Compartmentalization of functions and predicted miRNA regulation among contiguous regions of the nematode intestine

<p>The intestine of parasitic nematodes has proven an important target for therapies aimed at prevention and treatment of diseases caused by these pathogens in humans, animals and plants. We have developed a unique research model with the intestine of <i>Ascaris suum</i>, the large round worm of swine and humans, that will enhance biological research on this tissue. To expand utility of this model, we quantitatively compared expression of 15,382 coding RNAs and 277 noncoding, micro RNAs (miRNAs) among 3 contiguous regions of the adult <i>A. suum</i> intestine. Differentially expressed transcripts were identified among regions, with the largest number expressed at significantly higher levels in the anterior region, identifying this region as the most functionally unique compared to middle and posterior regions. We further identified 64 exon splice variants (from 47 genes) that are differentially expressed among these regions. A total of 2,063 intestinal mRNA transcripts were predicted to be targeted by intestinal miRNA, and negative correlation coefficients for miRNA:mRNA abundances predicted 22 likely influential miRNAs and 503 likely associated miRNA:mRNA pairs. <i>A. suum</i> intestinal miRNAs were identified that are conserved with intestinal miRNAs from <i>C. elegans</i> (10 mature sequences and 13 seed sequences conserved), and prospective intestinal miRNAs from the murine gastrointestinal nematode, <i>Heligmosomoides polygyrus</i> (5 mature and 11 seeds). Most of the conserved intestinal miRNAs were also high abundance miRNAs. The data provide the most comprehensive compilation of constitutively and differentially expressed genes along the length of the intestine for any nematode species. The information will guide prospective development of many hypotheses on nematode intestinal functions encoded by mRNAs, miRNAs and interactions between these RNA populations.</p

Additional file 1: of Improving eukaryotic genome annotation using single molecule mRNA sequencing

Figure demonstrating the differences in UTR lengths between AC-Orig and AC-PB for (A) 3’ UTRs and (B) 5’ UTRs. (TIFF 493 kb

Viral Discovery and Sequence Recovery Using DNA Microarrays

<div><p>Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.</p> </div

Prototypical Coronavirus Genome Structure

<p>Red bars indicate physical location of virus microarray DNA elements mapped to a generic coronavirus genome. Portions of the coronavirus genome sequenced by physical recovery and PCR methods are highlighted with homologies to known coronaviruses. Abbreviations: aa, amino acid; nt, nucleotide</p

Viral DNA Recovery and Sequencing Scheme

<p>Hybridized viral sequences were physically scraped from a DNA microarray spot, amplified, cloned, and subsequently sequenced.</p

Differentially-expressed brainstem microglia (BSM) and bone marrow monocyte (BMDM) transcripts.

<p>(<b>A</b>) A heat map was generated from unsupervised hierarchical clustering analysis that included all genes expressed (FPKM >1 in ≥1/6 samples). (<b>B</b>) Candidate genes selected for further validation for comparison to CX3CR1 and CCR2. </p

Transcript and protein validation of genes differentially expressed between BMDM and BSM.

<p>(<b>A</b>) BMDM-enriched transcripts, including <i>Sell</i> (p=0.0019), <i>Met</i> (p=0.0074), <i>Cd93</i> (p=0.0104), <i>Kit</i> (p=0.0377), and <i>Clec12a</i> (p=0.0049), were more highly expressed in independently-generated BMDM (n=5) relative to BSM (n=6) using TaqMan and SYBR Green qPCR. BSM-enriched transcripts, including <i>Mertk</i> (p=0.0001), <i>F11r</i> (p<0.0001), <i>P2ry13</i> (p=0.0002), <i>Cadm1</i> (p=0.0002), and <i>Cd81</i> (p=0.0001), were more highly expressed in BSM. (<b>B</b>) Flow cytometry analysis of BMDM (CD11b⁺ and CD115⁺ cells; R1, left panel) and BSM (CD11b⁺ CD45^low cells; R1, right panel) verified that SELL (<b>C</b>) and CLEC12A (<b>D</b>) were detected on BMDM, and not on BSM, while F11R (<b>E</b>) and CD81 (<b>F</b>) were detected on BSM and not BMDM. *, p<0.05; **, p<0.01; ***, p<0.001.</p

High-grade murine gliomas contain F11r⁺ microglia and macrophages.

<p>(<b>A</b>) Ntv-a Ink4a-Arf-/-;Gli-luc mice develop tumors following intracranial RCAS-PDGFB injection (n=4). Sell and F11r expression was examined by flow cytometry in lymphocytes (R1, CD11b^- CD45⁺ cells), microglia (R2, CD11b⁺ CD45^low cells), and macrophages (R3, CD11b⁺ CD45^high cells) within the tumor and in control naïve brains (n=4) in four separate experiments. (<b>B</b>) More F11r⁺ microglia and macrophages were identified in the gliomas relative to the control brains, most notably in the expansion of the R3 population (34% of positively labeled cells), which is typically a very small percentage in control brains (<2%). The majority of the labeled macrophages in the glioma are positive for F11r only (Q1; 94%), with few cells infiltrating the glioma positive for Sell only (Q3; 5%) or double positive for both F11r and Sell (Q2; 1.4%). (C) Bar graphs illustrate the mean (SEM) percentage and SEM for each immune cell population as well as their corresponding F11r and Sell surface expressions. White bars = control, black bars = glioma.</p

BMDM acquire F11r expression following brain infiltration.

<p>(<b>A</b>) GVHD was induced in recipient BALB/cJ mice following total body irradiation (TBI) and bone marrow transplantation (BMT) from T cell depleted (TCD) B6.SJL-<i>Ptprc</i>^<i>a</i><i>Pepc^b/BoyJ</i> donors. Following C57BL/6J mouse donor lymphocyte infusions (DLI), immune cell infiltration was assessed by flow cytometry at 1 week, 2 weeks, and 3 weeks post-DLI (n=6 GVHD and n=6 BMT-only control per time point). (<b>B</b>) Control BMT-only mice do not exhibit GVHD and lack substantial macrophage (R3) or lymphocyte (R1, and R4 in Figure S4) infiltration. There is a very small CD11b⁺, CD45.1^high monocyte population that expresses F11r in the control brain (<2%; R3, right panel). (<b>C</b>) Chimeric mice with GVHD have donor monocyte infiltration (CD11b⁺, CD45.1^high cells; R3) with negligible CD11b⁺, CD45.1^low cells (R2). F11r and Sell expression (right column) of positively-labeled infiltrating donor monocytes (R3) over the course of 3 weeks of GVHD demonstrates a shift from F11r⁺ Sell⁺ cells to F11r⁺ only cells. (D) Bar graphs illustrate the mean and SEM for each population of immune system cells over the course of the three weeks following DLI.</p

F11R expression correlates with GBM malignancy grade and survival.

<p>(<b>A</b>) Immunohistochemistry demonstrates that human bone marrow sections (n=3) have few F11R⁺ cells (arrows), while neurologically-normal post-mortem human frontal cortex brain sections contain numerous F11R⁺ mononuclear cells (n=3) (p= 0.0016). Endothelial cell labeling in the upper left quadrant of the brain section represent a positive control for staining. Scale bar = 50µm. Insets depict representative positively-labeled mononuclear cells. (<b>B</b>) A representative high-grade glioma, GBM, contains many F11R+ cells. Increased percentages of F11R⁺ cells (Kruskal-Wallis test/Dunn's Multiple Comparison Test, p<0.0001) are observed in high-grade glioma (AA, anaplastic astrocytoma, n=23; GBM, n=52) relative to low-grade tumors (PA, pilocytic astrocytoma, n=73) or normal brain (NB, n=23). (<b>C</b>) Kaplan-Meier curves and log rank test demonstrate that increased <i>F11R</i> expression negatively correlated with patient survival (GEO database: GSE16011, n=159, p=0.0037). (<b>D</b>) <i>F11R</i> was highly expressed in GSE16011 GBM samples assigned to Classical and Mesenchymal TCGA subtypes relative to the Proneural subtype (p=3.94E-12). </p