Using single-cell RNA sequencing to unravel cell lineage relationships in the respiratory tract.

The respiratory tract is lined by a pseudo-stratified epithelium from the nose to terminal bronchioles. This first line of defense of the lung against external stress includes five main cell types: basal, suprabasal, club, goblet and multiciliated cells, as well as rare cells such as ionocytes, neuroendocrine and tuft/brush cells. At homeostasis, this epithelium self-renews at low rate but is able of fast regeneration upon damage. Airway epithelial cell lineages during regeneration have been investigated in the mouse by genetic labeling, mainly after injuring the epithelium with noxious agents. From these approaches, basal cells have been identified as progenitors of club, goblet and multiciliated cells, but also of ionocytes and neuroendocrine cells. Single-cell RNA sequencing, coupled to lineage inference algorithms, has independently allowed the establishment of comprehensive pictures of cell lineage relationships in both mouse and human. In line with genetic tracing experiments in mouse trachea, studies using single-cell RNA sequencing (RNAseq) have shown that basal cells first differentiate into club cells, which in turn mature into goblet cells or differentiate into multiciliated cells. In the human airway epithelium, single-cell RNAseq has identified novel intermediate populations such as deuterosomal cells, 'hybrid' mucous-multiciliated cells and progenitors of rare cells. Novel differentiation dynamics, such as a transition from goblet to multiciliated cells have also been discovered. The future of cell lineage relationships in the respiratory tract now resides in the combination of genetic labeling approaches with single-cell RNAseq to establish, in a definitive manner, the hallmarks of cellular lineages in normal and pathological situations.

Genetic differences among Staphylococcus aureus isolates from dairy ruminant species: a single-dye DNA microarray approach.

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.

Gene expression profiling of human liver transplants identifies an early transcriptional signature associated with initial poor graft function.

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.

Copy-number analysis identified new prognostic marker in acute myeloid leukemia.

Recent advances in genomic technologies have revolutionized acute myeloid leukemia (AML) understanding by identifying potential novel actionable genomic alterations. Consequently, current risk stratification at diagnosis not only relies on cytogenetics, but also on the inclusion of several of these abnormalities. Despite this progress, AML remains a heterogeneous and complex malignancy with variable response to current therapy. Although copy-number alterations (CNAs) are accepted prognostic markers in cancers, large-scale genomic studies aiming at identifying specific prognostic CNA-based markers in AML are still lacking. Using 367 AML, we identified four recurrent CNA on chromosomes 11 and 21 that predicted outcome even after adjusting for standard prognostic risk factors and potentially delineated two new subclasses of AML with poor prognosis. ERG amplification, the most frequent CNA, was related to cytarabine resistance, a cornerstone drug of AML therapy. These findings were further validated in The Cancer Genome Atlas data. Our results demonstrate that specific CNA are of independent prognostic relevance, and provide new molecular information into the genomic basis of AML and cytarabine response. Finally, these CNA identified two potential novel risk groups of AML, which when confirmed prospectively, may improve the clinical risk stratification and potentially the AML outcome.

Novel role for CFTR in fluid absorption from the distal airspaces of the lung.

The active absorption of fluid from the airspaces of the lung is important for the resolution of clinical pulmonary edema. Although ENaC channels provide a major route for Na(+) absorption, the route of Cl(-) transport has been unclear. We applied a series of complementary approaches to define the role of Cl(-) transport in fluid clearance in the distal airspaces of the intact mouse lung, using wild-type and cystic fibrosis Delta F508 mice. Initial studies in wild-type mice showed marked inhibition of fluid clearance by Cl(-) channel inhibitors and Cl(-) ion substitution, providing evidence for a transcellular route for Cl(-) transport. In response to cAMP stimulation by isoproterenol, clearance was inhibited by the CFTR inhibitor glibenclamide in both wild-type mice and the normal human lung. Although isoproterenol markedly increased fluid absorption in wild-type mice, there was no effect in Delta F508 mice. Radioisotopic clearance studies done at 23 degrees C (to block active fluid absorption) showed approximately 20% clearance of (22)Na in 30 min both without and with isoproterenol. However, the clearance of (36)Cl was increased by 47% by isoproterenol in wild-type mice but was not changed in Delta F508 mice, providing independent evidence for involvement of CFTR in cAMP-stimulated Cl(-) transport. Further, CFTR played a major role in fluid clearance in a mouse model of acute volume-overload pulmonary edema. After infusion of saline (40% body weight), the lung wet-to-dry weight ratio increased by 28% in wild-type versus 64% in Delta F508 mice. These results provide direct evidence for a functionally important role for CFTR in the distal airspaces of the lung.

Genetic analysis of the beta subunit of the epithelial Na+ channel in essential hypertension.

Mutations of the last exon of the beta subunit of the amiloride-sensitive epithelial Na+ channel (betaENaC) can lead to Liddle's syndrome, a rare monogenic form of hypertension. The objective of this study was to test whether more subtle changes of betaENaC could be implicated in essential hypertension. After determination of the betaENaC coding gene organization (12 exons spanning 23.5 kb), a systematic screening of the last exon of the gene was performed in 525 subjects (475 whites, 50 Afro-Caribbeans), all probands of hypertensive families. This search was extended to the remaining 11 exons in a subset of 101 probands with low-renin hypertension. Seven amino acid changes were detected: G589S, T594M, R597H, R624C, E632G (last exon), G442V, and V434M (exon 8). These genetic variants were more frequent in subjects of African origin (44%) than in whites (1%). The functional properties of the variants were analyzed in Xenopus oocytes by two independent techniques, ie, electrophysiology and 22Na+ uptake. Small but not significant differences were observed between the variants and wild type. The clinical evaluation of the family bearing the G589S variant, which provided the highest relative ENaC activity, did not show a cosegregation between the mutation and hypertension. The present study illustrates the difficulty in establishing a relation of causality between a susceptibility gene and hypertension. Furthermore, it does not favor a substantial role of the betaENaC gene in essential hypertension.

Expression cloning of an epithelial amiloride-sensitive Na+ channel. A new channel type with homologies to Caenorhabditis elegans degenerins.

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.

Amiloride and its analogs as tools to inhibit Na+ transport via the Na+ channel, the Na+/H+ antiport and the Na+/Ca2+ exchanger.

Amiloride analogs inhibit a number of transmembrane Na+ transport systems: 1) the epithelium Na+ channel, 2) the Na+/H+ exchange system and 3) the Na+/Ca2+ exchange system. Structure--activity relationships using amiloride derivatives with selected modification of each of the functional groups of the molecule indicate that the 3 Na+ transporting systems have distinct pharmacological profiles. 5-N Disubstituted derivatives of amiloride, such as ethylisopropylamiloride are the most potent inhibitors of the Na+/H+ exchange system. Conversely, amiloride derivatives that are substituted on the guanidino moiety, such as phenamil, are potent inhibitors of the epithelium Na+ channel. It is thus possible, by using selected amiloride derivatives to inhibit selectively one or another of the Na+ transport systems.

The Na+/H+ antiport of eukaryotic cells: relationship between the kinetic properties of the system and its physiological function.

The Na+/H+ antiport is present in the plasma membrane of virtually all vertebrate cells and it plays a central role in cell homeostasis. The pharmacological properties and the characteristics of the interaction of extracellular Na+, Li+, H+ and of intracellular H+ with the Na+/H+ antiport are reviewed herein. The kinetic properties of the system are shown to be essential for defining its four main physiological functions: transepithelial ion transport, control of the pHi, control of the intracellular Na+ concentration, and control of the cell volume. The activity of the Na+/H+ antiport can be modulated by a large number of effectors which are thought to act via protein kinases. At least three mechanisms of activation of the Na+/H+ exchanger are defined from the analysis of the kinetic properties of the system. Activation of the Na+/H+ antiport leads to very different consequences, depending upon the activity of other ion transporting systems in the membrane.

Genotype-phenotype analysis of a newly discovered family with Liddle's syndrome.

OBJECTIVE: To investigate the clinical, biologic, and molecular abnormalities in a family with Liddle's syndrome and analyze the short- and long-term efficacies of amiloride treatment. PATIENTS: The pedigree consisted of one affected mother and four children, of whom three suffered from early-onset and moderate-to-severe hypertension. METHODS: In addition to the biochemical and hormonal measurements, genetic analysis of the carboxy terminus of the beta subunit of the epithelial sodium channel (beta ENaC) was conducted through single-strand conformation analysis and direct sequencing. The functional properties of the mutation were analyzed using the Xenopus expression system and compared with one mutation affecting the proline-rich sequence of the beta ENaC. RESULTS: Mild hypokalemia and suppressed levels of plasma renin and aldosterone were observed in all affected subjects. Administration of 10 mg/day amiloride for 2 months normalized the blood pressure and plasma potassium levels of all of the affected subjects, whereas their plasma and urinary aldosterone levels remained surprisingly low. A similar pattern was observed after 11 years of follow-up, but a fivefold increase in plasma aldosterone was observed under treatment with 20 mg/day amiloride for 2 weeks. Genetic analysis of the beta ENaC revealed a deletion of 32 nucleotides that had modified the open reading frame and introduced a stop codon at position 582. Expression of this beta 579del32 mutant caused a 3.7 +/- 0.3-fold increase in the amiloride-sensitive sodium current, without modification of the unitary properties of the channel. A similar increase was elicited by one mutation affecting the carboxy terminus of the beta ENaC. CONCLUSIONS: This new mutation leading to Liddle's syndrome highlights the importance of the carboxy terminus of the beta ENaC in the activity of the epithelial sodium channel. Small doses of amiloride are able to control the blood pressure on a long-term basis in this monogenic form of hypertension.

Polymorphisms of the gamma subunit of the epithelial Na+ channel in essential hypertension.

OBJECTIVE: The gamma subunit of the epithelial Na channel (gammaENaC) has been implicated in Liddle's syndrome. The objective of this study was to examine its status in essential hypertension. DESIGN AND METHODS: The search for molecular variants was performed using the SSCP technique after determination of the intron-exon boundaries of the transcribed sequence. We found an additional 205 bp intron splitting the published exon 10 in two. The last exon of gammaENaC was tested with samples from a series of 245 normotensive patients and 453 hypertensive subjects (383 Caucasians, 70 Afro-Caribbeans), all probands of hypertensive families in the HYPERGENE data set. The search was extended to the other 11 transcribed exons in a subset of 65 patients with low-renin profile. RESULTS: Four neutral polymorphisms were detected, three in the third exon of the gene (T387C, T474C, C549T) and one in the last exon (C1990G). These four variants were found with similar frequencies in hypertensive and normotensive Caucasian subjects as well as in patients with low-renin profile. Hypertensive Caucasians and hypertensive subjects of African ancestry also had similar frequencies. Lastly, we found two rare mutations, one the insertion of a proline residue at position 594 of the mature protein (594insP), the other an Arg-to-His substitution at position 631 (R631H). Compared to wild-type (1.00 +/- 0.42, n = 26), expression of the 594insP (1.10 +/- 0.43, n = 26) and R631H (0.97 +/- 0.43, n = 26) variants in Xenopus oocytes produced no significant increase in Na+ current. CONCLUSIONS: Screening of the entire coding sequence of gammaENaC does not suggest that this subunit is frequently involved in essential hypertension.

Differential expression of RNA and protein of the three pore-forming subunits of the amiloride-sensitive epithelial sodium channel in taste buds of the rat.

Salt taste signals from the rat anterior tongue are probably transduced via epithelial sodium channels (ENaCs) residing in the apical cellular pole of taste cells. The signals are blocked by mucosal amiloride in low microM concentrations. In contrast, the rat vallate papilla does not contribute to amiloride-blockable salt taste. Two approaches were used to probe for the three subunits of ENaC in the anterior and posterior tongue of the rats in sodium balance. (a) Immunohistochemistry with antibodies against ENaC subunits and against amiloride binding sites. In the anterior tongue, reactivity for alpha-, beta-, and gamma-subunits was present in taste buds and lingual epithelium. In the posterior tongue vallate papilla, reactivity for alpha-subunit and for amiloride binding sites was easily demonstrable, whereas that for beta-subunit and especially for gamma-subunit was weaker than in the anterior tongue. (b) RT-PCR techniques were used to probe for the presence of ENaC subunit mRNA. In isolated taste buds of the anterior tongue, mRNA of all three subunits was found, whereas in isolated taste buds of the vallate papilla only mRNA of the alpha-subunit was easily detectable. That of beta- and gamma-subunits was much less abundant. RNA of all three subunits was abundant only in taste buds of the anterior tongue. Therefore, subsets of elongated taste cells do express ENaC, but regional differences exist in the transcription and expression of subunits. The regional differences suggest that amiloride-sensitive salt taste, which requires all three subunits, is present in the anterior but not the posterior tongue of rats, as functional studies indicate.

Occurrence of ENaC subunit mRNA and immunocytochemistry of the channel subunits in taste buds of the rat vallate papilla.

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.

CDC25A targeting by miR-483-3p decreases CCND-CDK4/6 assembly and contributes to cell cycle arrest.

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.

MiR-210 promotes a hypoxic phenotype and increases radioresistance in human lung cancer cell lines.

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.

miR-210 is overexpressed in late stages of lung cancer and mediates mitochondrial alterations associated with modulation of HIF-1 activity.

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.

Functional characterization of ATAD2 as a new cancer/testis factor and a predictor of poor prognosis in breast and lung cancers.

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.

CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR-ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain.