Real-time Bioluminescence Imaging Research Articles

Bacterial transport mediated by micro-nanobubbles in porous media

Determining the role of micro-nanobubbles (MNBs) in controlling the risk posed by pathogens to soil and groundwater during reclaimed water irrigation requires clarification of the mechanism of how MNBs block pathogenic bacteria. In this study, real-time bioluminescence imaging was used to investigate the effects of MNBs on the transport and spatiotemporal distribution of bioluminescent Escherichia coli 652T7 strain in porous media. The presence of MNBs significantly increased the retention of bacteria in the porous media, decreasing the maximum relative effluent concentration (C/C0) by 78 % from 0.97 (without MNBs) to 0.21 (with MNBs). The results suggested that MNBs provided additional sites at the air-water interface (AWI) for bacterial attachment and acted as physical obstacles to reduce bacterial passage. These effects varied with environmental conditions such as solution ionic strength and pore water velocity. The results indicated that MNBs enhanced electrostatic attachment of bacteria at the AWI and their mechanical straining in pores. This study suggests that adding MNBs in pathogen-containing water is an effective measure for increasing filtration efficiency and reducing the risk of pathogenic contamination during agricultural irrigation.

Quantifying bacterial concentration in water and sand media during flow-through experiments using a non-invasive, real-time, and efficient method.

Monitoring the dynamics of bacteria in porous media is of great significance to understand the bacterial transport and the interplay between bacteria and environmental factors. In this study, we reported a non-invasive, real-time, and efficient method to quantify bioluminescent bacterial concentration in water and sand media during flow-through experiments. First, 27 column experiments were conducted, and the bacterial transport was monitored using a real-time bioluminescent imaging system. Next, we quantified the bacterial concentration in water and sand media using two methods-viable count and bioluminescent count. The principle of the bioluminescent count in sand media was, for a given bioluminescence image, the total number of bacteria was proportionally allocated to each segment according to its bioluminescence intensity. We then compared the bacterial concentration for the two methods and found a good linear correlation between the bioluminescent count and viable count. Finally, the effects of porous media surface coating, pore water velocity, and ionic strength on the bioluminescent count in sand media were investigated, and the results showed that the bioluminescence counting accuracy was most affected by surface coating, followed by ionic strength, and was hardly affected by pore water velocity. Overall, the study proved that the bioluminescent count was a reliable method to quantify bacterial concentration in water (106 to 2 × 108 cell mL-1) or sand media (5 × 106-5 × 108 cell cm-3). This approach also offers a new way of thinking for in situ bacterial enumeration in two-dimensional devices such as 2D flow cells, microfluidic devices, and rhizoboxes.

Visualizing lymphocytic choriomeningitis virus infection in cells and living mice

SummaryMammarenavirus are a large family of enveloped negative-strand RNA viruses that include several agents responsible for severe hemorrhagic fevers. Until now, no FDA-licensed drug has been admitted for treating an arenavirus infection, and only few effective anti-arenavirus drugs have been tested in vivo. In this work, we designed a recombinant reporter arenavirus lymphocytic choriomeningitis virus that stably expressed nanoluciferase (LCMV-Nluc). The LCMV-Nluc was proved to share similar biological properties with wild-type LCMV and the Nluc intensity reliably reflected viral replication both in vitro and in vivo. Replication of the Nluc-encoding virus in living mice can be visualized by real-time bioluminescent imaging, and bioluminescence can be detected in a variety of organs of infected mice. This work provides a novel approach that enables real-time study of the arenavirus infection and is a convenient and valuable tool for screening of compounds that are active against arenaviruses in vitro and in living mice.

Real-time bioluminescent imaging of spatiotemporal variation of microbial retention during transport through porous media under variably saturated flow conditions

This study investigated the transport and retention of bioluminescent Escherichia coli strain 652T7 under different pore water velocities (8.7 cm h−1 and 13.0 cm h−1) and pore water saturations (85% and 100%) utilizing a non-invasive, real-time bioluminescent imaging technique. Under saturated flow conditions, the concentrations of retained bioluminescent E. coli 652T7 decreased exponentially with distance from the source at the lower velocity but decreased non-exponentially at the higher velocity. Under unsaturated flow conditions, pore water velocity had no significant effect on bacterial breakthrough concentration; however, the concentrations of retained cells were maximal at a significant distance from the source (non-monotonic). The distance from source of the maximum concentration increased from 2.4-cm at 1.05 pore volumes to 4.3-cm at 3.15 pore volumes, indicating slow translation of bacterial down-gradient under unsaturated flow conditions. That conditions were modestly unfavorable to attachment at the solid-water interface (SWI) was indicated by deposition rate coefficients being greater (by a factor of four) for simulations versus experiments, and by significant repulsive barriers to attachment at both the SWI (260 kT) and the air–water interface (AWI, fully repulsive). The inferred slow translation under unsaturated flow conditions therefore reflects either accumulation without arrest in the secondary minimum at the SWI and/or capillary interaction at the AWI. This non-invasive bioluminescence method yielded real-time quantitative observation of bacterial distribution from source and demonstrated contrasting transport behaviors previously obtained solely via more laborious methods with limited spatio-temporal observation.

Circadian Oscillations Persist in Cervical and Esophageal Cancer Cells Displaying Decreased Expression of Tumor-Suppressing Circadian Clock Genes.

There is accumulating evidence for a link between circadian clock disruption and cancer progression. In this study, the circadian clock was investigated in cervical and esophageal cancers, to determine whether it is disrupted in these cancer types. Oncomine datamining revealed downregulation of multiple members of the circadian clock gene family in cancer patient tissue compared with matched normal epithelium. Real-time RT-PCR analysis confirmed significant downregulation of CLOCK, PER1, PER2, PER3, CRY1, CRY2, REV-ERBα, and RORα in esophageal tumor tissue. In cell line models, expression of several circadian clock genes was significantly decreased in transformed and cancer cells compared with noncancer controls, and protein levels were dysregulated. These effects were mediated, at least in part, by methylation, where CLOCK, CRY1, and RORα gene promoter regions were found to be methylated in cancer cells. Overexpression of CLOCK and PER2 in cancer cell lines inhibited cell proliferation and activation of RORα and REV-ERBα using agonists resulted in cancer cell death, while having a lesser effect on normal epithelial cells. Despite dysregulated circadian clock gene expression, cervical and esophageal cancer cells maintain functional circadian oscillations after Dexamethasone synchronization, as revealed using real-time bioluminescence imaging, suggesting that their circadian clock mechanisms are intact. IMPLICATIONS: This study is a first to describe dysregulated, yet oscillating, circadian clock gene expression in cervical and esophageal cancer cells, and knowledge of circadian clock functioning in these cancer types has the potential to inform chronotherapy approaches, where the timing of administration of chemotherapy is optimized on the basis of the circadian clock.

Abstract 379: Myeloperoxidase-produced HOCl is a paracrine effector linking myeloid cells to NF-κB signaling in melanoma, mediating anti-tumor responses during early melanoma progression

Abstract The myeloperoxidase (MPO) system of myeloid-derived cells (MDCs) is central to cellular innate immunity and the microbicidal activities of phagocytes, while also impacting the pathogenesis of many disorders, including cancer. Upon MDC activation, MPO is secreted into phagosomes, catalyzing the production of hypochlorous acid (HOCl). HOCl is a potent chlorinating oxidant with significant biological effects, but the mechanisms by which HOCl impacts cancer growth remain unresolved. Herein, we demonstrate that the myeloid lineage-restricted MPO-HOCl system has anti-tumor effects during early melanoma progression. Orthotopic melanomas grew slower in immuno-competent host mice wild type for MPO+/+ compared to syngeneic MPO-null (MPO-/-) animals. Using a specific and potent inhibitor of MPO, 4-aminobenzoic acid hydrazide (4-ABAH), melanoma tumor growth in MPO+/+ mice continuously administered 4-ABAH, pharmacologically mimicked the MPO-/- phenotype. Real-time intravital tumor imaging in vivo using skinfold window chamber animal models as well as live cell co-culture studies revealed a cell-cell proximity-dependent association between MDC-derived MPO activity and blockade of ligand-induced IκBα degradation in tumor cells. Real-time bioluminescent imaging and quantitative monitoring of the canonical NF-κB pathway was assessed in melanoma cells stably expressing bioluminescent reporters: a transcriptionally-activated NF-κB-promoter-driven Firefly luciferase (FLuc) reporter or a fusion reporter under the control of an NF-κB-responsive promoter fused to IκBα-FLuc reporter. HOCl was shown to directly inhibit IκB kinase (IKK) activity, thereby decreasing NF-κB transcriptional activation within melanomas, increasing circulating levels of the myeloid-attracting cytokines CXCL1 and CXCL5, enhancing local infiltration of CD8+ cytotoxic T cells, and dampening tumor growth. Overall, these data reveal a novel role for MDC-derived HOCl as a small molecule paracrine signaling factor that trans-inhibits IKK in melanoma cells, mediating anti-tumor responses during early tumor progression. Citation Format: Tracy W. Liu, Seth T. Gammon, Ping Yang, David T. Fuentes, David Piwnica-Worms. Myeloperoxidase-produced HOCl is a paracrine effector linking myeloid cells to NF-κB signaling in melanoma, mediating anti-tumor responses during early melanoma progression [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 379.

Real-Time Noninvasive Bioluminescence, Ultrasound and Photoacoustic Imaging in NFκB-RE-Luc Transgenic Mice Reveal Glia Maturation Factor-Mediated Immediate and Sustained Spatio-Temporal Activation of NFκB Signaling Post-Traumatic Brain Injury in a Gender-Specific Manner.

Neurotrauma especially traumatic brain injury (TBI) is the leading cause of death and disability worldwide. To improve upon the early diagnosis and develop precision-targeted therapies for TBI, it is critical to understand the underlying molecular mechanisms and signaling pathways. The transcription factor, nuclear factor kappa B (NFκB), which is ubiquitously expressed, plays a crucial role in the normal cell survival, proliferation, differentiation, function, as well as in disease states like neuroinflammation and neurodegeneration. Here, we hypothesized that real-time noninvasive bioluminescence molecular imaging allows rapid and precise monitoring of TBI-induced immediate and rapid spatio-temporal activation of NFκB signaling pathway in response to Glia maturation factor (GMF) upregulation which in turn leads to neuroinflammation and neurodegeneration post-TBI. To test and validate our hypothesis and to gain novel mechanistic insights, we subjected NFκB-RE-Luc transgenic male and female mice to TBI and performed real-time noninvasive bioluminescence imaging (BLI) as well as photoacoustic and ultrasound imaging (PAI). Our BLI data revealed that TBI leads to an immediate and sustained activation of NFκB signaling. Further, our BLI data suggest that especially in male NFκB-RE-Luc transgenic mice subjected to TBI, in addition to brain, there is widespread activation of NFκB signaling in multiple organs. However, in the case of the female NFκB-RE-Luc transgenic mice, TBI induces a very specific and localized activation of NFκB signaling in the brain. Further, our microRNA data suggest that TBI induces significant upregulation of mir-9-5p, mir-21a-5p, mir-34a-5p, mir-16-3p, as well as mir-155-5p within 24h and these microRNAs can be successfully used asTBI-specific biomarkers. To the best of our knowledge, this is one of the first and unique study of its kind to report immediate and sustained activation of NFκB signaling post-TBI in a gender-specific manner by utilizing real-time non-invasive BLI and PAI in NFκB-RE-Luc transgenic mice. Our study will prove immensely beneficial to gain novel mechanistic insights underlying TBI, unravel novel therapeutic targets, as well as enable us to monitor in real-time the response to innovativeTBI-specific precision-targetedgene and stem cell-based precision medicine.

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Entrainment of the Circadian Clock of the Enteric Bacterium Klebsiella aerogenes by Temperature Cycles.

SummaryThe gastrointestinal bacterium Klebsiella (née Enterobacter) aerogenes expresses an endogenously generated, temperature-compensated circadian rhythm in swarming motility. We hypothesized that this rhythm may be synchronized/entrained in vivo by body temperature (TB). To determine entrainment, cultures expressing bioluminescence were exposed to temperature cycles of 1°C (35°C–36°C) or 3°C (34°C–37°C) in amplitude at periods (T-cycles) of T = 22, T = 24, or T = 28 h. Bacteria entrained to all T-cycles at both amplitudes and with stable phase relationships. A high-amplitude phase response curve (PRC) in response to 1-h pulses of 3°C temperature spike (34°C–37°C) at different circadian phases was constructed, revealing a Type-0 phase resetting paradigm. Furthermore, real-time bioluminescence imaging revealed a spatiotemporal pattern to the circadian rhythm. These data are consistent with the hypothesis that the K. aerogenes circadian clock entrains to its host via detection of and phase shifting to the daily pattern of TB.

Abstract 1729: Investigating deregulated circadian clock machinery in cancer cells

Abstract The circadian clock plays an integral role in cellular functioning by temporally controlling gene expression, and there is accumulating evidence for a link between circadian clock disruption and cancer progression. In this study we aimed to investigate circadian clock gene expression and oscillation patterns in cervical and oesophageal cancers, in order to determine whether disruptions in circadian clock functioning occur in these cancer types. Microarray gene expression analysis revealed the circadian clock gene, Per2, to be one of the most significantly downregulated genes in cervical cancer patient tissue compared to normal epithelium. In addition, Oncomine data-mining revealed significant downregulation of not only Per2, but multiple members of the circadian clock gene family (Clock, Bmal1, Per1, Per2, Cry1, Rev-erbα and RORα) in cervical cancer tissue compared to normal. Real-time RT-PCR analysis of circadian clock genes in oesophageal cancer patient tissue compared to matched normal epithelium revealed significant downregulation of Clock, Per2, Cry1 and RORα in oesophageal tumour tissue. Significant positive correlations were observed where circadian clock genes were simultaneously dysregulated in tumour tissue specimens. In cell line models, Clock, Bmal1, Per2, Cry1 and RORα circadian clock genes were significantly downregulated in transformed cells, compared to their untransformed counterparts, as well as in most cervical and oesophageal cancer cell lines compared to non-cancer epithelial cells. Patterns of protein expression did not accurately match mRNA expression, likely due to extensive post-translational processing, but Clock and Cry1 protein levels were considerably reduced in the cancer cell lines compared to normal. Overexpression of circadian clock genes in cancer cell lines negatively affected cell proliferation, highlighting the tumour suppressor properties of these genes in cervical and oesophageal cancer cells. Despite downreglated expression of circadian clock genes, cervical and oesophageal cancer cells maintain functional circadian oscillations, after synchronisation with Dexamethasone, as shown using real-time bioluminescence imaging, suggesting that their circadian clock mechanisms are still intact. Together, this study is a first to describe deregulated circadian clock machinery in cervical and oesophageal cancer cells, although the cells maintain a functional circadian rhythm. The study has relevance to the field of chronotherapy, where elucidating differences in circadian clock functioning in normal and cancer cells could yield better insights into the timing of administration of chemotherapy, ultimately ensuring a better patient response. Citation Format: Pauline J. van der Watt, Kate Davis, Virna Leaner. Investigating deregulated circadian clock machinery in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1729.

Abstract 1514: Myeloid cell derived myeloperoxidase links cellular innate immunity to inhibition of NF-κB signaling in melanoma tumor cells

Abstract Myeloid-derived cells are considered the primary mediators of the cellular innate immune system where myeloperoxidase (MPO) is the major enzyme present in these cells. MPO is essential for fighting infections but little is known about the role of MPO in cancer. Using the murine melanoma cell line, B16F10, in an immune-competent orthotopic tumor model, tumor growth was compared in wild type (MPO+/+) and syngeneic MPO-deficient host (MPO-/-) mice. Survival studies demonstrated that B16F10 tumors grew slower in MPO+/+ animals (mean survival 27.4 days ± 4.5 days) compared to MPO-/- animals (mean survival 23.4 days ± 1.6 days; p ≤ 0.005). Using a specific and potent inhibitor of MPO, 4-aminobenzoic acid hydrazide (4ABAH), we pharmacologically mimicked the MPO-/- phenotype by continuously dosing MPO+/+ animals. B16F10 tumors grew faster in MPO+/+ animals treated with 4ABAH (mean survival 23.8 days ± 2.5 days; p ≤ 0.017). Utilizing intravital imaging with skinfold window chamber animal models, we evaluated in real-time the recruitment of MPO-expressing myeloid-derived cells during melanoma progression in MPO+/+ and MPO-/- animals. B16F10 tumor NF-κB signaling, and MPO-mediated activation from immune infiltrates were imaged simultaneously using a multi-spectral, multi-modal imaging strategy. Because NF-κB signaling is a central coordinator of the immune system and cancer development, the dynamics of NF-κB signaling in B16F10 tumor in vivo were assessed using a transcriptionally-activated NF-κB-promoter-driven Firefly luciferase reporter enabling real-time bioluminescent imaging and quantitative monitoring of NF-κB transcriptional activation. Mean NF-κB transcriptional activation within the tumor compartment in MPO-/- mice was 42.2% ± 1.6% greater compared to MPO+/+ animals. Intravital microscopy demonstrated heterogeneous activation levels of NF-κB within tumor cells in MPO+/+ animals. To explore whether the spatial heterogeneity was a consequence of myeloid-derived cell distribution within the microenvironment, myeloid-derived cells were labeled in vivo using an i.v. injection of fluorophore-labeled dextran and fluorophore-labeled αGr1 antibody. Remarkably, tumor cells in contact with myeloid cells in vivo demonstrated decreased NF-κB transcriptional activation compared to tumor cells not in contact with myeloid cells. These in vivo studies demonstrate that MPO-expressing myeloid-derived cells suppress NF-κB transcriptional activation within B16F10 tumors in a tight spatially-localized and proximity-dependent manner where MPO, expressed broadly by myeloid-derived cells, contributes to host protection and decreased tumor progression. We demonstrate that MPO-expressing myeloid-derived cells function as an anti-tumor component of the cellular innate immune response during early melanoma progression in a NF-κB-dependent manner. Citation Format: Tracy W. Liu, Seth T. Gammon, Ping Yang, David T. Fuentes, David Piwnica-Worms. Myeloid cell derived myeloperoxidase links cellular innate immunity to inhibition of NF-κB signaling in melanoma tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1514.

Real-Time Bioluminescence Analysis of Escherichia coli O157:H7 Survival on Livestock Meats Stored Fresh, Cold, or Frozen

Foodborne bacteria such as Escherichia coli O157:H7 can cause severe hemorrhagic colitis in humans following consumption of contaminated meat products. Contamination with pathogenic bacteria is frequently found in the food production environment, and adequate household storage conditions of purchased foods are vital for illness avoidance. Real-time monitoring was used to evaluate bacterial growth in ground horse, beef, and pork meats maintained under various storage conditions. Various levels of E. coli O157:H7 carrying the luxCDABE operon, which allows the cells to emit bioluminescence, were used to inoculate meat samples that were then stored at room temperature for 0.5 day, at 4°C (cold) for 7 or 9 days, or -20°C (frozen) for 9 days. Real-time bioluminescence imaging (BLI) of bacterial growth was used to assess bacterial survival or load. Ground horse meat BLI signals and E. coli levels were dose and time dependent, increasing during room temperature and -20°C storage, but stayed at low levels during 4°C storage. No bacteria survived in the lower level inoculum groups (101 and 103 CFU/g). With an inoculum of 107 CFU/g, pork meats had higher BLI signals than did their beef counterparts, displaying decreased BLI signals during 7 days storage at 4°C. Both meat types had higher BLI signals in the fat area, which was confirmed with isolated fat tissues in the beef meat. Beef lean and fat tissues contrasted with both pork fat and lean tissues, which had significantly higher BLI signals and bacterial levels. BLI appears to be a useful research tool for real-time monitoring of bacterial growth and survival in various stored livestock meats. The dependence of E. coli O157:H7 growth on meat substrate (fat or lean) and storage conditions may be used as part of an effective antibacterial approach for the production of safe ground horse, beef, and pork meats.

Real-time in vivo imaging reveals localised Nrf2 stress responses associated with direct and metabolism-dependent drug toxicity

The transcription factor Nrf2 coordinates an adaptive response to chemical and oxidative stress characterised by the upregulated expression of cytoprotective target genes. In order to understand the mechanistic relevance of Nrf2 as a marker of drug-induced stress it is important to know if this adaptive response is truly localised in the context of organ-specific drug toxicity. Here, we address this knowledge gap through real-time bioluminescence imaging of transgenic Nrf2-luciferase (Nrf2-luc) reporter mice following administration of the metabolism-dependent hepatotoxin acetaminophen (APAP) or the direct nephrotoxin cisplatin. We detected localised bioluminescence in the liver (APAP) and kidneys (cisplatin) in vivo and ex vivo, whilst qPCR, Taqman low-density array and immunoblot analysis of these tissues further revealed increases in the expression level of several endogenous Nrf2-regulated genes/proteins, including heme oxygenase 1 (Hmox1). Consistent with the toxic effects of APAP in the liver and cisplatin in the kidney, immunohistochemical analysis revealed the elevated expression of luciferase and Hmox1 in centrilobular hepatocytes and in tubular epithelial cells, respectively. In keeping with the role of reactive metabolite formation in APAP-induced chemical stress, both the hepatotoxicity and localised Nrf2-luc response were ameliorated by the cytochrome P450 inhibitor aminobenzotriazole. Together, these findings show that Nrf2 can reflect highly-localised cellular perturbations associated with relevant toxicological mechanisms.

Evaluation of chemical chaperones based on the monitoring of Bip promoter activity and visualization of extracellular vesicles by real-time bioluminescence imaging.

It is known that endoplasmic reticulum (ER) stress in cells and extracellular vesicles (EVs) plays a significant role in cancer cells, therefore the evaluation of compounds that can regulate ER stress and EV secretion would be a suitable system for further screening and development of new drugs. In this study, we evaluated chemical chaperones derived from natural products based on monitoring Bip/GRP78 promoter activity during cancer cell growth, at the level of the single cell, by a bioluminescence microscopy system that had several advantages compared with fluorescence imaging. It was found that several chemical chaperones, such as ferulic acid (FA), silybin, and rutin, affected the activity. We visualized EVs from cancer cells using bioluminescence imaging and showed that several EVs could be observed when using CD63 fused with NanoLuc luciferase, which has a much smaller molecular weight and higher intensity than conventional firefly luciferase. We then examined the effects of the chemical chaperones on EVs from cancer cells by bioluminescence imaging and quantified the expression of CD63 in these EVs. It was found that the chemical chaperones examined in this study affected CD63 levels in EVs. These results showed that imaging at the level of the single cell using bioluminescence is a powerful tool and could be used to evaluate chemical chaperones and EVs from cancer cells. This approach may produce new information in this field when taken together with conventional and classical methods.

A Trp53fl/flPtenfl/fl mouse model of undifferentiated pleomorphic sarcoma mediated by adeno-Cre injection and in vivo bioluminescence imaging.

Genetic mouse models of soft tissue sarcoma provide critical insights into disease pathophysiology, which are oftentimes unable to be extracted from human tumor samples or xenograft models. In this study we describe a mouse model of soft tissue sarcoma mediated by adenoviral-Cre recombinase injection into Trp53fl/fl/Ptenfl/fl lox-stop-lox luciferase mice. Injection of adenovirus expressing Cre recombinase, either subcutaneously or intramuscularly in two experimental groups, results in viral infection and gene recombination with 100% penetrance within the first 24 hours following injection. Luciferase expression measured by real-time bioluminescence imaging increases over time, with an initial robust increase following viral injection, followed by a steady rise over the next several weeks as primary tumors develop and grow. Intramuscular injections were more commonly associated with evidence of systemic viral distribution than subcutaneous injections. All mice developed soft tissue sarcomas at the primary injection site, with histological examination identifying 93% of tumors as invasive pleomorphic sarcomas based on microscopic morphology and immunohistochemical expression of sarcoma markers. A lymphocytic infiltrate was present in 64% of the sarcomas in this immunocompetent model and 71% of tumors expressed PD-L1. This is the first report of a viral-Cre mediated Trp53/Pten mouse model of undifferentiated pleomorphic sarcoma. The bioluminescence imaging feature, along with high penetrance of the model and its immunological characteristics, makes it suited for pre-clinical studies of soft tissue sarcoma.

Encapsulation of Antifungals in Micelles Protects Candida albicans during Gall-Bladder Infection.

Candida albicans is a dimorphic fungus that colonizes human mucosal surfaces with the potential to cause life-threatening invasive candidiasis. Studies on systemic candidiasis in a murine infection model using in vivo real-time bioluminescence imaging revealed persistence of C. albicans in the gall bladder under antifungal therapy. Preliminary analyses showed that bile conferred resistance against a wide variety of antifungals enabling survival in this cryptic host niche. Here, bile and its components were studied for their ability to reduce antifungal efficacy in order to elucidate the underlying mechanism of protection. While unconjugated bile salts were toxic to C. albicans, taurine, or glycine conjugated bile salts were well tolerated and protective against caspofungin and amphotericin B when exceeding their critical micellar concentration. Microarray experiments indicated that upregulation of genes generally known to mediate antifungal protection is not involved in the protection process. In contrast, rhodamine 6G and crystal violet in- and efflux experiments indicated encapsulation of antifungals in micelles, thereby reducing their bioavailability. Furthermore, farnesol sensing was abolished in the presence of conjugated bile salts trapping C. albicans cells in the hyphal morphology. This suggests that bioavailability of amphiphilic and hydrophobic compounds is reduced in the presence of bile. In contrast, small and hydrophilic molecules, such as cycloheximide, flucytosine, or sodium azide kept their antifungal properties. We therefore conclude that treatment of gall bladder and bile duct infections is hampered by the ability of bile salts to encapsulate antifungals in micelles. As a consequence, treatment of gall bladder or bile duct infections should favor the use of small hydrophilic drugs that are not solubilised in micelles.

Immune- and Nonimmune-Compartment-Specific Interferon Responses Are Critical Determinants of Herpes Simplex Virus-Induced Generalized Infections and Acute Liver Failure.

Herpes simplex virus 1 (HSV-1) infection is an incurable viral infection with the most significant morbidity and mortality occurring in neonates and patients with compromised immune systems. Severe pathologies from HSV include the blindness-inducing herpetic stromal keratitis, highly debilitating and lethal herpes simplex encephalitis, and generalized infections that can lead to herpes simplex virus-induced acute liver failure. While immune compromise is a known factor, the precise mechanisms that lead to generalized HSV infections are unknown. In this study, we used and developed a mouse model system in combination with real-time bioluminescence imaging to demonstrate the relative importance of the immune and nonimmune compartments for containing viral spread and promoting host survival after corneal infection. Our results shed light on the pathogenesis of HSV infections that lead to generalized infection and acute liver failure.

Real-Time Bioluminescence Imaging of Cell Distribution, Growth, and Differentiation in a Three-Dimensional Scaffold Under Interstitial Perfusion for Tissue Engineering.

Bioreactor systems allow safe and reproducible production of tissue constructs and functional analysis of cell behavior in biomaterials. However, current procedures for the analysis of tissue generated in biomaterials are destructive. We describe a transparent perfusion system that allows real-time bioluminescence imaging of luciferase expressing cells seeded in scaffolds for the study of cell-biomaterial interactions and bioreactor performance. A prototype provided with a poly(lactic) acid scaffold was used for "proof of principle" studies to monitor cell survival in the scaffold (up to 22 days). Moreover, using cells expressing a luciferase reporter under the control of inducible tissue-specific promoters, it was possible to monitor changes in gene expression resulting from hypoxic state and endothelial cell differentiation. This system should be useful in numerous tissue engineering applications, the optimization of bioreactor operation conditions, and the analysis of cell behavior in three-dimensional scaffolds.

Role of Estrogen Response Element in the Human Prolactin Gene: Transcriptional Response and Timing.

The use of bacterial artificial chromosome (BAC) reporter constructs in molecular physiology enables the inclusion of large sections of flanking DNA, likely to contain regulatory elements and enhancers regions that contribute to the transcriptional output of a gene. Using BAC recombineering, we have manipulated a 160-kb human prolactin luciferase (hPRL-Luc) BAC construct and mutated the previously defined proximal estrogen response element (ERE) located −1189 bp relative to the transcription start site, to assess its involvement in the estrogen responsiveness of the entire hPRL locus. We found that GH3 cell lines stably expressing Luc under control of the ERE-mutated hPRL promoter (ERE-Mut) displayed a dramatically reduced transcriptional response to 17β-estradiol (E2) treatment compared with cells expressing Luc from the wild-type (WT) ERE hPRL-Luc promoter (ERE-WT). The −1189 ERE controls not only the response to E2 treatment but also the acute transcriptional response to TNFα, which was abolished in ERE-Mut cells. ERE-WT cells displayed a biphasic transcriptional response after TNFα treatment, the acute phase of which was blocked after treatment with the estrogen receptor antagonist 4-hydroxy-tamoxifen. Unexpectedly, we show the oscillatory characteristics of hPRL promoter activity in individual living cells were unaffected by disruption of this crucial response element, real-time bioluminescence imaging showed that transcription cycles were maintained, with similar cycle lengths, in ERE-WT and ERE-Mut cells. These data suggest the −1189 ERE is the dominant response element involved in the hPRL transcriptional response to both E2 and TNFα and, crucially, that cycles of hPRL promoter activity are independent of estrogen receptor binding.