Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice

<p>Abstract</p> <p>Background</p> <p>Inducible conditional knockout animals are widely used to get insight in the function of genes and the pathogenesis of human diseases. These models frequently rely on Cre-mediated recombination of sequences flanked by Lox-P sites. To understand the consequences of gene disruption, it is essential to know the efficiency of the recombination process.</p> <p>Results</p> <p>Here, we describe a modification of the multiplex ligation-dependent probe amplification (MLPA), called extension-MLPA (eMLPA), which enables quantification of relatively small differences in DNA that are a consequence of Cre-mediated recombination. eMLPA, here applied on an inducible <it>Pkd1 </it>conditional deletion mouse model, simultaneously measures both the reduction of the floxed allele and the increase of the deletion allele in a single reaction thereby minimizing any type of experimental variation. Interestingly, with this method we were also able to observe the presence of the excised DNA fragment. This extra-chromosomal deletion-circle was detectable up to 5 months after activation of Cre.</p> <p>Conclusion</p> <p>eMLPA is a novel strategy which easily can be applied to measure the Cre-mediated recombination efficiency in each experimental case with high accuracy. In addition the fate of the deletion-circle can be followed simultaneously.</p

High Detection Rates of Enteropathogens in Asymptomatic Children Attending Day Care

BACKGROUND: Gastroenteritis morbidity is high among children under the age of four, especially amongst those who attend day care. OBJECTIVE: To determine the prevalence of a range of enteropathogens in the intestinal flora of children attending day care and to relate their occurrence with characteristics of the sampled child and the sampling season. METHODS: We performed three years of enteropathogen surveillance in a network of 29 child day care centers in the Netherlands. The centers were instructed to take one fecal sample from ten randomly chosen children each month, regardless of gastrointestinal symptoms at time of sampling. All samples were analyzed for the molecular detection of 16 enteropathogenic bacteria, parasites and viruses by real-time multiplex PCR. RESULTS: Enteropathogens were detected in 78.0% of the 5197 fecal samples. Of the total, 95.4% of samples were obtained from children who had no gastroenteritis symptoms at time of sampling. Bacterial enteropathogens were detected most often (most prevalent EPEC, 19.9%), followed by parasitic enteropathogens (most prevalent: D. fragilis, 22.1%) and viral enteropathogens (most prevalent: norovirus, 9.5%). 4.6% of samples related to children that experienced symptoms of gastroenteritis at time of sampling. Only rotavirus and norovirus were significantly associated with gastroenteritis among day care attendees. CONCLUSIONS: Our study indicates that asymptomatic infections with enteropathogens in day care attendees are not a rare event and that gastroenteritis caused by infections with these enteropathogens is only one expression of their presence

Aromatic Features in AGN: Star-Forming Infrared Luminosity Function of AGN Host Galaxies

We describe observations of aromatic features at 7.7 and 11.3 um in AGN of three types including PG, 2MASS and 3CR objects. The feature has been demonstrated to originate predominantly from star formation. Based on the aromatic-derived star forming luminosity, we find that the far-IR emission of AGN can be dominated by either star formation or nuclear emission; the average contribution from star formation is around 25% at 70 and 160 um. The star-forming infrared luminosity functions of the three types of AGN are flatter than that of field galaxies, implying nuclear activity and star formation tend to be enhanced together. The star-forming luminosity function is also a function of the strength of nuclear activity from normal galaxies to the bright quasars, with luminosity functions becoming flatter for more intense nuclear activity. Different types of AGN show different distributions in the level of star formation activity, with 2MASS> PG> 3CR star formation rates.Comment: Accepted for publication in ApJ, 24 pages, 13 figure

Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014.

Leishmaniasis is endemic in southern Europe, and in other European countries cases are diagnosed in travellers who have visited affected areas both within the continent and beyond. Prompt and accurate diagnosis poses a challenge in clinical practice in Europe. Different methods exist for identification of the infecting Leishmania species. Sixteen clinical laboratories in 10 European countries, plus Israel and Turkey, conducted a study to assess their genotyping performance. DNA from 21 promastigote cultures of 13 species was analysed blindly by the routinely used typing method. Five different molecular targets were used, which were analysed with PCR-based methods. Different levels of identification were achieved, and either the Leishmania subgenus, species complex, or actual species were reported. The overall error rate of strains placed in the wrong complex or species was 8.5%. Various reasons for incorrect typing were identified. The study shows there is considerable room for improvement and standardisation of Leishmania typing. The use of well validated standard operating procedures is recommended, covering testing, interpretation, and reporting guidelines. Application of the internal transcribed spacer 1 of the rDNA array should be restricted to Old World samples, while the heat-shock protein 70 gene and the mini-exon can be applied globally

Limited effects of growth hormone replacement in patients with GH deficiency during long-term cure of acromegaly

The aim of this study was to assess the effects of replacement with recombinant human growth hormone (rhGH) in patients with GH deficiency (GHD) after treatment of acromegaly. Intervention study. Sixteen patients (8 men, age 56 years), treated for acromegaly by surgery and radiotherapy, with an insufficient GH response to insulin-induced hypoglycaemia, were treated with 1 year of rhGH replacement. Study parameters were assessed at baseline and after 1 year of rhGH replacement. Study parameters were cardiac function, body composition, bone mineral density (BMD), fasting lipids, glucose, bone turnover markers, and Quality of Life (QoL). During rhGH replacement IGF-I concentrations increased from −0.4 ± 0.7 to 1.0 ± 1.5 SD (P = 0.001), with a mean daily dose of 0.2 ± 0.1 mg in men and 0.3 ± 0.2 mg in women. Nonetheless, rhGH replacement did not alter cardiac function, lipid and glucose concentrations, body composition or QoL. Bone turnover markers (PINP and β crosslaps) levels increased (P = 0.005 and P = 0.021, respectively), paralleled by a small, but significant decrease in BMD of the hip. The beneficial effects of rhGH replacement in patients with GHD during cure from acromegaly are limited in this study

The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder that frequently results in renal fallure due to progressive cyst development. The major locus, PKD1, maps to 16p13.3. We identified a chromosome translocation associated with ADPKD that disrupts a gene (PBP) encoding a 14 kb transcript in the PKD1 candidate region. Further mutations of the PBP gene were found in PKD1 patients, two deletions (one a de novo event) and a splicing defect, confirming that PBP is the PKD1 gene. This gene is located adjacent to the TSC2 locus in a genomic region that is reiterated more proximally on 16p. The duplicate area encodes three transcripts substantially homologous to the PKD1 transcript. Partial sequence analysis of the PKD1 transcript shows that it encodes a novel protein whose function is at present unknown

Meedoen en erbij horen: Maatschappelijke participatie van jongeren met een licht verstandelijke beperking in de gemeente Utrecht

Het project Meedoen en erbij horen is gericht op de maatschappelijke participatie van jongeren met een licht verstandelijke beperking. De problematiek van jongeren met een (licht) verstandelijke beperking wordt dikwijls onderschat. Daardoor ontbreekt het soms aan adequate ondersteuning bij hun persoonlijke ontwikkeling. Participatie in school, werk en sociale contacten kan stagneren. Dit kan gepaard gaan met schooluitval, gedragsproblemen en verslaving. En daardoor dreigend sociaal isolement. Doel van het project was om elementen vast te stellen die jongeren met een licht verstandelijke beperking helpen om meer deel uit te maken van de samenleving op de terreinen opleiding, werk en sociale participatie in de wijk. De focus werd gelegd op de sociale en informationele toegankelijkheid voor de jongeren met een licht verstandelijke beperking. Dit wil zeggen de bruikbaarheid, begrijpbaarheid, bereikbaarheid en beschikbaarheid van het ondersteuningsaanbod (Nachtergaele et al. 2017). In de taal van de gemeente Utrecht gaat het dan om “toegang tot voorzieningen in de sociale basis en in de Wet maatschappelijke ondersteuning”. In samenspraak met de gemeente Utrecht en medewerkers van lokale zorg- en welzijnsorganisaties zijn drie onderzoeksvragen centraal gesteld: 1. Hoe kunnen jongvolwassenen bereikt en adequaat ondersteund worden? 2. Wat vraagt dit van professionals en de samenwerking tussen professionals? 3. Wat voor soort sociale infrastructuur is hiervoor in de wijk nodig

Environmental contamination with Toxocara spp. eggs in public parks and playground sandpits of Greater Lisbon, Portugal.

Toxocarosis is a zoonotic parasitic disease transmitted from companion animals to humans. Environmental contamination with Toxocara eggs is considered to be the main source of human infections. In Portugal, knowledge regarding the current situation, including density, distribution and environmental contamination by Toxocara spp., is largely unknown. The present study investigated environmental contamination with Toxocara spp. eggs, in soil and faecal samples collected from public parks and playground sandpits in Greater Lisbon, Portugal. A total of 151 soil samples and 135 canine faecal samples were collected from 7 public sandpits and 12 public parks, over a 4 month-period. Soil samples were tested by a modified centrifugation and sedimentation/flotation technique and faecal samples were tested by an adaptation of the Cornell-Wisconsin method. Molecular analysis and sequencing were performed to discriminate Toxocara species in the soil. Overall, 85.7% of the sandpits (6/7) and 50.0% of the parks (6/12) were contaminated with Toxocara spp. eggs. The molecular analysis of soil samples showed that, 85.5% of the sandpits and 34.4% of the parks were contaminated with Toxocara cati eggs. Faecal analysis showed that 12.5% of the sandpits and 3.9% of the parks contained Toxocara canis eggs. In total, 53.0% of soil and 5.9% of faecal samples were positive for Toxocara spp. Additionally, 56.0% of the eggs recovered from the samples were embryonated after 60 days of incubation, therefore considered viable and infective. The average density was 4.2 eggs per hundred grams of soil. Public parks and playground sandpits in the Lisbon area were found to be heavily contaminated with T. cati eggs, representing a serious menace to public health as the studied areas represent common places where people of all ages, particularly children, recreate. This study sounds an alarm bell regarding the necessity to undertake effective measures such as reduction of stray animals, active faecal collection by pet owners, awareness campaigns and control strategies to decrease the high risk to both animal and human health