Bioanalytical applications of multicolour bioluminescence imaging: new tools for drug discovery and development

The subject of this thesis is multicolour bioluminescence analysis and how it can provide new tools for drug discovery and development.The mechanism of color tuning in bioluminescent reactions is not fully understood yet but it is object of intense research and several hypothesis have been generated. In the past decade key residues of the active site of the enzyme or in the surface surrounding the active site have been identified as responsible of different color emission. Anyway since bioluminescence reaction is strictly dependent from the interaction between the enzyme and its substrate D-luciferin, modification of the substrate can lead to a different emission spectrum too. In the recent years firefly luciferase and other luciferases underwent mutagenesis in order to obtain mutants with different emission characteristics. Thanks to these new discoveries in the bioluminescence field multicolour luciferases can be nowadays employed in bioanalysis for assay developments and imaging purposes. The use of multicolor bioluminescent enzymes expanded the potential of a range of application in vitro and in vivo. Multiple analysis and more information can be obtained from the same analytical session saving cost and time. This thesis focuses on several application of multicolour bioluminescence for high-throughput screening and in vivo imaging. Multicolor luciferases can be employed as new tools for drug discovery and developments and some examples are provided in the different chapters. New red codon optimized luciferase have been demonstrated to be improved tools for bioluminescence imaging in small animal and the possibility to combine red and green luciferases for BLI has been achieved even if some aspects of the methodology remain challenging and need further improvement. In vivo Bioluminescence imaging has known a rapid progress since its first application no more than 15 years ago. It is becoming an indispensable tool in pharmacological research. At the same time the development of more sensitive and implemented microscopes and low-light imager for a better visualization and quantification of multicolor signals would boost the research and the discoveries in life sciences in general and in drug discovery and development in particular

Effect of sex in the MRMT-1 model of cancer-induced bone pain

An overwhelming amount of evidence demonstrates sex-induced variation in pain processing, and has thus increased the focus on sex as an essential parameter for optimization of in vivo models in pain research. Mammary cancer cells are often used to model metastatic bone pain in vivo, and are commonly used in both males and females. Here we demonstrate that compared to male rats, female rats have an increased capacity for recovery following inoculation of MRMT-1 mammary cells, thus potentially causing a sex-dependent bias in interpretation of the data

La corruzione in Europa

[Dall'Introduzione] Attraverso l'analisi di numerosi casi di corruzione venuti alla luce nei diversi Paesi europei è stato costruito un quadro di analisi che ricompone gli elementi di base del fenomeno. Certamente molti altri elementi sono importanti nell'analisi della corruzione, ma questi costituiscono a nostro parere i caratteri fissi ed immancabili. Da questo primo lavoro è uscita una griglia di riferimento che può essere usata per comparare il fenomeno nei diversi Paesi europei dove sia il quadro legislativo che il tessuto sociale presentano differenze spesso di notevole portata. Gli elementi fondamentali che vanno presi in considerazione nell'esaminare il fenomeno corruzione sono sostanzialmente tre. Prima di tutto i settori che vengono aggrediti dalla corruzione. L'elenco che presentiamo è suscettibile di continue integrazioni perché questi possono variare a seconda delle politiche anticorruzione che vengono attuate e delle condizioni sociali ed economiche di un Paese. I settori elencati sono quelli in cui è stata riscontrata corruzione in uno o più Paesi europei. In secondo luogo i soggetti che agiscono sul mercato della corruzione. Vedremo poi che si tratta di soggetti con status socioeconomici o giuridici diversi. Interessante dal punto di vista dell'analisi è un soggetto bivalente, il mediatore, che interviene nel mercato della corruzione esattamente come accade nei mercati legali per favorire gli scambi. La sua presenza indica che il mercato della corruzione è abbastanza vasto e sviluppato da aver bisogno di soggetti che svolgono il ruolo di favorire e promuovere gli scambi. Terzo pilastro del sistema sono metodi, strumenti e meccanismi. Gli strumenti sono i mezzi di pagamento utilizzati per l'acquisto di corruzione. Metodi sono gli artifici contabili che servono a creare disponibilità extrabilancio, comunemente note col nome di fondi neri, che costituiscono la provvista finanziaria per operare sul mercato della corruzione. Per meccanismi si intendono gli apparati economici e/o giuridici messi in piedi allo scopo di creare canali per la raccolta o l'occultamento dei proventi della corruzione

Editorial: Small animal imaging: Technological and methodological advances to improve the translational power

Radiolog

Whole-body bioluminescence imaging of T-cell response in PDAC models

Introduction: The location of T-cells during tumor progression and treatment provides crucial information in predicting the response in vivo. Methods: Here, we investigated, using our bioluminescent, dual color, T-cell reporter mouse, termed TbiLuc, T-cell location and function during murine PDAC tumor growth and checkpoint blockade treatment with anti-PD-1 and anti-CTLA-4. Using this model, we could visualize T-cell location and function in the tumor and the surrounding tumor microenvironment longitudinally. We used murine PDAC clones that formed in vivo tumors with either high T-cell infiltration (immunologically ‘hot’) or low T-cell infiltration (immunologically ‘cold’). Results: Differences in total T-cell bioluminescence could be seen between the ‘hot’ and ‘cold’ tumors in the TbiLuc mice. During checkpoint blockade treatment we could see in the tumor-draining lymph nodes an increase in bioluminescence on day 7 after treatment. Conclusions: In the current work, we showed that the TbiLuc mice can be used to monitor T-cell location and function during tumor growth and treatment.</p

Bioluminescence imaging on-chip platforms for non-invasive high-content bioimaging

Incorporating non-invasive biosensing features in organ-on-chip models is of paramount importance for a wider implementation of these advanced in vitro microfluidic platforms. Optical biosensors, based on Bioluminescence Imaging (BLI), enable continuous, non-invasive, and in-situ imaging of cells, tissues or miniaturized organs without the drawbacks of conventional fluorescence imaging. Here, we report the first-of-its-kind integration and optimization of BLI in microfluidic chips, for non-invasive imaging of multiple biological readouts. The cell line HEK293T-GFP was engineered to express NanoLuc® luciferase under the control of a constitutive promoter and were cultured on-chip in 3D, in standard ECM-like hydrogels, to assess optimal cell detection conditions. Using real-time in-vitro dual-color microscopy, Bioluminescence (BL) and fluorescence (FL) were detectable using distinct imaging setups. Detection of the bioluminescent signals were observed at single cell resolution on-chip 20 min post-addition of Furimazine substrate and under perfusion. All hydrogels enabled BLI with higher signal-to-noise ratios as compared to fluorescence. For instance, agarose gels showed a ∼5-fold greater BL signal over background after injection of the substrate as compared to the FL signal. The use of BLI with microfluidic chip technologies opens up the potential for simultaneous in situ detection with continuous monitoring of multicolor cell reporters. Moreover, this can be achieved in a non-invasive manner. BL has great promise as a highly desirable biosensor for studying organ-on-chip platforms.</p

Optimized Longitudinal Monitoring of Stem Cell Grafts in Mouse Brain Using a Novel Bioluminescent/Near Infrared Fluorescent Fusion Reporter

Biodistribution and fate of transplanted stem cells via longitudinal monitoring has been successfully achieved in the last decade using optical imaging

Whole-body bioluminescence imaging of T-cell response in PDAC models

IntroductionThe location of T-cells during tumor progression and treatment provides crucial information in predicting the response in vivo.MethodsHere, we investigated, using our bioluminescent, dual color, T-cell reporter mouse, termed TbiLuc, T-cell location and function during murine PDAC tumor growth and checkpoint blockade treatment with anti-PD-1 and anti-CTLA-4. Using this model, we could visualize T-cell location and function in the tumor and the surrounding tumor microenvironment longitudinally. We used murine PDAC clones that formed in vivo tumors with either high T-cell infiltration (immunologically ‘hot’) or low T-cell infiltration (immunologically ‘cold’).ResultsDifferences in total T-cell bioluminescence could be seen between the ‘hot’ and ‘cold’ tumors in the TbiLuc mice. During checkpoint blockade treatment we could see in the tumor-draining lymph nodes an increase in bioluminescence on day 7 after treatment.ConclusionsIn the current work, we showed that the TbiLuc mice can be used to monitor T-cell location and function during tumor growth and treatment

Development of a New Hyaluronic Acid Based Redox-Responsive Nanohydrogel for the Encapsulation of Oncolytic Viruses for Cancer Immunotherapy

Oncolytic viruses (OVs) are emerging as promising and potential anti-cancer therapeutic agents, not only able to kill cancer cells directly by selective intracellular viral replication, but also to promote an immune response against tumor. Unfortunately, the bioavailability under systemic administration of OVs is limited because of undesired inactivation caused by host immune system and neutralizing antibodies in the bloodstream. To address this issue, a novel hyaluronic acid based redox responsive nanohydrogel was developed in this study as delivery system for OVs, with the aim to protect the OVs following systemic administration. The nanohydrogel was formulated by water in oil (W/O) nanoemulsion method and cross-linked by disulfide bonds derived from the thiol groups of synthesized thiolated hyaluronic acid. One DNA OV Ad[I/PPT-E1A] and one RNA OV Rigvir® ECHO-7 were encapsulated into the developed nanohydrogel, respectively, in view of their potential of immunovirotherapy to treat cancers. The nanohydrogels showed particle size of approximately 300–400 nm and negative zeta potential of around −13 mV by dynamic light scattering (DLS). A uniform spherical shape of the nanohydrogel was observed under the scanning electron microscope (SEM) and transmission electron microscope (TEM), especially, the successfully loading of OV into nanohydrogel was revealed by TEM. The crosslinking between the hyaluronic acid chains was confirmed by the appearance of new peak assigned to disulfide bond in Raman spectrum. Furthermore, the redox responsive ability of the nanohydrogel was determined by incubating the nanohydrogel into phosphate buffered saline (PBS) pH 7.4 with 10 μM or 10 mM glutathione at 37 °C which stimulate the normal physiological environment (extracellular) or reductive environment (intracellular or tumoral). The relative turbidity of the sample was real time monitored by DLS which indicated that the nanohydrogel could rapidly degrade within 10 h in the reductive environment due to the cleavage of disulfide bonds, while maintaining the stability in the normal physiological environment after 5 days. Additionally, in vitro cytotoxicity assays demonstrated a good oncolytic activity of OVs-loaded nanohydrogel against the specific cancer cell lines. Overall, the results indicated that the developed nanohydrogel is a delivery system appropriate for viral drugs, due to its hydrophilic and porous nature, and also thanks to its capacity to maintain the stability and activity of encapsulated viruses. Thus, nanohydrogel can be considered as a promising candidate carrier for systemic administration of oncolytic immunovirotherap

Intensive monitoring of conventional and surrogate quality parameters in a highly urbanized river affected by multiple combined sewer overflows

Abstract The paper reports results of four intensive campaigns carried out on the Seveso River (Milan metropolitan area, Italy) between 2014 and 2016, during intense precipitation events. Laboratory analyses were coupled with on-site, continuous measurements to assess the impact of pollutants on water quality based on both conventional and surrogate parameters. Laboratory data included total suspended solids, caffeine, total phosphorus and nitrogen, and their dissolved forms. Screening of trace metals (Cr, Cu, Pb, Ni, Cd) and PBDEs (polybromodiphenylethers) was carried out. Continuous measurements included water level, physico-chemical variables and turbidity. Nutrient concentrations were generally high (e.g. average total phosphorus &gt; 1,000 μg/L) indicating strong sewage contributions. Among monitored pollutants Cr, Cu, Pb, and Cd concentrations were well correlated to TSS, turbidity and discharge, being bound mostly to suspended particulate matter. A different behavior was found for Ni, that showed an early peak occurring before the flow peak, as a result of first flush events. PBDEs correlated well to nutrient concentrations, showing the highest peaks soon after activation of the combined sewer overflows, likely because of its accumulation in sewers. In addition to showing the existing correlations between quality parameters, the paper highlights the importance of surrogate parameters as indicators of anthropic pollution inputs