72 research outputs found
Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells
The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing the non-viral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4 and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed putative iPS cells showed long term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5 and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives of the three germ layers. Upon injection of putative iPS cells under the skin of immunodeficient mice, we observed teratomas in 3 of 6 cases. These results form the basis for in-depth studies towards the derivation of porcine iPS cells, which hold great promise for preclinical testing of novel cell therapies in the pig model
Association of Leptin Gene DNA Methylation With Diagnosis and Treatment Outcome of Anorexia Nervosa
Epigenetic alterations are increasingly implicated in the pathophysiology of anorexia nervosa (AN) but are as yet poorly understood. We investigated possible associations between the leptin gene (LEP) and the leptin receptor gene (LEPR) DNA promoter methylation and (1) a diagnosis of AN and (2) outcome after a 10 months psychotherapeutic outpatient treatment. 129 (LEPR: n = 135) patients with AN were investigated during the large scale psychotherapeutic Anorexia Nervosa Treatment Outpatient Study (ANTOP) trial, compared to 117 (LEPR: n = 119) age and height matched, normal-weight healthy controls. Blood samples were taken at baseline, the end of therapy (40 weeks) and the 12-months follow-up and compared to controls. Methylation was measured in whole blood via bisulfite sequencing. Within the promoter region 32 (LEP) and 39 CpG sites (LEPR) were analyzed. Two key findings were observed. First, LEP and LEPR methylation at baseline were lower in patients compared to controls (LEP: [%] AN: 30.94 ± 13.2 vs. controls: 34.53 ± 14.6); LEPR ([%] AN: 3.73 ± 5.4 vs. controls: 5.22 ± 8.3, mixed linear models: both P < 0.001). Second, lower DNA methylation of the LEP promoter, with a dynamic upregulation during treatment, was associated with a full recovery in AN patients (% change from baseline to follow-up in full recovery patients: +35.13% (SD: 47.56); mixed linear model: P < 0.0001). To test for potential predictive properties of mean LEP DNA methylation a LEP DNA methylation cut-off (31.25% DNA methylation) was calculated, which significantly discriminated full recovery vs. full syndrome AN patients. This cut-off was then tested in a group of previously unclassified patients (missing follow-up data of the Structured Interview for Anorexic and Bulimic disorders; n = 33). Patients below the cut-off (31.25% LEP DNA methylation) showed an increase in BMI over time, while those above the cut-off had a decrease in BMI (ANOVA at the 12-months follow-up: P = 0.0142). To our knowledge, this is the first study investigating epigenetic alterations in AN over time. Our findings indicate that LEP DNA methylation might be involved in the disease course of AN
Schmallenberg virus pathogenesis, tropism and interaction with the innate immune system of the host
Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV
Measurement of the cosmic ray spectrum above eV using inclined events detected with the Pierre Auger Observatory
A measurement of the cosmic-ray spectrum for energies exceeding
eV is presented, which is based on the analysis of showers
with zenith angles greater than detected with the Pierre Auger
Observatory between 1 January 2004 and 31 December 2013. The measured spectrum
confirms a flux suppression at the highest energies. Above
eV, the "ankle", the flux can be described by a power law with
index followed by
a smooth suppression region. For the energy () at which the
spectral flux has fallen to one-half of its extrapolated value in the absence
of suppression, we find
eV.Comment: Replaced with published version. Added journal reference and DO
Energy Estimation of Cosmic Rays with the Engineering Radio Array of the Pierre Auger Observatory
The Auger Engineering Radio Array (AERA) is part of the Pierre Auger
Observatory and is used to detect the radio emission of cosmic-ray air showers.
These observations are compared to the data of the surface detector stations of
the Observatory, which provide well-calibrated information on the cosmic-ray
energies and arrival directions. The response of the radio stations in the 30
to 80 MHz regime has been thoroughly calibrated to enable the reconstruction of
the incoming electric field. For the latter, the energy deposit per area is
determined from the radio pulses at each observer position and is interpolated
using a two-dimensional function that takes into account signal asymmetries due
to interference between the geomagnetic and charge-excess emission components.
The spatial integral over the signal distribution gives a direct measurement of
the energy transferred from the primary cosmic ray into radio emission in the
AERA frequency range. We measure 15.8 MeV of radiation energy for a 1 EeV air
shower arriving perpendicularly to the geomagnetic field. This radiation energy
-- corrected for geometrical effects -- is used as a cosmic-ray energy
estimator. Performing an absolute energy calibration against the
surface-detector information, we observe that this radio-energy estimator
scales quadratically with the cosmic-ray energy as expected for coherent
emission. We find an energy resolution of the radio reconstruction of 22% for
the data set and 17% for a high-quality subset containing only events with at
least five radio stations with signal.Comment: Replaced with published version. Added journal reference and DO
Measurement of the Radiation Energy in the Radio Signal of Extensive Air Showers as a Universal Estimator of Cosmic-Ray Energy
We measure the energy emitted by extensive air showers in the form of radio
emission in the frequency range from 30 to 80 MHz. Exploiting the accurate
energy scale of the Pierre Auger Observatory, we obtain a radiation energy of
15.8 \pm 0.7 (stat) \pm 6.7 (sys) MeV for cosmic rays with an energy of 1 EeV
arriving perpendicularly to a geomagnetic field of 0.24 G, scaling
quadratically with the cosmic-ray energy. A comparison with predictions from
state-of-the-art first-principle calculations shows agreement with our
measurement. The radiation energy provides direct access to the calorimetric
energy in the electromagnetic cascade of extensive air showers. Comparison with
our result thus allows the direct calibration of any cosmic-ray radio detector
against the well-established energy scale of the Pierre Auger Observatory.Comment: Replaced with published version. Added journal reference and DOI.
Supplemental material in the ancillary file
A Comparison of Shiga-Toxin 2 Bacteriophage from Classical Enterohemorrhagic Escherichia coli Serotypes and the German E. coli O104:H4 Outbreak Strain
Escherichia coli O104:H4 was associated with a severe foodborne disease outbreak originating in Germany in May 2011. More than 4000 illnesses and 50 deaths were reported. The outbreak strain was a typical enteroaggregative E. coli (EAEC) that acquired an antibiotic resistance plasmid and a Shiga-toxin 2 (Stx2)-encoding bacteriophage. Based on whole-genome phylogenies, the O104:H4 strain was most closely related to other EAEC strains; however, Stx2-bacteriophage are mobile, and do not necessarily share an evolutionary history with their bacterial host. In this study, we analyzed Stx2-bacteriophage from the E. coli O104:H4 outbreak isolates and compared them to all available Stx2-bacteriophage sequences. We also compared Stx2 production by an E. coli O104:H4 outbreak-associated isolate (ON-2011) to that of E. coli O157:H7 strains EDL933 and Sakai. Among the E. coli Stx2-phage sequences studied, that from O111:H- strain JB1-95 was most closely related phylogenetically to the Stx2-phage from the O104:H4 outbreak isolates. The phylogeny of most other Stx2-phage was largely concordant with their bacterial host genomes. Finally, O104:H4 strain ON-2011 produced less Stx2 than E. coli O157:H7 strains EDL933 and Sakai in culture; however, when mitomycin C was added, ON-2011 produced significantly more toxin than the E. coli O157:H7 strains. The Stx2-phage from the E. coli O104:H4 outbreak strain and the Stx2-phage from O111:H- strain JB1-95 likely share a common ancestor. Incongruence between the phylogenies of the Stx2-phage and their host genomes suggest the recent Stx2-phage acquisition by E. coli O104:H4. The increase in Stx2-production by ON-2011 following mitomycin C treatment may or may not be related to the high rates of hemolytic uremic syndrome associated with the German outbreak strain. Further studies are required to determine whether the elevated Stx2-production levels are due to bacteriophage or E. coli O104:H4 host related factors
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