Inhibitory properties of the hI and s5A α₁AT variants.

a<p>Calculated using the total concentration of α₁AT.</p>b<p>Calculated using the fractional concentration of α₁AT that forms an inhibitory complex with the proteinase.</p>c<p>Taken from the genomic analysis performed by Irving <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054766#pone.0054766-Irving1" target="_blank">[34]</a>. Each experiment was performed three times. Errors included represent the standard deviation of each dataset.</p

Schematic representation of α₁AT.

<p>Ribbon diagram of α₁AT (Protein Data Bank ID: 1QLP) with hI and s5A shown in black. The insets show a close-up view of hI and s5A with their residue side chains shown as sticks. Mutations at positions 299, 301, 305 and 334 were not characterized in this study.</p

Representative traces for the unfolding reactions of wild type α₁AT and the variants which showed the most significant difference.

<p>(a) GdnHCl-induced equilibrium unfolding curves of wild type α₁AT (solid line), the V333A variant (dashed line) and the K335A variant (dotted line). Data were obtained by monitoring the change in CD signal at 222 nm, and represent the average of three separate experiments, while lines represent three-state curve fits. (b) Kinetic traces of α₁AT variants unfolding in 4 M GdnHCl. Wild type α₁AT is shown in solid black, the K335A variant in dotted black and the V333A variant in gray. The inset depicts the trace for the K555A variant over a longer time period. Traces were obtained by monitoring the change in bis-ANS fluorescence above 450 nm, and represent the best of 10 experiments. Data were fit to two separate single exponential equations, and analyzed in the Proviewer package supplied with the instrument.</p

Polymerization properties of the hI and s5A α₁AT variants.

<p>The polymerization kinetics were determined at 60°C by following the changes in bis-ANS fluorescence at a protein concentration of 1 µM. <i>k</i>_cc is the rate of conformational change and <i>k</i>_agg the rate of aggregation. The rate of polymerization at 60°C was additionally determined at a protein concentration of 10 µM as a function of monomer loss (<i>k_-mon</i>) on native gels. Each experiment was performed three times. Errors included represent the standard deviation of each dataset.</p

Kinetic and thermodynamic stability properties of the hI and s5A α₁AT variants.

a<p><i>k</i>_N→I and <i>k</i>_I→U represent the rates for the N→I and the I→U transitions when the proteins were denatured in 4 M GdnHCl.</p>b<p>The GdnHCl-induced unfolding curves were analyzed using the three-state unfolding function to determine the midpoints of the N→I transition (<i>D</i>_mN→I) and the I→U transition (<i>D</i>_mI→U). ΔΔ<i>G</i>_N→I represents the change in free energy for the N→I transition (all experimental errors were within ±0.12 kcal/mol) and ΔΔ<i>G</i>_I→U the change in free energy for the I→U transition (all experimental errors were within ±0.12 kcal/mol).</p>c<p>The thermal denaturation curves obtained using far-UV CD were analyzed using the two-state unfolding function to determine the midpoint of transition. Each experiment was performed three times. Errors included represent the standard deviation of each dataset.</p

Oxidation of an Exposed Methionine Instigates the Aggregation of Glyceraldehyde-3-phosphate Dehydrogenase

Detail, upper half with arching bow; Half horse, half human, the centaur symbolizes the lower nature of mankind, the wild, instinctive side. The artist has chosen to render Keiron (Chiron), the wise centaur, who symbolizes nature’s healing capacity. The sculpture caused heated debate that lasted almost ten years before it finally was erected and unveiled in 1939. It is Fridman's best known work of sculpture; she was also a poet. Source: Wikipedia; http://en.wikipedia.org/wiki/Main_Page (accessed 7/18/2013

Retrotransposon instability dominates the acquired mutation landscape of mouse induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) can in principle differentiate into any cell of the body, and have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements and prevent transposable element-mediated mutagenesis. Here we apply short-read or Oxford Nanopore Technologies long-read genome sequencing to 38 bulk miPSC lines reprogrammed from 10 parental cell types, and 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs are rare, we find 83 de novo transposable element insertions, including examples intronic to Brca1 and Dmd. LINE-1 retrotransposons are profoundly hypomethylated in miPSCs, beyond other transposable elements and the genome overall, and harbor alternative protein-coding gene promoters. We show that treatment with the LINE-1 inhibitor lamivudine does not hinder reprogramming and efficiently blocks endogenous retrotransposition, as detected by long-read genome sequencing. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs

Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming

Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference, we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGoY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore, our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGoY hPSCs, underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs

Cell Type of Origin Dictates the Route to Pluripotency

Summary: Our current understanding of induced pluripotent stem cell (iPSC) generation has almost entirely been shaped by studies performed on reprogramming fibroblasts. However, whether the resulting model universally applies to the reprogramming process of other cell types is still largely unknown. By characterizing and profiling the reprogramming pathways of fibroblasts, neutrophils, and keratinocytes, we unveil that key events of the process, including loss of original cell identity, mesenchymal to epithelial transition, the extent of developmental reversion, and reactivation of the pluripotency network, are to a large degree cell-type specific. Thus, we reveal limitations for the use of fibroblasts as a universal model for the study of the reprogramming process and provide crucial insights about iPSC generation from alternative cell sources. : Nefzger et al. find that the molecular reprogramming trajectories of fibroblasts, neutrophils, and keratinocytes have a cell-type-specific component that only fully converges in induced pluripotent stem cells. The authors also identify universal changes shared by all three cell types, including two transcriptional waves and a conserved transcriptional program involving Egr1 downregulation. Keywords: reprogramming, induced pluripotent stem cells, fibroblasts, neutrophils, keratinocytes, transcriptional dynamics, Egr