BackgroundReelin is an extracellular glycoprotein of crucial importance in the developmental organisation of neurons in the mammalian cerebral cortex and other laminated brain regions. The pig possesses a gyrencephalic brain that bears resemblance to the human brain. In order to establish an animal model for neuronal migration disorders in the pig, we have studied the expression pattern and structure of Reelin during pig brain development.ResultsWe determined the sequence of pig Reelin mRNA and protein and identified a high degree of homology to human Reelin. A peak in Reelin mRNA and protein expression is present during the period of major neurogenesis and neuronal migration. This resembles observations for human brain development. Immunohistochemical analysis showed the highest expression of Reelin in the Cajal-Reztius cells of the marginal zone, in resemblance with observations for the developing brain in humans and other mammalian species.ConclusionsWe conclude that the pig might serve as an alternative animal model to study Reelin functions and that manipulation of the pig Reelin could allow the establishment of an animal model for human neuronal migration disorders.

TEM-15 and PBP3 substitutions impose an additive effect on resistance to third-generation cephalosporins in both H. influenzae and H. parainfluenzae. The effect of PBP3 substitutions on beta-lactam resistance in H. parainfluenzae can be addressed by transfer of ftsI genes in vitro.

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Disc diffusion testing of H. influenzae accurately identified β-lactamase-non-producing isolates with the N526K substitution by use of discs containing low amounts of penicillins. Cephalosporin-containing discs could detect mutational resistance in β-lactamase-producing isolates, but performed with reduced specificity.

bMutations in ftsI, encoding penicillin-binding protein 3, can cause decreased ␤-lactam susceptibility in Haemophilus influenzae. Sequencing of ftsI from clinical strains has indicated interspecies recombination of ftsI between H. influenzae and Haemophilus haemolyticus. This study documented apparently unrestricted homologous recombination of ftsI between H. influenzae and H. haemolyticus in vitro. Transfer of ftsI from resistant isolates conferred similar but not identical increases in the MICs of susceptible strains of H. influenzae and H. haemolyticus. Haemophilus influenzae is the major human pathogen of the genus Haemophilus (1, 2), and infections are usually treated with ␤-lactam antimicrobial agents. Amino acid substitutions in penicillin-binding protein 3 (PBP3), encoded by the ftsI gene, can confer decreased susceptibility to ␤-lactams in strains lacking ␤-lactamase genes (3, 4). Genetically defined ␤-lactamase-negative ampicillin-resistant (gBLNAR) isolates carry either the N526K (Ubukata group II) or the R517H substitution in PBP3, while the additional substitutions S385T and/or L389F (Ubukata group III) further reduce susceptibility (3, 5-7). A range of other amino acid substitutions has been identified, but their significance is unclear (3-5, 8, 9).Whether the worldwide spread of gBLNAR isolates is caused by clonal dissemination or horizontal gene transfer is controversial (8,10,11). H. influenzae and Haemophilus haemolyticus are close relatives, (1) and putative transfer of ftsI between these two species is indicated by mosaic structures of ftsI from clinical and nasopharyngeal carriage isolates (12,13).(Part of these data was presented on a poster at the 2014 International Pasteurellaceae Conference, Prato, Italy, 15 May 2014.)The present study was undertaken to examine putative species barriers and delineate transformation events after ftsI transfer under standardized in vitro conditions. ftsI genes (1,833 bp) plus flanking regions from two gBLNAR H. influenzae strains and two gBLNAR H. haemolyticus strains were amplified by PCR (primers used for amplification and sequencing are listed in Table S1 in the supplemental material) and used for electroporetic transformation of susceptible strains of H. influenzae and H. haemolyticus (one representative each) as previously described (3). Characteristics of the donor and recipient strains are listed in Table 1, and overall similarity of ftsI and PBP3 are given in Table S2 in the supplemental material. Transformants were selected on chocolate agar (Columbia agar [Oxoid] supplemented with 5% horse blood and Vitox [Oxoid]) containing 0.5 g/ml ampicillin (AMP) and screened for the presence of the N526K substitution using real-time PCR as previously described (14). We observed intraspecies ftsI transformation frequencies between 4.2 ϫ 10 Ϫ7 and 6.7 ϫ 10 Ϫ7 with H. influenzae strain Rd as the recipient and between 7 ϫ 10 Ϫ5 and 1 ϫ 10 Ϫ4 with H. haemolyticus strain ATCC 33390T as the recipient; we observed interspecies ftsI transformation frequencies betwee...