Gain of 20q11.21 in human pluripotent stem cells impairs TGF-β-dependent neuroectodermal commitment

Gain of 20q11.21 is one of the most common recurrent genomic aberrations in human pluripotent stem cells. Although it is known that overexpression of the antiapoptotic gene Bcl-xL confers a survival advantage to the abnormal cells, their differentiation capacity has not been fully investigated. RNA sequencing of mutant and control hESC lines, and a line transgenically overexpressing Bcl-xL, shows that overexpression of Bcl-xL is sufficient to cause most transcriptional changes induced by the gain of 20q11.21. Moreover, the differentially expressed genes in mutant and Bcl-xL overexpressing lines are enriched for genes involved in TGF-beta- and SMAD-mediated signaling, and neuron differentiation. Finally, we show that this altered signaling has a dramatic negative effect on neuroectodermal differentiation, while the cells maintain their ability to differentiate to mesendoderm derivatives. These findings stress the importance of thorough genetic testing of the lines before their use in research or the clinic

Trigonometric Parallaxes of Central Stars of Planetary Nebulae

Trigonometric parallaxes of 16 nearby planetary nebulae are presented, including reduced errors for seven objects with previous initial results and results for six new objects. The median error in the parallax is 0.42 mas, and twelve nebulae have parallax errors less than 20 percent. The parallax for PHL932 is found here to be smaller than was measured by Hipparcos, and this peculiar object is discussed. Comparisons are made with other distance estimates. The distances determined from these parallaxes tend to be intermediate between some short distance estimates and other long estimates; they are somewhat smaller than estimated from spectra of the central stars. Proper motions and tangential velocities are presented. No astrometric perturbations from unresolved close companions are detected.Comment: 24 pages, includes 4 figures. Accepted for A

Otx2 requires Lmx1b to control the development of mesodiencephalic dopaminergic neurons

Studying the development of mesodiencephalic dopaminergic (mdDA) neurons provides an important basis for better understanding dopamine-associated brain functions and disorders and is critical for establishing cell replacement therapy for Parkinson's disease. The transcription factors Otx2 and Lmx1b play a key role in the development of mdDA neurons. However, little is known about the genes downstream of Otx2 and Lmx1b in the pathways controlling the formation of mdDA neurons in vivo. Here we report on our investigation of Lmx1b as downstream target of Otx2 in the formation of mdDA neurons. Mouse mutants expressing Otx2 under the control of the En1 promoter (En1+/Otx2) showed increased Otx2 expression in the mid-hindbrain region, resulting in upregulation of Lmx1b and expansion of mdDA neurons there. In contrast, Lmx1b-/- mice showed decreased expression of Otx2 and impairments in several aspects of mdDA neuronal formation. To study the functional interaction between Otx2 and Lmx1b, we generated compound mutants in which Otx2 expression was restored in mice lacking Lmx1b (En1+/Otx2;Lmx1b-/-). In these animals Otx2 was not sufficient to rescue any of the aberrations in the formation of mdDA neurons caused by the loss of Lmx1b, but rescued the loss of ocular motor neurons. Gene expression studies in Lmx1b-/- embryos indicated that in these mutants Wnt1, En1 and Fgf8 expression are induced but subsequently lost in the mdDA precursor domain and the mid-hindbrain organizer in a specific, spatio-temporal manner. In summary, we demonstrate that Otx2 critically depends on Lmx1b for the formation of mdDA neurons, but not for the generation of ocular motor neurons. Moreover, our data suggest that Lmx1b precisely maintains the expression pattern of Wnt1, Fgf8 and En1, which are essential for mid-hindbrain organizer function and the formation of mdDA neurons

BMP/SMAD pathway promotes neurogenesis of midbrain dopaminergic neurons in vivo and in human induced pluripotent and neural stem cells

The embryonic formation of midbrain dopaminergic (mDA) neurons in vivo provides critical guidelines for the in vitro differentiation of mDA neurons from stem cells, which are currently being developed for Parkinson’s disease cell replacement therapy. Bone morphogenetic protein (BMP)/SMAD inhibition is routinely used during early steps of stem cell differentiation protocols, including for the generation of mDA neurons. However, the function of the BMP/SMAD pathway for in vivo specification of mammalian mDA neurons is virtually unknown. Here, we report that BMP5/7-deficient mice (Bmp5 -/- ; Bmp7 -/- ) lackmDAneurons due to reduced neurogenesis in the mDA progenitor domain. As molecular mechanisms accounting for these alterations in Bmp5 -/- ; Bmp7 -/- mutants, we have identified expression changes of the BMP/SMAD target genes MSX1/2 (msh homeobox 1/2) and SHH (sonic hedgehog). Conditionally inactivatingSMAD1in neural stem cells of mice in vivo (Smad1 Nes ) hampered the differentiation of progenitor cells intomDAneurons by preventing cell cycle exit, especially of TH + SOX6 + (tyrosine hydroxylase, SRY-box 6) and TH + GIRK2 + (potassium voltage-gated channel subfamily-J member-6) substantia nigra neurons. BMP5/7 robustly increased the in vitro differentiation of human induced pluripotent stem cells and induced neural stem cells to mDA neurons by up to threefold. In conclusion, we have identified BMP/SMAD signaling as a novel critical pathway orchestrating essential steps of mammalian mDA neurogenesis in vivo that balances progenitor proliferation and differentiation. Moreover, we demonstrate the potential of BMPs to improve the generation of stem-cell-derived mDA neurons in vitro, highlighting the importance of sequential BMP/SMAD inhibition and activation in this process

Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions

Trigonometric Parallaxes for Two Late-Type Subdwarfs: LSR1425+71 (sdM8.0) and the Binary LSR1610-00 (sd?M6pec)

Trigonometric parallax astrometry and BVI photometry are presented for two late-type subdwarf candidates, LSR1425+71 (sdM8.0) and LSR1610-00 (sd?M6pec). For the former we measure an absolute parallax of 13.37+/-0.51 mas yielding Mv=15.25+/-0.09. The astrometry for LSR1610-00 shows that this object is an astrometric binary with a period of 1.66+/-0.01 yr. The photocentric orbit is derived from the data; it has a moderate eccentricity (e ~ 0.44+/-0.02) and a semi-major axis of 0.28+/-0.01 AU based on our measured absolute parallax of 31.02+/-0.26 mas. Our radial velocity measure of -108.1+/-1.6 km/s for LSR1610-00 at epoch 2006.179, when coupled with the observation of -95+/-1 km/s at epoch 2005.167 by Reiners & Basri, indicates a systemic radial velocity of -101+/-1 km/s for the LSR1610-00AB pair. The galactic velocity components for LSR1425+71 and LSR1610-00AB -- (U,V,W)=(84+/-6, -202+/-13, 66+/-14) km/s and (U,V,W)=(36+/-2, -232+/-2, -61+/-2) km/s, respectively. For both stars, the velocities are characteristic of halo population kinematics. However, modeling shows that both stars have orbits around the galaxy with high eccentricity that pass remarkably close to the galactic center. LSR1425+71 has a luminosity and colors consistent with its metal-poor subdwarf spectral classification, while LSR1610-00 has a luminosity and most colors indicative of being only mildly metal-poor, plus a uniquely red B-V color. The companion to LSR1610-00 must be a low-mass, substellar brown dwarf. We speculate on the paradoxical nature of LSR1610-00 and possible sources of its peculiarities.Comment: Accepted for ApJ. 37 pages, including 8 figure