Association between genome-wide association studies reported SNPs and pediatric-onset Crohn’s disease in Canadian children

A recent pediatric-focused genome-wide association study has implicated three novel susceptibility loci for Crohn’ disease (CD).We aimed to investigate whether the three recently reported and other previously reported genes/loci were also associated with CD in Canadian children. A case–control design was implemented at three pediatric gastroenterology clinics in Canada. Children <19 years of age with a confirmed diagnosis of CD were recruited along with controls. Single nucleotide polymorphisms (SNPs) in 19 reported genes/loci were genotyped. Associations between individual SNPs and CD were examined. A total of 563 cases and 553 controls were studied. The mean (±SD) age of the cases was 12.3 (±3.2) years. Most cases were male (56.0%), had ileo-colonic disease (L3 ± L4, 48.8%) and inflammatory behavior (B1 ± p, 87.9%) at diagnosis. Allelic association analysis (two-tailed) showed that 8 of the 19 targeted SNPs were significantly associated with overall susceptibility for CD. Associations with one additional SNP was borderline non-significant. Significantly associated SNPs included SNPs rs1250550 (p = 0.026) and rs8049439 (p = 0.04), recently reported to be specifically associated with pediatric-onset CD.Based on the results, we confirmed associations between two of the three novel pediatric-CD loci and other regions reported for associations with either pediatric and/or adult-onset CD.     

Specific EEG Sleep Pattern in the Prefrontal Cortex in Primary Insomnia

Objective

To assess the specific prefrontal activity in comparison to those in the other main cortical areas in primary insomnia patients and in good sleepers.

Methods

Fourteen primary insomnia patients and 11 good sleepers were included in the analysis. Participants completed one night of polysomnography in the sleep lab. Power spectra were calculated during the NREM (Non-rapid eyes movements) and the REM (Rapid eyes movements) sleep periods at prefrontal, occipital, temporal and central electrode positions.

Results

During the NREM sleep, the power spectra did not differ between groups in the prefrontal cortex; while primary insomnia patients exhibited a higher beta power spectrum and a lower delta power spectrum compared to good sleepers in other areas. During the REM sleep, the beta1 power spectrum was lower in the prefrontal cortex in primary insomnia patients compared to good sleepers; while no significant difference between groups was obtained for the other areas.

Conclusions

The present study shows a specific prefrontal sleep pattern during the whole sleep period. In addition, we suggest that primary insomnia patients displayed a dysfunction in the reactivation of the limbic system during the REM sleep and we give additional arguments in favor of a sleep-protection mechanism displayed by primary insomnia patients.     

Entry of Spiroplasma citri into Circulifer haematoceps Cells Involves Interaction between Spiroplasma Phosphoglycerate Kinase and Leafhopper Actin

Transmission of the phytopathogenic mollicutes, spiroplasmas, and phytoplasmas by their insect vectors mainly depends on their ability to pass through gut cells, to multiply in various tissues, and to traverse the salivary gland cells. The passage of these different barriers suggests molecular interactions between the plant mollicute and the insect vector that regulate transmission. In the present study, we focused on the interaction between Spiroplasma citri and its leafhopper vector, Circulifer haematoceps. An in vitro protein overlay assay identified five significant binding activities between S. citri proteins and insect host proteins from salivary glands. One insect protein involved in one binding activity was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as actin. Confocal microscopy observations of infected salivary glands revealed that spiroplasmas colocated with the host actin filaments. An S. citri actin-binding protein of 44 kDa was isolated by affinity chromatography and identified by LC-MS/MS as phosphoglycerate kinase (PGK). To investigate the role of the PGK-actin interaction, we performed competitive binding and internalization assays on leafhopper cultured cell lines (Ciha-1) in which His₆-tagged PGK from S. citri or purified PGK from Saccharomyces cerevisiae was added prior to the addition of S. citri inoculum. The results suggested that exogenous PGK has no effect on spiroplasmal attachment to leafhopper cell surfaces but inhibits S. citri internalization, demonstrating that the process leading to internalization of S. citri in eukaryotic cells requires the presence of PGK. PGK, regardless of origin, reduced the entry of spiroplasmas into Ciha-1 cells in a dose-dependent manner.Phloem-feeding leafhoppers transmit plant pathogenic mollicutes, spiroplasmas, and phytoplasmas from plant to plant in a persistent propagative manner (26, 43). These phytopathogenic mollicutes are restricted to phloem and to certain vector tissues; thus, their vectors are phloem sap-feeding specialists. After being ingested from plant phloem by their insect vectors, they traverse the insect gut wall, move into the hemolymph, where they multiply, and invade the salivary glands (20, 33, 34, 36). During their movements in the insect vector until its transmission to a new host plant, spiroplasmas and phytoplasmas must traverse two major physical barriers, namely, the insect intestine and the salivary gland (35, 53). Until now, little was known about the molecular and cellular interactions contributing to the crossing of these physical barriers. Several lines of evidence suggest that host-pathogen interactions could be a prerequisite for invasion and colonization of insect vector organs (2, 48, 53). For human and animal pathogenic mollicutes, it is well established that successful colonization of the host cells requires adhesion as the first step. This event is mediated by surface proteins, and among these proteins adhesins play an important role (8, 44). Recently, it was reported that an antigenic membrane protein (Amp) of onion yellow phytoplasma interacts with the insect microfilament complex and that interaction plays an important role in determining the insect vector specificity (48). Several other immunodominant membrane proteins from various phytoplasmas have been mentioned in the literature as candidates for involvement in host-phytoplasma interactions (29, 30).Spiroplasma citri, the first phytopathogenic mollicute available in culture (45), has emerged as an outstanding model for studying spiroplasma interactions with its two hosts: the periwinkle plant and the insect vector Circulifer haematoceps. Following observations of membrane-bound cytoplasmic vesicles of midgut epithelium and salivary gland cells, S. citri was hypothesized to cross these physical barriers by receptor-mediated cell endocytosis (3, 33, 39). Several S. citri protein candidates have been identified as involved in transmission and, for a few of them, in an interaction with leafhopper vector proteins. Spiralin, the most abundant membrane protein, was suspected to be involved in the transmission for two reasons: (i) a S. citri spiralinless mutant was less effective in its transmissibility (19); (ii) spiralin acted in vitro as a lectin able to bind to glycoproteins of insect vectors and therefore might function as a ligand able to interact with leafhopper receptors (32). In addition, the ability of S. citri to be transmitted by C. haematoceps is clearly affected by disruption of a gene predicted to encode a lipoprotein with homology to a solute-binding protein of an ABC transporter (14). The proteome of nontransmissible S. citri strains specifically lacks adhesion-related proteins (ScARPs) and the membrane-associated protein P32 present in the proteome of transmissible strains (12, 13, 31). These proteins are encoded by plasmids pSci1 to -6 (46), which are present only in transmissible strains, and ScARPs share strong similarities with the adhesion-related protein SARP1 of S. citri strain BR3, in which the presence has been correlated to the ability for the spiroplasma to adhere to insect cells in vitro (9, 55). The specific interactions of S. citri with eukaryotic cells remain to be elucidated, but a combination of the effects of several proteins or a complex would be necessary to explain the invasion of a variety of host cell types by S. citri (33).Nevertheless, in the last sequence of events involved in insect vector transmission, the first contact and recognition for the efficient penetration of the salivary gland cells represents an essential step. In the present study, confocal images of infected salivary glands show the localization of S. citri cells along the actin filaments. We report the results of the first attempt to decipher the role of the spiroplasma''s phosphoglycerate kinase (PGK) in the internalization of S. citri in its insect vector''s cells.     

The Syk Kinase SmTK4 of Schistosoma mansoni Is Involved in the Regulation of Spermatogenesis and Oogenesis

The signal transduction protein SmTK4 from Schistosoma mansoni belongs to the family of Syk kinases. In vertebrates, Syk kinases are known to play specialized roles in signaling pathways in cells of the hematopoietic system. Although Syk kinases were identified in some invertebrates, their role in this group of animals has not yet been elucidated. Since SmTK4 is the first Syk kinase from a parasitic helminth, shown to be predominantly expressed in the testes and ovary of adult worms, we investigated its function. To unravel signaling cascades in which SmTK4 is involved, yeast two-/three-hybrid library screenings were performed with either the tandem SH2-domain, or with the linker region including the tyrosine kinase domain of SmTK4. Besides the Src kinase SmTK3 we identified a new Src kinase (SmTK6) acting upstream of SmTK4 and a MAPK-activating protein, as well as mapmodulin acting downstream. Their identities and colocalization studies pointed to a role of SmTK4 in a signaling cascade regulating the proliferation and/or differentiation of cells in the gonads of schistosomes. To confirm this decisive role we performed biochemical and molecular approaches to knock down SmTK4 combined with a novel protocol for confocal laser scanning microscopy for morphological analyses. Using the Syk kinase-specific inhibitor Piceatannol or by RNAi treatment of adult schistosomes in vitro, corresponding phenotypes were detected in the testes and ovary. In the Xenopus oocyte system it was finally confirmed that Piceatannol suppressed the activity of the catalytic kinase domain of SmTK4. Our findings demonstrate a pivotal role of SmTK4 in gametogenesis, a new function for Syk kinases in eukaryotes.     

Crystal structure of the Bacillus subtilis penicillin-binding protein 4a, and its complex with a peptidoglycan mimetic peptide

The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram-positive rod-shaped bacterium. PBP4a (encoded by the dacC gene) is a low-molecular mass PBP clearly exhibiting in vitro DD-carboxypeptidase activity. We have solved the crystal structure of this protein alone and in complex with a peptide (D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine) that mimics the C-terminal end of the Bacillus peptidoglycan stem peptide. PBP4a is composed of three domains: the penicillin-binding domain with a fold similar to the class A beta-lactamase structure and two domains inserted between the conserved motifs 1 and 2 characteristic of the penicillin-recognizing enzymes. The soaking of PBP4a in a solution of D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine resulted in an adduct between PBP4a and a D-alpha-aminopimelyl-epsilon-D-alanine dipeptide and an unbound D-alanine, i.e. the products of acylation of PBP4a by D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine with the release of a D-alanine. The adduct also reveals a binding pocket specific to the diaminopimelic acid, the third residue of the peptidoglycan stem pentapeptide of B. subtilis. This pocket is specific for this class of PBPs.     

Heredity--venturing beyond genetics

Our knowledge of heredity has recently undergone major upheaval. Heredity transmits considerably more than just genetic elements. First, the oocyte is full of maternal cytoplasmic components that subsequently are present in each new cell. Second, maternal cells can pass to the progeny, where they remain active into adult life (microchimerism). Here, we examine the notion that the transmission of characters involves at least two processes in addition to that of mendelian heredity, long considered to be the only hereditary mechanism. These processes all involve epigenetic processes, including the transmission of macromolecules, subcellular organelles, and living cells solely from the mother to her offspring, whether female or male, during pregnancy and lactation. We postulate that cytoplasmic heredity and maternal transmission of cells leading to a long-term state of microchimerism in progeny are two good examples of matrilineal, nonmendelian heredity. A mother's important contribution to the development and health of her progeny seems to possess many uncharted depths.     

Specificity towards oligomannoside and hybrid type glycans of the endo-β-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo--N-acetylglucosaminidase (EC 3.2.1.96) named Endo B, isolated from culture filtrates of the basidiomyceteSporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of theN-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43–49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)₁(GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₄(GlcNAc)₂ were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)₂(Man)₅(GlcNAc)₂ glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme.The oligomannoside-and theN-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule.Endo B represents a powerful tool for removing oligomannoside-andN-acetyllactosamine-type glycans fromN-glycopeptides andN-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.Abbreviations Gal d-galactose - Man d-mannose - GlcNAc N-acetyl-d-glucosamine - Con A concanavalin A - Asn asparagine - GLC gas liquid chromatography - TLC thin layer chromatography - Endo endo--N-acetylglucosaminidase - Endo B endo--N-acetylglucosaminidase isolated fromSporotrichum dimorphosporum - PBE polybuffer exchanger - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Resumption of cellular activity induced by cytokinin and gibberellin treatments in tomato flowers targeted for abortion in unfavorable light conditions

Mitotic activity and nuclear DNA synthesis in tomato ( Lycopersicon esculentum Mill., cv. King plus) flowers targeted for abortion under unfavorable light conditions are completely stopped 6 days after macroscopic appearance of the inflorescence. Ovular cells are arrested at the G₁ (80%) and G₂ (20%) stages of the cell cycle. Exogenous applications of a mixture of N⁶-benzyladenine (BA) and gibberellins A₄₊₇ (GA) directly on the inflorescence may prevent its failure. Nuclear DNA synthesis and mitoses resume in ovules of the flower 16 to 20 h after the BA+GA treatment. When applied alone, BA and GA are able to mimic the effect of the mixture upon the progression of ovular cells through their cycle. Sporogenesis processes are also set in motion by the exogenous plant growth regulators. The mechanism of action of cytokinins and gibberellins in the control of floral development is discussed.     

De novo identification of highly active fluorescent kappa opioid ligands from a rhodamine labeled tetrapeptide positional scanning library

Highly active fluorescent compounds having kappa opioid activity were identified following the screening in a kappa-specific radioligand binding assay of a positional scanning tetrapeptide combinatorial library in which every tetrapeptide was fluorescently labeled. Lissamine rhodamine B sulfonyl chloride was coupled to the N terminal of a mixture-based tetrapeptide positional scanning library made up of over 7.3 million tetrapeptides. Upon determination of the most active mixtures for each position of the library in the kappa binding assay, individual rhodamine labeled tetrapeptides were then synthesized and tested to determine their activities. Eight individual rhodamine labeled peptides were identified that were specific for the kappa opioid receptor, having binding affinities ranging from 5-20 nM. These peptides were poor inhibitors at the mu and delta receptors (K(i)>5,000 nM). Furthermore, neither rhodamine itself nor these same tetrapeptides lacking the N-terminal rhodamine had any significant activity at the kappa receptor, indicating that both the tetrapeptide sequence and the rhodamine moiety are required for receptor binding. This study has demonstrated that novel fluorescent compounds with intrinsic activity can be identified through the use of combinatorial chemistry.     

Sound-induced brain activity depends on stimulus subjective salience in female zebra finches

A key point in the study of acoustic perception is whether brain responsiveness to sounds depends on sound acoustic structure or sound perceptive salience. Songbirds provide some evidence that higher auditory regions are sensitive to the subjective importance of the stimulus for the subject. In the present paper, we compare brain activation elicited by mate versus non-mate calls in female zebra finches Taeniopygia guttata. Using playback, we examined the responsiveness of the caudal telencephalon by measuring the evoked expression of the immediate early gene ZENK. Our results show that mate calls elicit a significantly higher ZENK expression than the calls of another male in hippocampus, but not in auditory areas. Using a hierarchical ascending classification, we show that this difference in brain activation is not explained by call acoustic structure, but relies on call identity. Thus, these results give evidence for a genomic response to calls in hippocampus that differentiate between call identity, and not between call structure. Our study gives further insight into the implication of the hippocampus in sound recognition in female songbirds.