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During the last decades, the development of ever more powerful genetic, molecular and omic approaches has provided plant pathologists with a wide array of experimental tools for elucidating the intricacies of plant-pathogen interactions and proposing new control strategies. In the case of the Dutch elm disease (DED) pathosystem, these tools have been applied for advancing knowledge of the host (Ulmus spp.) and the causal agents (Ophiostoma ulmi, O. novo-ulmi and O. himal-ulmi). Genetic and molecular analyses have led to the identification, cloning and characterization of a few genes that contribute to parasitic fitness in the pathogens. Quantitative PCR and high-throughput methods, such as expressed sequence tag analysis, have been used for measuring gene expression and identifying subsets of elm genes that are differentially expressed in the presence of O. novo-ulmi. These analyses have also helped identify genes that were differentially expressed in DED fungi grown under defined experimental conditions. Until recently, however, functional analysis of the DED fungi was hampered by the lack of protocols for efficient gene knockout and by the unavailability of a full genome sequence. While the selective inactivation of Ophiostoma genes by insertional mutagenesis remains a challenge, an alternative approach based on RNA interference is now available for down-regulating the expression of targeted genes. In 2013, the genome sequences of O. ulmi and O. novo-ulmi were publicly released. The ongoing annotation of these genomes should spark a new wave of interest in the DED pathosystem, as it should lead to the formal identification of genes modulating parasitic fitness. A better understanding of DED, however, also requires that omic approaches are applied to the study of the other biotic components of this pathosystem.

Although cystic fibrosis (CF) pathophysiology is explained by a defect in CF transmembrane conductance regulator (CFTR) protein, the broad spectrum of disease severity is the consequence of environmental and genetic factors. Among them, oxidative stress has been demonstrated to play an important role in the evolution of this disease, with susceptibility to oxidative damage, decline of pulmonary function, and impaired lung antioxidant defense. Although oxidative stress has been implicated in the regulation of inflammation, its molecular outcomes in CF cells remain to be evaluated. To address the question, we compared the gene expression profile in NuLi-1 cells with wild-type CFTR and CuFi-1 cells homozygous for ΔF508 mutation cultured at air-liquid interface. We analyzed the transcriptomic response of these cell lines with microarray technology, under basal culture conditions and after 24 h oxidative stress induced by 15 μM 2,3-dimethoxy-1,4-naphtoquinone. In the absence of oxidative conditions, CuFi-1 gene profiling showed typical dysregulated inflammatory responses compared with NuLi-1. In the presence of oxidative conditions, the transcriptome of CuFi-1 cells reflected apoptotic transcript modulation. These results were confirmed in the CFBE41o- and corrCFBE41o- cell lines as well as in primary culture of human CF airway epithelial cells. Altogether, our data point to the influence of oxidative stress on cell survival functions in CF and identify several genes that could be implicated in the inflammation response observed in CF patients.

Nitric oxide (NO) plays an important role in innate host defense and inflammation. In response to infection, NO is generated by inducible nitric oxide synthase (iNOS), a gene product whose expression is highly modulated by different stimuli, including lipopolysaccharide (LPS) from gram-negative bacteria. We reported recently that LPS from Pseudomonas aeruginosa altered Na+transport in alveolar epithelial cells via a suramin-dependent process, indicating that LPS activated a purinergic response in these cells. To further study this question, in the present work, we tested whether iNOS mRNA and protein expression were modulated in response to LPS in alveolar epithelial cells. We found that LPS induced a 12-fold increase in iNOS mRNA expression via a transcription-dependent process in these cells. iNOS protein, NO, and nitrotyrosine were also significantly elevated in LPS-treated cells. Ca2+chelation and protein kinase C (PKCα-β1) inhibition suppressed iNOS mRNA induction by LPS, implicating Ca2+-dependent PKC signaling in this process. LPS evoked a significant increase of extracellular ATP. Because PKC activation is one of the signaling pathways known to mediate purinergic signaling, we evaluated the hypothesis that iNOS induction was ATP dependent. Although high suramin concentration inhibited iNOS mRNA induction, the process was not ATP dependent, since specific purinergic receptor antagonists could not inhibit the process. Altogether, these findings demonstrate that iNOS expression is highly modulated in alveolar epithelial cells by LPS via a Ca2+/PKCα-β1 pathway independent of ATP signaling.

Background. Chronic granulomatous disease is a rare inherited disorder of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The clinical course of the disease is marked by recurrent infections, includingBurkholderia cepacia complexinfection.Case Report. Here we report the case of a 21-year-old male hospitalized for aBurkholderia cepacia complexpneumonia. Despite the broad spectrum antibiotic treatment, fever continued and patient’s condition worsened. Anemia and thrombocytopenia developed together with hypofibrinogenemia. The patient died of multiple organ dysfunction 17 days after his admission. Autopsy revealed hemophagocytosis, suggesting the diagnosis of acquired hemophagocytic lymphohistiocytosis. DNA analysis showed a deletion in the p47phox gene, confirming the diagnosis of autosomal recessive chronic granulomatous disease.Discussion. In addition to chronic granulomatous disease, recent findings have demonstrated thatBurkholderia cepacia complexcan decrease activity of the NADPH oxidase. Interestingly, hemophagocytic lymphohistiocytosis is characterized by an impaired function of the T-cell mediated inflammation which is partly regulated by the NADPH oxidase. Physicians should therefore pay particular attention to this deadly association.

The mobility of transposable elements (TEs) can contribute to genome plasticity, under- or over-expression of genes and ectopic recombination. The data collected in this study provide evidence of stress-induced mobility of OPHIO1 and OPHIO2 transposons, recently detected in Ophiostoma ulmi and O. novo-ulmi, the causal agents of Dutch elm disease (DED). The analyses of OPHIO UTRs and TIRs indicated the presence of two potential binding site motifs and a heat shock protein (hsp) promoter which could be involved in the mobility of OPHIO1 following a heat shock stress. The exact position of the hsp promoter was determined by 5' RACE PCR. After confirmation of the expression by RT-PCR of both OPHIO1 and OPHIO2 transposases in the absence of stress factors, we tested two experimental procedures to induce mobility of OPHIO TEs: (1) an exogenous (cloned) copy of OPHIO1 was introduced into the O. novo-ulmi subsp. americana strain W2 (OPHIO1 free strain) to give mutant strain W2:OPHIO1. After exposure of W2:OPHIO1 to a 55 degrees C heat shock treatment, some of the survivors showed signs of incomplete transposition (excision without reinsertion) of OPHIO1. (2) The O. novo-ulmi subsp. novo-ulmi strain AST27, introgressed from O. ulmi and carrying a distinct endogenous copy of OPHIO2 (OPHIO2-int.), was subjected to a series of abiotic stress treatments. Although a promoter sequence could not be identified, both exposures to UV light and to a 4 degrees C cold treatment caused perfect excision of OPHIO2-int. In contrast to OPHIO1, heat shock stress did not induce OPHIO2-int. mobility. Taken together, these results allow us to hypothesize a potential interspecific invasion of OPHIO transposons due to their mobility in Ophiostoma spp.

Suppression subtractive hybridization cDNA libraries were prepared from asexual synnemata (S-lib) and sexual perithecia (P-lib) fruiting bodies of the Dutch elm disease pathogen Ophiostoma novo-ulmi subsp. novo-ulmi isolate H327 (mating-type MAT1-1) consisting of 630 and 401 cDNA clones, respectively. Both libraries were differentially screened in duplicate with forward and reverse subtracted probes. Up-regulated S-lib transcripts included those with homologies to phosphoenolpyruvate carboxykinase and aquaporin. Up-regulated P-lib transcripts included those with homologies to aspartyl proteinase, DNA lyase 2, and part of a mating-type (MAT) protein containing a DNA-binding domain of the high-mobility group (HMG) type. Phylogenetic analyses of HMG domains present within the putative O. novo-ulmi MAT protein and within MAT1-1-3 and MAT1-2-1 proteins of other ascomycete fungi identified the O. novo-ulmi protein as a homologue of the MAT1-1-3 protein, which represents part of the so far uncharacterized O. novo-ulmi MAT1-1 idiomorph. Reverse transcription – quantitative real-time PCR indicated up-regulation of the MAT1-1-3 homologue in O. novo-ulmi perithecia and synnemata. The present work identifies, for the first time, proteins involved in the formation of asexual and sexual fruiting bodies in O. novo-ulmi and should be of interest to researchers concerned with reproduction, mating type, and sexuality of filamentous ascomycete fungi.

To identify novel pathways that could modulate epithelial ion transport in CF, we used a system biology approach to identify genes that are differentially expressed in CF and Non-CF epithelial cells. Data analysis showed that expression of chitinase 3 like 1 gene (CHI3L1 also known as YKL-40), a secreted glycoprotein, was particularly elevated in CF epithelial cells. Moreover, we observed a higher concentration of circulating YKL-40 in dysglycemia CF patients. We demonstrated that a fasting YKL-40 concentration of 102 ng/mL was predictive of abnormal OGTT with a sensitivity of 74% and specificity of 83%, corresponding to a positive likelihood ratio weighted by prevalence of 7.75. We also observed a relationship between YKL-40 and inflammatory cytokines (especially IL-8) in the circulation, reflecting that YKL-40 participates in a systemic inflammatory response associated with cystic fibrosis. In order to explore the role of YKL-40-induced pathway, we combined YKL-40 coimmunoprecipitation and surface receptors protein arrays either on CF epithelial ( CFBE41o -) or macrophage ( THP-1 ) cell lines. Results demonstrated the presence of the CD36 scavenger receptor, on both cell types. CD36, which is able to bind to thrombospondin, oxidized LDL (oxLDL) and induces inflammatory responses, is also a marker of macrophage differentiation/activation. THP-1 monocyte-derived macrophages treated by 300 ng/µl of YKL-40 demonstrated an upregulation of inflammatory transcripts (IL6, IL10 and S100A12). We will present and discussed our results on the interaction between YKL-40, CD36 and inflammation in CF.

Transposable elements (TEs) are fundamental components of eukaryotic genomes and can contribute in various ways to genome plasticity and evolution. We describe here the first three DNA transposons in the Dutch elm disease (DED) pathogens Ophiostoma ulmi and O. novo-ulmi, named OPHIO1, OPHIO2 and OPHIO3. We demonstrate that OPHIO transposons, which show high homology to Fot1/pogo TEs within the Tc1/mariner superfamily, have different distribution patterns and specificity in the DED fungi and that interspecific hybrids could act as genetic bridges for transmission of TEs between closely related fungal species. OPHIO3 was found to have undergone repeat-induced point mutations (RIP). We have also developed a complementary method to Margolin’s ratios based on the computation of cumulative transition scores (CTS) in order to visualize rapidly RIP signatures on individual DNA strands of OPHIO transposons and TEs found in other ascomycete fungi.

Purpose: Cystic Fibrosis (CF) is the most common genetic disorder and, with improved survival, glucose abnormalities have emerged as a major comorbidity. Proprotein convertase subtilisin/kexin type 9 (PCSK9), a regulator of plasma LDL-cholesterol homeostasis, is associated with lipid and glucose metabolism in healthy individuals. Here we report on the link between PCSK9 and markers of metabolism in CF. Methods: Cross-sectional analysis was performed on CF patients (≥ 18 years, N=94) from the Montreal Cohort, without known diabetes, and on healthy individuals (N=19). The levels of PCSK9 and lipid markers were quantified and all subjects underwent a 2 h oral glucose tolerance test. Results: No significant differences in PCSK9 levels were found between healthy individuals and patients with CF, or between the groups with different degrees of glucose tolerance. No association was found between PCSK9 and markers of lipid metabolism; however, a positive correlation was found between PCSK9 and total insulin secretion and a negative one with insulin sensitivity in CF patients who had normal glucose tolerance. Conclusion: Circulating levels of PCSK9 in the CF population are comparable to those in the healthy population. There are no associations between PCSK9 levels and either glucose or lipid homeostasis parameters. Nevertheless, a statistically significant link was observed between PCSK9 and markers of insulin homeostasis, solely in CF patients who presented normal glucose tolerance. Further exploration of the relationship between PCSK9 and insulin homeostasis in CF patients with normal glucose tolerance is warranted.

The invasive fungal pathogens Ophiostoma ulmi and O. novo-ulmi have caused two successive pandemics of Dutch elm disease since the beginning of the 20th century. In nature, the highly aggressive O. novo-ulmi may hybridize with the less aggressive O. ulmi. Growth rate and molecular analyses were conducted on an unusual, moderately aggressive O. novo-ulmi isolate, AST27, carrying an introgressed pathogenicity gene, Pat1-m; on highly aggressive O. novo-ulmi isolate H327; on O. ulmi isolates Q412T and W9; and on progeny from laboratory crosses between H327 and AST27. Genetic analysis indicated that the Pat1 and Mat1 (mating type) loci were in different linkage groups corresponding to O. novo-ulmi H327 chromosomes 1 and 2, respectively. Most of the molecular differences between the nuclear genomes of H327 and AST27 occurred in the vicinity of Pat1 and Mat1. In addition, two putative quanti-tative trait loci, Mgr1 and Mgr2, which influence mycelial growth rate at 21°C and 28°C, the optima for O. novo-ulmi and O. ulmi, were linked to Mat1 and Pat1, respectively., Les champignons pathogènes envahissants Ophiostoma ulmi et O. novo-ulmi ont causé deux pandémies successives de maladie hollandaise de l’orme depuis le début du 20e siècle. On a mesuré la croissance et procédé à des analyses moléculaires chez l’isolat O. novo-ulmi AST27, modérément agressif et chez lequel le gène de pathogénie Pat1-m est introgressé; chez l’isolat O. novo-ulmi H327; chez les isolats O. ulmi Q412T et W9; ainsi que chez les descendants de croisements dirigés entre les isolats H327 et AST27. Les analyses génétiques indiquent que les loci Pat1 et Mat1 (type sexuel) sont situés sur des groupes de liaison différents qui correspondent respectivement aux chromosomes 1 et 2. Les différences moléculaires entre les génomes nucléaires des souches H327 et AST27 se retrouvent majoritairement dans le voisinage de Pat1 et de Mat1. De plus, deux loci présumés de caractères quantitatifs, Mgr1 et Mgr2, qui influencent la croissance mycélienne à 21 °C et à 28 °C (températures optimales pour O. novo-ulmi et O. ulmi) sont liés respectivement à Mat1 et à Pat1.