News release

Scientific Publication

Fluofarma is glad to announce a new scientific publication in the journal "Analytical Chemistry":

Recombinant Differential Anchorage Probes that Tower over the Spatial Dimension of Intracellular Signals for High Content Screening and Analysis


Laura Schembri^a, Marion Zanese^b, Gaelle Depierre-Plinet^b, Muriel Petit^b, Assia Elkaoukabi-Chaibi^b, Loic Tauzin^b, Cristina Florean^a,c, Lydia Lartigue^a, Chantal Medina^a, Christophe Rey^a, Francis Belloc^d, Josy Reiffers^a, François Ichas^a,b and Francesca De Giorgi*^a,b

a INSERM U916, Institut Bergonié, 229 cours de l'Argonne, 33076 Bordeaux, France
b Fluofarma, 2 rue Robert Escarpit, 33600 Pessac, France
c Università di Padova, 35121 Padova, Italia
d CHU de Bordeaux, Hôpital du Haut Lévèque, 33600 Pessac, France
* Corresponding author : francesca.degiorgi-ichas@inserm.fr


Abstract
Recombinant fluorescent probes allow the detection of molecular events inside living cells. Many of them exploit the intracellular space to provide positional signals and, thus, require detection by single cell imaging. We describe here a novel strategy based on probes capable of encoding the spatial dimension of intracellular signals into “all-or-none” fluorescence intensity changes (differential anchorage probes, DAPs). The resulting signals can be acquired in single cells at high throughput by automated flow cytometry, (i) bypassing image acquisition and analysis, (ii) providing a direct quantitative readout, and (iii) allowing the exploration of large experimental series. We illustrate our purpose with DAPs for Bax and the effector caspases 3 and 7, which are keys players in apoptotic cell death, and show applications in basic research, high content multiplexed library screening, compound characterization, and drug profiling.

Analytical Chemistry advance online publication October 29, 2009; doi: 10.1021/ac9015227

Press Release

Roche adopts Fluofarma’s high-content screening platform
Bordeaux (France), October 19, 2009 – Fluofarma, a leading company in High Content Screening (HCS) technologies, which offers services and counseling to the Pharmaceutical industry, announced today that it entered into a multi-year agreement with Roche.
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Publication

Fluofarma cited in a Biocompare featured article “Flow Cytometry Gets a Makeover”:

"If a cytometry system that adds imaging doesn’t suit your fancy, Fluofarma has a high-content screening (HCS) platform that includes fully automated flow cytometry, cell culture, and data analysis. They also offer a proprietary line of recombinant fluorescent biosensors for use in HCS. 'The combination of specifically engineered probes with analytical flow cytometry dramatically broadens the application field of this detection technology,' says Bruno Brisson, Fluofarma’s chief business officer, 'especially when image segmentation is difficult, and when acquiring more than a few hundreds of cells per condition is required. We have demonstrated that the use of these instruments as the pivotal detection unit of versatile HCS detection platforms is possible, and that it can supplant HCS by imaging in many key applications'."

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Scientific Publication

Fluofarma is glad to announce a new scientific publication in the journal Cell Research:

Outer membrane VDAC1 controls permeability transition of the inner mitochondrial membrane in cellulo during stress-induced apoptosis

Flora Tomasello^a,b, Angela Messina^b,c, Lydia Lartigue^a, Laura Schembri^a, Chantal Medina^a, Simona Reina^b,c, Didier Thoraval^d, Marc Crouzet^d, François Ichas^a,e, Vito De Pinto^b,c and Francesca De Giorgi^a,e

a INSERM U916, Institut Bergonié, 229 cours de l'Argonne, 33076 Bordeaux, France
b Dipartimento Scienze Chimiche, Università di Catania, Catania, Italia
c Istituto Nazionale di Biomembrane e Biosistemi, Sezione di Catania, Roma, Italia
d CNRS UMR 5095, Université Bordeaux 2, 146 rue Leo Saignat, 33076 Bordeaux, France
e Fluofarma, 2 rue Robert Escarpit, 33600 Pessac, France

Abstract:
Voltage-dependent anion channel (VDAC)1 is the main channel of the mitochondrial outer membrane (MOM) and it has been proposed to be part of the permeability transition pore (PTP), a putative multiprotein complex candidate agent of the mitochondrial permeability transition (MPT). Working at the single live cell level, we found that overexpression of VDAC1 triggers MPT at the mitochondrial inner membrane (MIM). Conversely, silencing VDAC1 expression results in the inhibition of MPT caused by selenite-induced oxidative stress. This MOM-MIM crosstalk was modulated by Cyclosporin A and mitochondrial Cyclophilin D, but not by Bcl-2 and Bcl-xl, indicative of PTP operation. VDAC1-dependent MPT engages a positive feedback loop involving reactive oxygen species and p38-MAPK, and secondarily triggers a canonical apoptotic response including Bax activation, cytochrome c release and caspase 3 activation. Our data thus support a model of the PTP complex involving VDAC1 at the MOM, and indicate that VDAC1-dependent MPT is an upstream mechanism playing a causal role in oxidative stress-induced apoptosis.

Cell Research advance online publication 11 August 2009; doi: 10.1038/cr.2009.98

Scientific Publication

 Fluofarma is glad to announce a new scientific publication in the New York Academy of Sciences

Differential effects of the new glucocorticoid receptor antagonist ORG 34517 and RU486 (mifepristone) on glucocorticoid receptor nuclear translocation in the AtT20 cell line.

Peeters BW^a*, Ruigt GS^a, Craighead M^b, Kitchener P^c

a ORGANON, Department of Clinical Research, OSS, The Netherlands
b ORGANON, Department of Molecular Pharmacology, Newhouse Scotland, UK
c FLUOFARMA, 2 rue Robert Escarpit, 33600 Pessac, France

*Author for correspondence (e-mail: ard.peeters@organon.com)

Abstract:
Glucocorticoid agonists bind to cytoplasmic glucocorticoid receptors (GRs) and subsequently translocate as an agonist-GR complex into the nucleus. In the nucleus the complex regulates the transcription of target genes. A number of GR antagonists (RU486, progesterone, RU40555) have also been shown to induce receptor translocation. These compounds should be regarded as partial agonists. For the nonselective progesterone receptor antagonists, RTI3021-012 and RTI3021-022, it was shown that GR antagonism is possible without the induction of GR translocation. In the present studies, the new GR antagonist, ORG 34517, was investigated for its potential to induce GR translocation and to antagonize corticosterone-induced GR translocation in the AtT20 (mouse pituitary) cell line. ORG 34517 was compared to RU486. In contrast to RU486, ORG 34517 (at doses up to 3 x 10(-7) M) did not induce GR translocation, but was able to block corticosterone (3 x 10(-8) M) induced GR translocation. ORG 34517 can be regarded as a true competitive GR antagonist without partial agonistic activities.

Annals of the New York Academy of Sciences; 2008, vol. 1148, pp. 536-541

Scientific Publication

Fluofarma is glad to announce a new scientific publication in the Journal of Cell Science:

An intracellular wave of cytochrome c propagates and precedes Bax redistribution during apoptosis

Lydia Lartigue^a,*, Chantal Medina^a,*, Laura Schembri^a, Paul Chabert^a, Marion Zanese^a,b, Flora Tomasello^a, Renée Dalibart^a, Didier Thoraval^c, Marc Crouzet^c, François Ichas^a,b,+and Francesca De Giorgi^a,b

 
a INSERM U916, Institut Bergonié, 229 cours de l Argonne, 33000 Bordeaux, France
b FLUOFARMA, 2 rue Robert Escarpit, 33600 Pessac, France
c CNRS UMR 5095, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France

*These authors contributed equally to this work
+Author for correspondence (e-mail: francois.ichas@inserm.fr)

Abstract:

Bax is considered to be pivotal in inducing cytochrome c release (CCR) from mitochondria during apoptosis. Indeed, Bax redistributes to the mitochondrial outer membrane (MOM) upon activation and forms homo-multimersthat are capable of permeabilizing the MOM. Our attempts to image this sequence of events in single live cells resulted in unexpected observations. Bax redistribution exhibited two distinct components: an early minor redistribution that was silent in terms of homomultimerization and a major late redistribution that was synchronous with the formation of Bax multimers, but that proceeded belatedly, i.e. only after caspase 3/7 (C3/7) had already been activated. Intriguingly, neither of these two components of redistribution correlated with CCR, which turned out to be spatially organized, propagating as a traveling wave at constant velocity. Strikingly, propagation of the CCR wave (1) preceded signs of in situ Bax conformational activation; (2) appeared to be independent of autocatalytic loops involving a positive feedback of either C3/7, Ca2+ mobilization or mitochondrial permeability transition; and (3) was triggered by diffuse stimulation with the synthetic Bak activator BH3I-1 but then proceeded independently of Bak activation. Thus, the CCR wave not only questions the exact role of Bax redistribution in cell death, but also indicates the existence of yet unidentified positive-feedback loops that ensure a spatiotemporal control of apoptosis at the subcellular scale.

J Cell Sci. 2008 Oct 7. Accepted 21 July 2008

Scientific Publication

Fluofarma is glad to announce a new scientific publication in the journal Biochimica et Biophysica Acta - Molecular Cell Research:


High content analysis of γ-secretase activity reveals variable dominance of presenilin mutations linked to familial Alzheimer's disease

Cristina Florean^{a, b}, Enrico Zampese^a, Marion Zanese^{b, c}, Lucia Brunello^a, François Ichas^{b, c}, Francesca De Giorgi^{b, c} and Paola Pizzo^a,

^aDepartment Biomedical Sciences and CNR Institute of Neuroscience, University of Padua, Via G. Colombo, 3, 35121 Padua, Italy
^bINSERM U916, VINCO, Institut Bergonié, 229 cours de l'Argonne, 33076 Bordeaux, France
^cFLUOFARMA, 2 rue Robert Escarpit, 33600 Pessac, France
 

Abstract
γ-Secretase mediates the intramembranous proteolysis of amyloid precursor protein (APP), Notch and other cellular substrates and is considered a prime pharmacological target in the development of therapeutics for Alzheimer's disease (AD). We describe here an efficient, new, simple, sensitive and rapid assay to quantify γ-secretase activity in living cells by flow cytometry using two membrane-bound fluorescent probes, APP-GFP or C99-GFP, as substrates for γ-secretase. The principle of the assay is based on the fact that the soluble intracellular domain of GFP-tagged APP (AICD-GFP) is released from the membrane into the cytosol following γ-secretase cleavage. Using this feature, enzymatic activity of γ-secretase could be deduced from the extent of the membrane retention of the probe observed after plasma membrane permeabilization and washout of the cleaved fraction. By applying two well-known γ-secretase inhibitors (DAPT and L-685,458), we validated our assay showing that the positional GFP-based probes for γ-secretase activity behave properly when expressed in different cell lines, providing the basis for the further development of a high-throughput and high content screening for AD targeted drug discovery. Moreover, by co-expression of different familiar AD-linked mutated forms of presenilin – the key component of the γ-secretase complex – in cells devoid of any endogenous γ-secretase, our method allowed us to evaluate in situ the contribution of different presenilin variants to the modulation of the enzyme.