The role of TGFβ signaling in load-induced bone formation.

<p>Load represses TGFβ signaling through Smad2/3 in osteocytes, which is required for the activation of SOST expression through an indirect, Runx2-independent mechanism. Loss of function mutations in the TGFβ type II receptor impair load-mediated repression of SOST and new bone formation.</p

TGFβ induces <i>SOST</i> expression through Smad3.

<p>Treating UMR-106 cells with TGFβ1 (5 ng/ml) for 2, 8, or 24 h results in an increase in <i>SOST</i> mRNA, while inhibiting TGFβ signaling with SB431542 results in a decrease in <i>SOST</i> expression (A). Smad3 overexpression with pRK5-Smad3 induces the <i>SOST</i> promoter-reporter construct, 3XECR-hSOSTpLuc, in a TGFβ dose-dependent manner within 24 h of TGFβ treatment (B). Blocking translation with cycloheximide (CHX) or transcription with actinomycin-D (ActD) for 2 h prevents TGFβ (5 ng/ml) induction of <i>SOST</i> expression (C). siRNA mediated knockdown of <i>Runx2</i> did not prevent TGFβ (5 ng/ml) induction of <i>SOST</i> expression (D). (For panel C, * represents p<0.05 computed by comparing samples with added TGFβ to samples without added TGFβ in each treatment group; for all other panels, * p<0.05 computed by comparing samples to untreated cells).</p

Quantitative measures of micro-computed tomography analyses.

<p>Values represent means and standard deviations for n = 5 mice. An asterisk (*) represents significant differences between loaded and nonloaded bones (p<0.05). Bolded values indicate parameters that differ significantly between WT and DNTβRII mice (p<0.05).</p

Characterization of Impurities in Therapeutic RNAs at the Single Nucleotide Level

The chemistry of guide RNA (gRNA) affects the performance of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing technique. However, the literature is very scarce about the study of gRNA degradation and in particular at the single nucleotide level. In this work, we developed a workflow to characterize the impurities of large RNAs at the single nucleotide level and identified the residues prone to degradation. Our strategy involves (i) the reduction of RNA length, (ii) a chromatographic mode able to capture subtle changes in impurity polarity, and (iii) a streamlined data treatment. To illustrate the approach, stressed gRNA samples were analyzed by coupling an immobilized ribonuclease T1 cartridge to a hydrophilic interaction liquid chromatography (HILIC) column hyphenated with tandem mass spectrometry (MS/MS). Critical findings were made possible by the presented technology. In particular, the desulfurization of phosphorothioate (PS) linkages was the major degradation observed at the single nucleotide level while no change in purity profile could be observed when using conventional ion-pairing reversed-phase (IPRP) liquid chromatography. To our knowledge, this is the first time that several impurity types are screened for a large RNA molecule using an automated online digestion analysis approach

An Efficient Light-Driven P450 BM3 Biocatalyst

P450s are heme thiolate enzymes that catalyze the regio- and stereoselective functionalization of unactivated C–H bonds using molecular dioxygen and two electrons delivered by the reductase. We have developed hybrid P450 BM3 heme domains containing a covalently attached Ru­(II) photosensitizer in order to circumvent the dependency on the reductase and perform P450 reactions upon visible light irradiation. A highly active hybrid enzyme with improved stability and a modified Ru­(II) photosensitizer is able to catalyze the light-driven hydroxylation of lauric acid with total turnover numbers of 935 and initial reaction rate of 125 mol product/(mol enzyme/min)

Anomalously Large Polarization Effect Responsible for Excitonic Red Shifts in PbSe Quantum Dot Solids

The formation of solid thin films from colloidal semiconductor quantum dots (QDs) is often accompanied by red shifts in excitonic transitions, but the mechanisms responsible for the red shifts are under debate. We quantitatively address this issue using optical absorption spectroscopy of two-dimensional (2D) and three-dimensional (3D) arrays of PbSe QDs with controlled inter-QD distance, which was determined by the length of alkanedithiol linking molecules. With decreasing inter-QD distance, the first and second exciton absorption peaks show increasing red shifts. Using thin films consisting of large and isolated QDs embedded in a matrix of small QDs, we determine that a dominant contribution to the observed red shift is due to changes in polarization of the dielectric environment surrounding each QD (∼88%), while electronic or transition dipole coupling plays a lesser role. However, the observed red shifts are more than 1 order of magnitude larger than theoretical predictions based on the dielectric polarization effect for spherical QDs. We attribute this anomalously large polarization effect to deviations of the exciton wave functions from eigenfunctions of the idealized spherical quantum well model