Influence of Sp1/Sp3 expression on corneal epithelial cells proliferation and differentiation properties in reconstructed tissues

PURPOSE : Primary cultured epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. However, as such cells are passaged in culture, they often lose their ability to proliferate by progressing toward terminal cell differentiation, a process likely to be determined by altered expression of transcription factors that have functions critical for cell adhesion and differentiation. This study was designed to determine whether the variable life span of primary cultured human corneal epithelial cells (HCECs) might be the consequence of varying expression levels of the well-known transcription factors Sp1 and Sp3 (Sp1/Sp3). METHODS : HCECs were obtained from donor eyes and cultured on irradiated Swiss-3T3. Sp1/Sp3 expression was monitored by Western blot and electrophoretic mobility shift assay (EMSA). The Sp1/Sp3 regulatory influence was evaluated by transfection of HCECs with a recombinant plasmid bearing the Sp1/Sp3-dependent poly(ADP-ribose) polymerase (rPARP) promoter fused to the CAT reporter gene. HCECs that expressed various levels of Sp1/Sp3 were also used for the production of corneal substitutes. RESULTS : Expression of Sp1/Sp3 was dramatically inconsistent between HCECs isolated from the eyes of different donors. Both factors were highly expressed during one passage and then totally disappeared as cells terminally differentiated. Proper stratification of HCECs on reconstructed tissue substitutes could be obtained only with cells that also had a delayed peak of Sp1/Sp3 expression when cultured in vitro. CONCLUSIONS : Expression of Sp1/Sp3 may represent a good predictor for selecting HCECs that are most likely to proliferate, stratify, and differentiate properly when used for the production of reconstructed corneal substitutes

Transcriptional regulation of the human α6 integrin gene by the transcription factor NFI during corneal wound healing

Purpose. Wound healing of the corneal epithelium is highly influenced by regulation of integrin gene expression. A recent study demonstrated that laminin (LM), a major constituent of the extracellular matrix (ECM), reduces expression of the human α6 integrin subunit gene by altering the properties of the transcription factor (TF) Sp1. In this work, a target site was identified for the TF nuclear factor I (NFI) on the human α6 gene, and its regulatory influence was characterized in corneal epithelial cells. Methods. Plasmids bearing the α6 promoter fused to the CAT gene were transfected into human (HCECs) and rabbit (RCECs) corneal epithelial cells grown on LM. The DNA-binding site for NFI in the α6 promoter was identified by DNase I footprinting. Expression and DNA binding of NFI was monitored by Western blot, RT-PCR, and electrophoretic mobility shift assays (EMSAs), and its function was investigated through RNAi and NFI overexpression assays. Results. All NFI isoforms were found to be expressed in HCECs and RCECs. Transfection analyses revealed that NFI is a repressor of α6 expression in both types of cells. LM increases expression of NFI, whereas inhibition of each NFI isoform increases promoter activity suggesting that NFI is a key repressor of α6 transcription. In addition, the negative influence of NFI appears to be potentiated by the degradation of Sp1 when cells are grown on LM. Conclusions. Repression of α6 expression therefore contributes to the final steps of corneal wound healing by both reducing proliferation and allowing attachment of the epithelium to the basal membrane

Sensing of Oxygen Concentration in a Microfluidic Device mimicking Liver 3D Microarchitecture

International audienceWe designed a microfluidic structure which closely reproduces liver microarchitecture, constraining primary rat hepatocytes at a high density and in three dimensions (3D), and in which a gradient of oxygen can be generated. The device includes an oxygen sensitive membrane that could map the oxygen consumption of hepatocytes. INTRODUCTION Compared to classical two-dimensional cell culture, microfluidic devices or/and 3D culture conditions were evidenced to increase the period of time during which primary hepatocytes retain their functions [1]. Moreover, microfluidic techniques offer the opportunity to mimic the in vivo hepatocyte zonation, by subjecting hepatocytes to oxygen gradients [1-2]. Such oxygen gradients that can be estimated by numerical simulations, were recently experimentally assessed using an oxygen sensitive fluorescent membrane [3]. We proposed to include the oxygen sensitive membrane within a miniaturized fluidic device mimicking several hepatic cords in series, and inducing a gradient of oxygen on those. Moreover each of those hepatic cord units was inducing 3D organization of hepatocytes, due to the 72 µm height of culture chambers in which they can aggregate

Tetrahydrobiopterin improves recognition memory in the triple-transgenic mouse model of Alzheimer's disease, without altering amyloid-β and tau pathologies

*Background: Alzheimer's disease (AD) is a multifactorial disease, implying that multi-target treatments may be necessary to effectively cure AD. Tetrahydrobiopterin (BH4) is an enzymatic cofactor required for the synthesis of monoamines and nitric oxide that also exerts antioxidant and anti-inflammatory effects. Despite its crucial role in the CNS, the potential of BH4 as a treatment in AD has never been scrutinized. *Objective: Here, we investigated whether BH4 peripheral administration improves cognitive symptoms and AD neuropathology in the triple-transgenic mouse model of AD (3xTg-AD), a model of age-related tau and amyloid-beta (A beta) neuropathologies associated with behavior impairment. *Methods: Non-transgenic (NonTg) and 3xTg-AD mice were subjected to a control diet (5% fat - CD) or to a high-fat diet (35% fat - HFD) from 6 to 13 months to exacerbate metabolic disorders. Then, mice received either BH4 (15 mg/kg/day, i.p.) or vehicle for ten consecutive days. *Results: This sub-chronic administration of BH4 rescued memory impairment in 13-month-old 3 xTg-AD mice, as determined using the novel object recognition test. Moreover, the HFD-induced glucose intolerance was completely reversed by the BH4 treatment in 3xTg-AD mice. However, the HFD or BH4 treatment had no significant impact on A beta and tau neuropathologies. *Conclusion: Overall, our data suggest a potential benefit from BH4 administration against AD cognitive and metabolic deficits accentuated by HFD consumption in 3xTg-AD mice, without altering classical neuropathology. Therefore, BH4 should be considered as a candidate for drug repurposing, at least in subtypes of cognitively impaired patients experiencing metabolic disorders.IdEx BordeauxProgram Initiative d’Excellenc

Role of Retinoid X Receptors (RXRs) and dietary vitamin A in Alzheimer's disease: Evidence from clinicopathological and preclinical studies.

BACKGROUND Vitamin A (VitA), via its active metabolite retinoic acid (RA), is critical for the maintenance of memory function with advancing age. Although its role in Alzheimer's disease (AD) is not well understood, data suggest that impaired brain VitA signaling is associated with the accumulation of β-amyloid peptides (Aβ), and could thus contribute to the onset of AD. METHODS We evaluated the protective action of a six-month-long dietary VitA-supplementation (20 IU/g), starting at 8 months of age, on the memory and the neuropathology of the 3xTg-AD mouse model of AD (n = 11-14/group; including 4-6 females and 7-8 males). We also measured protein levels of Retinoic Acid Receptor β (RARβ) and Retinoid X Receptor γ (RXRγ) in homogenates from the inferior parietal cortex of 60 participants of the Religious Orders study (ROS) divided in three groups: no cognitive impairment (NCI) (n = 20), mild cognitive impairment (MCI) (n = 20) and AD (n = 20). RESULTS The VitA-enriched diet preserved spatial memory of 3xTg-AD mice in the Y maze. VitA-supplementation affected hippocampal RXR expression in an opposite way according to sex by tending to increase in males and decrease in females their mRNA expression. VitA-enriched diet also reduced the amount of hippocampal Aβ40 and Aβ42, as well as the phosphorylation of tau protein at sites Ser396/Ser404 (PHF-1) in males. VitA-supplementation had no effect on tau phosphorylation in females but worsened their hippocampal Aβ load. However, the expression of Rxr-β in the hippocampus was negatively correlated with the amount of both soluble and insoluble Aβ in both males and females. Western immunoblotting in the human cortical samples of the ROS study did not reveal differences in RARβ levels. However, it evidenced a switch from a 60-kDa-RXRγ to a 55-kDa-RXRγ in AD, correlating with ante mortem cognitive decline and the accumulation of neuritic plaques in the brain cortex. CONCLUSION Our data suggest that (i) an altered expression of RXRs receptors is a contributor to β-amyloid pathology in both humans and 3xTg-AD mice, (ii) a chronic exposure of 3xTg-AD mice to a VitA-enriched diet may be protective in males, but not in females

Investigation financière et juricomptabilité. Guide des bonnes pratiques

First French book about investigative and forensic accounting. The authors are listed forensic experts, in Canada and France. A normative and practiclal book, well-experienced.Le présent ouvrage a pour objectif de présenter au lecteur les « bonnes pratiques » de juricomptabilité, de sorte à agir efficacement et en sécurité lorsque l'urgence se présente. Deux dossiers permettront principalement d'illustrer concrètement chacune des étapes de l'investigation financière. L'ouvrage a par ailleurs une dimension internationale, présentant les façons de faire en Amérique du Nord et en France

Can amino acids be used to inhibit germination or deplete the soil seedbank of Phelipanche ramosa (L.) Pomel?

International audiencePhelipanche ramosa is a weed holoparasite which became a major agronomic problem for several crops in France. The yield and quality losses may be complete and lead to the abandonment of crops in the most infested fields. Chemical weed control using synthetic herbicides does not have a direct effect on this weed because the first half of its reproductive cycle occurs on the root system of its host. The aim of this study is to contribute to the definition of an integrated weed management program of P. ramosa, especially focusing on the use of the possible direct effect of amino acids on seed of broomrapes as a potential biocontrol solution. We studied a set of 14 amino acids in in vitro conditions to investigate whether some amino acids can have an inhibitory effect on the germination of the P. ramosa seeds, whether this effect may be permanent after the removal of the amino acid, and whether some amino acids can cause a high dormancy or seed mortality rate. Our main results showed (1) a strong inhibitory effect of four amino acids on the seed germination concerning arginine, lysine, phenylalanine and tryptophan, and (2) that this effect appeared temporary for arginine and lysine, but permanent for phenylalanine and tryptophan. Our study thus highlighted the interest of phenylalanine and tryptophan as they caused both high inhibition of germination and high seed mortality of P. ramosa. Based on these results, we suggest three options for the use of these amino acids that could provide concrete answers to farmers

Differences in seed germination response of two populations of Phelipanche ramosa (L.) Pomel to a set of GR24 concentrations and durations of stimulation

International audiencePhelipanche ramosa is a major weed holoparasite characterized by a broad host range with a suboptimal development on numerous hosts, suggesting inter- or intra-species specificities. Seeds of P. ramosa germinate after exposure to exogenous chemicals exuded by surrounding host roots such as strigolactones, the concentrations of these germination stimulants varying between hosts. In France, P. ramosa is characterized by genetically differentiated populations presenting varying germination rates and a host specificity. The objective of our study was to investigate the sensitivity of seeds of two P. ramosa populations harvested on tobacco and oilseed rape, to a set of GR24 concentrations, a synthetic strigol analogue. The assessment of the germination rate was based on in vitro experiments. Seeds of P. ramosa were placed in Petri dishes with various concentrations of GR24. The cumulative number of germinated seeds of P. ramosa was counted several times after application of the treatment. Cumulative germination curves were analysed using a three-parameter log-logistic model and a time-to-event approach. The results show that the germination rate of P. ramosa seeds depends on the GR24 concentration and the duration of stimulation, but also that the response to these two factors varies greatly according to the origin of the P. ramosa seeds. The difference in germination speed between P. ramosa populations further shows distinct responses at the intraspecific level, thus suggesting that the specialization of P. ramosa probably occurs at least from the first stage of the holoparasite cycle

Sensing of Oxygen Concentration in a Microfluidic Device mimicking Liver 3D Microarchitecture

International audienceWe designed a microfluidic structure which closely reproduces liver microarchitecture, constraining primary rat hepatocytes at a high density and in three dimensions (3D), and in which a gradient of oxygen can be generated. The device includes an oxygen sensitive membrane that could map the oxygen consumption of hepatocytes. INTRODUCTION Compared to classical two-dimensional cell culture, microfluidic devices or/and 3D culture conditions were evidenced to increase the period of time during which primary hepatocytes retain their functions [1]. Moreover, microfluidic techniques offer the opportunity to mimic the in vivo hepatocyte zonation, by subjecting hepatocytes to oxygen gradients [1-2]. Such oxygen gradients that can be estimated by numerical simulations, were recently experimentally assessed using an oxygen sensitive fluorescent membrane [3]. We proposed to include the oxygen sensitive membrane within a miniaturized fluidic device mimicking several hepatic cords in series, and inducing a gradient of oxygen on those. Moreover each of those hepatic cord units was inducing 3D organization of hepatocytes, due to the 72 µm height of culture chambers in which they can aggregate

F-Specific RNA Bacteriophages Model the Behavior of Human Noroviruses during Purification of Oysters: the Main Mechanism Is Probably Inactivation Rather than Release

International audienc