The chelate ligand dipyrido [3,2-a:2',3'-c] phenazine (dppz), its 11,12-dimethyl derivative dmdppz, and corresponding complexes with [Ru(bpy)2]2+ were studied in multiply reduced states by low-temperature cyclic voltammetry and UV/vis and EPR spectroscopy. The (dm)dppz ligands are reduced in two reversible steps, followed by a very moisture-sensitive third step. Highly resolved EPR and 'H-ENDOR spectra of the intermediate anion radicals were obtained and analyzed. The results are interpreted using a HMO/McLachlan perturbation approach of x spin populations and orbital energies. Three low-lying unoccupied x molecular orbitals can be identified as phenazinetype (bi, lowest) and as the ^(b,) and xfo) orbitals of the a-diimine moiety. Complexes with the N(4),N(3)-bound [Ru(bpy)2]2+ fragment show at least six reversible one-electron reduction steps in rigorously dried DMF at 200 K; the first four persistent reduced states were characterized by EPR and UV/vis spectroscopy. The EPR spectra of the first three reduced states of the complexes show a signal which proves the occupation of the phenazine-localized x* orbital of (dm)dppz by a single electron, the stepwise reduction of the bpy ligands resulting in temperaturedependent intensity loss of that EPR signal. The very basic quadruply reduced state exhibits EPR characteristics which are typical for Ru(II)-bound a-diimine anion radicals. All assignments are supported by UV/vis spectra and analyses of redox potential values. Because the very easily protonated higher reduced states are not sufficiently persistent for EPR and UV/vis characterization, further assignments could thus be based only on the analysis of redox potential values. The particular composite electronic structure of the complexes with differing redox and "optical" orbitals is related to their "light switch" behavior, i.e. to the absence of luminescence quenching in a nonaqueous environment.

Background: Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. Objective: To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1-548 under the control of human HTT promoter. Methods: Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. Results: One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old siblings showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa. Conclusions:The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study.

Hybrid sterility is a hallmark of speciation, but the underlying molecular mechanisms remain poorly understood. Here, we report that speciation may regularly proceed through a stage at which gene flow is completely interrupted, but hybrid sterility occurs only in male hybrids whereas female hybrids reproduce asexually. We analysed gametogenic pathways in hybrids between the fish species Cobitiselongatoides and C. taenia and revealed that male hybrids were sterile owing to extensive asynapsis and crossover reduction among heterospecific chromosomal pairs in their gametes, which was subsequently followed by apoptosis. We found that polyploidization allowed pairing between homologous chromosomes and therefore partially rescued the bivalent formation and crossover rates in triploid hybrid males. However, it was not sufficient to overcome sterility. In contrast, both diploid and triploid hybrid females exhibited premeiotic genome endoreplication, thereby ensuring proper bivalent formation between identical chromosomal copies. This endoreplication ultimately restored female fertility but it simultaneously resulted in the obligate production of clonal gametes, preventing any interspecific gene flow. In conclusion, we demonstrate that the emergence of asexuality can remedy hybrid sterility in a sex-specific manner and contributes to the speciation process.

Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9) system has become a revolutionary tool for gene editing. Since viral delivery systems have significant side effects, and naked DNA delivery is not an option, the nontoxic, non-viral delivery of CRISPR/Cas9 components would significantly improve future therapeutic delivery. In this study, we aim at characterizing nanoparticles to deliver plasmid DNA encoding for the CRISPR-Cas system in eukaryotic cells in vitro. CRISPR/Cas9 complexed polyethylenimine (PEI) magnetic nanoparticles (MNPs) were generated. We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to evaluate efficient homology-directed repair (HDR) and non-homologous end joining (NHEJ) events following transfection with NPs. MNPs have been synthesized by co-precipitation with the average particle size around 20 nm in diameter. The dynamic light scattering and zeta potential measurements showed that NPs exhibited narrow size distribution and sufficient colloidal stability. Genome editing events were as efficient as compared to standard lipofectamine transfection. Our approach tested non-viral delivery of CRISPR/Cas9 and DNA template to perform HDR and NHEJ in the same assay. We demonstrated that PEI-MNPs is a promising delivery system for plasmids encoding CRISPR/Cas9 and template DNA and thus can improve safety and utility of gene editing.

Background: The aim of this experimental study on New Zealand's white rabbits was to find differences in the results of treating the distal physeal femoral defect by the transplantation of autologous or allogeneic mesenchymal stem cells (MSCs). After the excision of a created bone bridge in the distal physis of the right femur, modified composite scaffold with MSCs was transplanted into the defect. In animal Group A (n = 11) autogenous MSCs were implanted; in animal Group B (n = 15) allogeneic MSCs were implanted. An iatrogenic physeal defect of the left femur of each animal not treated by MSCs transplantation served as control. The rabbits were euthanized four months after the transplantation. The treatment results were evaluated morphometrically (femoral length and valgus deformity measurement) and histologically (character and quality of the new cartilage).