Approche variationnelle pour la déconvolution rapide de données 3D en microscopie biphotonique

National audienceBetter understanding of biological processes requires new, improved, high resolution imagery techniques. The present work concerns the restoration of data acquired with two-photon microscopy in biological tissue, in-vivo in particular. Two main challenges to tackle are: the large dimensionality of the acquired data and the incomplete knowledge of the impulse response of the system. We propose here an experimental setting to estimate it based on the observation of fluorescent micro-beads. The non-blind formulation of the related inverse problem of image restoration is then solved by minimizing a penalized criterion using an efficient convex optimization strategy based on the Majoration-Minimization approach. The effectiveness of the proposed approach is not only shown on simulated data but also on real data.Les technologies d'imagerie permettant d'obtenir des images à l'échelle cellulaire sont devenues incontournables pour mieux comprendre les processus biologiques. Nous nous intéressons ici à l'imagerie biphotonique et plus particulièrement, à la reconstruction des images acquises par un tel système dans un contexte in-vivo. Deux difficultés majeures sont : les gros volumes de données que représentent les acquisitions et la mauvaise connaissance de la réponse impulsionnelle de l'instrument. Nous proposons dans ce travail d'estimer cette dernière à l'aide d'un montage expérimental basé sur l'observation de micro-billes fluorescentes. Le problème inverse de restauration d'image est ensuite résolu dans un cadre non aveugle, en le formulant sous la forme d'un critère pénalisé minimisé à l'aide d'une méthode d'optimisation convexe efficace fondée sur la technique de Majoration-Minimisation. L'efficacité de l'approche proposée est démontrée sur données simulées et réelles

The Nrf2-Antioxidant Response Element Signaling Pathway Controls Fibrosis and Autoimmunity in Scleroderma

Systemic sclerosis (SSc) is an autoimmune disease with fibrosis of the skin and internal organs and vascular alterations. Dysregulations in the oxidant/antioxidant balance are known to be a major factor in the pathogenesis of the disease. Indeed, reactive oxygen species (ROS) trigger neoepitopes leading to a breach of immune tolerance and autoimmune responses, activate fibroblasts to proliferate and to produce excess of type I collagen. ROS also alter endothelial cells leading to vascular dysfunction. Glutathione (GSH) is the most potent antioxidant system in eukaryotic cells. Numerous studies have reported a defect in GSH in SSc animal models and humans, but the origin of this defect remains unknown. The transcription factor NRF2 is a key player in the antioxidant defense, as it can induce the transcription of antioxidant and cytoprotective genes, including GSH, through its interaction with the antioxidant response elements. In this work, we investigated whether NRF2 could be implicated in the pathogenesis of SSc, and if this pathway could represent a new therapeutic target in this orphan disease with no curative medicine. Skin biopsies from 11 patients and 10 controls were harvested, and skin fibroblasts were extracted. Experimental SSc was induced both in BALB/c and in nrf2−/− mice by daily intradermal injections of hypochloric acid. In addition, diseased BALB/c mice were treated with an nrf2 agonist, dimethyl fumarate, or placebo. A drop in nrf2 and target genes mRNA levels was observed in skin fibroblasts of SSc patients compared to controls. Moreover, the nrf2 pathway is also downregulated in skins and lungs of SSc mice. In addition, we observed that nrf2−/− mice have a more severe form of SSc with increased fibrosis and inflammation compared to wild-type SSc mice. Diseased mice treated with the nrf2 agonist dimethyl fumarate (DMF) exhibited reduced fibrosis and immune activation compared to untreated mice. The ex vivo treatment of skin fibroblasts from SSc mice with DMF restores GSH intracellular content, decreases ROS production and cell proliferation. These results suggest that the nrf2 pathway is highly dysregulated in human and SSc mice with deleterious consequences on fibrosis and inflammation and that Nrf2 modulation represents a therapeutic target in SSc

Serum Osteopontin Levels Are Decreased in Focal Adenomyosis

We investigated whether serum osteopontin (OPN) levels are different according to specific phenotypes of adenomyosis and endometriosis. We conducted a prospective laboratory study in a university referral center for endometriosis between May 2005 and May 2013 and included 148 nonpregnant women, younger than 42 years, undergoing surgery for a benign gynecological condition and who had a preoperative pelvic magnetic resonance imaging (MRI). The presence of focal and/or diffuse adenomyosis was determined by pelvic MRI, and women were classified into 3 groups: no-adenomyosis (No-AM), isolated diffuse adenomyosis (DIF-AM), and focal adenomyosis with or without diffuse adenomyosis (FOC-AM). After complete surgical exploration of the pelvic cavity, the presence and type of endometriosis was surgically determined and histologically confirmed. We distinguished 4 phenotypes: no endometriosis, superficial peritoneal endometriosis (SUP), ovarian endometrioma, and deep infiltrating endometriosis (DIE). Osteopontin levels were measured by enzyme-linked immunosorbent assay in serum samples obtained in all participants in the month preceding surgery. Our results show lower OPN levels in women with focal adenomyosis compared to adenomyosis-free controls. Our results also show a decrease in OPN levels in women with associated DIE and focal adenomyosis compared to women with SUP. Various serum biomarkers have been studied in the context of endometriosis severity and subtypes, whereas data on serum markers of adenomyosis are scarce. Both entities are often associated, and adenomyosis could be a confounding factor influencing results. Future research on serum biomarkers should describe subtypes of adenomyosis and endometriosis and analyze results according to well-defined subtypes

Activation of the MAPK/ERK Cell-Signaling Pathway in Uterine Smooth Muscle Cells of Women With Adenomyosis

We investigated whether the myometrium might be intrinsically different in women with adenomyosis. We studied whether the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPKs/ERKs) and phosphoinositide 3-kinase/mammalian target of rapamycin/AKT (PI3K/mTOR/AKT) cell-signaling pathways, implicated in the pathogenesis of endometriosis, might also be activated in uterine smooth muscle cells (uSMCs) of women with adenomyosis and measured the production of reactive oxygen species (ROS), proinflammatory mediators that modulate cell proliferation and have been shown to activate the MAPK/ERK pathway in endometriosis. The uSMC cultures were derived from myometrium biopsies obtained during hysterectomy or myomectomy in women with adenomyosis and controls with leiomyoma. Proliferation of uSMCs and in vitro activation of the MAPK/ERK cell-signaling pathway were increased in women with adenomyosis compared to controls. The activation of the PI3K/mTOR/AKT pathway was not significant. The ROS production and ROS detoxification pathways were not different between uSMCs of women with adenomyosis and controls suggesting an ROS-independent activation of the MAPK/ERK pathway. Our results also provide evidence that protein kinase inhibitors and the rapanalogue temsirolimus can control proliferation of uSMCs in vitro suggesting an implication of the MAPK/ERK and the PI3K/mTOR/AKT pathways in proliferation of uSMCs in women with adenomyosis and leiomyomas

Unbiased In Silico Analysis of Gene Expression Pinpoints Circulating miRNAs Targeting KIAA1324, a New Gene Drastically Downregulated in Ovarian Endometriosis

Objective: To identify circulating miRNAs associated with ovarian endometriosis (OMA), and to analyze candidate genes targeted by these miRNAs. Methods: Putative regulating miRNAs were identified through an original bioinformatics approach. We first queried the miRWalk 2.0 database to collect putative miRNA targets. Then, we matched it to a transcriptomic dataset of OMA. Moving from gene expression in the tissue to possible alterations in the patient plasma, a selection of these miRNAs was quantified by qRT-PCR in plasma samples from 93 patients with isolated OMA and 95 patients surgically checked as free from endometriosis. Then, we characterized the genes regulated by more than one miRNA and validated them by immunohistochemistry and transfection experiments on endometrial cell primary cultures obtained from endometrial biopsies of 10 women with and without endometriosis with miRNA mimics. Stromal and epithelial cells were isolated and cultured separately and gene expression levels were measured by RT-qPCR. Results: Eight miRNAs were identified by bioinformatics analysis. Two of them were overexpressed in plasma from OMA patients: let-7b-5p and miR-92a-3p (p < 0.005). Three miRNAs, let-7b and miR-92a-3p, and miR-93-5p potentially targeted KIAA1324, an estrogen-responsive gene and one of the most downregulated genes in OMA. Transfection experiments with mimics of these two miRNAs showed a strong decrease in KIAA1324 expression, up to 40%. Immunohistochemistry revealed a moderate-to-intense staining for KIAA1324 in the eutopic endometrium and a faint-to-moderate staining in the ectopic endometrium for half of the samples, which is concordant with the transcriptomic data. Discussion and Conclusion: Our results suggested that KIAA1324 might be involved in endometriosis through the downregulating action of two circulating miRNAs. As these miRNAs were found to be overexpressed, their quantification in plasma could provide a tool for an early diagnosis of endometriosis

Chalcone-Coumarin Derivatives as Potential Anti-Cancer Drugs: An in vitro and in vivo Investigation

International audienceCancer cells display an overproduction of reactive oxygen species resulting from an exaggerated intrinsic oxidative stress. However, the concept of deleterious oxidants versus beneficial antioxidants has recently evolved. Indeed, molecules like natural coumarins have shown anti-oxidant or pro-oxidant properties depending on their intracellular concentration. Therefore, we have investigated the structure-activity relationship of a variety of coumarin derivatives in terms of cytotoxicity towards human and murine carcinoma cell lines (HT29, HepG2, A549, MCF7, OVCAR and CT26). Amongst those compounds, (E)-7-methoxy-4-(3-oxo-3phenylprop- 1-enyl)-2H-chromen-2-one and (E)-7-hydroxy-4-(3-(4-hydroxyphenyl)-3-oxoprop-1-enyl)-2H-chromen-2-one displayed the most potent cytotoxic effect on colon cancer cells, CT26, (IC50= 4.9 mu M) linked to their pro-oxidant properties. Those compounds triggered the in vitro production of reactive oxygen species by tumor cells, leading to their death through a necrotic process. In vivo, molecules also slowed down tumor growth by 65.7% and 35.4%, respectively, without inducing significant side effects

Chalcone-Coumarin derivatives as potential anti-cancer drugs: an in vitro and in vivo investigation

Cancer cells display an overproduction of reactive oxygen species resulting from an exaggerated intrinsic oxidative stress. However, the concept of deleterious oxidants versus beneficial antioxidants has recently evolved. Indeed, molecules like natural coumarins have shown anti-oxidant or pro-oxidant properties depending on their intracellular concentration. Therefore, we have investigated the structure-activity relationship of a variety of coumarin derivatives in terms of cytotoxicity towards human and murine carcinoma cell lines (HT29, HepG2, A549, MCF7, OVCAR and CT26). Amongst those compounds, (E)-7-methoxy-4-(3-oxo-3- phenylprop-1-enyl)-2H-chromen-2-one and (E)-7-hydroxy-4-(3-(4-hydroxyphenyl)-3-oxoprop-1-enyl)-2H-chromen-2-one displayed the most potent cytotoxic effect on colon cancer cells, CT26, (IC50=4.9µM) linked to their pro-oxidant properties. Those compounds triggered the in vitro production of reactive oxygen species by tumor cells, leading to their death through a necrotic process. In vivo, molecules also slowed down tumor growth by 65.7% and 35.4%, respectively, without inducing significant side effects

Chalcone-Coumarin Derivatives as Potential Anti-Cancer Drugs: An in vitro and in vivo Investigation

International audienceCancer cells display an overproduction of reactive oxygen species resulting from an exaggerated intrinsic oxidative stress. However, the concept of deleterious oxidants versus beneficial antioxidants has recently evolved. Indeed, molecules like natural coumarins have shown anti-oxidant or pro-oxidant properties depending on their intracellular concentration. Therefore, we have investigated the structure-activity relationship of a variety of coumarin derivatives in terms of cytotoxicity towards human and murine carcinoma cell lines (HT29, HepG2, A549, MCF7, OVCAR and CT26). Amongst those compounds, (E)-7-methoxy-4-(3-oxo-3phenylprop- 1-enyl)-2H-chromen-2-one and (E)-7-hydroxy-4-(3-(4-hydroxyphenyl)-3-oxoprop-1-enyl)-2H-chromen-2-one displayed the most potent cytotoxic effect on colon cancer cells, CT26, (IC50= 4.9 mu M) linked to their pro-oxidant properties. Those compounds triggered the in vitro production of reactive oxygen species by tumor cells, leading to their death through a necrotic process. In vivo, molecules also slowed down tumor growth by 65.7% and 35.4%, respectively, without inducing significant side effects

Peritoneal NKG2D ligands levels measured by ELISA in patients with endometriosis and in controls after exploration of the abdominopelvic cavity.

<p>Samples with undetectable levels of NKG2D ligands have been excluded. The peritoneal fluid MICA and MICB ratio levels among groups was significantly different by the Mann Whitney test (P = 0.035 and P<0.001 respectively). Peritoneal NKG2D ligands ratio values are represented on a logarithmic scale as a scatter dot plot. The medians with their interquartile range are reported.</p