Network-driven discovery yields new insight into Shox2-dependent cardiac rhythm control.

The homeodomain transcription factor SHOX2 is involved in the development and function of the heart's primary pacemaker, the sinoatrial node (SAN), and has been associated with cardiac conduction-related diseases such as atrial fibrillation and sinus node dysfunction. To shed light on Shox2-dependent genetic processes involved in these diseases, we established a murine embryonic stem cell (ESC) cardiac differentiation model to investigate Shox2 pathways in SAN-like cardiomyocytes. Differential RNA-seq-based expression profiling of Shox2+/+ and Shox2-/- ESCs revealed 94 dysregulated transcripts in Shox2-/- ESC-derived SAN-like cells. Of these, 15 putative Shox2 target genes were selected for further validation based on comparative expression analysis with SAN- and right atria-enriched genes. Network-based analyses, integrating data from the Mouse Organogenesis Cell Atlas and the Ingenuity pathways, as well as validation in mouse and zebrafish models confirmed a regulatory role for the novel identified Shox2 target genes including Cav1, Fkbp10, Igfbp5, Mcf2l and Nr2f2. Our results indicate that genetic networks involving SHOX2 may contribute to conduction traits through the regulation of these genes.

Keto-enol/enolate equilibria in the N-acetylamino-p-methylacetophenone system. Effect of a beta-nitrogen substituent.

The cis-enol of N-acetylamino-p-methylacetophenone was generated flash photolytically and its rates of ketonization in aqueous HClO(4) and NaOH solutions as well as in HCO(2)H, CH(3)CO(2)H, H(2)PO(4)(-), (CH(2)OH)(3)CNH(3)(+), and NH(4)(+) buffers were measured. Rates of enolization of N-acetylamino-p-methylacetophenone to the cis-enol were also measured by hydrogen exchange of its methylene protons, and combination of the enolization and ketonization data gave the keto-enol equilibrium constant pK(E) = 5.33, the acidity constant of the enol ionizing as an oxygen acid pQ(a)(E)= 9.12, and the acidity constant of the ketone ionizing as a carbon acid pQ(a)(K)= 14.45. Comparison of these results with corresponding values for p-methylacetophenone itself shows that the N-acetylamino substituent raises all three of these equilibrium constants: K(E) by 3 orders of magnitude, Q(a)(E) by 1 order of magnitude, and Q(a)(K)by 4 orders of magnitude. This substituent also retards the rate of H+ catalyzed enol ketonization by 4 orders of magnitude. The origins of these substituent effects are discussed.

Structural, CV and IR spectroscopic evidences for preorientation in PET-active phthalimido carboxylic acids.

Hydrogen bond and potassium cation mediated preorientation were detected for phthalimido acetic acid and the corresponding acetate. Evidence for these phenomena came from X-ray structure analysis as well as cyclic voltammetric and IR spectroscopic measurements. These interactions rationalize the photoinduced electron transfer (PET) reactivity of the substrates in photodecarboxylation reactions.

Time-resolved spectroscopy of sulfur- and carboxy-substituted N-alkylphthalimides.

The photophysical and photochemical properties of N-phthaloyl-methionine (1), S-methyl-N-phthaloyl-cysteine methyl ester (2) and N-phthaloyltranexamic acid (3) were studied by time-resolved UV/Vis spectroscopy, using laser pulses at 248 or 308 nm. The quantum yield of fluorescence is low (phi(f)< 10(-2)) for 1-3 in fluid and glassy media, whereas that of phosphorescence is large (0.3-0.5) in ethanol at - 196 degrees C. The triplet properties were examined in several solvents, at room temperature and below. The spectra and decay kinetics are similar, but the population of the pi(pi*) triplet state, as measured by T-T absorption, is much lower for 1 and 2 than for 3 or N-methyltrimellitimide (5') at ambient temperatures. The quantum yield (phi(delta)) of singlet molecular oxygen O2(1deltag) formation is substantial for 3 and 5' in several air- or oxygen-saturated solvents at room temperature, but small for 2 and 1. The quantum yield of decomposition is substantial (0.2-0.5) for 3 and small (<0.05) for 2 and 1. It is postulated that photoinduced charge separation in the spectroscopically undetectable 3n,pi* state may account for the cyclization products of 1 and 2. In aqueous solution, this also applies for 3, whereas in organic solvents cyclization involves mainly the lower lying 3pi,(pi*) state. Triplet acetone, acetophenone and xanthone are quenched by 1-3 in acetonitrile; the rate constant is close to the diffusion-controlled limit, but smaller for benzophenone. While the energy transfer from the triplet ketone occurs for 3, a major contribution of electron transfer to the N-phthalimide derivative is suggested for 1 and 2, where the radical anion of benzophenone or 4-carboxybenzophenone is observed in alkaline aqueous solution.

Photocyclization of 2-azabicyclo[3.3.0]octane-3-carboxylate derivatives: induced and noninduced diastereoselectivity.

[reaction: see text] The decarboxylative photocyclization of potassium 2-azabicyclo[3.3.0]octanoate ethylene-linked to phthalimide leads to a mixture of two diastereoisomeric products. The overall noninduced diastereoselectivity is remarkably high, indicating a preference of spin-orbit coupling controlled 1,7-biradical combination process. Memory of chirality vanished due to free rotation about the connecting spacer bonds.

Induction of morphological transformation and micronuclei in Syrian hamster embryo fibroblasts by 1,2-dioxetanes. Correlation with single-strand breaks in HL-60 cells.

The photochemical genotoxic and cell-transforming potential of 4-hydroxymethyl-3,3,4-trimethyl-1,2-dioxetane (HTMD) and 3-(N-[4-pyridino]carbamoyl)methyl-3,4,4-trimethyl-1,2-dioxetane , (APD), in mammalian cell was studied. Both dioxetanes, which are efficient sources of triplet-excited ketones on thermal decomposition, induced morphological transformation in Syrian hamster embryo (SHE) fibroblasts. Unscheduled DNA synthesis in SHE and in HeLa cells could not be detected with these dioxetanes, but the number of micronuclei scored after the first mitosis was dose-dependently increased. Single-strand breaks but not Micrococcus luteus u.v.-endonuclease sensitive sites were observed by alkaline elution in HL-60 cells when treated with sub-lethal doses of HTMD and APD. A possible mechanism for the transformation mediated by DNA and chromosomal damage as well as the intermediacy of triplet carbonyls in these events are discussed.