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Multipotency of skeletal muscle stem cells on their native substrate and the expression of Connexin 43 during adoption of adipogenic and osteogenic fate
Skeletal muscle contains a resident stem cell population called Satellite cells (SC) which have a huge capacity to regenerate damaged tissue. Transplantation of a single fiber consisting of less than ten cells is able to generate tens of thousands of myonuclei within a matter of a few weeks (Collins et al., 2005). SC take their name from their peripheral position relative to the muscle fiber (Mauro, 1961). They are located under the basal lamina, in direct contact with the sarcolemma of muscle
fibers. In undamaged muscle, they are relatively metabolically inactive as indicated by their low cytoplasmic content and they exist in a quiescent state. However, they express certain markers including Pax7 that
aid their identification (Zammit et al., 2006). Upon muscle damage, SC become activated by inducing a number of genes including MyoD that encodes a member of the Myogenic Determination Factor family (MRF) of transcriptions factors (Zammit et al., 2006). Activation of SC permits cell division as well as migration. Initially, SC migrate under the basal lamina but then take up a supra-basal position by remodeling their overlying extra-cellular matrix (Otto et al., 2011). Activated SC can either revert to their quiescent state by down-regulating MyoD while maintaining Pax7 or can commit to myogenic differentiation by shutting off Pax7
expression and inducing the expression o
The Core Protein of Classical Swine Fever Virus Is Dispensable for Virus Propagation In Vitro
Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C). Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF) and bovine viral diarrhea (BVD). Recent findings describe the ability of NS3 of classical swine fever virus (CSFV) to compensate for disabling size increase of core protein (Riedel et al., 2010). NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008). These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS) within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447Δc), virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447) and Vp447Δc. Upon infection of the natural host, Vp447Δc was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general
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
Core Protein of Pestiviruses Is Processed at the C Terminus by Signal Peptide Peptidase
The core protein of pestiviruses is released from the polyprotein by viral and cellular proteinases. Here we report on an additional intramembrane proteolytic step that generates the C terminus of the core protein. C-terminal processing of the core protein of classical swine fever virus (CSFV) was blocked by the inhibitor (Z-LL)(2)-ketone, which is specific for signal peptide peptidase (SPP). The same effect was obtained by overexpression of the dominant-negative SPP D(265)A mutant. The presence of (Z-LL)(2)-ketone reduced the viability of CSFV almost 100-fold in a concentration-dependent manner. Reduction of virus viability was also observed in infection experiments using a cell line that inducibly expressed SPP D(265)A. The position of SPP cleavage was determined by C-terminal sequencing of core protein purified from virions. The C terminus of CSFV core protein is alanine(255) and is located in the hydrophobic center of the signal peptide. The intramembrane generation of the C terminus of the CSFV core protein is almost identical to the processing scheme of the core protein of hepatitis C viruses
Exosomes isolation and identification from equine mesenchymal stem cells
Abstract Background Mesenchymal stem cells are used for different therapeutic approaches, e.g. for osteoarthritis, lesions of the tendon as well as for bone defects. Current research on the mechanism of stem cells on the repair of damaged tissue suggest an important role of a cell-to-cell communication through secreted extracellular vesicles, mainly represented by exosomes. To enhance the scarce knowledge on the functional role of exosomes we compared as a first step different techniques to isolate and identify exosomes from the supernatant of equine adipose derived mesenchymal stem cells for further characterization and usage in functional assays. Results It was possible to obtain exosomes secreted from equine adipose derived mesenchymal stem cells with three common techniques: a stepwise ultracentrifugation at 100.000 g, an ultrafiltration with 3 kDa exclusion membranes and a charge-based precipitation method. The mean sizes and amounts of exosomes isolated with the different techniques were measured by the nanoparticle tracking analysis. The diameter ranged between 116.2 nm (ultracentrifugation), 453.1 nm (precipitation) and 178.7 nm (ultrafiltration), the counts of particles / ml ranged between 9.6 × 108 (ultracentrifugation), 2.02 × 109 (precipitation) and 52.5 × 109 (ultrafiltration). Relevant marker for exosomes, tetraspanins CD9, CD63 and CD81 were detectable by immunofluorescence staining of the investigated exosomes secreting mesenchymal stem cells. In addition, transmission electron microscopy and immunogold labeling with CD9 and CD90 was performed to display the morphological shape of exosomes and existence of marker relevant for exosomes (CD9) and mesenchymal stem cells (CD90). Western blot analysis of CD9 and CD90 of exosomes ensured the specificity of the rare available respectively cross reacting antibodies against equine antigens. Conclusion Exosomes generated by equine mesenchymal stem cells can be obtained by ultrafiltration and ultracentrifugation in an equal quality for in vitro experiments. Especially for later therapeutic usage we recommend ultrafiltration due to a higher concentration without aggregation of extracellular vesicles in comparison to exosomes obtained by ultracentrifugation
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