Crosstalk between immune cells and the microbiota in mucosal tissues can set an individual on a trajectory toward health or disease. Little is known about these early-life events in the human respiratory tract. We examined bacterial colonization and immune system maturation in the lower airways over the first year of life. The lower respiratory tract microbiota forms within the first 2 postnatal months. Within the first weeks, three microbial profiles are evident, broadly distinguished as dysbiotic or diverse, and representing different microbial virulence potentials, including proteolysis of immunoglobulin A (IgA) that is critical for mucosal defense. Delivery mode determines microbiota constituents in preterm, but not term, births. Gestational age is a key determinant of immune maturation, with airway cells progressively increasing expression of proallergic cytokine interleukin-33 and genes linked with IgA. These data reveal microbial and immunological development in human airways, and may inform early-life interventions to prevent respiratory diseases.

Human neocentromeres are fully functional centromeres that arise epigenetically from non-centromeric precursor sequences that are devoid of alpha-satellite DNA. Using chromatin immunoprecipitation (ChIP) and BAC-array analysis, we have previously described a 330 kb binding domain for CENP-A (a histone H3 variant that confers centromere-specific nucleosomal property) at the 10q25 neocentromere found on a chromosome 10-derived marker chromosome mardel(10). For the further detailed analysis of the CENP-A-associated chromatin, we have generated a high-resolution genomic array consisting of PCR fragments with an average size of 8 kb, providing an approximately 20-fold increment in analytical resolution. ChIP and PCR-array analysis reveals seven distinct CENP-A-binding clusters within the 330 kb domain, demonstrating the interspersion of CENP-A-associated nucleosomal blocks within the neocentromeric chromatin. Independent ChIP-PCR analysis verified this distribution profile and indicated that histone H3-containing nucleosomes directly intervene the CENP-A-binding clusters. The CENP-A-binding clusters are uneven in size, with the central cluster (>50 kb) being significantly larger than the flanking ones (10-30 kb), and the flanking clusters arranged in an interesting hierarchical and symmetrical configuration of alternating larger and smaller sizes around the central cluster. In silico sequence analysis indicates an approximately 2.5-fold increase in the prevalence of L1 retroelements within the CENP-A-binding clusters when compared with the non-CENP-A-binding regions. These results provide insight into the possible role of retroelements in determining the positioning of CENP-A binding at human neocentromeres, and that a hierarchical and symmetrical arrangement of CENP-A-binding clusters of varying sizes may be an important structural requirement for mammalian kinetochore assembly and/or to provide stability to withstand polar microtubule forces.

BackgroundDried blood (Guthrie card) spots provide an efficient way to collect and store blood specimens. DNA from this source has been utilised for a number of molecular analyses including genome-wide association studies, but only few studies have tested the feasibility of using it for epigenetic applications, particularly at a genome-wide level.ResultsIn this study, we demonstrate the successful use of DNA isolated from archived dried blood spots for the Infinium HumanMethylation450 Beadchip, along with DNA from matched frozen buffy coats. We obtained high quality and reproducible genome-wide DNA methylation profiles using both sample types. We also report high correlations (r > 0.9907) between DNA obtained from matched dried blood spots and frozen buffy coats, sufficient to distinguish between unrelated individuals.ConclusionsWe, thus, demonstrate that DNA from archived dried blood spots is suitable for genome-wide DNA methylation profiling.