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Transport of lung liquid is essential for both normal pulmonary physiologic processes and for resolution of pathologic processes. The large internal surface area of the lung is lined by alveolar epithelial type I (TI) and type II (TII) cells; TI cells line >95% of this surface, TII cells + and K + (Rb + ) transport in TI cells isolated from adult rat lungs and compared the results to those of concomitant experiments with isolated TII cells. TI cells take up Na + in an amiloride-inhibitable fashion, suggesting the presence of Na + channels; TI cell Na + uptake, per microgram of protein, is ≈2.5 times that of TII cells. Rb + uptake in TI cells was ≈3 times that in TII cells and was inhibited by 10 −4 M ouabain, the latter observation suggesting that TI cells exhibit Na + -, K + -ATPase activity. By immunocytochemical methods, TI cells contain all three subunits (α, β, and γ) of the epithelial sodium channel ENaC and two subunits of Na + -, K + -ATPase. By Western blot analysis, TI cells contain ≈3 times the amount of αENaC/μg protein of TII cells. Taken together, these studies demonstrate that TI cells not only contain molecular machinery necessary for active ion transport, but also transport ions. These results modify some basic concepts about lung liquid transport, suggesting that TI cells may contribute significantly in maintaining alveolar fluid balance and in resolving airspace edema.

Le battement coordonne des cils vibratiles permet un mouvement liquidien efficace, qui joue un role important dans divers processus physiologiques comme l’evacuation par l’appareil respiratoire des particules inhalees piegees dans le mucus (clai-rance mucociliaire), la circulation du liquide cephalo-rachidien ou la migra-tion de l’embryon dans le tractus geni-tal [1, 2]. Les dyskinesies ciliaires, la mucoviscidose, l’asthme, les broncho-pneumopathies chroniques obstructives ou encore l’hydrocephalie sont autant de maladies ou un dysfonctionnement primaire ou secondaire des cils vibrati-les peut contribuer a l’aggravation de symptomes respiratoires, cerebraux, ou reproductifs [1, 2]. L’elucidation des mecanismes gouvernant la synthese de ces cils vibratiles apparait ainsi comme une question fondamentale de biolo-gie avec des implications biomedicales evidentes. La fabrication des cils vibratiles (mul-ticiliogenese) intervient au cours du developpement et lors de la regenera-tion de certains epitheliums speciali-ses, notamment mucociliaires. La mul-ticiliogenese necessite : (1) la sortie du cycle cellulaire ; (2) la production de centaines de corps basaux grâce a la multiplication massive de centrioles (centriologenese) ; (3) la migration et l’ancrage des corps basaux a la sur-face apicale ou ils jouent le role de centre organisateur des microtubules, a la base de chaque cil [3]. Quels sont les mecanismes controlant l’elabora-tion de multiples cils vibratiles dans une meme cellule ? Afin de repondre a cette question, nous nous sommes interesses a deux exemples d’epithelium mucociliaire : le premier borde les voies aeriennes des mammiferes ; le second correspond a l’epiderme d’embryons d’amphibiens. Tous deux disposent a la fois de cellules secretrices de mucus et de cellules multiciliees [4-6]

Single cell RNA sequencing approaches are instrumental in studies of cell-to-cell variability. 5΄ selective transcriptome profiling approaches allow simultaneous definition of the transcription start size and have advantages over 3΄ selective approaches which just provide internal sequences close to the 3΄ end. The only currently existing 5΄ selective approach requires costly and labor intensive fragmentation and cell barcoding after cDNA amplification. We developed an optimized 5΄ selective workflow where all the cell indexing is done prior to fragmentation. With our protocol, cell indexing can be performed in the Fluidigm C1 microfluidic device, resulting in a significant reduction of cost and labor. We also designed optimized unique molecular identifiers that show less sequence bias and vulnerability towards sequencing errors resulting in an improved accuracy of molecule counting. We provide comprehensive experimental workflows for Illumina and Ion Proton sequencers that allow single cell sequencing in a cost range comparable to qPCR assays.

Incidence of cutaneous squamous cell carcinomas (cSCCs) constantly increases in the Caucasian population. Developing preferentially on precancerous lesions such as actinic keratoses due to chronic sunlight exposure, cSCCs result from the malignant transformation of keratinocytes. Although a resection of the primary tumor is usually curative, a subset of aggressive cSCCs shows a high risk of recurrence and metastases. The characterization of the molecular dysfunctions involved in cSCC development should help to identify new relevant targets against these aggressive cSCCs. In that context, we have used small RNA sequencing to identify 100 microRNAs (miRNAs) whose expression was altered during chemically induced mouse skin tumorigenesis. The decreased expression of the miR-193b/365a cluster during tumor progression suggests a tumor suppressor role. Ectopic expression of these miRNAs in tumor cells indeed inhibited their proliferation, clonogenic potential and migration, which were stimulated in normal keratinocytes when these miRNAs were blocked with antisense oligonucleotides. A combination of in silico predictions and transcriptome analyses identified several target genes of interest. We validated KRAS and MAX as direct targets of miR-193b and miR-365a. Repression of these targets using siRNAs mimicked the effects of miR-193b and miR-365a, suggesting that these genes might mediate, at least in part, the tumor-suppressive action of these miRNAs.

The amiloride-sensitive epithelial Na+channel (ENaC) is an apical membrane protein complex involved in active Na+absorption and in control of fluid composition in airways. There are no data reporting the distribution of its pore-forming α-, β-, and γ-subunits in the developing human lung. With use of two different rabbit polyclonal antisera raised against β- and γ-ENaC, immunohistochemical localization of the channel was performed in fetal (10–35 wk) and in adult human airways. Both subunits were detected after 17 wk of gestation on the apical domain of bronchial ciliated cells, in glandular ducts, and in bronchiolar ciliated and Clara cells. After 30 wk, the distribution of β- and γ-subunits was similar in fetal and adult airways. In large airways, the two subunits were detected in ciliated cells, in cells lining glandular ducts, and in the serous gland cells. In the distal bronchioles, β- and γ-subunits were identified in ciliated and Clara cells. Ultrastructural immunogold labeling confirmed the identification of β- and γ-ENaC proteins in submucosal serous cells and bronchiolar Clara cells. Early expression of ENaC proteins in human fetal airways suggests that Na+absorption might begin significantly before birth, even if secretion is still dominant.

Epithelial Na+ channels (ENaCs) are thought to mediate the amiloride-blockable salt taste. The rat vallate papilla does not contribute to amiloride-blockable salt taste, yet the presence of ENaC-mRNA in this tissue has been reported. Is ENaC actually contained in the taste cells, or is it merely present in the supporting lingual epithelium? To avoid contamination by ENaC contained in the lingual epithelium, we physically isolated taste buds from the vallate papilla and used mRNA purification followed by reverse transcriptase polymerase chain reaction (RT-PCR) to investigate the presence of ENaC-type message in the isolated buds. mRNA of alpha-, beta- and gamma-subunits was detected, the alpha-signal being the strongest. These results provide first molecular evidence for the presence of ENaC subunits in taste buds that were isolated from the posterior tongue and were free of epithelial contamination. In addition, we used immunohistochemistry to show ENaC-like reactivity in posterior tongue taste cells. Interestingly, the immunoreactivity was not predominantly apical but was intracellular and close to or at the basolateral membrane. The function of basolateral ENaC-type channels is unknown. Possibly, the channels are normally closed or of very low open probability in the resting state.

Amiloride sensitivity is a common characteristic of structurally related cationic channels that are associated with a wide range of physiological functions. In Caenorhabditis elegans, neuronal and muscular degenerins are involved in mechanoperception. In animal epithelia, a Na + -selective channel participates in vectorial Na + transport. In the snail nervous system, an ionotropic receptor for the peptide FMRFamide forms a Na + -selective channel. In mammalian brain and/or in sensory neurons, ASIC channels form H + -activated cation channels involved in nociception linked to acidosis. We have now cloned a new member of this family from Drosophila melanogaster. The corresponding protein displays low sequence identity with the previously cloned members of the super-family but it has the same structural organization. Its mRNA was detected from late embryogenesis (14–17 hours) and was present in the dendritic arbor subtype of the Drosophila peripheral nervous system multiple dendritic (md) sensory neurons. While the origin and specification of md neurons are well documented, their roles are still poorly understood. They could function as stretch or touch receptors, raising the possibility that this Drosophila gene product, called dmdNaC1, could also be involved in mechanotransduction.

The Helix aspersa Phe-Met-Arg-Phe-amide (FMRFamide)-gated sodium channel is formed by homomultimerization of several FMRFamide-activated Na+ channel (FaNaCh) proteins. FaNaCh is homologous to the subunits that compose the amiloride-sensitive epithelial sodium channel, to Caenorhabditis elegans degenerins, and to acid-sensing ionic channels. FaNaCh properties were analyzed in stably transfected human embryonic kidney cells (HEK-293). The channel was functional with an EC50 for FMRFamide of 1 microM and an IC50 (25 degreesC) for amiloride of 6.5 microM as assessed by 22Na+ uptake measurements. The channel activity was associated with the presence of a protein at the cell surface with an apparent molecular mass of 82 kDa. The 82-kDa form was derived from an incompletely glycosylated form of 74 kDa found in the endoplasmic reticulum. Formation of covalent bonds between subunits of the same complex were observed either after formation of intersubunit disulfide bonds following cell homogenization and solubilization with Triton X-100 or after use of bifunctional cross-linkers. This resulted in the formation of covalent multimers that contained up to four subunits. Hydrodynamic properties of the solubilized FaNaCh complex also indicated a maximal stoichiometry of four subunits per complex. It is likely that epithelial Na+ channels, acid-sensing ionic channels, degenerins, and the other proteins belonging to the same ion channel superfamily also associate within tetrameric complexes.

Fibrosis, or tissue scarring, is defined as excessive and persistent accumulation of extracellular matrix components in response to chronic tissue injury. Fibrosis is a pathological feature characterizing nearly all forms of chronic organ failure. Fibroproliferative disorders of liver, kidney, heart, and lung are frequently associated with considerable morbidity and mortality worldwide. Limited therapeutic options are available; none is yet effective in stopping the ultimate progression of the disease. This has prompted investigations for new molecular targets. Recent studies have shown aberrant expression of miRNAs (fibromiRs) during the development of fibrosis. The challenge now is to understand how these aberrantly expressed miRNAs collaborate to drive fibrogenesis. Progress in understanding how fibromiRs contribute to tissue fibrosis is necessary to translate molecular discoveries into new therapeutics for fibroproliferative diseases.

The Fragile X-Related 1 gene (FXR1) is a paralog of the Fragile X Mental Retardation 1 gene (FMR1), whose absence causes the Fragile X syndrome, the most common form of inherited intellectual disability. FXR1P plays an important role in normal muscle development, and its absence causes muscular abnormalities in mice, frog, and zebrafish. Seven alternatively spliced FXR1 transcripts have been identified and two of them are skeletal muscle-specific. A reduction of these isoforms is found in myoblasts from Facio-Scapulo Humeral Dystrophy (FSHD) patients. FXR1P is an RNA–binding protein involved in translational control; however, so far, no mRNA target of FXR1P has been linked to the drastic muscular phenotypes caused by its absence. In this study, gene expression profiling of C2C12 myoblasts reveals that transcripts involved in cell cycle and muscular development pathways are modulated by Fxr1-depletion. We observed an increase of p21—a regulator of cell-cycle progression—in Fxr1-knocked-down mouse C2C12 and FSHD human myoblasts. Rescue of this molecular phenotype is possible by re-expressing human FXR1P in Fxr1-depleted C2C12 cells. FXR1P muscle-specific isoforms bind p21 mRNA via direct interaction with a conserved G-quadruplex located in its 3′ untranslated region. The FXR1P/G-quadruplex complex reduces the half-life of p21 mRNA. In the absence of FXR1P, the upregulation of p21 mRNA determines the elevated level of its protein product that affects cell-cycle progression inducing a premature cell-cycle exit and generating a pool of cells blocked at G0. Our study describes a novel role of FXR1P that has crucial implications for the understanding of its role during myogenesis and muscle development, since we show here that in its absence a reduced number of myoblasts will be available for muscle formation/regeneration, shedding new light into the pathophysiology of FSHD., Author Summary Muscle development is a complex process controlled by the timely expression of genes encoding crucial regulators of the muscle cell precursors called myoblasts. We know from previous studies that inactivation of the Fragile X related 1 (FXR1) gene in various animal models (mouse, frog, and zebrafish) causes muscular and cardiac abnormalities. Also, FXR1P is reduced in a human myopathy called Fascio-Scapulo Humeral Dystrophy (FSHD), suggesting its critical role in muscle that findings presented in this study contribute to elucidating. Cell-cycle arrest is a prerequisite to differentiation of myoblasts into mature myotubes, which will form the muscle. One key regulator is the p21/Cdkn1a/Cip1/Waf1 protein, which commands myoblasts to stop proliferating, and this action is particularly important during muscle regeneration. In this study, we have identified FXR1P as a novel regulator of p21 expression. We show that FXR1P absence in mouse myoblasts and FSHD-derived myopathic myoblasts increases abnormally the levels of p21, causing a premature cell cycle exit of myoblasts. Our study predicts that FXR1P absence leads to a reduced number of myoblasts available for muscle formation and regeneration. This explains the drastic effects of FXR1 inactivation on muscle and brings a better understanding of the molecular/cellular bases of FSHD.

International audience; The resistance of hypoxic cells to radiotherapy and chemotherapy is a major problem in the treatment of cancer. Recently, an additional mode of hypoxia-inducible factor (HIF)-dependent transcriptional regulation, involving modulation of a specific set of micro RNAs (miRNAs), including miR-210, has emerged. We have recently shown that HIF-1 induction of miR-210 also stabilizes HIF-1 through a positive regulatory loop. Therefore, we hypothesized that by stabilizing HIF-1 in normoxia, miR-210 may protect cancer cells from radiation. We developed a non-small cell lung carcinoma (NSCLC)-derived cell line (A549) stably expressing miR-210 (pmiR-210) or a control miRNA (pmiR-Ctl). The miR-210-expressing cells showed a significant stabilization of HIF-1 associated with mitochondrial defects and a glycolytic phenotype. Cells were subjected to radiation levels ranging from 0 to 10 Gy in normoxia and hypoxia. Cells expressing miR-210 in normoxia had the same level of radioresistance as control cells in hypoxia. Under hypoxia, pmiR-210 cells showed a low mortality rate owing to a decrease in apoptosis, with an ability to grow even at 10 Gy. This miR-210 phenotype was reproduced in another NSCLC cell line (H1975) and in HeLa cells. We have established that radioresistance was independent of p53 and cell cycle status. In addition, we have shown that genomic double-strand breaks (DSBs) foci disappear faster in pmiR-210 than in pmiR-Ctl cells, suggesting that miR-210 expression promotes a more efficient DSB repair. Finally, HIF-1 invalidation in pmiR-210 cells removed the radioresistant phenotype, showing that this mechanism is dependent on HIF-1. In conclusion, miR-210 appears to be a component of the radioresistance of hypoxic cancer cells. Given the high stability of most miRNAs, this advantage could be used by tumor cells in conditions where reoxygenation has occurred and suggests that strategies targeting miR-210 could enhance tumor radiosensitization.

Disruption of contact inhibition and serum afflux that occur after a tissue injury activate cell cycle, which then stops when confluence is reached again. Although the events involved in cell cycle entry have been widely documented, those managing cell cycle exit have remained so far ill defined. We have identified that the final stage of wound closure is preceded in keratinocytes by a strong accumulation of miR-483-3p, which acts as a mandatory signal triggering cell cycle arrest when confluence is reached. Blocking miR-483-3p accumulation strongly delays cell cycle exit, maintains cells into a proliferative state and retards their differentiation program. Using two models of cell cycle synchronization (i.e. mechanical injury and serum addition), we show that an ectopic upregulation of miR-483-3p blocks cell cycle progression in early G1 phase. This arrest results from a direct targeting of the CDC25A phosphatase by miR-483-3p, which can be impeded using an anti-miRNA against miR-483-3p or a protector that blocks the complex formation between miR-483-3p and the 3'-untranslated region (UTR) of CDC25A transcript. We show that the miRNA-induced silencing of CDC25A increases the tyrosine phosphorylation status of CDK4/6 cyclin-dependent kinases which, in turn, abolishes CDK4/6 capacity to associate with D-type cyclins. This prevents CDK4/6 kinases' activation, impairs downstream events such as cyclin E stimulation and sequesters cells in early G1. We propose this new regulatory process of cyclin-CDK association as a general mechanism coupling miRNA-mediated CDC25A invalidation to CDK post-transcriptional modifications and cell cycle control.

As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases., Author Summary Fibrosis is the final common pathway in virtually all forms of chronic organ failure, including lung, liver, and kidney, and is a leading cause of morbidity and mortality worldwide. Fibrosis results from the excessive activity of fibroblasts, in particular a differentiated form known as myofibroblast that is responsible for the excessive and persistent accumulation of scar tissue and ultimately organ failure. Idiopathic Lung Fibrosis (IPF) is a chronic and often rapidly fatal pulmonary disorder of unknown origin characterized by fibrosis of the supporting framework (interstitium) of the lungs. Given the poor prognosis of IPF patients, new insights into the biology of (myo)fibroblasts is of major interest to develop new therapeutics aimed at reducing (myo)fibroblast activity to slow or even reverse disease progression, thereby preserving organ function and prolonging life. MicroRNAs (miRNAs), a class of non-coding RNA recently identified, are associated with normal cellular processes; and deregulation of miRNAs plays a causative role in a vast array of complex diseases. In this study, we identified a particular miRNA: miR-199a-5p that governs lung fibroblast activation and ultimately lung fibrosis. Overall we showed that miR-199a-5p is a major regulator of fibrosis with strong therapeutic potency to treat fibroproliferative diseases such as IPF.

The peptide Phe-Met-Arg-Phe-NH2 (FMRFamide) and structurally related peptides are present both in invertebrate and vertebrate nervous systems. Although they constitute a major class of invertebrate peptide neurotransmitters, the molecular structure of their receptors has not yet been identified. In neurons of the snail Helix aspersa, as well as in Aplysia bursting and motor neurons, FMRFamide induces a fast excitatory depolarizing response due to direct activation of an amiloride-sensitive Na+ channel. We have now isolated a complementary DNA from Helix nervous tissue; when expressed in Xenopus oocytes, it encodes an FMRFamide-activated Na+ channel (FaNaCh) that can be blocked by amiloride. The corresponding protein shares a very low sequence identity with the previously cloned epithelial Na+ channel subunits and Caenorhabditis elegans degenerins, but it displays the same overall structural organization. To our knowledge, this is the first characterization of a peptide-gated ionotropic receptor.

Polyclonal antibodies have been raised against the α, β and γ subunits of the amiloride-sensitive Na+ channel. The three subunits were detected by immunohistochemistry at the apical membrane of epithelial cells from the distal colon, the lung and the distal segments of the kidney tubules. No significant labelling was detected in lung alveoli, suggesting that it is not a major site of expression of the Na+ channel. Effects of a low Na+ diet or of dexamethasone treatment were measured at the mRNA level and at the protein level by immunohistochemistry. In the colon, steroids controlled Na+ channel activity via the stimulation of the transcription of β and γ subunits. The α mRNA was constitutively expressed. However, while neither α, β nor γ proteins were detected in the colon of control animals, they were all detected in the colon of steroid-treated animals. In the lung, Na+ channel expression was regulated by glucocorticoids the circulating level of which was sufficiently high to induce a maximal expression of the three subunits, even in control animals. Adrenalectomy drastically reduced expression of the three subunits. A surprising finding was the apparent absence of steroid effects on α, β and γ subunit expression in the kidney. Neither the expression of the mRNAs nor the expression of the proteins were significantly altered by aldosterone or by dexamethasone. These results could be due to mixed gluco -and mineralocorticoid regulations in different segments of the kidney tubule, but their interpretation also requires regulations that are apparently not found in the lung or colon.

Specificity of interaction between a microRNA (miRNA) and its targets crucially depends on the seed region located in its 5'-end. It is often implicitly considered that two miRNAs sharing the same biological activity should display similarity beyond the strict six nucleotide region that forms the seed, in order to form specific complexes with the same mRNA targets. We have found that expression of hsa-miR-147b and hsa-miR-210, though triggered by different stimuli (i.e. lipopolysaccharides and hypoxia, respectively), induce very similar cellular effects in term of proliferation, migration and apoptosis. Hsa-miR-147b only shares a "minimal" 6-nucleotides seed sequence with hsa-miR-210, but is identical with hsa-miR-147a over 20 nucleotides, except for one base located in the seed region. Phenotypic changes induced after heterologous expression of miR-147a strikingly differ from those induced by miR-147b or miR-210. In particular, miR-147a behaves as a potent inhibitor of cell proliferation and migration. These data fit well with the gene expression profiles observed for miR-147b and miR-210, which are very similar, and the gene expression profile of miR-147a, which is distinct from the two others. Bioinformatics analysis of all human miRNA sequences indicates multiple cases of miRNAs from distinct families exhibiting the same kind of similarity that would need to be further characterized in terms of putative functional redundancy. Besides, it implies that functional impact of some miRNAs can be masked by robust expression of miRNAs belonging to distinct families.

International audience; Epithelial cell contribution to the natural history of childhood allergic respiratory disease remains poorly understood. Our aims were to identify epithelial pathways that are dysregulated in different phenotypes of respiratory allergy. We established gene expression signatures of nasal brushings from children with dust mite-allergic rhinitis, associated or not associated with controlled or uncontrolled asthma. Supervised learning and unsupervised clustering were used to predict the different subgroups of patients and define altered signalling pathways. These profiles were compared with those of primary cultures of human nasal epithelial cells stimulated with either interleukin (IL)-4, IL-13, interferon (IFN)-α, IFN-β or IFN-γ, or during in vitro differentiation. A supervised method discriminated children with allergic rhinitis from healthy controls (prediction accuracy 91%), based on 61 transcripts, including 21 T-helper cell (Th) type 2-responsive genes. This method was then applied to predict children with controlled or uncontrolled asthma (prediction accuracy 75%), based on 41 transcripts: nine of them, which were down-regulated in uncontrolled asthma, are directly linked to IFN. This group also included GSDML, which is genetically associated with asthma. Our data revealed a Th2-driven epithelial phenotype common to all children with dust mite allergic rhinitis. It highlights the influence of epithelially expressed molecules on the control of asthma, in association with atopy and impaired viral response.

International audience; In the postnatal and adult mouse forebrain, a mosaic of spatially separated neural stem cells along the lateral wall of the ventricles generates defined types of olfactory bulb neurons. To understand the mechanisms underlying the regionalization of the stem cell pool, we focused on the transcription factor Pax6, a determinant of the dopaminergic phenotype in this system. We found that, although Pax6 mRNA was transcribed widely along the ventricular walls, Pax6 protein was restricted to the dorsal aspect. This dorsal restriction was a result of inhibition of protein expression by miR-7a, a microRNA (miRNA) that was expressed in a gradient opposing Pax6. In vivo inhibition of miR-7a in Pax6-negative regions of the lateral wall induced Pax6 protein expression and increased dopaminergic neurons in the olfactory bulb. These findings establish miRNA-mediated fine-tuning of protein expression as a mechanism for controlling neuronal stem cell diversity and, consequently, neuronal phenotype.

Molecular cloning of the amiloride-sensitive Na+ channel has permitted analysis of the mechanisms of its stimulation by steroids. In rat lung cells in primary culture, where its mRNA has been detected, the activity of an amiloride-sensitive channel, highly selective for Na+, is controlled by corticosteroids. Dexamethasone (0.1 microM) or aldosterone (1 microM) induced, after a minimum 10 h treatment, a large increase of the amiloride-induced hyperpolarization and of the amiloride-sensitive current. A parallel increase in the amount of the mRNA was observed. The corresponding gene is thus a target for steroid action. Using synthetic specific agonists and antagonists for mineralo- and glucocorticoid receptors, it has been shown that the steroid action on Na+ channel expression is mediated via glucocorticoid receptors. Triiodothyronine, known to modulate steroid action in several tissues, had no effect on both the amiloride-sensitive Na+ current and the level of the mRNA for the Na+ channel protein, but potentiates the stimulatory effect of dexamethasone. The increase in Na+ channel activity observed in the lung around birth can thus be explained by a direct increase in transcription of the Na+ channel gene.

Cystic fibrosis is a major inherited disorder involving abnormalities of fluid and electrolyte transport in a number of different organs. Epithelial cells of cystic fibrosis patients have a decreased capacity to secrete chloride in response to cAMP-mobilizing agents because of the mutation of a single gene. The gene product, the cystic fibrosis transmembrane conductance regulator or CFTR, is a chloride channel. The most frequent mutation is a deletion of phenylalanine in position 508 (delta F508-CFTR) that reduces both the expression of the CFTR protein at the cell surface, and the activity of the Cl- channel. This work presents the properties of NS004, a substituted benzimidazolone, which is the first activator of normal and mutant CFTR-associated chloride channels to be described. NS004 activated CFTR and delta F508-CFTR Cl- channels expressed in Xenopus oocytes, and increased 125I efflux (via the Cl- channel) from Vero cells expressing CFTR and delta F508-CFTR. Application of NS004 to the external side of outside-out patches excised from these CFTR- and delta F508-CFTR-expressing cells induced a marked and reversible increase in channel activity.

International audience; The molecular mechanism responsible for the antitumor activity of histone deacetylase inhibitors (HDACi) remains elusive. As HDACi have been described to alter miRNA expression, the aim of this study was to characterize HDACi-induced miRNAs and to determine their functional importance in the induction of cell death alone or in combination with other cancer drugs. Two HDACi, trichostatin A and vorinostat, induced miR-129-5p overexpression, histone acetylation and cell death in BCPAP, TPC-1, 8505C, and CAL62 cell lines and in primary cultures of papillary thyroid cancer (PTC) cells. In addition, miR-129-5p alone was sufficient to induce cell death and knockdown experiments showed that expression of this miRNA was required for HDACi-induced cell death. Moreover, miR-129-5p accentuated the anti-proliferative effects of other cancer drugs such as etoposide or human α-lactalbumin made lethal for tumor cells (HAMLET). Taken together, our data show that miR-129-5p is involved in the antitumor activity of HDACi and highlight a miRNA-driven cell death mechanism.

International audience; The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences.

International audience; Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. However, the mechanisms governing motile cilia biosynthesis remain poorly elucidated. We identify miR-449 microRNAs as evolutionarily conserved key regulators of vertebrate multiciliogenesis. In human airway epithelium and Xenopus laevis embryonic epidermis, miR-449 microRNAs strongly accumulated in multiciliated cells. In both models, we show that miR-449 microRNAs promote centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. We established Notch1 and its ligand Delta-like 1(DLL1) as miR-449 bona fide targets. Human DLL1 and NOTCH1 protein levels were lower in multiciliated cells than in surrounding cells, decreased after miR-449 overexpression and increased after miR-449 inhibition. In frog, miR-449 silencing led to increased Dll1 expression. Consistently, overexpression of Dll1 mRNA lacking miR-449 target sites repressed multiciliogenesis, whereas both Dll1 and Notch1 knockdown rescued multiciliogenesis in miR-449-deficient cells. Antisense-mediated protection of miR-449-binding sites of endogenous human Notch1 or frog Dll1 strongly repressed multiciliogenesis. Our results unravel a conserved mechanism whereby Notch signalling must undergo miR-449-mediated inhibition to permit differentiation of ciliated cell progenitors.

International audience; C. burnetii is a Gram-negative intracellular Y-proteobacteria that causes the zoonotic disease Q fever. Q fever can manifest as an acute or chronic illness. Different typing methods have been previously developed to classify C. burnetii isolates to explore its pathogenicity. Here, we report a comprehensive genomotyping method based on the presence or absence of genes using microarrays. The genomotyping method was then tested in 52 isolates obtained from different geographic areas, different hosts and patients with different clinical manifestations. The analysis revealed the presence of 10 genomotypes organized into 3 groups, with a topology congruent with that obtained through multi-spacer typing. We also found that only 4 genomotypes were specifically associated with acute Q fever, whereas all of the genomotypes could be associated to chronic human infection. Serendipitously, the genomotyping results revealed that all hard tick isolates, including the Nine Mile strain, belong to the same genomotype.

International audience; ABSTRACT: BACKGROUND: In severe obesity, as well as in normal development, the growth of adipose tissue is the result of an increase in adipocyte size and numbers, which is underlain by the stimulation of adipogenic differentiation of precursor cells. A better knowledge of the pathways that regulate adipogenesis is therefore essential for an improved understanding of adipose tissue expansion. As microRNAs (miRNAs) have a critical role in many differentiation processes, our study aimed to identify the role of miRNA-mediated gene silencing in the regulation of adipogenic differentiation. RESULTS: We used deep sequencing to identify small RNAs that are differentially expressed during adipogenesis of adipose tissue-derived stem cells. This approach revealed the un-annotated miR-642a-3p as a highly adipocyte-specific miRNA. We then focused our study on the miR-30 family, which was also up-regulated during adipogenic differentiation and for which the role in adipogenesis had not yet been elucidated. Inhibition of the miR-30 family blocked adipogenesis, whilst over-expression of miR-30a and miR-30d stimulated this process. We additionally showed that both miR-30a and miR-30d target the transcription factor RUNX2, and stimulate adipogenesis via the modulation of this major regulator of osteogenesis. CONCLUSIONS: Overall, our data suggest that the miR-30 family plays a central role in adipocyte development. Moreover, as adipose tissue-derived stem cells can differentiate into either adipocytes or osteoblasts, the down-regulation of the osteogenesis regulator RUNX2 represents a plausible mechanism by which miR-30 miRNAs may contribute to adipogenic differentiation of adipose tissue-derived stem cells.

Extensive sequencing efforts, combined with ad hoc bioinformatics developments, have now led to the identification of 1222 distinct miRNAs in human (derived from 1368 distinct genomic loci) and of many miRNAs in other multicellular organisms. The present chapter is aimed at describing a general experimental strategy to identify specific miRNA expression profiles and to highlight the functional networks operating between them and their mRNA targets, including several miRNAs deregulated in cystic fibrosis and during differentiation of airway epithelial cells.

The colon and lung amiloride-binding proteins were cloned from rat tissues. Two sizes of transcripts were identified. The 2.7-kb transcript codes for an 85-kDa protein, whereas the 1.2-kb transcript codes for a 25-kDa polypeptide. The 2.7-kb transcript was detected in the proximal and distal colon and in duodenum, liver, placenta and thymus. The 1.2-kb transcript was the only form present in lung and spleen, and it was also detected in placenta and colon. The short form corresponds to the 3' terminus of the longer one. It is formed by alternative transcription under the control of an internal promoter. Cells stably transfected with cDNAs encoding these two proteins were used for binding studies using [3H]phenamil, a potent blocker of the epithelial Na+ channel, derived from amiloride. Both the long and short forms of the protein bind amiloride and some of its derivatives, but they have distinct pharmacologies. The order of potency of the different amiloride derivatives to inhibit [3H]phenamil binding was phenamil (K0.5 = 10 nM) > benzamil (K0.5 = 43 nM) > amiloride (K0.5 = 1.4 microM) approximately ethylisopropylamiloride (K0.5 = 1.6 microM) for the long form, whereas it was phenamil (K0.5 = 68 nM) > amiloride (K0.5 = 3.2 microM) approximately ethylisopropylamiloride (K0.5 = 4 microM) approximately benzamil (K0.5 = 6.3 microM) for the short form. Although the binding proteins described here are distinct from the pore-forming protein of the epithelial Na+ channel, the pharmacological profile of the long form of the ABP is identical to that described previously in pig and human kidney, and similar to that expected for an epithelial Na+ channel. The pharmacological profile of the short form resembles that previously described for an amiloride-binding protein in pneumocytes. Results presented in this paper suggest that previously purified preparations showing Na+ channel activity contain different forms of the amiloride-binding protein, possibly associated with other proteins. The similarity between amiloride-binding proteins and a protein identified in seminal vesicles suggests that amiloride-binding proteins are the first members of a new family of epithelia-specific proteins.

A complementary DNA encoding an amiloride-sensitive Na+ channel has been cloned and characterized from rat colon. The protein encoded by the cDNA has a sequence of 699 amino acids (79 kDa) containing several putative membrane spanning domains and potential phosphorylation sites. It forms a channel that has the electrophysiological and pharmacological properties characteristic of the epithelial Na+ channel. Homologies (including in transmembrane domains) have been found between a part of the channel sequence and the Mec4 gene product of Caenorhabditis elegans, a protein associated with mutation-induced neuronal degeneration.

ABCC4 is a member of the ATP-binding cassette transporter family known to transport prostaglandin E2 and other molecules across cellular membranes. A mutation in ABCC4 is now shown to cause defects in ciliogenesis, revealing a role for prostaglandin signalling in regulating cilia dynamics.

International audience; Following the identification of a set of hypoxia-regulated microRNAs (miRNAs), recent studies have highlighted the importance of miR-210 and of its transcriptional regulation by the transcription factor hypoxia-inducible factor-1 (HIF-1). We report here that miR-210 is overexpressed at late stages of non-small cell lung cancer. Expression of miR-210 in lung adenocarcinoma A549 cells caused an alteration of cell viability associated with induction of caspase-3/7 activity. miR-210 induced a loss of mitochondrial membrane potential and the apparition of an aberrant mitochondrial phenotype. The expression profiling of cells overexpressing miR-210 revealed a specific signature characterized by enrichment for transcripts related to 'cell death' and 'mitochondrial dysfunction', including several subunits of the electron transport chain (ETC) complexes I and II. The transcript coding for one of these ETC components, SDHD, subunit D of succinate dehydrogenase complex (SDH), was validated as a bona fide miR-210 target. Moreover, SDHD knockdown mimicked miR-210-mediated mitochondrial alterations. Finally, miR-210-dependent targeting of SDHD was able to activate HIF-1, in line with previous studies linking loss-of-function SDH mutations to HIF-1 activation. miR-210 can thus regulate mitochondrial function by targeting key ETC component genes with important consequences on cell metabolism, survival and modulation of HIF-1 activity. These observations help explain contradictory data regarding miR-210 expression and its putative function in solid tumors.Cell Death and Differentiation advance online publication, 1 October 2010; doi:10.1038/cdd.2010.119.

International audience; Cancer cells frequently express genes normally active in male germ cells. ATAD2 is one of them encoding a conserved factor harbouring an AAA type ATPase domain and a bromodomain. We show here that ATAD2 is highly expressed in testis as well as in many cancers of different origins and that its high expression is a strong predictor of rapid mortality in lung and breast cancers. These observations suggest that ATAD2 acts on upstream and basic cellular processes to enhance oncogenesis in a variety of unrelated cell types. Accordingly, our functional studies show that ATAD2 controls chromatin dynamics, genome transcriptional activities and apoptotic cell response. We could also highlight some of the important intrinsic properties of its two regulatory domains, including a functional cross-talk between the AAA ATPase domain and the bromodomain. Altogether, these data indicate that ATAD2 overexpression in somatic cells, by acting on basic properties of chromatin, may contribute to malignant transformation.

International audience; The prevalence of obesity has steadily increased over the last few decades. During this time, populations of industrialized countries have been exposed to diets rich in fat with a high content of linoleic acid and a low content of alpha-linolenic acid compared to recommended intake. To assess the contribution of dietary fatty acids, male and female mice fed a high-fat diet (35% energy as fat, linoleic acid/alpha-linolenic acid ratio of 28) were mated randomly and maintained after breeding on the same diet for successive generations. Offspring showed, over four generations, a gradual enhancement in fat mass due to combined hyperplasia and hypertrophy with no change in food intake. Transgenerational alterations in adipokine levels were accompanied by hyperinsulinemia. Gene expression analyses of the stromal vascular fraction of adipose tissue, over generations, revealed discrete and steady changes in certain important players such as CSF3, Nocturnin and sFRP4. Thus, under conditions of genome stability and with no change in the regimen over four generations, we show that a Western-like fat diet induces a gradual fat mass enhancement, in accordance with the increasing prevalence of obesity observed in humans.

International audience; Half of the human genome consists of repetitive DNA sequences. Recent studies in various organisms highlight the role of chromatin regulation of repetitive DNA in gene regulation as well as in maintainance of chromosomes and genome integrity. Hence, repetitive DNA sequences might be potential "sensors" for chromatin changes associated with pathogenesis. Therefore, we developed a new genomic tool called RepArray. RepArray is a repeat-specific microarray composed of a representative set of human repeated sequences including transposon-derived repeats, simple sequences repeats, tandemly repeated sequences such as centromeres and telomeres. We showed that combined to anti-methylcytosine immunoprecipitation assay, the RepArray can be used to generate repeat-specific methylation maps. Using cell lines impaired chemically or genetically for DNA methyltransferases activities, we were able to distinguish different epigenomes demonstrating that repeats can be used as markers of genome-wide methylation changes. Besides, using a well-documented system model, the thermal stress, we demonstrated that RepArray is also a fast and reliable tool to obtain an overview of overall transcriptional activity on whole repetitive compartment in a given cell type. Thus, the RepArray represents the first valuable tool for systematic and genome-wide analyses of the methylation and transcriptional status of the repetitive counterpart of the human genome.

Imatinib is used to treat chronic myelogenous leukemia (CML), but resistance develops in all phases of this disease. The purpose of the present study was to identify the mode of resistance of newly derived imatinib-resistant (IM-R) and PD166326-resistant (PD-R) CML cells. IM-R and PD-R clones exhibited an increase in viability and a decrease in caspase activation in response to various doses of imatinib and PD166326, respectively, as compared with parental K562 cells. Resistance involved neither mutations in BCR-ABL nor increased BCR-ABL, MDR1 or Lyn expression, all known modes of resistance. To gain insight into the resistance mechanisms, we used pangenomic microarrays and identified 281 genes modulated in parental versus IM-R and PD-R cells. The gene signature was similar for IM-R and PD-R cells, accordingly with the cross-sensitivity observed for both inhibitors. These genes were functionally associated with pathways linked to development, cell adhesion, cell growth, and the JAK-STAT cascade. Especially relevant were the increased expression of the tyrosine kinases AXL and Fyn as well as CD44 and HMGA2. Small interfering RNA experiments and pharmacologic approaches identified FYN as a candidate for resistance to imatinib. Our findings provide a comprehensive picture of the transcriptional events associated with imatinib and PD166326 resistance and identify Fyn as a new potential target for therapeutic intervention in CML. [Mol Cancer Ther 2009;8(7):1924–33]

Dax-1 (Nr0b1) is an orphan member of the nuclear hormone receptor superfamily that has a key role in adrenogonadal development and function. Recent studies have also implicated Dax-1 in the transcriptional network controlling embryonic stem (ES) cell pluripotency. Here, we show that Dax-1 expression is affected by differentiating treatments and pharmacological activation of β-catenin–dependent transcription in mouse ES cells. Furthermore, Dax-1 knockdown induced upregulation of multilineage differentiation markers, and produced enhanced differentiation and defects in ES viability and proliferation. Through RNA interference and transcriptome analysis, we have identified genes regulated by Dax-1 in mouse ES cells at 24 and 48 hours after knockdown. Strikingly, the great majority of these genes are upregulated, showing that the prevalent function of Dax-1 is to act as a transcriptional repressor in mouse ES cells, as confirmed by experiments using the Gal4 system. Genes involved in tissue differentiation and control of proliferation are significantly enriched among Dax-1–regulated transcripts. These data show that Dax-1 is an essential element in the molecular circuit involved in the maintenance of ES cell pluripotency and have implications for the understanding of stem cell function in both physiological (adrenal gland) and clinical (Ewing tumors) settings where Dax-1 plays a pivotal role in development and pathogenesis, respectively. Disclosure of potential conflicts of interest is found at the end of this article.

International audience

Background Whole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER), the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood. Results We successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences) on the model Rickettsiales ER to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of ER's cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome ER microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from ER microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R2 = 0.7). Moreover, SCOTS method is crucial for microarrays analysis of ER, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively. Conclusions We conclude that this SCOTS method has a key importance for the transcriptomic analysis of ER and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both ER pathogenicity and the adaptation of obligate intracellular bacteria to their environment.

International audience; MicroRNAs (miRNAs) are small non-protein-coding RNA that negatively control mRNA expression at a post-transcriptional level. They regulate various cellular functions and bioinformatic data suggest that they collectively control about 30% of human mRNAs. MiRNAs have been recently implicated in several carcinogenic processes, where they can act either as oncogenes or as tumor suppressors. This is the case in lung cancer, i.e. the leading cause of cancer deaths in Western countries, in which about 40-45 miRNAs have been found to be aberrantly expressed, thereby constituting a specific miRNA signature. Some of these miRNAs can play an important role in lung carcinogenesis. Indeed, some transcripts of the let-7 family that are significantly down-regulated in lung tumors have been identified as tumor suppressors through their ability to control several oncogenic pathways, including the RAS pathway. Identification of a growing number of other potential oncogenic or tumor suppressor miRNAs in lung cancers is in constant progress. Recent evidence supports the use of specific miRNA signatures to predict clinical outcome. This review aims to report the current knowledge about the role of miRNAs in lung cancer carcinogenesis, their potential for improving diagnosis and prognosis and their impact on future therapeutic strategies.

Intestinal epithelial cells were isolated from a fetus with cystic fibrosis (CF) and transfected with a plasmid vector recombined with the ori- mutant of SV40. A population of proliferative cells was then subcloned and designated as CFI-3. These cells had a doubling time of 24 h and were maintained in culture for up to 25 passages. At passage 8, CFI-3 cells did not produce any tumors in nude mice. Northern blot and immunofluorescence studies indicated that the extended lifespan of CFI-3 cells results in genomic insertion of SV40 LT. Intestinal CFI-3 cells are epithelial, according to the expression of the human cytokeratin 18 gene and poorly differentiated by phase-contrast and electron microscopy. Functional membrane receptors activated by vasoactive intestinal peptide (VIP), its natural analogue pituitary adenylate cyclase activating peptide (PACAP-38), and isoproterenol were observed in CFI-3 cells. Restriction fragment length polymorphism analysis of the PstI KM19 site revealed that the cftr locus was identical in the chorionic villi and in CFI-3 cells. The manifestation of CF in this family was not related to the common mutation delta F508, since this fetus was heterozygous for the substitutions S549N and N1303K. Chloride transport, assessed by the 125I efflux, was induced in CFI-3 cells by the calcium inophore ionomycin, but not by the adenylate cyclase activator forskolin, and was inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid. These results were confirmed in patch clamp studies in which the cpt cAMP analogue failed to stimulate membrane currents, while the calcium ionophore ionomycin stimulated inward currents. We conclude that intestinal CFI-3 cells retain the CF phenotype relating to defective regulation of Cl- channels, and therefore constitute a suitable model, 1) for elucidating the function of CFTR protein, 2) developing new therapeutic agents, and 3) correcting the CF defect by gene replacement therapy in vitro.

Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating sheep, goats and cows. The present study was carried out to compare 65 S. aureus isolates mainly obtained from nasal carriage and subclinical mastitis in dairy sheep and 43 isolates obtained from subclinical mastitis from 22 goats and 21 cows. A DNA microarray, containing probes against 190 true or putative virulence factors, was used to detect the presence of the virulence genes. Their presence/absence was independently assessed by PCR for the genes of interest. Sheep isolates obtained from the nostrils or the udders did not show any significant tissue specific virulence factor. The dominant pulse-field electrophoresis profile (OV/OV'), associated with spa clonal complex spa-CC 1773, matched mainly with the agr group III and was only found in ovine and caprine isolates. This clone was more specifically characterized by the prevalence of the following virulence genes: lpl4, ssl6, bsaA1, bsaB, bsaP, SAV0812. Moreover, seven virulence-associated genes (lpl1, sel, sec, tst, lukF-PV-like component, lukM, SAV0876) were associated with isolates from small ruminants, while the egc cluster, fhuD1, abiF and SAV2496 with bovine isolates. This genomic study suggests the existence of lineage- and host-specific genes leading to the development of host-specific pathogenic traits of S. aureus isolates.

Liver ischemia-reperfusion injury occurring in orthotopic liver transplantation (OLT) may be responsible for early graft failure. Molecular mechanisms underlying initial poor graft function (IPGF) have been poorly documented in human. The purpose of this study was to identify the major transcriptional alterations occurring in human livers during OLT. Twenty-one RNA extracts derived from liver transplant biopsies taken after graft reperfusion were compared with 7 RNA derived from normal control livers. Three hundred seventy-one genes were significantly modulated and classified in molecular pathways relevant to liver metabolism, inflammatory response, cell proliferation and liver protection. Grafts were then subdivided into two groups based on their peak levels of serum aspartate amino transferase within 72 h after OLT (group 1, non-IPGF: 14 patients; group 2, IPGF: 7 patients). The two corresponding data sets were compared using a supervised prediction method. A new set of genes able to correctly classify 71% of the patients was defined. These genes were functionally associated with oxidative stress, inflammation and inhibition of cell proliferation. This study provides a comprehensive picture of the transcriptional events associated with human OLT and IPGF. We anticipate that such alterations provide a framework for the elucidation of the molecular mechanisms leading to IPGF.

BACKGROUND: Hypertension and chronic renal failure (CRF) are considered models of accelerated arterial stiffening. Arterial stiffness increases further when CRF is associated with hypertension. We hypothesized that, in patients with mild CRF, aortic gene expression profile would include genes involved in arterial calcifications and enlargement. METHOD: We analysed human aorta with the 'GeneChip Microarray' technology, in patients with or without CRF, scheduled for a coronary artery bypass graft. RESULTS: Nine of 25 patients had high-quality RNA and were included in the study. Among the 101 transcripts differentially expressed between CRF patients and controls, 97 transcripts were overexpressed in CRF patients. Two genes had the highest overexpression in CRF patients: lumican (LUM), involved in the regulation of collagen fibrillogenesis; and ornithine decarboxylase (ODC1), involved in polyamine biosynthesis, smooth muscle cell growth and proliferation. Immunohistochemical staining revealed an increased amount of LUM and ODC1 in the vascular smooth muscle cells (VSMCs) of CRF compared to non-CRF aortic sections. Eight genes were implicated in the regulation of the cytoskeleton (including capping protein muscle Z-line 1 alpha and moesin) and cell migration, and five genes were implicated in extracellular matrix function and apoptosis. A trend towards an upregulation of candidate genes involved in arterial calcifications was observed in CRF patients, but did not reach statistical significance. Carotid-femoral pulse wave velocity was not correlated with gene expression level. CONCLUSION: In conclusion, these results show that patients at an early stage of CRF have a specific gene expression profile of aortic tissue and suggest that genes implicated in collagen fibrillogenesis, and VSMCs migration and proliferation, particularly LUM and ODC1, may play a role.

Matrix metalloproteinases (MMPs) have been implicated in the hepatic injury induced after cold ischemia-warm reperfusion (CI-WR), by altering the extracellular matrix (ECM), but their precise role remains unknown. The hepatic MMP expression was evaluated after 2 conditions of CI (4 degrees C for 24 and 42 hours: viable and nonviable livers) followed by different periods of WR, using isolated perfused rat livers. CI-WR induced moderate changes in hepatic MMP transcript levels not influenced by CI duration, whereas gelatinase activities accumulated in liver effluents. Therefore, the protective effect of a new phosphinic MMP inhibitor, RXP409, was tested after prolonged CI. RXP409 (10 microM) was added to the University of Wisconsin solution, and livers were preserved for 42 hours (4 degrees C), then reperfused for 1 hour in Krebs solution (37 degrees C), containing 20% erythrocytes. Liver viability parameters were recorded, and the extent of cell necrosis was evaluated on liver biopsies, using trypan blue nuclear uptake. Treatment with RXP409 significantly improved liver function (transaminase release and bile secretion) and liver injury. In particular, the MMP inhibitor significantly modified the extent of cell death from large clusters of necrotic hepatocytes as found in control livers (2%-60% of liver biopsies; mean, 26% +/- 9%) to isolated necrotic hepatocytes as found in treated livers (0.2%-12%; mean, 3% +/- 2%) (P < 0.05). CONCLUSION: These data demonstrate that MMPs, by altering the ECM, play a major role in liver CI-WR injury leading to extensive hepatocyte necrosis and that their inhibition might prove to be a new strategy in improving preservation solutions.

Phenamil, an analog of amiloride, is a potent blocker of the epithelial Na+ channel. It has been used to purify the porcine kidney amiloride-binding protein. Synthetic oligonucleotides derived from partial sequences have been used to screen a human kidney cDNA library and to isolate the cDNA encoding the human amiloride-binding protein. The primary structure was deduced from the DNA sequence analysis. The protein is 713 residues long, with a 19-amino acid signal peptide. The mRNA was expressed in 293-S and NIH 3T3 cells, yielding a glycoprotein (i) that binds amiloride and amiloride analogs with affinities similar to the amiloride receptor associated with the apical Na+ channel in pig kidney membranes and (ii) that is immunoprecipitated with monoclonal antibodies raised against pig kidney amiloride-binding protein.

This paper describes a large-scale purification procedure of the amiloride binding component of the epithelium Na+ channel. [3H]Phenamil was used as a labeled ligand to follow the purification. The first two steps are identical with those previously described [Barbry, P., Chassande, O., Vigne, P., Frelin, C., Ellory, C., Cragoe, E. J., Jr.,Lazdunski, M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4836-4840]. A third step was a hydroxyapatite column. The purified material consisted of a homodimer of two 88-kDa proteins that migrated anomalously in SDS-PAGE to give an apparent Mr of 105,000. Deglycosylation by treatment with neuraminidase and endoglycosidase F or with neuraminidase and glycopeptidase F indicated that less than 5% of the mass of the native receptor was carbohydrate. Sedimentation analysis of the purified Na+ channel in H2O and D2O sucrose gradients and gel filtration experiments led to an estimated molecular weight of the [3H]phenamil receptor protein-detergent-phospholipid complex of 288,000 and of the native [3H]phenamil receptor protein of 158,000. [3H]Br-benzamil is another labeled derivative of amiloride that recognized binding sites that had the same pharmacological properties as [3H]phenamil binding sites and that copurified with them. Upon irradiation of kidney membranes, [3H]Br-benzamil incorporated specifically into a 185-kDa polypeptide chain under nonreducing electrophoretic conditions and a 105-kDa protein under reducing conditions. The same labeling pattern was observed at the different steps of the purification. Reconstitution of the purified phenamil receptor into large unilamellar vesicles was carried out. A low but significant phenamil- and amiloride-sensitive electrogenic Na+ transport was observed.

International audience; The study of protein function mostly relies on perturbing regulatory networks by acting upon protein expression levels or using transdominant negative agents. Here we used the Escherichia coli global transcription regulator Fur (ferric uptake regulator) as a case study to compare the perturbations exerted by a gene knockout , the expression of a dominant negative allele of a gene, and the expression of peptide aptamers that bind a gene product. These three perturbations caused phenotypes that differed quantitatively and qualitatively from one another. The Fur peptide aptamers inhibited the activity of their target to various extents and reduced the virulence of a pathogenic E. coli strain in Drosophila. A genome-wide transcriptome analysis revealed that the "penetrance" of a peptide aptamer was comparable to that of a dominant negative allele but lower than the penetrance of the gene knockout. Our work shows that comparative analysis of phenotypic and transcriptome responses to different types of perturbation can help decipher complex regulatory networks that control various biological processes.

The Rickettsia genus is composed of Gram-negative bacteria responsible for Typhus and spotted fevers. Because of the limitations imposed by their obligate intracellular location, the molecular mechanisms responsible for their pathogenicity remain poorly understood. Several rickettsial genomes are now available, thus providing the foundation for a new era of post-genomic research. Here, using Rickettsia conorii as model, we developed a suitable method for microarray-based transcriptome analysis of rickettsiae. Total RNA was extracted from infected Vero cells using a protocol preserving its integrity, as observed by Bioanalyzer (Agilent) profiles. By a subtractive hybridization method, the samples were subsequently depleted of eukaryotic RNA that represents up to 90% of the whole extract and that hampers fluorochrome labeling of rickettsial nucleic acids. To obtain the amount of material required for microarray hybridization, the bacterial RNA was then amplified using random primers. Hybridizations were carried out on microarrays specific for R. conorii but containing a limited number of selected targets. Our results show that this method yielded reproducible signals. Transcriptional changes observed following exposure of R. conorii to a nutrient stress were verified by real-time quantitative PCR and by quantitative reverse transcription PCR starting from amplified cDNA and total RNA as templates, respectively. We conclude that this approach has great potential for the study of mechanisms behind the virulence and intracellular survival of members of the genus Rickettsia.