatment with PAW decreased the bioluminescence to a lesser extent. In addition, direct PAW induced a greater decrease in bioluminescence compared to indirect treatments for both single and multiple exposures. In contrast to the PG, PAW treatments induced a lower bactericidal effect with 0.11 to 0.22-log reduction for indirect PAW and 0.2 to 0.32-log for direct PAW. Our results also indicate that antimicrobial activity of PAW decreased slowly within 20 min of its preparation. The rapid decrease in bioluminescence followed by a partial recovery in a repeatable pattern suggests an incomplete inactivation, and that the reducing power of the cell helps them to survive. Moreover, the complete and partial oxidation of NADH solutions in vitro by PG and PAW, respectively, strongly suggest that the lux fluorophore FMNH2 and other reducing cofactors could be the target of such treatment before other cell components. This hypothesis was supported by the tendency to recover luminescence by potentially replenishing the pool of FMNH2 after plasma treatment. It is also important to consider that the reducing power of the cell (NADH, NADPH, and FMNH2) is crucial for cell viability mostly due to reducing potential for critical metabolic reactions. Therefore, in situ bioluminescence monitoring technology can potentially serve as a unique approach to elucidate the mechanism of bacteria inactivation in real time. Industrial relevance The present study developed three dielectric barrier discharge (DBD) plasma setups to produce plasma gas and plasma activated water, which can disinfect both food products and their contact surfaces regardless of geometry. Our in situ bioluminescent technology elucidated bacterial inactivation mechanisms of plasma treatments, which may potentially suggest sufficient exposures to plasma resulting in safe food products without deteriorating their quality. The results will help food manufacturers apply new plasma-based disinfection methods with appropriate treatments.

Toxicological and pharmacological profiling of organically and non-organically cultivated Cymbopogon citratus

BackgroundWith the rapid depletion of forests, impairing the availability of raw drugs, Ayurveda, has reached a very critical phase. Consequently, cultivation of medicinal plants is essential to ensure their availability to the industry. In view of the above concept, organic farming of medicinal plants needs scientific validity. ObjectivesThe present study includes the organic and non-organic cultivation of Cymbopogon citratus, followed by toxicological and pharmacological profiling of extracts. Materials and methodsC. citratus was simultaneously cultivated organically (OCC) and non-organically (NCC). Toxicity profile of aqueous extracts was recorded on prokaryotes using bioluminescent bacteria, Vibrio harveyi and evaluated its type 2 anti-diabetic activity. ResultsOCC have shown the higher mean values of height, number of germplasms and root lengths compared to NCC. The higher level of toxicity was shown by NCC with decrease in bioluminescence with increasing concentration of extract. In acute type 2 anti-diabetic study, OCC showed prominent decrease in blood glucose at postprandial condition (6th h) (48.86% OCC-200). The order of sub-chronic anti-diabetic activity was observed as positive control > OCC-200 > NCC-200, while OCC at 200 mg/kg corrected the altered lipid profile and antioxidant status with significant increase in body weights of animals. Histopathological examination of pancreas showed the enlargement of pancreatic islets and formation of neo islets with degenerative changes in OCC treated animals. ConclusionThe study confirms that organically grown C. citratus is better in terms of nourishment, biological activity and safety measures.

Reconstructing promoter activity from Lux bioluminescent reporters.

The bacterial Lux system is used as a gene expression reporter. It is fast, sensitive and non-destructive, enabling high frequency measurements. Originally developed for bacterial cells, it has also been adapted for eukaryotic cells, and can be used for whole cell biosensors, or in real time with live animals without the need for euthanasia. However, correct interpretation of bioluminescent data is limited: the bioluminescence is different from gene expression because of nonlinear molecular and enzyme dynamics of the Lux system. We have developed a computational approach that, for the first time, allows users of Lux assays to infer gene transcription levels from the light output. This approach is based upon a new mathematical model for Lux activity, that includes the actions of LuxAB, LuxEC and Fre, with improved mechanisms for all reactions, as well as synthesis and turn-over of Lux proteins. The model is calibrated with new experimental data for the LuxAB and Fre reactions from Photorhabdus luminescens—the source of modern Lux reporters—while literature data has been used for LuxEC. Importantly, the data show clear evidence for previously unreported product inhibition for the LuxAB reaction. Model simulations show that predicted bioluminescent profiles can be very different from changes in gene expression, with transient peaks of light output, very similar to light output seen in some experimental data sets. By incorporating the calibrated model into a Bayesian inference scheme, we can reverse engineer promoter activity from the bioluminescence. We show examples where a decrease in bioluminescence would be better interpreted as a switching off of the promoter, or where an increase in bioluminescence would be better interpreted as a longer period of gene expression. This approach could benefit all users of Lux technology.

Cerenkov Radiation-Induced Photoimmunotherapy with 18F-FDG.

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of antibodies for targeting tumors with toxicity induced by photoabsorbers after irradiation with NIR light. A limitation of NIR-PIT is the inability to deliver NIR light to a tumor located deep inside the body. Cerenkov radiation (CR) is the ultraviolet and blue light that is produced by a charged particle traveling through a dielectric medium faster than the speed of light in that medium and is commonly produced during radioactive decay. Here, we demonstrate the feasibility of using CR generated by 18F-FDG accumulated in tumors to induce photoimmunotherapy. Methods: Using A431-luc cells, we evaluated the therapeutic effects of CR-PIT in vitro and in vivo using bioluminescence imaging. Results: CR-PIT showed significant suppression of tumor size, but the decrease of bioluminescence after CR-PIT was not observed consistently over the entire time course. Conclusion: Although CR-PIT can induce tumor killing deep within body, it is less effective than NIR-PIT, possibly related to the relatively lower efficiency of short wavelength light than NIR.

Magnetic single-walled carbon nanotubes as efficient drug delivery nanocarriers in breast cancer murine model: noninvasive monitoring using diffusion-weighted magnetic resonance imaging as sensitive imaging biomarker.

PurposeTargeting doxorubicin (DOX) by means of single-walled carbon nanotube (SWCNT) nanocarriers may help improve the clinical utility of this highly active therapeutic agent. Active targeting of SWCNTs using tumor-specific antibody and magnetic attraction by tagging the nanotubes with iron oxide nanoparticles can potentially reduce the unnecessary side effects and provide enhanced theranostics. In the current study, the in vitro and in vivo efficacy of DOX-loaded SWCNTs as theranostic nanoprobes was evaluated in a murine breast cancer model.MethodsIron-tagged SWCNTs conjugated with Endoglin/CD105 antibody with or without DOX were synthetized and extensively characterized. Their biocompatibility was assessed in vitro in luciferase (Luc2)-expressing 4T1 (4T1-Luc2) murine breast cancer cells using TiterTACS™ Colorimetric Apoptosis Detection Kit (apoptosis induction), poly (ADP-ribose) polymerase (marker for DNA damage), and thiobarbituric acid-reactive substances (oxidative stress generation) assays, and the efficacy of DOX-loaded SWCNTs was evaluated by measuring the radiance efficiency using bioluminescence imaging (BLI). Tumor progression and growth were monitored after 4T1-Luc2 cells inoculation using noninvasive BLI and magnetic resonance imaging (MRI) before and after subsequent injection of SWCNT complexes actively and magnetically targeted to tumor sites.ResultsSignificant increases in apoptosis, DNA damage, and oxidative stress were induced by DOX-loaded SWCNTs. In addition, a tremendous decrease in bioluminescence was observed in a dose- and time-dependent manner. Noninvasive BLI and MRI revealed successful tumor growth and subsequent attenuation along with metastasis inhibition following DOX-loaded SWCNTs injection. Magnetic tagging of SWCNTs was found to produce significant discrepancies in apparent diffusion coefficient values providing a higher contrast to detect treatment-induced variations as noninvasive imaging biomarker. In addition, it allowed their sensitive noninvasive diagnosis using susceptibility-weighted MRI and their magnetic targeting using an externally applied magnet.ConclusionEnhanced therapeutic efficacy of DOX delivered through antibody-conjugated magnetic SWCNTs was achieved. Further, the superiority of apparent diffusion coefficient measurements using diffusion-weighted MRI was found to be a sensitive imaging biomarker for assessment of treatment-induced changes.

APOBEC3A catabolism of electroporated plasmid DNA in mouse muscle.

The mouse is widely used as a model for DNA therapy and vaccination even though the efficiency of DNA delivery in higher mammals and humans is much less. The human APOBEC3 (A3) enzymes impact viral genomes by cytidine deamination, which introduces multiple uridine residues into single-stranded DNA, a process known as genetic editing. This initiates rapid DNA catabolism via a uracil DNA glycosylase dependent pathway. In tissue culture, A3A, A3C and A3B can hyperedit transfected plasmid DNA. We explored plasmid catabolism in vivo initiated by A3A, the most efficient of the human enzymes and one that is functionally conserved across most mammals. As rodents do not encode an A3A enzyme, it was possible to explore DNA degradation in the mouse model. Human A3A genetically edits co-electroporated luciferase plasmid DNA in mouse skeletal muscle that initiates DNA degradation resulting in approximately fourfold decrease in bioluminescence. Part of the degradation occurs in the nucleus as indicated by complex hyperedited DNA molecules. As human A3A is strongly upregulated by interferon α and DNA sensing pathways, it is a strong candidate enzyme for restricting plasmid DNA in higher mammals.

Bioluminescent assay for evaluating antimicrobial activity in insect haemolymph

We describe an antibacterial assay based on bioluminescence of two Gram negative bacteria, Photorhabdus luminescens and transformed Escherichia coli, which can be used as a real-time measurement of antibacterial activity in insect haemolymph. This method is based on the production of the bioluminescence signal depending on the viability of bacterial cells. We observed a significant rapid dose-dependent decrease in bioluminescence using both bacterial species, and Bombyx mori or Galleria mellonella haemolymph, which was confirmed by the decrease in bacterial viability determined by plating. The humoral origin of the antibacterial activity observed in whole haemolymph was confirmed for haemolymph plasma without haemocytes. Antibacterial activity directed against Gram negative bacteria was recorded in unaffected insect larvae as well as after septic injury; increased antibacterial activity of haemolymph was detected in the latter case confirming the inducibility of antimicrobial agents. We think it is likely that this method could be widely used for determining antibacterial activity in insects and other invertebrates.

RNA-Induced Silencing Complex-Bound Small Interfering RNA Is a Determinant of RNA Interference-Mediated Gene Silencing in Mice

Deeper knowledge of pharmacokinetic and pharmacodynamic (PK/PD) concepts for RNA therapeutics is important to streamline the drug development process and for rigorous selection of best performing drug candidates. Here we characterized the PK/PD relationship for small interfering RNAs (siRNAs) targeting luciferase by examining siRNA concentration in plasma and liver, the temporal RNA-induced silencing complex binding profiles, mRNA reduction, and protein inhibition measured by noninvasive bioluminescent imaging. A dose-dependent and time-related decrease in bioluminescence was detected over 25 days after a single treatment of a lipid nanoparticle-formulated siRNA targeting luciferase messenger RNA. A direct relationship was observed between the degree of in vivo mRNA and protein reduction and the Argonaute2 (Ago2)-bound siRNA fraction but not with the total amount of siRNA found in the liver, suggesting that the Ago2-siRNA complex is the key determinant of target inhibition. These observations were confirmed for an additional siRNA that targets endogenously expressed Sjögren syndrome antigen B (Ssb) mRNA, indicating that our observations are not limited to a transgenic mouse system. Our data provide detailed information of the temporal regulation of siRNA liver delivery, Ago2 loading, mRNA reduction, and protein inhibition that are essential for the rapid and cost-effective clinical development of siRNAs therapeutics.

Bioavailability and toxicity of copper in soils: Integrating chemical approaches with responses of microbial biosensors

Abstract Copper (Cu) is a trace element essential for the healthy functioning of soil biological systems. However, at elevated concentrations Cu can be a potential toxicant. Consequently, an understanding of Cu availability and toxicity to soil biota is essential for effective ecological assessment of metal impacts in soil. The present study measured the response of a constitutive and Cu specific luminescence marked Pseudomonas fluorescens to Cu analytically determined in five soils. Conditioned Cu amended and historically contaminated soils were used. The free ion, soil pore water and solid-phase Cu were defined analytically by standard techniques frequently used to assess environmental availability of metals in soils, i.e. exchange resin, single and sequential metal extractions. The response of the constitutively expressed biosensor (i.e. a decrease in bioluminescence in the presence of Cu) assessed directly the toxicity of soil pore water Cu. The expression of bioluminescence by the bioreporter (i.e. an induction of bioluminescence by the presence of Cu) enabled quantification of Cu bioavailability in soil pore waters. Analytically determined Cu 2+ concentrations strongly linked Cu toxicity to the biosensor responses and thus supported the conventional view of free metal ion toxicity in soil solutions. However, in two soils, other forms of Cu than free ion caused a response of the bioreporter, hence were bioavailable. Relationships between biological responses and analytically defined solid-phase Cu fractions identified pools of labile, potentially bioavailable Cu. As no single chemical method could be used to quantify bioavailable fraction of solid-phase Cu in all soils, the bioreporter provided analytical techniques with inherent biological relevance. This study demonstrates that Cu in soils with aged amendments of inorganic salts is more chemically available than in soils treated with sludge containing Cu. These differences are due to the concentration and form of Cu in the aqueous phase and the corresponding extractability of Cu in the soil solid phase. Bioreporter and biosensor validate the performance of rigorous analytical soil chemistry approaches. Their value in soil pollution should be considered a complementary technique rather than an alternative as their performance in complex environmental matrixes is yet to be validated.

Abstract SY28-04: Rational incorporation of novel agents into multimodality treatment of glioma and neuroblastoma

Abstract SY28-04: Rational incorporation of novel agents into multimodality treatment of glioma and neuroblastoma

Quorum Sensing Influences Vibrio harveyi Growth Rates in a Manner Not Fully Accounted For by the Marker Effect of Bioluminescence

BackgroundThe light-emitting Vibrios provide excellent material for studying the interaction of cellular communication with growth rate because bioluminescence is a convenient marker for quorum sensing. However, the use of bioluminescence as a marker is complicated because bioluminescence itself may affect growth rate, e.g. by diverting energy.Methodology/Principal FindingsThe marker effect was explored via growth rate studies in isogenic Vibrio harveyi (Vh) strains altered in quorum sensing on the one hand, and bioluminescence on the other. By hypothesis, growth rate is energy limited: mutants deficient in quorum sensing grow faster because wild type quorum sensing unleashes bioluminescence and bioluminescence diverts energy. Findings reported here confirm a role for bioluminescence in limiting Vh growth rate, at least under the conditions tested. However, the results argue that the bioluminescence is insufficient to explain the relationship of growth rate and quorum sensing in Vh. A Vh mutant null for all genes encoding the bioluminescence pathway grew faster than wild type but not as fast as null mutants in quorum sensing. Vh quorum sensing mutants showed altered growth rates that do not always rank with their relative increase or decrease in bioluminescence. In addition, the cell-free culture fluids of a rapidly growing Vibrio parahaemolyticus (Vp) strain increased the growth rate of wild type Vh without significantly altering Vh's bioluminescence. The same cell-free culture fluid increased the bioluminescence of Vh quorum mutants.Conclusions/SignificanceThe effect of quorum sensing on Vh growth rate can be either positive or negative and includes both bioluminescence-dependent and independent components. Bioluminescence tends to slow growth rate but not enough to account for the effects of quorum sensing on growth rate.

Bacterial biosensors for rapid and effective monitoring of biodegradation of organic pollutants in wastewater effluents

Significant amounts of toxic substances which are hazardous to animals, plants, microorganisms, and other living organisms including humans are released annually into aquatic and terrestrial environments, mostly from improper wastewater discharges. Early detection of such pollutants in wastewater effluents and proper monitoring before their final release into the environment is therefore necessary. In this study, two whole-cell bacterial biosensors were constructed by transforming competent cells of Shigella flexneri and Shigella sonnei with pLUX plasmids and evaluated for their potential to monitor wastewater samples undergoing degradation by measuring bioluminescence response using a microplate luminometer. Both bacterial biosensors were found to be extremely sensitive to the wastewater samples, with different patterns, concomitant with those of the COD removals demonstrated at the different days of the degradation. Generally higher bioluminescence values were obtained at the later days of the degradation period compared to the initial values, with up to 571.76% increase in bioluminescence value obtained at day 5 for 0.1% (v/v) effluent concentration. Also, a steady decrease in bioluminescence was observed for the bacterial biosensors with increasing time of exposure to the wastewater effluent for all the sampling days. These biosensor constructs could therefore be applicable to indicate the bioavailability of pollutants in a way that chemical analysis cannot, and for in situ monitoring of biodegradation. This has great potential to offer a risk assessment strategy in predicting the level of bioremediation required during municipal wastewater treatment before their final discharge into the aquatic milieu.