Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data.

MOTIVATION: High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. Results: We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays.

Characterization of the staphylococcal cassette chromosome mec insertion site in 108 isolates lacking the mecA gene and identified as methicillin-resistant Staphylococcus aureus by the Xpert MRSA assay.

During a 3-year period, 848 patients were detected as carriers of methicillin-resistant Staphylococcus aureus (MRSA) by the Xpert MRSA assay (Cepheid). Among them, 108 patients (12.7 %) were colonized with strains showing methicillin-susceptible phenotypes and absence of the mecA gene, despite being positive with the rapid polymerase chain reaction (PCR) assay. DNA sequences of the staphylococcal cassette chromosome mec (SCCmec) insertion site of these "false-positive" strains was determined by direct sequencing of the genomic DNA. More than half (53.7 %) of the strains had DNA sequences unrelated to either SCC or SCCmec and one-third had DNA sequences related to non-mec SCC. Only 10.2 % of the strains carried sequences related to SCCmec, suggesting that a sequence containing the mecA gene was lost from an SCCmec. These findings differ from the general idea that all methicillin-susceptible S. aureus having positive Xpert MRSA assay results are essentially MRSA that lost the mecA gene.

Safety and accuracy of robot-assisted versus fluoroscopy-guided pedicle screw insertion for degenerative diseases of the lumbar spine: a matched cohort comparison.

Object Recent years have been marked by efforts to improve the quality and safety of pedicle screw placement in spinal instrumentation. The aim of the present study is to compare the accuracy of the SpineAssist robot system with conventional fluoroscopy-guided pedicle screw placement. Methods Ninety-five patients suffering from degenerative disease and requiring elective lumbar instrumentation were included in the study. The robot cohort (Group I; 55 patients, 244 screws) consisted of an initial open robot-assisted subgroup (Subgroup IA; 17 patients, 83 screws) and a percutaneous cohort (Subgroup IB, 38 patients, 161 screws). In these groups, pedicle screws were placed under robotic guidance and lateral fluoroscopic control. In the fluoroscopy-guided cohort (Group II; 40 patients, 163 screws) screws were inserted using anatomical landmarks and lateral fluoroscopic guidance. The primary outcome measure was accuracy of screw placement on the Gertzbein-Robbins scale (Grade A to E and R [revised]). Secondary parameters were duration of surgery, blood loss, cumulative morphine, and length of stay. Results In the robot group (Group I), a perfect trajectory (A) was observed in 204 screws (83.6%). The remaining screws were graded B (n = 19 [7.8%]), C (n = 9 [3.7%]), D (n = 4 [1.6%]), E (n = 2 [0.8%]), and R (n = 6 [2.5%]). In the fluoroscopy-guided group (Group II), a completely intrapedicular course graded A was found in 79.8% (n = 130). The remaining screws were graded B (n = 12 [7.4%]), C (n = 10 [6.1%]), D (n = 6 [3.7%]), and E (n = 5 [3.1%]). The comparison of "clinically acceptable" (that is, A and B screws) was neither different between groups (I vs II [p = 0.19]) nor subgroups (Subgroup IA vs IB [p = 0.81]; Subgroup IA vs Group II [p = 0.53]; Subgroup IB vs Group II [p = 0.20]). Blood loss was lower in the robot-assisted group than in the fluoroscopy-guided group, while duration of surgery, length of stay, and cumulative morphine dose were not statistically different. Conclusions Robot-guided pedicle screw placement is a safe and useful tool for assisting spine surgeons in degenerative spine cases. Nonetheless, technical difficulties remain and fluoroscopy backup is advocated.

Hereditary hepatic and systemic amyloidosis caused by a new deletion/insertion mutation in the apolipoprotein AI gene.

We report a Spanish family with autosomal-dominant non-neuropathic hereditary amyloidosis with a unique hepatic presentation and death from liver failure, usually by the sixth decade. The disease is caused by a previously unreported deletion/insertion mutation in exon 4 of the apolipoprotein AI (apoAI) gene encoding loss of residues 60-71 of normal mature apoAI and insertion at that position of two new residues, ValThr. Affected individuals are heterozygous for this mutation and have both normal apoAI and variant molecules bearing one extra positive charge, as predicted from the DNA sequence. The amyloid fibrils are composed exclusively of NH2-terminal fragments of the variant, ending mainly at positions corresponding to residues 83 and 92 in the mature wild-type sequence. Amyloid fibrils derived from the other three known amyloidogenic apoAI variants are also composed of similar NH2-terminal fragments. All known amyloidogenic apoAI variants carry one extra positive charge in this region, suggesting that it may be responsible for their enhanced amyloidogenicity. In addition to causing a new phenotype, this is the first deletion mutation to be described in association with hereditary amyloidosis and it significantly extends the value of the apoAI model for investigation of molecular mechanisms of amyloid fibrillogenesis.

Identification of novel functional TBP-binding sites and general factor repertoires.

Our current knowledge of the general factor requirement in transcription by the three mammalian RNA polymerases is based on a small number of model promoters. Here, we present a comprehensive chromatin immunoprecipitation (ChIP)-on-chip analysis for 28 transcription factors on a large set of known and novel TATA-binding protein (TBP)-binding sites experimentally identified via ChIP cloning. A large fraction of identified TBP-binding sites is located in introns or lacks a gene/mRNA annotation and is found to direct transcription. Integrated analysis of the ChIP-on-chip data and functional studies revealed that TAF12 hitherto regarded as RNA polymerase II (RNAP II)-specific was found to be also involved in RNAP I transcription. Distinct profiles for general transcription factors and TAF-containing complexes were uncovered for RNAP II promoters located in CpG and non-CpG islands suggesting distinct transcription initiation pathways. Our study broadens the spectrum of general transcription factor function and uncovers a plethora of novel, functional TBP-binding sites in the human genome.

Identification of HIV integration sites in infected host genomic DNA.

The integration of the Human Immunodeficiency Virus (HIV) genetic information into the host genome is fundamental for its replication and long-term persistence in the host. Isolating and characterizing the integration sites can be useful for obtaining data such as identifying the specific genomic location of integration or understanding the forces dictating HIV integration site selection. The methods outlined in this article describe a highly efficient and precise technique for identifying HIV integration sites in the host genome on a small scale using molecular cloning techniques and standard sequencing or on a massive scale using 454 pyrosequencing.

Effects of thyroid hormones and aldosterone on mineralocorticoid binding sites in the toad bladder.

In the urinary bladder of the toad Bufo marinus triiodothyronine selectively inhibits the late effect of aldosterone on Na+ transport. We have investigated whether T3 might mediate its antimineralocorticoid action by controlling: i) the level of aldosterone binding sites in the soluble (cytosolic) pool isolated from tissues treated with T3 (60 nM) for up to 20 hr of incubation; ii) the kinetics of uptake of 3H-aldosterone into cytoplasmic and nuclear fractions after 2 or 20 hr of exposure to T3. The number and the affinity of Type I (high affinity, low capacity) and Type II (low affinity, high capacity) cytosolic binding sites (measured at 0 degrees C) did not vary significantly after 18 hr of exposure to T3, while aldosterone-dependent Na+ transport was significantly inhibited. In addition, T3 did not modify the kinetics of uptake (90 min) of 3H-aldosterone into cytoplasmic and nuclear fractions of toad bladder incubated in vitro at 25 degrees C. By contrast, aldosterone itself was able to down-regulate its cytosolic and nuclear binding sites after an 18-hr exposure to the steroid hormone (10 or 80 nM). T3 slightly (20%) but significantly potentiated the down regulation of nuclear binding sites. In conclusion, T3 does not appear to have major effects on the regulation of the aldosterone receptor, which could explain in a simple manner its antimineralocorticoid action.

NS4B Self-Interaction through Conserved C-Terminal Elements Is Required for the Establishment of Functional Hepatitis C Virus Replication Complexes.

Hepatitis C virus (HCV) is an important human pathogen, persistently infecting more than 170 million individuals worldwide. Studies of the HCV life cycle have become possible with the development of cell culture systems supporting the replication of viral RNA and the production of infectious virus. However, the exact functions of individual proteins, especially of nonstructural protein 4B (NS4B), remain poorly understood. NS4B triggers the formation of specific, vesicular membrane rearrangements, referred to as membranous webs, which have been reported to represent sites of HCV RNA replication. However, the mechanism of vesicle induction is not known. In this study, a panel of 15 mutants carrying substitutions in the highly conserved NS4B C-terminal domain was generated. Five mutations had only a minor effect on replication, but two of them enhanced assembly and release of infectious virus. Ten mutants were replication defective and used for selection of pseudoreversions. Most of the pseudoreversions also localized to the highly conserved NS4B C-terminal domain and were found to restore replication competence upon insertion into the corresponding primary mutant. Importantly, pseudoreversions restoring replication competence also restored heterotypic NS4B self-interaction, which was disrupted by the primary mutation. Finally, electron microscopy analyses of membrane alterations induced by NS4B mutants revealed striking morphological abnormalities, which were restored to wild-type morphology by the corresponding pseudoreversion. These findings demonstrate the important role of the C-terminal domain in NS4B self-interaction and the formation of functional HCV replication complexes.

Transglutaminase 1-deficient recessive lamellar ichthyosis associated with a LINE-1 insertion in Jack Russell terrier dogs.

BACKGROUND: Congenital, nonepidermolytic cornification disorders phenotypically resembling human autosomal recessive ichthyosis have been described in purebred dog breeds, including Jack Russell terrier (JRT) dogs. One cause of gene mutation important to humans and dogs is transposon insertions. OBJECTIVES: To describe an autosomal recessive, severe nonepidermolytic ichthyosis resembling lamellar ichthyosis (LI) in JRT dogs due to insertion of a long interspersed nucleotide element (LINE-1) in the transglutaminase 1 (TGM1) gene. METHODS: Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Phenotypic information and genotyping with a canine microsatellite marker suggested TGM1 to be a candidate gene. Genomic DNA samples and cDNA generated from epidermal RNA were examined. Consequences of the mutation were evaluated by Western blotting, quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme activity from cultured keratinocytes. RESULTS: Affected dogs had generalized severe hyperkeratosis. Histological examination defined laminated to compact hyperkeratosis without epidermolysis; ultrastructurally, cornified envelopes were thin. Affected dogs were homozygous for a 1980-bp insertion within intron 9 of TGM1. The sequence of the insertion was that of a canine LINE-1 element. Quantitative RT-PCR indicated a significant decrease in TGM1 mRNA in affected dogs compared with wild-type. TGM1 protein was markedly decreased on immunoblotting, and membrane-associated enzyme activity was diminished in affected dogs. CONCLUSIONS: Based on morphological and molecular features, this disease is homologous with TGM1-deficient LI in humans, clinically models LI better than the genetically modified mouse and represents its first spontaneous animal model. This is the first reported form of LI due to transposon insertion.

Insertion vélamenteuse du cordon et grossesse gémellaire

Problématique : L'insertion vélamenteuse du cordon ombilical est reportée dans environ 1% des grossesses uniques et semble associée à de sévères complications obstétricales. Mais l'étude de cette insertion reste encore lacunaire en ce qui concerne les grossesses gémellaires. Depuis quelques années, ces dernières sont en augmentation et doivent être considérées comme des grossesses à risque. En effet, les jumeaux occupent une place toujours plus importante dans les unités de néonatologie, du fait de la morbidité particulière de ces grossesses. Ces dernières peuvent de plus s'accompagner d'autres anomalies qui peuvent influencer la croissance des jumeaux déjà prétérités par rapport aux bébés uniques. Parmi celles-ci, l'insertion vélamenteuse du cordon. Existe-il une association entre grossesse gémellaire et insertion vélamenteuse ? Quelles seraient les conséquences de ce type d'insertion sur l'évolution de la grossesse ?¦Objectifs : Evaluer l'incidence de la survenue d'une insertion vélamenteuse du cordon ombilical dans les grossesses gémellaires par rapport aux grossesses uniques et la différence de cette incidence entre placentas monochoriaux et bichoriaux. Analyser les conséquences majeures qui peuvent lui être associées.¦Méthode : Analyse rétrospective de tous les placentas de grossesses gémellaires reçus pour examen à l'institut universitaire de pathologie anatomique (IUPA) de Lausanne entre janvier 2000 et septembre 2010, ainsi que du suivi périnatal des enfants issus de ces grossesses.¦Résultats : 722 placentas issus de grossesses gémellaires ont été reçus et analysés à l'IUPA. L'insertion vélamenteuse du cordon est rencontrée dans plus de 17% des grossesses gémellaires. Elle représente plus de 9% de toutes les insertions lors de ces grossesses. Elle est retrouvée dans 35% des cas lorsque le placenta est monochorial et dans 10% des cas lorsque le placenta est bichorial.¦Notre étude a démontré une influence significative de l'insertion vélamenteuse sur l'âge gestationnel, le poids du placenta, la croissance foetale (poids > taille > PC), la durée d'hospitalisation et la mortalité. La monochorialité est le seul facteur de risque à avoir été mis en évidence.¦Conclusion : Avec l'augmentation de l'âge maternel et le recours de plus en plus fréquent aux traitements de fertilité, le nombre de grossesses gémellaires devrait s'accroitre dans les années à venir et, avec elles, le nombre d'insertions vélamenteuses du cordon. Cependant, le diagnostic d'une telle insertion ne peut être que suspecté lors d'un examen ultrasonographique en cours de grossesse. Il est donc difficile de l'identifier avant la naissance et ainsi de prévoir ses conséquences. Mais en étant conscient des complications éventuelles que peut engendrer cette insertion, nous serions plus à même d'y faire face.

What is the influence of vaccination's routes on the regression of tumors located at mucosal sites?

Tumor-regressions following tumor-associated-antigen vaccination in animal models contrast with the limited clinical outcomes in cancer patients. Most animal studies however used subcutaneous-tumor-models and questions arise as whether these are relevant for tumors growing in mucosae; whether specific mucosal-homing instructions are required; and how this may be influenced by the tumor.

Covalent homodimers of murine secretory component induced by epitope substitution unravel the capacity of the polymeric Ig receptor to dimerize noncovalently in the absence of IgA ligand.

Recombinant secretory immunoglobulin A containing a bacterial epitope in domain I of the secretory component (SC) moiety can serve as a mucosal delivery vehicle triggering both mucosal and systemic responses (Corthésy, B., Kaufmann, M., Phalipon, A., Peitsch, M., Neutra, M. R., and Kraehenbuhl, J.-P. (1996) J. Biol. Chem. 271, 33670-33677). To load recombinant secretory IgA with multiple B and T epitopes and extend its biological functions, we selected, based on molecular modeling, five surface-exposed sites in domains II and III of murine SC. Loops predicted to be exposed at the surface of SC domains were replaced with the DYKDDDDK octapeptide (FLAG). Another two mutants were obtained with the FLAG inserted in between domains II and III or at the carboxyl terminus of SC. As shown by mass spectrometry, internal substitution of the FLAG into four of the mutants induced the formation of disulfide-linked homodimers. Three of the dimers and two of the monomers from SC mutants could be affinity-purified using an antibody to the FLAG, mapping them as candidates for insertion. FLAG-induced dimerization also occurred with the polymeric immunoglobulin receptor (pIgR) and might reflect the so-far nondemonstrated capacity of the receptor to oligomerize. By co-expressing in COS-7 cells and epithelial Caco-2 cells two pIgR constructs tagged at the carboxyl terminus with hexahistidine or FLAG, we provide the strongest evidence reported to date that the pIgR dimerizes noncovalently in the plasma membrane in the absence of polymeric IgA ligand. The implication of this finding is discussed in terms of IgA transport and specific antibody response at mucosal surfaces.

Qualités des sites internet anglophones traitant du trouble obsessionnel complusif

Enjeux et contexte : Dans les questions de santé mentale, internet constitue de plus en plus une source d'information pour les personnes souffrant de troubles psychiatriques ainsi que de leurs proches. D'autre part, le trouble obsessionnel compulsif revêt une importance grandissante et bénéficie d'un intérêt croissant en raison de sa fréquence et de la charge qu'il représente pour le patient et pour la société. Les patients souffrant de trouble obsessionnel compulsif, qu'ils soient diagnostiqués ou non, ainsi que leurs proches peuvent être amenés à rechercher une information de bonne qualité sur net sur le sujet. Cette étude vise à évaluer la qualité de l'information issue de l'internet concernant les sites anglophones traitant du trouble obsessionnel compulsif et de comparer les résultats des requêtes en utilisant un moteur de recherche général (Google) à celles obtenues avec un moteur de recherche spécialisé (Omni Medical Search). Des mots-clés relatifs au trouble obsessionnel compulsif ont été introduits dans Google et Omni Medical Search. Les sites retenus ont été évalués selon leur responsabilité, interactivité, lisibilité et la qualité de leur contenu. Le Label HON et la version brève de l'échelle DISCERN ont été utilisés comme indicateurs possibles de la qualité du contenu. Sur les 235 adresses retrouvées, 53 sites retenus ont été analysés. Résultats : La qualité du contenu des sites examinés est relativement bonne. L'utilisation d'un moteur de recherche spécialisé ne constitue pas un avantage par comparaison au moteur général utilisé par la grande majorité des internautes. Un score > 16 de la version brève du DISCERN est associé à une meilleure qualité du contenu. En conclusion : cette étude montre que le contenu des sites web concernant le trouble obsessionnel compulsif est acceptable. L'utilisation d'un moteur de recherche spécialisé n'offre pas d'avantage par rapport à Google. Comme implications pratiques : internet renferme des sites de haute qualité sur le trouble obsessionnel compulsif. L'accès à ces sites ne nécessite pas l'utilisation d'un moteur de recherche spécialisé. En revanche, une discussion entre le patient et le soignant à propos de l'information disponible sur internet demeure indispensable Perspectives : en dépit des limitations de notre étude, on peut dire que l'information contenue dans le web concernant le trouble obsessionnel compulsif est acceptable. Le contenu et la présentation de cette information pourraient être améliorés. Quant à l'internaute qui cherche une information de qualité, il pourrait être guidé par deux éléments : le HON et la version brève du DISCERN.

Polymorphic mutations associated with the emergence of the multinucleoside/tide resistance mutations 69 insertion and Q151M.

BACKGROUND: We hypothesized that polymorphic mutations exist that are associated with the emergence of the multinucleoside resistance mutations (MNR), 69 insertion and Q151M. METHODS: The Swiss HIV Cohort Study was screened, and the frequencies of polymorphic mutations in HIV-1 (subtype B) were compared between patients detected with the 69 insertion (n = 17), Q151M (n = 29), ≥2 thymidine analogue mutations (TAM) 1 (n = 400) or ≥2 TAM 2 (n = 249). Logistic regressions adjusted for the antiretroviral treatment history were performed to analyze the association of the polymorphic mutations with MNR. RESULTS: The 69 insertion and TAM 1 were strongly associated and occurred in 94.1% (16 of 17) together. The 69 insertion seemed to emerge as a consequence of the TAM 1 pathway (median years until detection: 6.8 compared with 4.4 for ≥2 TAM 1, P Wilcoxon = 0.009). Frequencies of 8 polymorphic mutations (K43E, V60I, S68G, S162C, T165I, I202V, R211K, F214L) were significantly different between groups. Logistic regression showed that F214L and V60I were associated with the emergence of Q151M/TAM 2 opposed to 69 insertion/TAM 1. S68G, T165I, and I202V were associated with Q151M instead of TAM 2. CONCLUSIONS: Besides antiretroviral therapy, polymorphic mutations may contribute to the emergence of specific MNR mutations.

Characterization of spontaneous gacS and gacA regulatory mutants of Pseudomonas fluorescens biocontrol strain CHAO.

In Pseudomonasfluorescens strain CHAO, the response regulator gene gacA controls expression of extracellular enzymes and antifungal secondary metabolites, which are important for this strain's biocontrol activity in the plant rhizosphere. Two Tn5 insertion mutants of strain CHA0 that had the same pleiotropic phenotype as gacA mutants were complemented by the gacS sensor kinase gene of P. syringae pv. syringae as well as that of P. fluorescens strain Pf-5, indicating that both transposon insertions had occurred in the gacS gene of strain CHA0. This conclusion was supported by Southern hybridisation using a gacS probe from strain Pf-5. Overexpression of the wild-type gacA gene partially compensated for the gacS mutation, however, the overexpressed gacA gene was not stably maintained, suggesting that this is deleterious to the bacterium. Strain CHA0 grown to stationary phase in nutrient-rich liquid media for several days accumulated spontaneous pleiotropic mutants to levels representing 1.25% of the population; all mutants lacked key antifungal metabolites and extracellular protease. Half of 44 spontaneous mutants tested were complemented by gacS, the other half were restored by gacA. Independent point and deletion mutations arose at different sites in the gacA gene. In competition experiments with mixtures of the wild type and a gacA mutant incubated in nutrient-rich broth, the mutant population temporarily increased as the wild type decreased. In conclusion, loss of gacA function can confer a selective advantage on strain CHA0 under laboratory conditions.