Recent studies highlighting mesenchymal stem cell (MSC) epigenetic memory suggest that a different differentiation medium may be required depending on the tissue of origin. As synovial-derived stem ...cells (SDSCs) attract interest we aimed to investigate the influence of TGF-β1, BMP-2 and dexamethasone on SDSC chondrogenesis in vitro. We demonstrate that dexamethasone-free medium led to enhanced chondrogenic differentiation at both the mRNA and matrix level. The greatest
/
ratio was detected in cells exposed to a combination medium containing 10 ng/mL BMP-2 and 1 ng/mL TGF-β1 in the absence of dexamethasone, and this was reflected in the total amount of glycosaminoglycans produced. In summary, dexamethasone-free medium containing BMP-2 and TGF-β1 may be the most suitable when using SDSCs for cartilage tissue regeneration.
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An experiment (E166) at the Stanford Linear Accelerator Center has demonstrated a scheme in which a multi-GeV electron beam passed through a helical undulator to generate multi-MeV, circularly ...polarized photons which were then converted in a thin target to produce positrons (and electrons) with longitudinal polarization above 80% at 6 MeV. The results are in agreement with GEANT4 simulations that include the dominant polarization-dependent interactions of electrons, positrons, and photons in matter.
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CMK, CTK, FMFMET, IJS, NUK, PNG, UM
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
4.
Undulator-based production of polarized positrons Alexander, G.; Barley, J.; Batygin, Y. ...
Nuclear instruments & methods in physics research. Section A, Accelerators, spectrometers, detectors and associated equipment,
11/2009, Volume:
610, Issue:
2
Journal Article
Peer reviewed
Open access
Full exploitation of the physics potential of a future International Linear Collider will require the use of polarized electron and positron beams. Experiment E166 at the Stanford Linear Accelerator ...Center (SLAC) has demonstrated a scheme in which an electron beam passes through a helical undulator to generate photons (whose first-harmonic spectrum extended to 7.9
MeV) with circular polarization, which are then converted in a thin target to generate longitudinally polarized positrons and electrons. The experiment was carried out with a 1-m-long, 400-period, pulsed helical undulator in the Final Focus Test Beam (FFTB) operated at 46.6
GeV. Measurements of the positron polarization have been performed at five positron energies from 4.5 to 7.5
MeV. In addition, the electron polarization has been determined at 6.7
MeV, and the effect of operating the undulator with a ferrofluid was also investigated. To compare the measurements with expectations, detailed simulations were made with an upgraded version of G
eant4
that includes the dominant polarization-dependent interactions of electrons, positrons, and photons with matter. The measurements agree with calculations, corresponding to 80% polarization for positrons near 6
MeV and 90% for electrons near 7
MeV.
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A proof-of-principle experiment has been carried out in the Final Focus Test Beam (FFTB) at SLAC to demonstrate production of polarized positrons in a manner suitable for implementation at the ILC. A ...helical undulator of 2.54 mm period and 1-m length produced circularly polarized photons of first harmonic endpoint energy of 8 MeV when traversed by a 46.6 GeV electron beam. The polarized photons were converted to polarized positrons in a 0.2-radiation-length tungsten target. The polarization of these positrons was measured at several energies, with a peak value in excess of 80%, by transmission polarimetry of photons obtained on reconversion of the positrons in a second tungsten target.
Astrocytic volume regulation and neurotransmitter uptake are critically dependent on the intracellular anion concentration, but little is known about the mechanisms controlling internal anion ...homeostasis in these cells. Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride‐sensitive dye MQAE to measure intracellular chloride concentrations in murine Bergmann glial cells in acute cerebellar slices. We found Bergmann glial Cl−int to be controlled by two opposing transport processes: chloride is actively accumulated by the Na+‐K+‐2Cl− cotransporter NKCC1, and chloride efflux through anion channels associated with excitatory amino acid transporters (EAATs) reduces Cl−int to values that vary upon changes in expression levels or activity of these channels. EAATs transiently form anion‐selective channels during glutamate transport, and thus represent a class of ligand‐gated anion channels. Age‐dependent upregulation of EAATs results in a developmental chloride switch from high internal chloride concentrations (51.6 ± 2.2 mM, mean ± 95% confidence interval) during early development to adult levels (35.3 ± 0.3 mM). Simultaneous blockade of EAAT1/GLAST and EAAT2/GLT‐1 increased Cl−int in adult glia to neonatal values. Moreover, EAAT activation by synaptic stimulations rapidly decreased Cl−int. Other tested chloride channels or chloride transporters do not contribute to Cl−int under our experimental conditions. Neither genetic removal of ClC‐2 nor pharmacological block of K+‐Cl− cotransporter change resting Bergmann glial Cl−int in acute cerebellar slices. We conclude that EAAT anion channels play an important and unexpected role in adjusting glial intracellular anion concentration during maturation and in response to cerebellar activity. GLIA 2017;65:388–400
Main Points
Bergmann glial intracellular chloride concentrations were determined in acute murine cerebellar slices using fluorescence lifetime imaging (FLIM) with MQAE.
Chloride is accumulated by NKCC1, whereas EAAT anion channels reduce Cl−int.
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Striated muscle responds to mechanical overload by rapidly up-regulating the expression of the cardiac ankyrin repeat protein, CARP, which then targets the sarcomere by binding to ...titin N2A in the I-band region. To date, the role of this interaction in the stress response of muscle remains poorly understood. Here, we characterise the molecular structure of the CARP-receptor site in titin (UN2A) and its binding of CARP. We find that titin UN2A contains a central three-helix bundle fold (ca 45 residues in length) that is joined to N- and C-terminal flanking immunoglobulin domains by long, flexible linkers with partial helical content. CARP binds titin by engaging an α-hairpin in the three-helix fold of UN2A, the C-terminal linker sequence, and the BC loop in Ig81, which jointly form a broad binding interface. Mutagenesis showed that the CARP/N2A association withstands sequence variations in titin N2A and we use this information to evaluate 85 human single nucleotide variants. In addition, actin co-sedimentation, co-transfection in C2C12 cells, proteomics on heart lysates, and the mechanical response of CARP-soaked myofibrils imply that CARP induces the cross-linking of titin and actin myofilaments, thereby increasing myofibril stiffness. We conclude that CARP acts as a regulator of force output in the sarcomere that preserves muscle mechanical performance upon overload stress.
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The
Escherichia coli peptide binding protein OppA is an essential component of the oligopeptide transporter Opp. Based on studies on its orthologue from
Salmonella typhimurium, it has been proposed ...that OppA binds peptides between two and five amino acids long, with no apparent sequence selectivity. Here, we studied peptide binding to
E. coli OppA directly and show that the protein has an unexpected preference for basic peptides. OppA was expressed in the periplasm, where it bound to available peptides. The protein was purified in complex with tightly bound peptides. The crystal structure (up to 2.0 Å) of OppA liganded with the peptides indicated that the protein has a preference for peptides containing a lysine. Mass spectrometry analysis of the bound peptides showed that peptides between two and five amino acids long bind to the protein and indeed hinted at a preference for positively charged peptides. The preference of OppA for peptides with basic residues, in particular lysines, was corroborated by binding studies with peptides of defined sequence using isothermal titration calorimetry and intrinsic protein fluorescence titration. The protein bound tripeptides and tetrapeptides containing positively charged residues with high affinity, whereas related peptides without lysines/arginines were bound with low affinity. A structure of OppA in an open conformation in the absence of ligands was also determined to 2.0 Å, revealing that the initial binding site displays a negative surface charge, consistent with the observed preference for positively charged peptides. Taken together,
E. coli OppA appears to have a preference for basic peptides.
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► The
E. coli peptide binding protein OppA is part of the oligopeptide permease Opp. ► The substrate preferences of OppA have been defined. ► OppA has a preference for positively charged peptides.
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