Isolation of genetic mutation leading to abnormal phenotype in human through ultra-high-throughput sequencing (UHTS)

The introduction of Next Generation Sequencing (NGS) facilitated the task of localizing DNA variation and identifying the genetic cause of yet unsolved Mendelian disorders. Using Whole Exome Capture method and NGS, we identified the causative genetic aberration responsible for a number of monogenic disorders previously undetermined. Due to the novelty of the NGS method we benchmarked different algorithms to assess their merits and defects. This allowed us to establish a pipeline that we successfully used to pinpoint genes responsible for a form of West's syndrome, a Complex Intellectual Disability syndrome associated with patellar dislocation and celiac disease, and correcting some erroneous molecular diagnosis of Alport's syndrome in a Saudi Arabian family.

The prevalence of erectile dysfunction and its association with antiretroviral therapy in HIV-infected men: the Swiss HIV Cohort Study.

BACKGROUND: Here, we aimed to determine the prevalence of erectile dysfunction (ED) among HIV-infected men and its association with components of antiretroviral therapy. METHODS: Cross-sectional data on sexual dysfunction were collected in the Swiss HIV Cohort Study (SHCS) between December 2009 and November 2010. Multilevel logistic regression models were used to estimate the association between ED and exposure to 24 different antiretroviral drugs from four drug classes. RESULTS: During the study period, 5,194 of 5,539 eligible men in the SHCS had a follow-up visit; 4,064 men answered a question on ED for the first time. Among these men, ED was experienced often by 459 (11%), sometimes by 543 (13%), rarely by 389 (10%), never by 2,526 (62%) and 147 (4%) did not know. ED was associated with older age, an earlier HIV diagnosis and depression. No association was found with any drug class; however, ED was associated with cumulative exposure to either zalcitabine (OR 1.29 per year of use; 95% CI 1.07, 1.55) or enfuvirtide (OR 1.28; 95% CI 1.08, 1.52). CONCLUSIONS: Around 1 in 10 men in the SHCS reported often experiencing ED. We found no association between ED and any drug class, but those exposed to zalcitabine or enfurvitide (drugs no longer or rarely used) were more likely to report ED; this second association was probably not causal.

Obesity and central obesity as risk factors for incident dementia and its subtypes: a systematic review and meta-analysis

While dementia affects 6-10% of persons 65 years or older, industrialized countries have witnessed an alarming rise in obesity. However, obesity's influence on dementia remains poorly understood. The conducted a systematic review and meta-analysis. PUBMED search (1995-2007) resulted in 10 relevant prospective cohort studies of older adults (40-80 years at baseline) with end points being dementia and predictors including adiposity measures, such as body mass index (BMI) and waist circumference (WC). There was a significant U-shaped association between BMI and dementia (P= 0.034), with dementia risk increased for obesity and underweight. Pooled odds ratios (OR) and 95% confidence intervals (CI) for underweight, overweight and obesity compared with normal weight in relation to incident dementia were: 1.36 (1.07, 1.73), 0.88 (0.60, 1.27) and 1.42 (0.93, 2.18) respectively. Pooled ORs and 95% CI for obesity and incident Alzheimer's disease (AD) and vascular dementia were 1.80 (1.00, 3.29) vs. 1.73 (0.47, 6.31) and were stronger in studies with long follow-up (>10 years) and young baseline age (

The role of CTCFL/BORIS in Meiosis

The complexity of mammalian genome organization demands a complex interplay of DNA and proteins to orchestrate proper gene regulation. CTCF, a highly conserved, ubiquitously expressed protein has been postulated as a primary organizer of genome architecture because of its roles in transcriptional activation/repression, insulation and imprinting. Diverse regulatory functions are exerted through genome wide binding via a central eleven zinc finger DNA binding domain and an array of diverse protein-protein interactions through N- and C- terminal domains. CTCFL has been identified as a paralog of CTCF expressed only in spermatogenic cells of the testis. CTCF and CTCFL have a highly homologous DNA-binding domain, while the flanking amino acid sequences exhibit no significant similarity. Genome- wide mapping of CTCF binding sites has been carried out in many cell types, but no data exist for CTCFL apart from a few identified loci. The lack of high quality antibodies prompted us to generate an endogenously flag-tagged CTCFL mouse model using BAC recombination. IHC staining using anti-flag antibodies confirmed CTCFL localization to type Β spermatogonia and preleptotene spermatocytes and a mutually exclusive pattern of expression with CTCF. ChIP followed by high-throughput sequencing identified 10,382 binding sites showing 70% overlap but representing only 20% of CTCF sites. Consensus sequence analysis identified a significantly longer binding motif with prominently less ambiguity of base calling at every position. The significant difference between CTCF and CTCFL genomic binding patterns proposes that their binding to DNA is differentially regulated. Analysis of CTCFL binding to methylated regions on a genome wide scale identified approximately 1,000 loci. Methylation-independent binding of CTCFL might be at least one of the mechanisms that ensures distinct binding patterns of CTCF and CTCFL since CTCF binding is methylation- sensitive. Co-localization of CTCF with cohesin has been well established and analysis of CTCFL and SMC3 overlap identified around 3,300 binding sites from which two related but distinct consensus sequence motifs were derived. Because virtually all data for cohesin binding originate from mitotically proliferating cells, the anticipated overlap is expected to be considerably higher in meiotic cells. Meiosis-specific cohesin subunit Rec8 is specific for spermatocytes and 6 out of the 12 identified binding sites are also bound by CTCFL. In conclusion, this was the first genome-wide mapping of CTCFL binding sites in spermatocytes, the only cell type where CTCF is not expressed. CTCFL has a unique binding site repertoire distinct from CTCF, binds to methylated sequences and shows a significant overlap with cohesin binding sites. Future efforts will be oriented towards deciphering the role CTCFL plays in conversion of chromatin structure and function from mitotic to meiotic chromosomes. - La complexité de l'organisation du génome des mammifères exige une interaction particulière entre ADN et protéines pour orchestrer une régulation appropriée de l'expression des gènes. CTCFL, une protéine ubiquitaire très conservée, serait le principal organisateur de l'architecture du génome de par son rôle dans l'activation / la répression de la transcription, la protection et la localisation des gènes. Diverses régulations sont opérées, d'une part au travers d'interactions à différents endroits du génome par le biais d'un domaine protéique central de liaison à l'ADN à onze doigts de zinc, et d'autre part par des interactions protéine-protéine variées au niveau de leur domaine N- et C-terminal. CTCFL a été identifié comme un paralogue de CTCF exprimé uniquement dans les cellules spermatiques du testicule. CTCFL et CTCF ont un domaine de liaison à l'ADN très homologue, tandis que les séquences d'acides aminés situées de part et d'autre de ce domaine ne présentent aucune similitude. Une cartographie générale des sites de liaison au CTCF a été réalisée pour de nombreux types cellulaires, mais il n'existe aucune donnée pour CTCFL à l'exception de l'identification de quelques loci. L'absence d'anticorps de bonne qualité nous a conduit à générer un modèle murin portant un CTCFL endogène taggué grâce à un procédé de recombinaison BAC. Une coloration IHC à l'aide d'anticorps anti-FLAG a confirmé la présence de CTCFL au niveau des spermatogonies de type Β et des spermatocytes au stade préleptotène, et une distribution mutuellement exclusive avec CTCF. Une méthode de Chromatine Immunoprecipitation (ChIP) suivie d'un séquençage à haut débit a permis d'identifier 10.382 sites de liaison montrant 70% d'homologie mais ne représentant que 20% des sites CTCF. L'analyse de la séquence consensus révèle un motif de fixation à l'ADN nettement plus long et qui comporte bien moins de bases aléatoires à chaque position nucléotidique. La différence significative entre les séquences génomiques des sites de liaison au CTCF et CTCFL suggère que leur fixation à l'ADN est régulée différemment. Appliquée à l'échelle du génome, l'étude de l'interaction de CTCFL avec des régions méthylées de l'ADN a permis d'identifier environ 1.000 loci. Contrairement à CTCFL, la liaison de CTCF dépend de l'état de méthylation de l'ADN ; cette modification épigénétique constitue donc au moins un des mécanismes de régulation expliquant une localisation de CTCF et CTCFL à des sites distincts du génome. La co- localisation de CTCF avec la cohésine étant établie, l'analyse de la superposition des séquences de CTCFL avec la sous-unité SMC3 identifie environ 3.300 sites de liaison parmi lesquels deux mêmes motifs consensus distincts par leur séquence sont mis en évidence. La presque quasi-totalité des données sur la cohésine ayant été établie à partir de cellules en prolifération mitotique, il est probable que la similitude au sein des séquences consensus soit encore plus grande dans le cas des cellules en méiose. La sous-unité Rec8 de la cohésine propre à l'état de méiose est spécifiquement exprimée dans les spermatocytes. Or 6 des 12 sites de liaison identifiés sont également utilisés par CTCFL. Pour conclure, ce travail constitue la première cartographie à l'échelle du génome des sites de liaison de CTCFL dans les spermatocytes, seul type cellulaire où CTCFL n'est pas exprimé. CTCFL possède un répertoire unique de sites de fixation à l'ADN distinct de CTCF, se lie à des séquences méthylées et présente un nombre important de sites de liaison communs avec la cohésine. Les perspectives futures sont d'élucider le rôle de CTCFL dans le remodelage de la structure de la chromatine et de définir sa fonction dans le processus de méiose.

Development of a Bacillus sphaericus tablet formulation and its evaluation as a larvicide in the biological control of Culex quinquefasciatus

This study aimed to analyze the final fermentation culture of Bacillus sphaericus 2362, standardize it and develop an active tablet formulation for use in urban mosquito breeding sites. It was performed in three phases: analysis and standardization of a B. sphaericus fermented culture; physical, chemical, and biological analysis of the active powder (solubility, residual humidity, particle size, resting angle, flowing off time, compacted density, and biological activity against Culex quinquefasciatus larvae); and the development of fast-disintegrating tablets. Five formulations with differing compositions were developed and a UV protector was added to the selected formulation. The formulation products with or without UV protector, as well as the active powder caused 100% larval mortality from 1 day to 2 months after a single treatment under simulated field conditions. These results show that the UV protector does not affect the initial larvicide activity of B. sphaericus, nor its persistence over a period of two months.

Decoupled post-glacial history in mutualistic plant-insect interactions: insights from the yellow loosestrife (Lysimachia vulgaris) and its associated oil-collecting bees (Macropis europaea and M-fulvipes)

AimWe take a comparative phylogeographical approach to assess whether three species involved in a specialized oil-rewarding pollination system (i.e. Lysimachia vulgaris and two oil-collecting bees within the genus Macropis) show congruent phylogeographical trajectories during post-glacial colonization processes. Our working hypothesis is that within specialized mutualistic interactions, where each species relies on the co-occurrence of the other for survival and/or reproduction, partners are expected to show congruent evolutionary trajectories, because they are likely to have followed parallel migration routes and to have shared glacial refugia. LocationWestern Palaearctic. MethodsOur analysis relies on the extensive sampling of 104 Western Palaearctic populations (totalling 434, 159 and 74 specimens of Lysimachiavulgaris, Macropiseuropaea and Macropisfulvipes, respectively), genotyped with amplified fragment length polymorphism. Based on this, we evaluated the regional genetic diversity (Shannon diversity and allele rarity index) and genetic structure (assessed using structure, population networks, isolation-by-distance and spatial autocorrelation metrics) of each species. Finally, we compared the general phylogeographical patterns obtained. ResultsContrary to our expectations, the analyses revealed phylogeographical signals suggesting that the investigated organisms demonstrate independent post-glacial trajectories as well as distinct contemporaneous demographic parameters, despite their mutualistic interaction. Main conclusionsThe mutualistic partners investigated here are likely to be experiencing distinct and independent evolutionary dynamics because of their contrasting life-history traits (e.g. dispersal abilities), as well as distinct hubs and migration routes. Such conditions would prevent and/or erase any signature of co-structuring of lineages in space and time. As a result, the lack of phylogeographical congruence driven by differences in life-history traits might have arisen irrespective of the three species having shared similar Pleistocene glacial refugia.

Role of protease-activated receptor-2 in inflammation, and its possible implications as a putative mediator of periodontitis

Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. This receptor is widely distributed throughout the body and seems to be importantly involved in inflammatory processes. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and bacterial proteases, such as gingipain produced by Porphyromonas gingivalis. This review describes the current stage of knowledge of the possible mechanisms that link PAR2 activation with periodontal disease, and proposes future therapeutic strategies to modulate the host response in the treatment of periodontitis.

Anti Bullying Procedures for Primary and Post Primary Schools - Appendix 4 Checklist for annual review of the anti-bullying policy and its implementation

The Board of Management (the Board) must undertake an annual review of the school’s anti-bullying policy and its implementation. The following checklist must be used for this purpose. The checklist is an aid to conducting this review and is not intended as an exhaustive list. In order to complete the checklist, an examination and review involving both quantitative and qualitative analysis, as appropriate across the various elements of the implementation of the school’s anti-bullying policy will be required.

The effect of Bulgarian propolis against Trypanosoma cruzi and during its interaction with host cells

Propolis has shown activity against pathogenic microorganisms that cause diseases in humans and animals. The ethanol (Et-Blg) and acetone (Ket-Blg) extracts from a Bulgarian propolis, with known chemical compositions, presented similar activity against tissue culture-derived amastigotes. The treatment of Trypanosoma cruzi-infected skeletal muscle cells with Et-Blg led to a decrease of infection and of the intracellular proliferation of amastigotes, while damage to the host cell was observed only at concentration 12.5 times higher than those affecting the parasite. Ultrastructural analysis of the effect of both extracts in epimastigotes revealed that the main targets were the mitochondrion and reservosomes. Et-Blg also affected the mitochondrion-kinetoplast complex in trypomastigotes, offering a potential target for chemotherapeutic agents.

Localization of Brugia malayi (sub-periodic) adults in different organs of Mastomys coucha and its influence on microfilaraemia and host antibody response

Lymphatic filariasis caused by nematode parasites Wuchereria bancrofti or Brugia malayi is a spectral disease and produces wide range of immune responses and varying levels ofmicrofilaraemia in infected individuals. The relationship between the immune response of host and the developmental stage of the parasite as well as the microfilariae (mf) density and specific location of the adult worms is yet to be understood. As an experimental model, B. malayi adapted in the experimental animal Mastomys coucha has been used widely for various studies in filariasis. The present study was to assess microfilaraemia as well as the humoral immune response of M. coucha during various stages of B. malayi development and their localization in different organs. The result showed that the density of mf in the circulating blood of the experimental animal depended upon the number of female worms as well as the location and co-existence of male and female worms. The mf density in the blood increased with the increase in the number of females. The clearance of inoculated infective stage (L3) or single sex infection or segregation of male and female to different organs of infected host resulted in amicrofilaraemic condition. With respect to antibody response, those animals cleared L3 after inoculation and those with adult worm as well as mf showed low antibody levels. But those with developmental fourth stage and/or adult worms without mf showed significantly higher antibody levels.

Dynamics of feeding and defecation in Triatoma vitticeps (Stal, 1859) (Hemiptera, Reduviidae, Triatominae) and its potential in the transmission of Trypanosoma cruzi

Adults of Triatoma vitticeps infected by flagellates similar to Trypanosoma cruzi are frequently captured by the inhabitants of rural areas in the Brazilian state of Espírito Santo. The dynamics of feeding and defecation were observed in three groups of adult triatomines, consisting of sylvatic T. vitticeps and laboratory-reared specimens of this species and T. infestans. Triatomines were observed from the moment they were presented with an immobilized chicken as a bloodmeal source until 240 min after feeding had ended. Mean times between the end of feeding and defecation for T. infestans, wild T. vitticeps and laboratory-reared specimens of the latter species were 1.2, 21.1, and 64 min respectively. All T. infestans defecated within 10 min of feeding, while only 29.9 of wild and 52.8% laboratory-reared specimens of T. vitticeps did so within this period. These results may explain the low efficiency of T. vitticeps in T. cruzi transmission to man. The shorter time between feeding and defecation in laboratory-reared T. vitticeps may indicate a change in behaviour of this species as a result of adaptation to an artificial environment.

A new Eimeria (Apicomplexa: Eimeriidae), possessing mitra-shaped oocysts, from the Neotropical chelid turtle Batrachemys heliostemma (Testudines: Chelidae), and its comparison with Eimeria mitraria (Laveran & Mesnil 1902)

Eimeria jirkamoraveci sp. n. is described from faeces of two specimens of the toad-headed, side-necked turtle Batrachemys heliostemma collected at Iquitos in Peru. Oocysts are ovoid to almost spherical, 10.6 (8-12) × 8.9 (7-10) mum, without micropyle, polar granule and oocyst residuum. One conically stretched end and three blunt conical tubercles at the opposite end of oocyst give it mitra-like appearance. Sporocysts are elongated, ellipsoidal, 7.2 (6-8) × 4.1 (4-4.5) mum, with a small, knob-like Stieda body and sporocyst residuum composed of fine granules. To avoid possible conspecificity, the described new species is thoroughly compared with the most similar coccidium, E. mitraria, collected from its type host, Chinemys reevesii.