Bioassays to Monitor Taspase1 Function for the Identification of Pharmacogenetic Inhibitors

Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamideand 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance

Use of a CMOS image sensor for beta-emitting radionuclide measurements

Radio-Guided Surgery enables the surgeon to identify tumor residuals in real time, with millimetric resolution. This technique takes advantage of a radiopharmaceutical as tracer and a probe as detector. The employment of β - emitters as tracers, instead of γ or β + , reduces the background from the healthy tissues and the medical exposition risk. The performances of a CMOS sensor, the MT9V011, originally designed for visible light imaging, as β - radiation detector have been investigated. In particular, we have studied the impact of an infra-red filter placed in front of the sensor in the existing commercial devices, and we have determined the sensitivity and spatial resolution of the sensor, in the presence of a localized ionization source of variable size. Finally, by evaluating the sensitivity to clinical-like activities and geometries we conclude that even with the small size of the sensor, that can potentially be increased, the acquisition time required to discriminate a tumor residual from healthy tissues is of a few seconds for transverse dimensions greater than 3 mm

Populism and Government: Continuity and Paradoxes in the Yellow-Green Experiment

Domestic and comparative constitutional analyses routinely describe the government as the most under-theorised among constitutional organs. This chapter aims to consider whether and how the Italian government has been affected by the rise of populist parties and movements or, more vaguely, by populist arguments and discourse. At first glance, an obvious starting point is the link between the trend towards the presidentialisation of the executives and the institutional programmes of populism. What has been described as the first experiment in purely populist government in Europe seems to contradict such claims and to have revived some long-term characters of the Italian executive. As scholars have put it, electoral laws and European integration are the factors that have most contributed to defining and shaping the role of government over the last three decades: in a nutshell, they have contributed, among other things, to strengthening the institutional role and visibility of the Prime Minister. Still, the Italian government which was formed in 2018 was based on a “contract” signed by the leaders of the Five Star Movement and the League, and to whose drafting Giuseppe Conte, who later went on to be appointed to the premiership, was largely foreign

MALIZIUSELLA / Specchia et Capotosti. TIENEME STRETTA A TE / Da Positano et Leoni. BAMBINA INNAMORATA / Bracchi et d'Anzi. LA MIA STORIA / Testoni et Bassi ; Dallara (Tony), chant et ses "Campioni", champions

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