Development and validation of a luminescence-based, medium-throughput assay for drug screening in Schistosoma mansoni

Schistosomiasis, one of the world's greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs. METHODOLOGY/PRINCIPAL FINDINGS: The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery. CONCLUSIONS: The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies

Sialidase NEU3 Dinamically Associates to Different Membrane Domains Specifically Modifying Their Ganglioside Pattern and Triggering Akt Phosphorylation

Lipid rafts are known to regulate several membrane functions such as signaling, trafficking and cellular adhesion. The local enrichment in sphingolipids and cholesterol together with the low protein content allows their separation by density gradient flotation after extraction with non-ionic detergent at low temperature. These structures are also referred to as detergent resistant membranes (DRM). Among sphingolipids, gangliosides play important roles in different biological events, including signal transduction and tumorigenesis. Sialidase NEU3 shows high enzymatic specificity toward gangliosides. Moreover, the enzyme is present both at the cell surface and in endosomal structures and cofractionates with caveolin. Although changes in the expression level of NEU3 have been correlated to different tumors, little is known about the precise distribution of the protein and its ability in modifying the ganglioside composition of DRM and non-DRM, thus regulating intracellular events. By means of inducible expression cell system we found that i) newly synthesized NEU3 is initially associated to non-DRM; ii) at steady state the protein is equally distributed between the two membrane subcompartments, i.e., DRM and non-DRM; iii) NEU3 is degraded via the proteasomal pathway; iv) the enzyme specifically modifies the ganglioside composition of the membrane areas where it resides; and v) NEU3 triggers phosphorylation of Akt, even in absence of exogenously administered EGF. Taken together our data demonstrate that NEU3 regulates the DRM ganglioside content and it can be considered as a modulator of Akt phosphorylation, further supporting the role of this enzyme in cancer and tumorigenesis

Nat Metab.

Bile acids (BAs) are signalling molecules that mediate various cellular responses in both physiological and pathological processes. Several studies report that BAs can be detected in the brain1, yet their physiological role in the central nervous system is still largely unknown. Here we show that postprandial BAs can reach the brain and activate a negative-feedback loop controlling satiety in response to physiological feeding via TGR5, a G-protein-coupled receptor activated by multiple conjugated and unconjugated BAs2 and an established regulator of peripheral metabolism3,4,5,6,7,8. Notably, peripheral or central administration of a BA mix or a TGR5-specific BA mimetic (INT-777) exerted an anorexigenic effect in wild-type mice, while whole-body, neuron-specific or agouti-related peptide neuronal TGR5 deletion caused a significant increase in food intake. Accordingly, orexigenic peptide expression and secretion were reduced after short-term TGR5 activation. In vitro studies demonstrated that activation of the Rho–ROCK–actin-remodelling pathway decreases orexigenic agouti-related peptide/neuropeptide Y (AgRP/NPY) release in a TGR5-dependent manner. Taken together, these data identify a signalling cascade by which BAs exert acute effects at the transition between fasting and feeding and prime the switch towards satiety, unveiling a previously unrecognized role of physiological feedback mediated by BAs in the central nervous system.Développment d'une infrastructure française distribuée coordonnéeLa signalisation des acides biliaires dans le cerveau et son rôle dans le contrôle métaboliqueInnovations instrumentales et procédurales en psychopathologie expérimentale chez le rongeu

Overexpression of sialidase NEU3 increases the cellular radioresistance potential of U87MG glioblastoma cells

The plasma membrane-associated sialidase NEU3 is known to play important roles in different physiological and pathophysiological processes such as proliferation, cellular differentiation and tumorigenesis. Up-regulation of NEU3 has been associated to several tumors and recently it was demonstrated that its down-modulation in glioblastoma cells promotes cell invasiveness. To date, no information concerning the possible role played by NEU3 in relation to tumor radioresistance is available. Here we show that overexpression of NEU3 in glioblastoma U87MG cells activates PI3K/Akt signaling pathway resulting in an increased radioresistance capacity and in an improved efficiency of double strand DNA-repair mechanisms after irradiation. Our results demonstrate for the first time that NEU3 contributes to the radioresistance features of U87MG cells, bringing to evidence a novel rand peculiar role of the enzyme in cancer biology

The Slc25a47 locus is a novel determinant of hepatic mitochondrial function implicated in liver fibrosis

Background & Aims: Transporters of the SLC25 mitochondrial carrier superfamily bridge cytoplasmic and mitochondrial metabolism by channeling metabolites across mitochondrial membranes and are pivotal for metabolic homeostasis. Despite their physiological relevance as gatekeepers of cellular meta-bolism, most of the SLC25 family members remain uncharac-terized. We undertook a comprehensive tissue distribution analysis of all Slc25 family members across metabolic organs and identified SLC25A47 as a liver-specific mitochondrial carrier. Methods: We used a murine loss-of-function model to unravel the role of this transporter in mitochondrial and hepatic ho-meostasis. We performed extensive metabolic phenotyping and molecular characterization of newly generated Slc25a47hep-/-and Slc25a47-Fgf21(hep-)/-mice. Results: Slc25a47hep-/-mice displayed a wide variety of metabolic abnormalities, as a result of sustained energy deficiency in the liver originating from impaired mitochondrial respiration. This mitochondrial phenotype was associated with an activation of the mitochondrial stress response (MSR) in the liver, and the development of fibrosis, which was exacerbated upon feeding a high-fat high-sucrose diet. The MSR induced the secretion of several mitokines, amongst which FGF21 played a preponderant role on systemic physiology. To dissect the FGF21-dependent and-independent physiological changes induced in Slc25a47hep-/-mice, we generated a double Slc25a47-Fgf21(hep)-/-mouse model and demonstrated that several aspects of the hypermetabolic state were driven by hepatic secretion of FGF21. On the other hand, the metabolic fuel inflexibility observed in Slc25a47(hep)-/-mice could not be rescued with the genetic removal of Fgf21. Conclusion: Collectively, our data place the Slc25a47 locus at the center of mitochondrial homeostasis, which upon dysfunction triggers robust liver-specific and systemic adaptive stress responses. The prominent role of the Slc25a47 locus in hepatic fibrosis identifies this carrier, or its transported metabolite, as a potential target for therapeutic intervention.Lay summary: Herein, we report the importance of a locus containing a liver-specific gene coding for a mitochondrial transport protein called SLC25A47. Mitochondria are the powerhouses of cells. They are crucial for metabolism and energy generation. We show that mice with genetic disruption of the Slc25a47 locus cannot maintain mitochondrial homeostasis (balance), leading to wide-ranging problems in the liver that have far-reaching physiological consequences. (C) 2022 The Author(s). Published by Elsevier B.V

LD₅₀ of hit compounds on schistosomula.

<p>LD₅₀ of hit compounds on schistosomula.</p

PI and FDA uptake correlates with schistosomula survival.

<p>Schistosomula were incubated with serial dilutions of GA and PI and FDA fluorescence signals in treated schistosomula were measured. Left axis: FDA raw data for each concentration (n = 5) normalized between 50 μM GA-treated control (0% survival) and DMSO treated schistosomula (100% survival). Right axis: PI raw data for each concentration (n = 5) normalized between 50 μM GA-treated control (100% death) and DMSO treated schistosomula (0% death). The error bars represent the standard error, while the dotted lines are the 95% confidence interval of the fitted sigmoid curve. The calculated LD₅₀ was 3.5 μM. The percentage of live and dead parasites, calculated as described in Materials and Methods, are shown.</p

ATP luminescent signal correlates with schistosomula viability.

<p>Schistosomula ranging from 25–200 were incubated with serial dilutions of GA and ATP luminescence signals were measured. Raw data (RLU) for each concentration (n = 5) are reported on the y-axis. The error bars represent the standard error. The table reports the calculated LD₅₀ at different parasite concentrations.</p

ATP quantitation correlates with the number of schistosomula.

<p>Correlation between the number of schistosomula (X-axis, logarithmic scale) and the ATP signals (Y-axis, linear scale). A semi-log plot was used to better visualize the data; a linear correlation between schistosomula numbers and ATP signals is represented by the portion of curve comprised between dotted vertical lines. The LLoQ (Lower Limit of Quantification) was defined as a signal greater than three times the background signal. The ULoQ (Upper Limit of Quantification) was defined as the highest signal lying on the linear correlation. RLU = Relative Luminescent Units.</p

Robust penetration of the CTG reagent with preservation of the overall schistosomula morphology.

<p>Representative fluorescence (A), bright field (B) and merge (C) confocal laser microscopy images of schistosomula treated with DMSO and incubated with CTG and the membrane-impermeant DNA dye CellTox green are shown. Scale bar = 25 μm.</p