Dynamic expression of epoxyeicosatrienoic acid synthesizing and metabolizing enzymes in the primate corpus luteum

Epoxyeicosatrienoic acids (EpETrEs), produced from arachidonic acid via cytochrome P450 (CYP) epoxygenases, regulate inflammation, angiogenesis, cellular proliferation, ion transport and steroidogenesis. EpETrE actions are regulated through their metabolism to diols (dihydroxyeicosatrienoic acids; DiHETrE) via the enzyme soluble epoxide hydrolase (EPHX2). We set out to determine, therefore, whether EpETrE generating (epoxygenases CYP2C8, 2C9, 2C19, 2J2, 1A2 and 3A4) and metabolizing (EPHX2) enzymes are expressed in the primate corpus luteum (CL). CL were isolated from rhesus macaques during the early (day 3-5 post-LH surge), mid (day 6-8), mid-late (day 10-12), late (day 14-16) and very-late (day 17-19: menses) luteal phase of natural menstrual cycles. EPHX2 mRNA levels peaked in mid-late CL (5-fold when compared with early CL, P<0.05) and remained elevated in the late CL. Ablation of pituitary LH secretion and luteal steroid synthesis significantly reduced (P<0.05) EPHX2 mRNA levels in the mid-late CL, with progestin replacement being insufficient to restore its level of expression to control values. EPHX2 protein was localized to large and small luteal cells, as well as vascular endothelial cells. The EpETrE-generating CYP epoxygenase 2J2, 2C9 and 3A4 genes were also expressed in the macaque CL. While CYP2J2 mRNA levels did not significantly change through the luteal phase, CYP2C9 and CYP3A4 levels were significantly (P<0.05) higher in the mid-late phase when compared with the early phase. CYP2C9, 2J2 and 3A4 proteins were each localized to the large luteal cells, with 2C9 and 2J2 also being present in the small luteal, stromal and endothelial cells. These studies demonstrate for the first time that an EpETrE generating and metabolizing system exists in the primate CL, with the latter being regulated by LH and steroid hormone(s).Fil: Irusta, Griselda. State University of Oregon; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Murphy, M. J.. Oregon Health and Science University; Estados UnidosFil: Perez, W. D.. Oregon Health and Science University; Estados UnidosFil: Hennebold J.D.. Oregon Health and Science University; Estados Unido

Rapid, accurate mapping of transgene integration in viable rhesus macaque embryos using enhanced-specificity tagmentation-assisted PCR.

Genome engineering is a powerful tool for in vitro research and the creation of novel model organisms and has growing clinical applications. Randomly integrating vectors, such as lentivirus- or transposase-based methods, are simple and easy to use but carry risks arising from insertional mutagenesis. Here we present enhanced-specificity tagmentation-assisted PCR (esTag-PCR), a rapid and accurate method for mapping transgene integration and copy number. Using stably transfected HepG2 cells, we demonstrate that esTag-PCR has higher integration site detection accuracy and efficiency than alternative tagmentation-based methods. Next, we performed esTag-PCR on rhesus macaque embryos derived from zygotes injected with piggyBac transposase and transposon/transgene plasmid. Using low-input trophectoderm biopsies, we demonstrate that esTag-PCR accurately maps integration events while preserving blastocyst viability. We used these high-resolution data to evaluate the performance of piggyBac-mediated editing of rhesus macaque embryos, demonstrating that increased concentration of transposon/transgene plasmid can increase the fraction of embryos with stable integration; however, the number of integrations per embryo also increases, which may be problematic for some applications. Collectively, esTag-PCR represents an important improvement to the detection of transgene integration, provides a method to validate and screen edited embryos before implantation, and represents an important advance in the creation of transgenic animal models

Novel Cleft Susceptibility Genes in Chromosome 6q

Cleft lip/palate is a defect of craniofacial development. In previous reports, chromosome 6q has been suggested as a candidate region for cleft lip/palate. A multipoint posterior probability of linkage analysis of multiplex families from the Philippines attributed an 88% probability of harboring a cleft-susceptibility gene to a narrower region on bands 6q14.2-14.3. We genotyped 2732 individuals from families and unrelated individuals with and without clefts to investigate the existence of possible cleft-susceptibility genes in this region. We found association of PRSS35 and SNAP91 genes with cleft lip/palate in the case-control cohort and in Caucasian families. Haplotype analyses support the individual associations with PRSS35. We found Prss35 expression in the head and palate of mouse embryos at critical stages for palatogenesis, whereas Snap91 was expressed in the adult brain. We provide further evidence of the involvement of chromosome 6q in cleft lip/palate and suggest PRSS35 as a novel candidate gene

A modified flavonoid accelerates oligodendrocyte maturation and functional remyelination

Myelination delay and remyelination failure following insults to the central nervous system (CNS) impede axonal conduction and lead to motor, sensory and cognitive impairments. Both myelination and remyelination are often inhibited or delayed due to the failure of oligodendrocyte progenitor cells (OPCs) to mature into myelinating oligodendrocytes (OLs). Digestion products of the glycosaminoglycan hyaluronan (HA) have been implicated in blocking OPC maturation, but how these digestion products are generated is unclear. We tested the possibility that hyaluronidase activity is directly linked to the inhibition of OPC maturation by developing a novel modified flavonoid that functions as a hyaluronidase inhibitor. This compound, called S3, blocks some but not all hyaluronidases and only inhibits matrix metalloproteinase activity at high concentrations. We find that S3 reverses HA-mediated inhibition of OPC maturation in vitro, an effect that can be overcome by excess recombinant hyaluronidase. Furthermore, we find that hyaluronidase inhibition by S3 accelerates OPC maturation in an in vitro model of perinatal white matter injury. Finally, blocking hyaluronidase activity with S3 promotes functional remyelination in mice with lysolecithin-induced demyelinating corpus callosum lesions. All together, these findings support the notion that hyaluronidase activity originating from OPCs in CNS lesions is sufficient to prevent OPC maturation, which delays myelination or blocks remyelination. These data also indicate that modified flavonoids can act as selective inhibitors of hyaluronidase activity and can promote OPC maturation, making them excellent candidates to accelerate myelination or promote remyelination following perinatal and adult CNS insults