Metabolic enhancement of the one carbon metabolism (OCM) in bovine oocytes IVM increases the blastocyst rate: evidences for a OCM checkpoint.

The one carbon metabolism (OCM) has a primary role in the process of oocyte maturation. In this study bovine oocytes were cultured for 24 h, up to MII stage, with standard medium supplemented or not with 8 metabolic enhancers of the OCM and the MII and blastocyst rate were compared. Additional analyses were performed on matured oocytes, cumulus cells, zygotes and blastocysts. The OCM supplementation increased the blastocyst rate derived from in vitro fertilization. The mitochondrial mass and DNMT3a protein expression were increased whereas DNA fragmentation decreased in matured oocytes. DNA methylation in female pronucleus of zygotes was increased. The supplementation did not directly affect the redox balance as ROS and GSH in matured oocytes and homocysteine in the spent medium were unchanged. The supplementation of the oocytes with metabolic enhancers of the OCM may increase the yield from the culture, likely due to improved DNA methylation and epigenetic programming. The lack of effects on MII rate with huge differences appearing at the blastocyst stage suggest the existence of a OCM metabolic check point that hampers oocytes progression to blastocyst post-fertilization, if they were not properly primed at the time of maturation.

Do neprilysin inhibitors walk the line? Heart ameliorative but brain threatening!

Sacubitril/valsartan (Entresto™; LCZ696) is the first angiotensin receptor-neprilysin inhibitor (ARNI) drug approved by the US and EU for heart failure (HF) and especially recommended for hypertensive HF (HHF). Sacubitril inhibits the enzyme neprilysin (NEP) which produces both beneficial and adverse effects in the human body. While LCZ696 causes beneficial cardiovascular effects, it may induce memory and cognitive dysfunction, or even exacerbate Alzheimer's disease (AD). This article reviewed data reported by experimental and clinical studies that examined NEP inhibitors and their dementia-related side effects. Based on the literature, LCZ696 increases the risk of memory and cognitive dysfunctions, and clinical trials failed to show compelling evidence for LCZ696 safety for the brain. Together, it was concluded that more experimental and clinical studies with particular focus on LCZ696 side effects on ß-amyloid (Aß) degradation are needed to assess LCZ696 safety for the cognitive function, especially in case of long-term administration.

Incorporation of F-MWCNTs into electrospun nanofibers regulates osteogenesis through stiffness and nanotopography.

Nanotopography and stiffness are major physical cues affecting cell fate. However, the current nanofiber modifications techniques are limited by their ability to control these two physical cues irrespective of each other without changing the materials' surface chemistry. For this reason, the isolated effects of topography and stiffness on osteogenic regulation in electrospun nanofibers have been studied incompletely. Here, we investigated 1. how functionalized multiwall carbon nanotubes (F-MWCNTs) loaded in Polycaprolactone (PCL) nanofibers control their physical properties and 2. whether the resulting unique structures lead to distinctive phenotypes in bone progenitor cells. Changes in material properties were measured by high-resolution electron microscopes, protein adsorption and tensile tests. The effect of the developed structures on human mesenchymal stem cell (MSC) osteogenic differentiation was determined by extensive quantification of early and late osteogenic marker genes. It was found that F-MWCNT loading was an effective method to independently control the PCL nanofiber surface nanoroughness or stiffness, depending on the applied F-MWCNT concentration. Collectively, this suggests that stiffness and topography activate distinct osteogenic signaling pathway. The current strategy can help our further understanding of the mechano-biological responses in osteoprogenitor cells, which could ultimately lead to improved design of bone substitute biomaterials.

Production of Minicircle DNA Vectors Using Site-Specific Recombinases.

Minicircle DNA vectors are plasmid derivatives free of bacterial elements. These vectors are mostly provided from common plasmids via recombination by site-specific recombinases in E. coli. Absence of bacterial backbone in minicircle vectors results in high-level and persistent expression of transgene in comparison with conventional plasmids and provides promising vehicles for gene therapy and vaccination. Here we describe the production of replicative minicircle DNA vectors using the PBAD/araC system expressing ΦC31 integrase in E. coli.

Construction of a new minicircle DNA carrying an enhanced green florescent protein reporter gene for efficient expression into mammalian cell lines.

The presence of a bacterial backbone in conventional eukaryotic expression plasmids may cause undesirable effects by triggering the immune responses in mammals and repression of episomal transgene expression. To avoid these problems, researchers have proposed the use of minicircle DNAs which are episomal vectors that have lost their bacterial backbone using a site-specific recombinase mediated recombination. In the present study, we have constructed a new minicircle DNA vector that carries an enhanced green florescent protein (EGFP) reporter gene using phage ΦC31 integrase-mediated recombination and homing endonuclease ISceI-mediated purification in E. coli. ΦC31 integrase expression was under the control of the araBAD promoter, whereas ISceI endonuclease was controlled by the tac promoter. This vector was transfected into CHO-K1 cells, which showed transient expression of EGFP up to 14 generations. Similar results were obtained upon transient transfection into HEK cells. In addition, PCR results on genomic DNA, demonstrated the EGFP-minicircle was episomal and did not integrate into the host genome. Our constructed parental plasmid expresses EGFP and could be used for the generation of episomal minicircle DNA with intent to carry out transient transfection of interested DNA fragments into the eukaryotic cells for various purposes.