Gene silencing during development of in vitro-produced female bovine embryos

In early development, female embryos (XX) produce twice the transcripts of X-linked genes compared with male embryos (XY). During the course of development, inactivation of the X chromosome equilibrates gene dosage, making the development of female embryos viable. Moreover, the biotechnologies used for producing embryos in vitro seem to work better with male embryos, making it easier for them to reach the blastocyst stage and allow for complete gestation. We investigated the expression of three X-linked genes that are involved in development, XIST, G6PD, and HPRT, and of the transcript interferon-tau, in male and female bovine blastocysts produced by nuclear transfer (NT) and by in vitro fertilization (IVF). Oocytes that had been matured in vitro were enucleated and reconstructed with somatic cells from adult animals at 18 h post-maturation. After fusion (two pulses of 2.25 kv/cm) and chemical activation (5.0 mu M ionomycin for 5 min and 2.0 mM 6-DMAP for 3 h), the oocytesomatic cell units were cultivated in CR2 with a monolayer of granulosa cells at 38.8 degrees C, in a humidified 5% CO(2) atmosphere. IVF embryos were inseminated, after centrifugation in a Percoll gradient, with 2 x 10(6) sperm/mL TALP medium supplemented with BSA and PHE and cultivated under the same conditions as the cloned embryos. We used real-time PCR to analyze the gene expression of individual blastocysts compared to expression of the housekeeping gene, GAPDH. The gene XIST was expressed in female embryos and not in male embryos produced by IVF, though it was expressed at low levels in male embryos produced by NT. Unlike previous reports, we found lower levels of the transcript of G6PD in females than in males, suggesting double silencing or other mechanisms of control of this gene. Female embryos produced by IVF expressed the HPRT gene at a higher level than female embryos produced by NT, suggesting that gene silencing proceeds faster in NT-produced female embryos due to ""inactivation memory"" from the nucleus donor. In conclusion, male and female embryos express different levels of X-chromosome genes and failures of these genes that are essential for development could reduce the viability of females. Nuclear transfer can modify this relation, possibly due to epigenetic memory, leading to frequent failures in nuclear reprogramming.

Ticagrelor vs. clopidogrel in patients with acute coronary syndromes and diabetes: a substudy from the PLATelet inhibition and patient Outcomes (PLATO) trial

Patients with diabetes mellitus (DM) have high platelet reactivity and are at increased risk of ischaemic events and bleeding post-acute coronary syndromes (ACS). In the PLATelet inhibition and patient Outcomes (PLATO) trial, ticagrelor reduced the primary composite endpoint of cardiovascular death, myocardial infarction, or stroke, but with similar rates of major bleeding compared with clopidogrel. We aimed to investigate the outcome with ticagrelor vs. clopidogrel in patients with DM or poor glycaemic control. We analysed patients with pre-existing DM (n = 4662), including 1036 patients on insulin, those without DM (n = 13 951), and subgroups based on admission levels of haemoglobin A1c (HbA1c; n = 15 150). In patients with DM, the reduction in the primary composite endpoint (HR: 0.88, 95% CI: 0.76-1.03), all-cause mortality (HR: 0.82, 95% CI: 0.66-1.01), and stent thrombosis (HR: 0.65, 95% CI: 0.36-1.17) with no increase in major bleeding (HR: 0.95, 95% CI: 0.81-1.12) with ticagrelor was consistent with the overall cohort and without significant diabetes status-by-treatment interactions. There was no heterogeneity between patients with or without ongoing insulin treatment. Ticagrelor reduced the primary endpoint, all-cause mortality, and stent thrombosis in patients with HbA1c above the median (HR: 0.80, 95% CI: 0.70-0.91; HR: 0.78, 95% CI: 0.65-0.93; and HR: 0.62, 95% CI: 0.39-1.00, respectively) with similar bleeding rates (HR: 0.98, 95% CI: 0.86-1.12). Ticagrelor, when compared with clopidogrel, reduced ischaemic events in ACS patients irrespective of diabetic status and glycaemic control, without an increase in major bleeding events.

Serum Starvation and Full Confluency for Cell Cycle Synchronization of Domestic Cat (Felis catus) Foetal Fibroblasts

Nuclear transfer of domestic cat can be used as a tool to develop reproductive biotechnologies in wild felids. The importance of cell cycle phase during the nuclear transfer has been a matter of debate since the first mammalian clone was produced. The cell cycle phase of donor cells interferes on maintenance of correct ploidy and genetic reprogramming of the reconstructed embryo. The use of G0/G1 arrested donor cells has been shown to improve nuclear transfer efficiency. The present study was conducted to test the hypothesis that domestic cat foetal fibroblasts cultured up to the fifth passage and submitted to full confluency provide a higher percentage of cells at G0/G1 stage than fibroblasts cultured in serum starved media. Results demonstrated that serum starvation increased (p < 0.05) the percentage of G0/G1 fibroblasts when compared with control. Moreover, the combined protocol using confluency and serum starvation was more efficient (p < 0.05) synchronizing cells at G0/G1 stage than serum starvation or confluency alone for the first 3 days of treatment. In conclusion, serum starvation and full confluency act in a synergistic manner to improve domestic cat foetal fibroblast cell cycle synchronization at the G0/G1 stage.