Pharmokinetics of r-metHuGDNF.

<p>(A) ELISA to show amount of total human GDNF protein (pg/mg of total protein) within the striatum after 2 weeks and 4 weeks following acute CED of 0.1, 0.2, 0.6, and 1.0 µg/µL r-metHuGDNF into the striatum. The flow rate employed was 1 µL/minute. Each bar indicates the mean ± SD. **<i>p</i><0.01, r-metHuGDNF infused hemispheres versus aCSF control infused hemispheres by one-way ANOVA and Dunnett post-hoc test. (B) Representative images of immunostaining for GDNF after 2 weeks and 4 weeks following acute CED of 0.1, 0.2, 0.6, and 1.0 µg/µL r-metHuGDNF into the striatum. Scale bar = 250 µm.</p

Immunostaining for Glial Fibrillary Acidic Protein (GFAP).

<p>GFAP-immunopositive cells were observed in the tissue surrounding the needle track area at both 2 and 4 weeks (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056186#pone-0056186-g003" target="_blank">Figure 3</a>). There was no detectable correlation between the degree of gliosis adjacent to the needle track and the infused concentration of r-metHuGDNF at any time point. Scale bar = 500 µm.</p

Expression of GAP43 mRNA and synaptophysin.

<p>Levels of GAP43 mRNA were analysed following Q-PCR analysis (A). Synaptophysin protein levels were measured using Sandwich ELISA (B). Each bar indicates the mean ± SD. **<i>p</i><0.01, r-metHuGDNF infused hemispheres versus aCSF control infused hemispheres.</p

Immunostaining for activated caspase 3 to demonstrate apoptosis.

<p>An acute dose of GDNF (0.1, 0.2, 0.6 and 1.0 µg/µL) or aCSF vehicle control was infused into the striatum and tissue was collected after 2 weeks and 4 weeks. Representative images showing cells immuno-positive for activated caspase 3 (red) and NeuN (green). Scale bar = 100 µm.</p

Number of hemispheres infused with r-metHuGDNF for qPCR, ELISA, and immunohistochemical analysis.

<p>Number of hemispheres infused with r-metHuGDNF for qPCR, ELISA, and immunohistochemical analysis.</p

Immunostaining for NeuN to show neuronal cell loss.

<p>Neuonal cell loss was seen around the r-metHuGDNF infused area after 2 weeks and 4 weeks at 0.6 µg/µL and 1.0 µg/µL. A lack of NeuN (red) was generally visible where GDNF (green) had been infused at these concentrations (see inlay). Nuclei are marked in blue with the use of DAPI. Scale bar = 250 µm.</p

Immunostaining for Iba1, a protein upregulated during the activation of microglia.

<p>Immunostaining is shown 2 weeks and 4 weeks after r-metHuGDNF infusion. Iba1 (red) was seen in all infused concentrations at 2 weeks. The inlay shows the presence of perivascular microglia. GDNF is shown in green and nuclei marker, DAPI, in blue. Scale bar = 250 µm.</p

Additional file 1: Table S1. of Impact of prematurity and nutrition on the developing gut microbiome and preterm infant growth

Number of significant associations identified in the initial linear mixed-effects regression analysis. Specifically, for each covariate listed in this table, we performed linear mixed-effects regression analyses in which the response variables are microbial taxa abundance and the regressors are as follows: (1) gestational age at birth, (2) post-menstral age, (3) this covariate (main), and (4) the interation between the main covariate and PMA. Regression t tests were used to assess the statistical significance of associations. Benjamini-Hochberg multiple testing procedure was used to control false discovery rate at 0.05 level. Listed in this table are the numbers of taxa that are significantly associated with each regressors in these initial regression analyses. (DOCX 17 kb

Additional file 3: Table S2. of Impact of prematurity and nutrition on the developing gut microbiome and preterm infant growth

Confusion table of phases vs. Dirichlet multinomial mixture components. Each sample was classified as representing a specific phase based on the ratios of Bacilli, Gammaproteobacteria, and Clostridia, as described. Independently, each sample was classified as representing a Dirichlet multinomial mixture (DMM) component based on the abundances of all classes of bacteria present. Each row of the table above indicates the number of samples classified as a given phase, and each column indicates the number of samples classified as a given DMM component. Row/column intersections indicate the number of samples classified as the corresponding phase (row) and DMM component (column). DMM components are numbered automatically from the most common to the least common, while phases are numbered according to their order in a model of temporal progression. Phase 1 is equivalent to DMM component 3; phase 2 corresponds to DMM component 2; and phase 3 corresponds to both DMM components 1 and 4. Colors on the row and column labels indicate these correspondence relationships, and colors internal to the table identify sets of samples where the phase-based and DMM component classifications are in agreement. (DOCX 18 kb

Additional file 2: of Impact of prematurity and nutrition on the developing gut microbiome and preterm infant growth

Figure S1. Composition bar charts by subject and sampling method. Figure S2. Alpha diversity by subject and sampling method observed OTUs. Figure S3. Number of Dirichlet components vs. model fit. Figure S4. Weighted UniFrac Principal Coordinate Analyses of phase and Dirichlet component. (A) Weighted UniFrac Principal Coordinate Analysis plot colored by phase. (B) Weighted UniFrac Principal Coordinate Analysis plot colored by Dirichlet Component. Figure S5. Total functional variance based on the fitted microbiome abundance at the species level relative to postmenstrual age (PMA). Figure S6. Linear regression analysis of meconium samples and Phase 1 rectal samples. Comment on Figure 3F. (DOCX 1141 kb