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This article discusses a new bioinformatics research tool that is being developed to identify genes involved in prenatal syndromes based on ultrasound phenotypes. The tool uses an internal database that links ultrasound fetal phenotypes to genes curated by experts in prenatal diagnosis of rare diseases. The tool was tested on 10 cases of CHARGE Syndrome and accurately identified the gene and syndrome in 90% of the cases. Comparative analyses with other available solutions showed that the research tool outperformed other apps in finding the correct gene and/or syndrome. The article concludes that early gene detection of rare syndromes can be significantly improved using research tools specifically designed for prenatal phenotypes. [Extracted from the article]

Les non-dits ont fait l’objet de nombreuses recherches en français comme en japonais. Cependant, les omissions de la conclusion en fin de séquence explicative et situées après les connecteurs donc (français) et dakara (japonais) n’ont jamais été traitées. Or, cette configuration discursive confère un cadre d’analyse permettant d’expliquer la structuration discursive des non-dits. Cette étude contrastive de données orales (ESLO et CSJ) de locuteurs natifs des deux langues susnommées explicite les différents choix sémantico-discursifs opérés pour encoder l’implicite. Il s’avère que le français s’appuie davantage sur l’explicitation des liens qui relient la forme logique présentée alors que le japonais, sur les sous-entendus inférables à partir des structures discursives. Ainsi, chacune des langues a sa propre manière d’articuler les dits et non-dits du discours et invite des procédés cognitifs de résolution des inférences différentes.

De nombreuses études portant sur la causalité se sont intéressées à parce que, car et puisque. Cependant, comme à valeur causale mérite une plus grande attention, car sa structure syntaxique se démarque des autres connecteurs en introduisant la cause avant la conséquence. Cette particularité pourrait-elle induire une perspective différente du rapport de causalité ? En s’appuyant sur le corpus oral ESLO (Enquêtes SocioLinguistiques à Orléans), cet article présente une étude contrastive de comme et parce que qui met en évidence leurs distributions quantitative, syntaxique et discursive à l’oral. Les résultats montrent que parce que peut être dépendant et indépendant syntaxiquement et pragmatiquement. Il peut assurer différentes relations discursives (Explication, Résultat, Arrière-plan), mais les locuteurs l’emploient essentiellement pour exprimer la première. En revanche, comme est moins fréquent, mais révèle une plus grande variété d’emplois discursifs dans le corpus, malgré des contraintes linguistiques plus importantes que parce que. Les tests de substitution le démontrent. Pour finir, les cas litigieux de relations causales et d’arrière-plan avec comme ont révélé une propension à introduire une cause cadrative alors que parce que indique plutôt l’origine de la conséquence. Ainsi chaque connecteur confère intrinsèquement une perspective différente du rapport de causalité.

Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the μLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/μl for 50 kb fragments and an analytical time of 50 min. Then, μLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with μLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with μLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: A multi-centre field trial was conducted to evaluate the efficacy of afoxolaner based chewables (NexGard® or NexGard Spectra®) for the treatment of generalised demodicosis caused by Demodex canis in dogs under field conditions in France, Italy and Poland., Methods: Client-owned dogs, diagnosed positive for Demodex mites by pre-treatment skin scrapings and presenting clinical signs of generalised demodicosis were included. Dogs were orally treated with afoxolaner three times at monthly intervals. Of the 50 dogs enrolled, 48 completed the whole study. Efficacy of the treatments was assessed monthly by Demodex mite counts and physical examination with special regard to the severity and extension of skin lesions., Results: Treatments were well tolerated in all dogs and resulted in a rapid reduction of mites, with all post-treatment mite counts significantly lower than baseline. The number of mites was reduced by 87.6%, 96.5% and 98.1% on Days 28, 56 and 84, respectively. In addition, the skin lesion severity and extent scores as well as the pruritus were all significantly lower at all post-treatment visits compared to the pre-treatment assessment., Conclusions: This clinical field study demonstrated that monthly administrations of afoxolaner in NexGard® or NexGard Spectra®, offered a convenient and reliable solution for the treatment of canine generalised demodicosis.

The aim of this study was the evaluation of the safety and efficacy of unilateral subretinal injection of the adeno-associated vector (AAV) serotypes 2 and 4 (AAV2/4) RPE65-RPE65 vector in patients with Leber congenital amaurosis (LCA) associated with RPE65 gene deficiency. We evaluated ocular and general tolerance and visual function up to 1 year after vector administration in the most severely affected eye in nine patients with retinal degeneration associated with mutations in the RPE65 gene. Patients received either low (1.22 × 10 10 to 2 × 10 10 vector genomes [vg]) or high (between 3.27 × 10 10 and 4.8 × 10 10 vg) vector doses. An ancillary study, in which six of the original nine patients participated, extended the follow-up period to 2-3.5 years. All patients showed good ophthalmological and general tolerance to the rAAV2/4-RPE65-RPE65 vector. We observed a trend toward improved visual acuity in patients with nystagmus, stabilization and improvement of the visual field, and cortical activation along visual pathways during fMRI analysis. OCT analysis after vector administration revealed no retinal thinning, except in cases of macular detachment. Our findings show that the rAAV2/4.RPE65.RPE65 vector was well tolerated in nine patients with RPE65-associated LCA. Efficacy parameters varied between patients during follow-up., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The genetic relationships between behavior and digestive efficiency were studied in 860 chickens from a cross between two lines divergently selected on digestive efficiency. At 2 weeks of age each chick was video-recorded in the home pen to characterize general activity and feeding behavior. Tonic immobility and open-field tests were also carried out individually to evaluate emotional reactivity (i.e. the propensity to express fear responses). Digestive efficiency was measured at 3 weeks. Genetic parameters of behavior traits were estimated. Birds were genotyped on 3379 SNP markers to detect QTLs. Heritabilities of behavioral traits were low, apart from tonic immobility (0.17-0.18) and maximum meal length (0.14). The genetic correlations indicated that the most efficient birds fed more frequently and were less fearful. We detected 14 QTL (9 for feeding behavior, 3 for tonic immobility, 2 for frequency of lying). Nine of them co-localized with QTL for efficiency, anatomy of the digestive tract, feed intake or microbiota composition. Four genes involved in fear reactions were identified in the QTL for tonic immobility on GGA1.

Background: The development of alternative approaches in ectoparasite management is currently required. Essential oils have been demonstrated to exhibit fumigant and topical toxicity to a number of arthropods. The aim of the present study was to assess the potential efficacy of ten essential oils against Sarcoptes scabiei., Methods: The major chemical components of the oils were identified by GC-MS analysis. Contact and fumigation bioassays were performed on Sarcoptes mites collected from experimentally infected pigs. For contact bioassays, essential oils were diluted with paraffin to get concentrations at 10, 5, and even 1% for the most efficient ones. The mites were inspected under a stereomicroscope 10, 20, 30, 40, 50, 60, 90, 120, 150, and 180min after contact. For fumigation bioassay, a filter paper was treated with 100 μL of the pure essential oil. The mites were inspected under a stereomicroscope for the first 5min, and then every 5min until 1h., Results: Using contact bioassays, 1% clove and palmarosa oil killed all the mites within 20 and 50min, respectively. The oils efficacy order was: clove > palmarosa > geranium > tea tree > lavender > manuka > bitter orange > eucalyptus > Japanese cedar. In fumigation bioassays, the efficacy order was: tea tree > clove > eucalyptus > lavender > palmarosa > geranium > Japanese cedar > bitter orange > manuka. In both bioassays, cade oil showed no activity., Conclusion: Essential oils, especially tea tree, clove, palmarosa, and eucalyptus oils, are potential complementary or alternative products to treat S. scabiei infections in humans or animals, as well as to control the mites in the environment.

Skeletal integrity in meat-type chickens is affected by many factors including rapid growth rate, nutrition and genetics. To investigate the genetic basis of bone and mineral metabolism, a QTL detection study was conducted in an intercross between two lines of meat-type chickens divergently selected for their high (D +) or low (D -) digestive efficiency. Tibia size (length, diameter, volume) and ash content were determined at 3 weeks of age as well as phosphorus (P) retention and plasma concentration. Heritability of these traits and their genetic correlations with digestive efficiency were estimated. A QTL mapping study was performed using 3379 SNP markers. Tibia size, weight, ash content and breaking strength were highly heritable (0.42 to 0.61). Relative tibia diameter and volume as well as P retention were strongly and positively genetically correlated with digestive efficiency (0.57 to 0.80). A total of 35 QTL were identified (9 for tibia weight, 13 for tibia size, 5 for bone strength, 5 for bone mineralization, 2 for plasma P concentration and 1 for P retention). Six QTL were genome-wide significant, and 3 QTL for tibia relative volume, weight and ash weight on chromosome 6 were fixed, the positive allele coming from the D-line. For two QTL for ash content on chromosome 18 and relative tibia length on chromosome 26, the confidence intervals were small enough to identify potential candidate genes. These findings support the evidence of multiple genetic loci controlling bone and mineral metabolism. The identification of candidate genes may provide new perspectives in the understanding of bone regulation, even beyond avian species.

Background: Improving feed efficiency is a major goal in poultry production in order to reduce production costs, increase the possibility of using alternative feedstuffs and decrease the volume of manure. However, in spite of their economic and environmental impact, very few quantitative trait loci (QTL) have been reported on these traits. Thus, we undertook the detection of QTL on 820 meat-type chickens from a F2 cross between D- and D+ lines that were divergently selected on low or high digestive efficiency at 3 weeks of age. Birds were measured for growth between 0 and 23 days, feed intake and feed conversion ratio between 9 and 23 days, breast and abdominal fat yields at 23 days, and the anatomy of their digestive tract (density, relative weight and length of the duodenum, jejunum, ileum, and ratio of proventriculus to gizzard weight) was examined. To evaluate excretion traits, fresh and dry weight, water content, pH, nitrogen to phosphorus ratio from 0 to 23 days, and pH of gizzard and jejunum contents at 23 days were measured. A set of 3379 single nucleotide polymorphisms distributed on 28 Gallus gallus (GGA) autosomes, the Z chromosome and one unassigned linkage group was used for QTL detection., Results: Using the QTLMap software developed for linkage analyses by interval mapping, we detected 16 QTL for feed intake, 13 for feed efficiency, 49 for anatomy-related traits, seven for growth, six for body composition and ten for excretion. Nine of these QTL were genome-wide significant (four for feed intake on GGA1, one for feed efficiency on GGA2, and four for anatomy on GGA1, 2, 3 and 4). GGA16, 19, and 26 carried many QTL for different types of traits that co-localize at the same position., Conclusions: This study identified several QTL regions that are involved in the control of digestive efficiency in chicken. Further studies are needed to identify the genes that underlie these effects, and to validate these in other commercial populations and for different breeding environments.

Objectives: Feed efficiency and its digestive component, digestive efficiency, are key factors in the environmental impact and economic output of poultry production. The interaction between the host and intestinal microbiota has a crucial role in the determination of the ability of the bird to digest its food and to the birds' feed efficiency. We therefore investigated the phenotypic and genetic relationships between birds' efficiency and the composition of the cecal microbiota in a F2 cross between broiler lines divergently selected for their high or low digestive efficiency., Methods: Analyses were performed on 144 birds with extreme feed efficiency values at 3 weeks, with feed conversion values of 1.41±0.05 and 2.02±0.04 in the efficient and non-efficient groups, respectively. The total numbers of Lactobacillus, L. salivarius, L. crispatus, C. coccoides, C. leptum and E. coli per gram of cecal content were measured., Results: The two groups mainly differed in larger counts of Lactobacillus, L. salivarius and E. coli in less efficient birds. The equilibrium between bacterial groups was also affected, efficient birds showing higher C. leptum, C. coccoides and L. salivarius to E. coli ratios. The heritability of the composition of microbiota was also estimated and L. crispatus, C. leptum, and C. coccoides to E. coli ratios were moderately but significantly heritable (0.16 to 0.24). The coefficient of fecal digestive use of dry matter was genetically and positively correlated with L. crispatus, C. leptum, C. coccoides (0.50 to 0.76) and negatively with E. coli (-0.66). Lipid digestibility was negatively correlated with E. coli (-0.64), and AMEn positively correlated with C. coccoides and with the C. coccoides to Lactobacillus ratio (0.48 to 0.64). We also detected 14 Quantitative Trait Loci (QTL) for microbiota on the host genome, mostly on C. leptum and Lactobacillus. The QTL for C. leptum on GGA6 was close to genome-wide significance. This region mainly includes genes involved in anti-inflammatory responses and in the motility of the gastrointestinal tract.

Abstract Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.

Background: Improving digestive efficiency is a major goal in poultry production, to reduce production costs, make possible the use of alternative feedstuffs and decrease the volume of manure produced. Since measuring digestive efficiency is difficult, identifying molecular markers associated with genes controlling this trait would be a valuable tool for selection. Detection of QTL (quantitative trait loci) was undertaken on 820 meat-type chickens in a F2 cross between D- and D+ lines divergently selected on low or high AMEn (apparent metabolizable energy value of diet corrected to 0 nitrogen balance) measured at three weeks in animals fed a low-quality diet. Birds were measured for 13 traits characterizing digestive efficiency (AMEn, coefficients of digestive utilization of starch, lipids, proteins and dry matter (CDUS, CDUL, CDUP, CDUDM)), anatomy of the digestive tract (relative weights of the proventriculus, gizzard and intestine and proventriculus plus gizzard (RPW, RGW, RIW, RPGW), relative length and density of the intestine (RIL, ID), ratio of proventriculus and gizzard to intestine weight (PG/I); and body weight at 23 days of age. Animals were genotyped for 6000 SNPs (single nucleotide polymorphisms) distributed on 28 autosomes, the Z chromosome and one unassigned linkage group., Results: Nine QTL for digestive efficiency traits, 11 QTL for anatomy-related traits and two QTL for body weight at 23 days of age were detected. On chromosome 20, two significant QTL at the genome level co-localized for CDUS and CDUDM, i.e. two traits that are highly correlated genetically. Moreover, on chromosome 16, chromosome-wide QTL for AMEn, CDUS, CDUDM and CDUP, on chromosomes 23 and 26, chromosome-wide QTL for CDUS, on chromosomes 16 and 26, co-localized QTL for digestive efficiency and the ratio of intestine length to body weight and on chromosome 27 a chromosome-wide QTL for CDUDM were identified., Conclusions: This study identified several regions of the chicken genome involved in the control of digestive efficiency. Further studies are necessary to identify the underlying genes and to validate these in commercial populations and breeding environments.

The bacterium Flavobacterium psychrophilum is a serious problem for salmonid farming worldwide. This study investigates by multilocus sequence typing (MLST) the population structure of this pathogen in Japan where it is also a major concern for ayu, a popular game fish related to salmoniforms. A total of 34 isolates collected across the country and 80 isolates sampled in a single model river by electrofishing were genotyped. The data accounting for 15 fish species allowed identifying 35 distinct sequence types (ST) in Japan. These ST are distinct from those reported elsewhere, except for some ST found in rainbow trout and coho salmon, two fish that have been the subject of intensive international trade. The pattern of polymorphism is, however, strikingly similar across geographical scales (model river, Japan, world) in terms of the fraction of molecular variance linked to the fish host (~50%) and of pairwise nucleotide diversity between ST (~5 Kbp(-1)). These observations go against the hypothesis of a recent introduction of F. psychrophilum in Japan. Two findings were made that are important for disease control: 1) at least two independent F. psychrophilum lineages infect ayu and 2) co-infections of the same individual fish by different strains occur.

Polled and Multisystemic Syndrome (PMS) is a novel developmental disorder occurring in the progeny of a single bull. Its clinical spectrum includes polledness (complete agenesis of horns), facial dysmorphism, growth delay, chronic diarrhea, premature ovarian failure, and variable neurological and cardiac anomalies. PMS is also characterized by a deviation of the sex-ratio, suggesting male lethality during pregnancy. Using Mendelian error mapping and whole-genome sequencing, we identified a 3.7 Mb deletion on the paternal bovine chromosome 2 encompassing ARHGAP15, GTDC1 and ZEB2 genes. We then produced control and affected 90-day old fetuses to characterize this syndrome by histological and expression analyses. Compared to wild type individuals, affected animals showed a decreased expression of the three deleted genes. Based on a comparison with human Mowat-Wilson syndrome, we suggest that deletion of ZEB2, is responsible for most of the effects of the mutation. Finally sperm-FISH, embryo genotyping and analysis of reproduction records confirmed somatic mosaicism in the founder bull and male-specific lethality during the first third of gestation. In conclusion, we identified a novel locus involved in bovid horn ontogenesis and suggest that epithelial-to-mesenchymal transition plays a critical role in horn bud differentiation. We also provide new insights into the pathogenicity of ZEB2 loss of heterozygosity in bovine and humans and describe the first case of male-specific lethality associated with an autosomal locus in a non-murine mammalian species. This result sets PMS as a unique model to study sex-specific gene expression/regulation.

The fur of common rabbits is constituted of 3 types of hair differing in length and diameter while that of rex animals is essentially made up of amazingly soft down-hair. Rex short hair coat phenotypes in rabbits were shown to be controlled by three distinct loci. We focused on the "r1" mutation which segregates at a simple autosomal-recessive locus in our rabbit strains. A positional candidate gene approach was used to identify the rex gene and the corresponding mutation. The gene was primo-localized within a 40 cM region on rabbit chromosome 14 by genome scanning families of 187 rabbits in an experimental mating scheme. Then, fine mapping refined the region to 0.5 cM (Z = 78) by genotyping an additional 359 offspring for 94 microsatellites present or newly generated within the first defined interval. Comparative mapping pointed out a candidate gene in this 700 kb region, namely LIPH (Lipase Member H). In humans, several mutations in this major gene cause alopecia, hair loss phenotypes. The rabbit gene structure was established and a deletion of a single nucleotide was found in LIPH exon 9 of rex rabbits (1362delA). This mutation results in a frameshift and introduces a premature stop codon potentially shortening the protein by 19 amino acids. The association between this deletion and the rex phenotype was complete, as determined by its presence in our rabbit families and among a panel of 60 rex and its absence in all 60 non-rex rabbits. This strongly suggests that this deletion, in a homozygous state, is responsible for the rex phenotype in rabbits.

A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 x 10¹¹ particles/ml; 95% confidence interval [CI], 7.89 x 10¹¹ to 1.05 x 10¹² particles/ml), vector genomes ({X}, 3.28 x 10¹⁰ vector genomes/ml; 95% CI, 2.70 x 10¹⁰ to 4.75 x 10¹⁰ vector genomes/ml), transducing units ({X}, 5.09 x 10⁸ transducing units/ml; 95% CI, 2.00 x 10⁸ to 9.60 x 10⁸ transducing units/ml), and infectious units ({X}, 4.37 x 10⁹ TCID₅₀ IU/ml; 95% CI, 2.06 x 10⁹ to 9.26 x 10⁹ TCID₅₀ IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.

Background: Aminopeptidase B (Ap-B; EC 3.4.11.6) catalyzes the cleavage of basic residues at the N-terminus of peptides and processes glucagon into miniglucagon. The enzyme exhibits, in vitro, a residual ability to hydrolyze leukotriene A4 into the pro-inflammatory lipid mediator leukotriene B4. The potential bi-functional nature of Ap-B is supported by close structural relationships with LTA4 hydrolase (LTA4H ; EC 3.3.2.6). A structure-function analysis is necessary for the detailed understanding of the enzymatic mechanisms of Ap-B and to design inhibitors, which could be used to determine the complete in vivo functions of the enzyme., Results: The rat Ap-B cDNA was expressed in E. coli and the purified recombinant enzyme was characterized. 18 mutants of the H325EXXHX18E348 Zn2+-binding motif were constructed and expressed. All mutations were found to abolish the aminopeptidase activity. A multiple alignment of 500 sequences of the M1 family of aminopeptidases was performed to identify 3 sub-families of exopeptidases and to build a structural model of Ap-B using the x-ray structure of LTA4H as a template. Although the 3D structures of the two enzymes resemble each other, they differ in certain details. The role that a loop, delimiting the active center of Ap-B, plays in discriminating basic substrates, as well as the function of consensus motifs, such as RNP1 and Armadillo domain are discussed. Examination of electrostatic potentials and hydrophobic patches revealed important differences between Ap-B and LTA4H and suggests that Ap-B is involved in protein-protein interactions., Conclusion: Alignment of the primary structures of the M1 family members clearly demonstrates the existence of different sub-families and highlights crucial residues in the enzymatic activity of the whole family. E. coli recombinant enzyme and Ap-B structural model constitute powerful tools for investigating the importance and possible roles of these conserved residues in Ap-B, LTA4H and M1 aminopeptidase catalytic sites and to gain new insight into their physiological functions. Analysis of Ap-B structural model indicates that several interactions between Ap-B and proteins can occur and suggests that endopeptidases might form a complex with Ap-B during hormone processing.