Automated colitis detection from endoscopic biopsies as a tissue screening tool in diagnostic pathology

<p>We present a method for identifying colitis in colon biopsies as an extension of our framework for the automated identification of tissues in histology images. Histology is a critical tool in both clinical and research applications, yet even mundane histological analysis, such as the screening of colon biopsies, must be carried out by highly-trained pathologists at a high cost per hour, indicating a niche for potential automation. To this end, we build upon our previous work by extending the histopathology vocabulary (a set of features based on visual cues used by pathologists) with new features driven by the colitis application. We use the multiple-instance learning framework to allow our pixel-level classifier to learn from image-level training labels. The new system achieves accuracy comparable to state-of-the-art biological image classifiers with fewer and more intuitive features.</p

NOL11, Implicated in the Pathogenesis of North American Indian Childhood Cirrhosis, Is Required for Pre-rRNA Transcription and Processing

<div><p>The fundamental process of ribosome biogenesis requires hundreds of factors and takes place in the nucleolus. This process has been most thoroughly characterized in baker's yeast and is generally well conserved from yeast to humans. However, some of the required proteins in yeast are not found in humans, raising the possibility that they have been replaced by functional analogs. Our objective was to identify non-conserved interaction partners for the human ribosome biogenesis factor, hUTP4/Cirhin, since the R565W mutation in the C-terminus of hUTP4/Cirhin was reported to cause North American Indian childhood cirrhosis (NAIC). By screening a yeast two-hybrid cDNA library derived from human liver, and through affinity purification followed by mass spectrometry, we identified an uncharacterized nucleolar protein, NOL11, as an interaction partner for hUTP4/Cirhin. Bioinformatic analysis revealed that NOL11 is conserved throughout metazoans and their immediate ancestors but is not found in any other phylogenetic groups. Co-immunoprecipitation experiments show that NOL11 is a component of the human ribosomal small subunit (SSU) processome. siRNA knockdown of NOL11 revealed that it is involved in the cleavage steps required to generate the mature 18S rRNA and is required for optimal rDNA transcription. Furthermore, abnormal nucleolar morphology results from the absence of NOL11. Finally, yeast two-hybrid analysis shows that NOL11 interacts with the C-terminus of hUTP4/Cirhin and that the R565W mutation partially disrupts this interaction. We have therefore identified NOL11 as a novel protein required for the early stages of ribosome biogenesis in humans. Our results further implicate a role for NOL11 in the pathogenesis of NAIC.</p> </div

NOL11 is required for optimal rDNA transcription.

<p>Cells were transfected with the indicated siRNA and a plasmid containing firefly luciferase under the control of the rDNA promoter (pHrD-IRES-Luc). Transfection efficiency was normalized by co-transfection with a plasmid containing <i>Renilla</i> luciferase. Luciferase levels were measured using a 20/20n luminometer (Turner BioSystems) and the Dual Luciferase Assay System (Promega) 24 h after transfection. Each experiment was performed in triplicate. Average relative luciferase activity is shown with the standard deviation indicated by the error bars.</p

Loss of NOL11 causes abnormal nucleolar morphology.

<p>The indicated siRNAs were transfected into MCF-10A cells and nucleolar morphology was determined 72 h later by staining nucleoli with an anti-fibrillarin antibody (72B9; top panels). Hoechst was used to stain chromatin. Images from the blue channel only are shown in the bottom panels. Blue = Hoechst, Red = fibrillarin.</p

RNA polymerase I transcription is required for NOL11 association with the SSU processome.

<p>HeLa cells were lysed and the lysate was clarified by centrifugation. The known SSU processome protein, fibrillarin, was immunoprecipitated using an anti-fibrillarin antibody (72B9) conjugated to beads. Total (5%) and immunoprecipitated proteins were separated by SDS-PAGE and transferred to a PVDF membrane. Association of NOL11 with the SSU processome was assayed by Western blotting for NOL11 with an anti-NOL11 antibody. Cells were either not treated (−ActD) or treated (+ActD) with 0.1 µg/ml ActD for 1 h before harvesting. B = beads alone, T = total protein, IP = immunoprecipitation.</p

Proteins that co-purify with hUTP4/Cirhin identified by mass spectrometry.

<p>Only proteins that were identified in both biological duplicates and had at least five unique peptides identified between duplicates are included. The full list can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002892#pgen.1002892.s001" target="_blank">Table S1</a>.</p>+<p>Bait protein used for affinity purification.</p>*<p>Proteins that are repeatedly isolated with other bait proteins.</p

hUTP4/Cirhin interacts with NOL11.

<p>(A) hUTP4/Cirhin was screened against a human liver cDNA library using the yeast two-hybrid methodology. Two positive clones were recovered (clones 5 and 6), both of which were C-terminal fragments of nucleolar protein 11 (NOL11). (B) Directed Y2H analysis confirms that hUTP4/Cirhin interacts with the two positive clones as well as with full-length NOL11 (NOL11-FL). EV is the empty Y2H prey vector. Growth of a blue yeast colony indicates interaction. (C) NOL11 is conserved throughout metazoans. BLAST searches using human NOL11 (NP_056277.2) as a query were used to identify NOL11 orthologs in other species. BioEdit <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002892#pgen.1002892-Hall1" target="_blank">[57]</a> was used to calculate percent identity and similarity.</p

NOL11 is required for pre-rRNA processing in HeLa cells.

<p>(A) Diagram of pre-rRNA processing in human cells. Cleavage sites are indicated with labeled arrows. The positions of oligonucleotide probes used for Northern blotting are indicated with lettered lines. (B) hUTP4/Cirhin and NOL11 were successfully knocked down by siRNAs. After siRNA treatment, total protein was extracted from HeLa cells, separated by SDS-PAGE, and transferred to a PVDF membrane. Western blotting was used to check for the presence of hUTP4/Cirhin (left) or NOL11 (right). (C) Northern blot analysis of pre-rRNA processing. After siRNA treatment, total RNA was extracted from HeLa cells, separated on an agarose/formaldehyde gel, and transferred to a Hybond XL nylon membrane. RNA was detected with the indicated oligonucleotide probe or with methylene blue.</p

The NAIC mutation disrupts the interaction between NOL11 and a C-terminal fragment of hUTP4/Cirhin.

<p>(A) Diagram of the motifs in hUTP4/Cirhin predicted by the SMART database <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002892#pgen.1002892-Letunic1" target="_blank">[61]</a> and the hUTP4/Cirhin mutants used in this study. Triangles represent WD40 repeats. The lightning bolt indicates the NAIC mutation (R565W) and the arrows show the locations of truncations at P374, K514, and D557. (B) Western blot showing expression of MYC-tagged hUTP4/Cirhin wild type (WT) and mutants (R565W, P374X, K514X, D557X, 374-end, 374-end R565W) from the Y2H bait vector. An anti-Mpp10 antibody was used to assess gel loading. (C) Y2H analysis between NOL11 and hUTP4/Cirhin mutants shows that NOL11 interacts with the C-terminus of hUTP4/Cirhin but not with the C-terminus of hUTP4/Cirhin containing the NAIC mutation. 10-fold serial dilutions were spotted onto medium lacking leucine, tryptophan, and histidine; growth on this medium indicates an interaction. (D) Immuno-fluorescence microscopy showing the subcellular localization of hUTP4/Cirhin mutants. V5-tagged hUTP4/Cirhin mutants were transfected into HT1080-3D1 cells and detected with an anti-V5 antibody. An anti-UBF antibody was used to co-stain nucleoli. DAPI was used to stain chromatin. Blue = DAPI, Red = hUTP4/Cirhin, Green = UBF.</p

Subcellular localization of nucleolar proteins in WDR43 depleted HeLa cells.

<p>IF analysis of nucleolar protein localization in control siRNA (A, C, E, G) and <i>WDR43</i> (B, D, F, H) siRNA treated HeLa cells. WDR43 depleted cells exhibited mislocalized expression of TCOF1 (B), Mpp10 (D), Nucleolin (F) and Fibrillarin (H) (arrows), as compared to their respective control GFP siRNA treated cells (see arrows). Scale bar = 10 µm.</p