One by One: Accumulating Evidence by using Meta-Analytical Procedures for Single-Case Experiments

This paper presents a unilevel and multilevel approach for the analysis and meta-analysis of single-case experiments (SCEs). We propose a definition of SCEs and derive the specific features of SCEs’ data that have to be taken into account when analysing and meta-analysing SCEs. We discuss multilevel models of increasing complexity and propose alternative and complementary techniques based on probability combining and randomisation test wrapping. The proposed techniques are demonstrated with real-life data and corresponding R code.

Inferring demographic parameters in bacterial genomic data using Bayesian and hybrid phylogenetic methods

Background: Recent developments in sequencing technologies make it possible to obtain genome sequences from a large number of isolates in a very short time. Bayesian phylogenetic approaches can take advantage of these data by simultaneously inferring the phylogenetic tree, evolutionary timescale, and demographic parameters (such as population growth rates), while naturally integrating uncertainty in all parameters. Despite their desirable properties, Bayesian approaches can be computationally intensive, hindering their use for outbreak investigations involving genome data for a large numbers of pathogen isolates. An alternative to using full Bayesian inference is to use a hybrid approach, where the phylogenetic tree and evolutionary timescale are estimated first using maximum likelihood. Under this hybrid approach, demographic parameters are inferred from estimated trees instead of the sequence data, using maximum likelihood, Bayesian inference, or approximate Bayesian computation. This can vastly reduce the computational burden, but has the disadvantage of ignoring the uncertainty in the phylogenetic tree and evolutionary timescale. Results: We compared the performance of a fully Bayesian and a hybrid method by analysing six whole-genome SNP data sets from a range of bacteria and simulations. The estimates from the two methods were very similar, suggesting that the hybrid method is a valid alternative for very large datasets. However, we also found that congruence between these methods is contingent on the presence of strong temporal structure in the data (i.e. clocklike behaviour), which is typically verified using a date-randomisation test in a Bayesian framework. To reduce the computational burden of this Bayesian test we implemented a date-randomisation test using a rapid maximum likelihood method, which has similar performance to its Bayesian counterpart. Conclusions: Hybrid approaches can produce reliable inferences of evolutionary timescales and phylodynamic parameters in a fraction of the time required for fully Bayesian analyses. As such, they are a valuable alternative in outbreak studies involving a large number of isolates.

319 APPROACHES TO IMPROVE INTRACYTOPLASMIC SPERM INJECTION MEDIATED TRANSGENESIS AND MAXIMIZE THE USE OF SEX-SORTED SPERM IN BOVINE

Intracytoplasmic sperm injection (ICSI) mediated transgenesis is an effective tool for transgenic animal production. However, ICSI in cattle remains inefficient. In this work, we assayed approaches to improve egfp expressing blastocysts production by ICSI: the sperm pretreatment with heparin and l-glutathione (Hep-GSH), the use of sex-sorted sperm (SS), the refrozen/thawing of SS sperm, and the combination of these. Quality of ICSI blastocysts was analysed by studying the expression of 4 genes, and the rates of DNA fragmentation. Cumulus-oocyte complexes from slaughtered cow ovaries were in vitro-matured for 21 h. Nonsorted (NS) and sex-sorted (SS) frozen straws were thawed. Some of them were incubated with 80 μM Hep-15 mM GSH for 20 h (Hep-GSH+). The Hep-GSH-control group was not pretreated. Semen samples were co-incubated with 50 ng µL–1 of pCX-EGFP for 5 min before ICSI. Moreover, the SS sperm that are usually discarded after ICSI were cryopreserved and used for ICSI after a second thawing (ICSI SS refrozen). The ICSI NS, sham, and diploid parthenogenetic (Diplo PA) controls were included. Oocytes were activated with 5 µM ionomycin for 4 min, TCM-199 for 3 h (except for diploid PA), and 1.9 mM DMAP for 3 h. Cleavage and blastocyst/egfp expression rates were evaluated on Days 2 and 7 post-ICSI, respectively. Results are shown in Table 1. Relative expression of HMGN1, GLUT5, AQP3, and OCT4 genes from ICSI NS Hep-GSH+ and IVF blastocysts were compared by qPCR. Data were analysed by the pair-wise fixed reallocation randomisation test. None of the 4 genes showed differences between groups. The DNA fragmented nucleus index/blastocyst cell numbers were determined by TUNEL assay, not showing differences between groups (Kruskal–Wallis test, P ≤ 0.05). Means ± s.d. were 29 ± 17/91 ± 27 for ICSI Hep-GSH+; 27 ± 15/63 ± 34 for ICSI Hep-GSH–; 28 ± 17/68 ± 17 for ICSI SS, 28 ± 13/75 ± 24 for ICSI SS refrozen; and 21 ± 13/105 ± 59 for IVF SS control. The Hep-GSH pretreatment can increase blastocyst and transgene expressing blastocysts rates after TM-ICSI, except when SS semen is used. Interestingly, the use of SS sperm for ICSI can be maximized by cryopreservation and reuse of discarded sperm cells. The parameters analysed in this work indicate that the proposed approaches do not affect blastocyst quality. Therefore, Hep-GSH pretreatment of NS sperm and refrozen SS sperm could be applied for TM-ICSI in bovine for the production of transgenic animals. Table 1.In vitro development and egfp expression of ICSI embryos fertilized with nonsorted (NS) and sex-sorted (SS) sperm pretreated with Hep-GSH, refrozen, or both