Organoplatinum(II) Complexes Self-Assemble and Recognize AT-Rich Duplex DNA Sequences.

The specific recognition of AT-rich DNA sequences opens up the door to promising diagnostic and/or therapeutic strategies against gene-related diseases. Here, we demonstrate that amphiphilic PtII complexes of the type [Pt(dmba)(N∧N)]NO3 (dmba = N,N-dimethylbenzylamine-κN, κC; N∧N = dpq (3), dppz (4), and dppn (5)) recognize AT-rich oligonucleotides over other types of DNA, RNA, and model proteins. The crystal structure of 4 shows the presence of significant π-stacking interactions and a distorted coordination sphere of the d8 PtII atom. Complex 5, containing the largest π-conjugated ligand, forms supramolecular assemblies at high concentrations under aqueous environment. However, its aggregation can be promoted in the presence of DNA at concentrations as low as 10 µM in a process that "turns on" its excimer emission around 600 nm. Viscometry, gel electrophoresis, and theoretical calculations demonstrate that 5 binds to minor groove when self-assembled, while the monomers of 3 and 4 intercalate into the DNA. The complexes also inhibit cancer cell growth with low-micromolar IC50 values in 2D tissue culture and suppress tumor growth in 3D tumor spheroids with a multicellular resistance (MCR) index comparable to that of cisplatin.

Incubation of boar spermatozoa in viscous media by addition of methylcellulose improves sperm quality and penetration rates during in vitro fertilization.

This work was designed to study whether viscous media can improve the in vitro sperm functionality in pigs by using methylcellulose as a thickener. Viscosity of porcine oviductal fluid (POF) was compared with culture medium (Tyrode's) supplemented with methylcellulose (MET 0, 0.5 and 1% w/v). Spermatozoa were incubated in the different media (0, 1 and 2 h) and sperm motion parameters, lipid membrane disorder, plasma membrane integrity and reactive oxygen species (ROS) formation were assessed. Fertilization results were assessed i) preincubating spermatozoa in the viscous media followed by gamete coculture in a non-viscous medium; and ii) gamete coculture in the viscous media. Viscosity of POF from early luteal phase was higher than late follicular phase. Medium without methylcellulose presented constant viscosity with increased shear rate, while viscosity of the POF and media with methylcellulose was reduced by increased shear rates. Methylcellulose improved sperm linearity, straightness and the proportion of fast-linear spermatozoa. Moreover, methylcellulose increased the rate of viable spermatozoa with intact acrosome and low lipid disorder, reducing the ROS generation. Preincubation in viscous media increased the penetration rate and the mean number of spermatozoa bound to the zona pellucida (both with 0.5 and 1% MET) and reduced monospermy with 1% MET. On the other hand fertilization in the viscous media reduced penetration rate and increased monospermy. The efficiency of the IVF system was not improved with the use of viscous media. The results show the relevance of increasing viscosity thus making the in vitro media more comparable to physiological conditions.

Shape Analysis of DNA-Au Hybrid Particles by Analytical Ultracentrifugation.

Current developments in nanotechnology have increased the demand for nanocrystal assemblies with well-defined shapes and tunable sizes. DNA is a particularly well-suited building block in nanoscale assemblies because of its scalable sizes, conformational variability, and convenient self-assembly capabilities via base pairing. In hybrid materials, gold nanoparticles (AuNPs) can be assembled into nanoparticle structures with programmable interparticle distances by applying appropriate DNA sequences. However, the development of stoichiometrically defined DNA/NP structures is still challenging since product mixtures are frequently obtained and their purification and characterization is the rate-limiting step in the development of DNA-NP hybrid assemblies. Improvements in nanostructure fractionation and characterization techniques offer great potential for nanotechnology applications in general. This study reports the application of analytical ultracentrifugation (AUC) for the characterization of anisotropic DNA-linked metal-crystal assemblies. On the basis of transmission electron microscopy data and the DNA primary sequence, hydrodynamic bead models are set up for the interpretation of the measured frictional ratios and sedimentation coefficients. We demonstrate that the presence of single DNA strands on particle surfaces as well as the shape factors of multiparticle structures in mixtures can be quantitatively described by AUC. This study will significantly broaden the possibilities to analyze mixtures of shape-anisotropic nanoparticle assemblies. By establishing insights into the analysis of nanostructure mixtures based on fundamental principles of sedimentation, a wide range of potential applications in basic research and industry become accessible.

The Protein Acetyltransferase PatZ from Escherichia coli Is Regulated by Autoacetylation-induced Oligomerization.

Lysine acetylation is an important post-translational modification in the metabolic regulation of both prokaryotes and eukaryotes. In Escherichia coli, PatZ (formerly YfiQ) is the only known acetyltransferase protein and is responsible for acetyl-CoA synthetase acetylation. In this study, we demonstrated PatZ-positive cooperativity in response to acetyl-CoA and the regulation of acetyl-CoA synthetase activity by the acetylation level. Furthermore, functional analysis of an E809A mutant showed that the conserved glutamate residue is not relevant for the PatZ catalytic mechanism. Biophysical studies demonstrated that PatZ is a stable tetramer in solution and is transformed to its octameric form by autoacetylation. Moreover, this modification is reversed by the sirtuin CobB. Finally, an in silico PatZ tetramerization model based on hydrophobic and electrostatic interactions is proposed and validated by three-dimensional hydrodynamic analysis. These data reveal, for the first time, the structural regulation of an acetyltransferase by autoacetylation in a prokaryotic organism.