NUAK1 promotes organ fibrosis via YAP and TGF-ß/SMAD signaling.

Fibrosis is a central pathway that drives progression of multiple chronic diseases, yet few safe and effective clinical antifibrotic therapies exist. In most fibrotic disorders, transforming growth factor-ß (TGF-ß)-driven scarring is an important pathologic feature and a key contributor to disease progression. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are two closely related transcription cofactors that are important for coordinating fibrogenesis after organ injury, but how they are activated in response to tissue injury has, so far, remained unclear. Here, we describe NUAK family kinase 1 (NUAK1) as a TGF-ß-inducible profibrotic kinase that is up-regulated in multiple fibrotic organs in mice and humans. Mechanistically, we show that TGF-ß induces a rapid increase in NUAK1 in fibroblasts. NUAK1, in turn, can promote profibrotic YAP and TGF-ß/SMAD signaling, ultimately leading to organ scarring. Moreover, activated YAP and TAZ can induce further NUAK1 expression, creating a profibrotic positive feedback loop that enables persistent fibrosis. Using mouse models of kidney, lung, and liver fibrosis, we demonstrate that this fibrogenic signaling loop can be interrupted via fibroblast-specific loss of NUAK1 expression, leading to marked attenuation of fibrosis. Pharmacologic NUAK1 inhibition also reduced scarring, either when initiated immediately after injury or when initiated after fibrosis was already established. Together, our data suggest that NUAK1 plays a critical, previously unrecognized role in fibrogenesis and represents an attractive target for strategies that aim to slow fibrotic disease progression.

Next-generation RNA sequencing of archival formalin-fixed paraffin-embedded urothelial bladder cancer.

UNLABELLED: Molecular profiling of individual cancers is key to personalised medicine. While sequencing technologies have required stringent sample collection and handling, recent technical advances offer sequencing from tissues collected in routine practice and tissues already stored in archives. In this paper, we establish methods for whole-transcriptome RNA sequencing (RNA-seq) from formalin-fixed paraffin-embedded tissues. We obtain average RNA-seq reads of >100 million per sample using the Illumina HiSeq2000 platform. We find high concordance with results from matching fresh frozen samples (>0.8 Spearman correlation). For validation, we compared low- and high-grade bladder cancer transcriptomes in 49 tumour samples after transurethral resection of bladder tumour. We found 947 differentially expressed protein-coding genes. While high-grade lesions exhibited distinct intertumour transcriptome heterogeneity, the transcriptome of low-grade tumours was homogeneous. PATIENT SUMMARY: In this report, we show that it is now possible to use universally available bladder cancer samples that have been fixed in formalin to perform high-quality transcriptome analysis. This ability will facilitate the development of transcriptome-wide tests based on gene expression correlated with clinical outcome.

A pilot evaluation of a novel immunohistochemical assay for topoisomerase II-alpha and minichromosome maintenance protein 2 expression (ProEx C) in cervical adenocarcinoma in situ, adenocarcinoma, and benign glandular mimics.

The histopathologic distinction of cervical adenocarcinoma in situ (AIS) and invasive adenocarcinoma (AC) from some benign endocervical lesions can be challenging. The ProEx C antibody reagent targets nuclear proteins (minichromosome maintenance protein 2, MCM2 and topoisomerase II-alpha, TOP2A), which are over expressed during the aberrant S-phase induction of HPV infected and neoplastic cells. In this immunohistochemical study the utility of the ProEx C reagent in distinguishing AIS and AC from a variety of non-neoplastic glandular lesions was examined. ProEx C immunohistochemical staining was performed on sections from formalin-fixed, paraffin-embedded tissue of 65 cervical tissues including 48 non-neoplastic cervices (normal [n=10], microglandular hyperplasia [n=10], tubal metaplasia [n=11], cervical endometriosis [n=7], reactive endocervix [n=10]) and 17 cervices with glandular malignancy (AIS [n=12] and AC [n=5]). Both intensity and prevalence of immunoreactivity was scored. The median and distribution of scores for both prevalence and intensity was compared for AIS versus each of the 5 benign cervical lesions using a Mann-Whitney U test. The median and distribution of prevalence of immunohistochemical staining for AIS was different from all benign mimics, but the intensity of staining for AIS did overlap with some mimics as it was not significantly different from endometriosis, microglandular hyperplasia, and reactive endocervix. ProEx C reagent has potential as an adjunctive testing tool in the histopathologic diagnosis of both AIS and AC, particularly in difficult cases with small biopsies or foci of disease.

PGDS, a novel technique combining chromogenic in situ hybridization and immunohistochemistry for the assessment of ErbB2 (HER2/neu) status in breast cancer.

Given the important prognostic and predictive utility of v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ErbB2) [human epidermal growth factor receptor-2 (HER2/neu)] in breast cancer, it is recommended that ErbB2 testing be performed on all invasive breast cancers at the time of diagnosis. A consensus, however, has not yet been reached as to the optimal method of evaluating ErbB2 status. Immunohistochemistry to detect protein overexpression and fluorescence in situ hybridization (FISH) to detect ErbB2 gene amplification are the most frequently used methods. As no one detection method fulfills all necessary requirements of reliability, reproducibility, and ease of use, we developed a novel approach in the form of a simple assay we refer to as protein and gene double staining (PGDS) which simultaneously evaluates protein overexpression and gene amplification by combining immunohistochemistry with chromogenic in situ hybridization (CISH). A total of 134 invasive breast carcinomas, including 81 cases with a full-face section and 53 cases included in a tissue microarray (TMA), were assessed by PGDS, and the results were correlated with ErbB2 gene amplification status as determined by FISH. ErbB2 gene copy number determined by CISH analysis in the PGDS assay showed excellent concordance with that of FISH (correlation coefficient 0.82; P<0.001 with full-face section cases, and 0.98; P<0.001 with cases in a TMA). The overall concordance rate for gene amplification status between PGDS and FISH was 90.12% in cases with a full-face section and 92.45% with TMA cases. Perfect correlation was seen between the PGDS assay and FISH in cases that were considered either nonamplified or highly amplified by the dual assay. Of the 17 cases that showed low amplification by PGDS, 5 were classified as nonamplified by FISH. Correction for chromosome 17 copy number in the FISH assessment contributed to the discordance between CISH and FISH results. This newly developed PGDS method represents a novel approach to ErbB2 status determination that combines the assessment of both protein overexpression and gene amplification in one simple assay. It is likely that this assay will aid in immunohistochemical calibration and will also increase the sensitivity and specificity of ErbB2 testing.

Dynamic regulation of Drosophila nuclear receptor activity in vivo.

Nuclear receptors are a large family of transcription factors that play major roles in development, metamorphosis, metabolism and disease. To determine how, where and when nuclear receptors are regulated by small chemical ligands and/or protein partners, we have used a 'ligand sensor' system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live developing animals. Transgenic lines were established that express the ligand binding domain of each nuclear receptor fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, the fusion proteins show tissue- and stage-specific patterns of activation. We show that these responses accurately reflect the presence of endogenous and exogenously added hormone, and that they can be modulated by nuclear receptor partner proteins. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. We also see dynamic changes in activation that are indicative of sweeping changes in ligand and/or co-factor production. The screening of a small compound library using this system identified the angular psoralen angelicin and the insect growth regulator fenoxycarb as activators of the Ultraspiracle (USP) ligand-binding domain. These results demonstrate the utility of this system for the functional dissection of nuclear receptor pathways and for the development of new receptor agonists and antagonists that can be used to modulate metabolism and disease and to develop more effective means of insect control.

Intestinal flora translocation and overgrowth in upper gastrointestinal tract induced by hepatic failure.

AIM:To explore the relationship between endoinfection caused by intestinal flora translocation and multiple organ dysfunction in hepatic failure.METHODS:By using the quantitative bacteria culture, bacteria colony was counted in GI tract, bile duct and mesenteric lymphonodus in rat hepatic failure model.RESULTS:Intestinal flora migrated up to the upper GI tract and overgrew in stomach and jejunum in rats with hepatic failure.The number of bacteria colonies in the specimens of stomach, jejunum and ileum were 4.7X10(4)/mL, 2.1X10(5)/mL, 5.5X10(6)/mL in experiment group and 4.6X10(2)/mL, 6.1X10(1)/mL, 2.4X10(3)/mL in control group respectively (P < 0.05 ). Bacteria in bile duct and mesenteric lymphonodus of hepatic failure rats were also cultured. Extensive damages of gastrointestinal mucosa caused by bacterial overgrowth were observed.CONCLUSION:Intestinal flora translocation and overgrowth in stomach and jejunum formed an endoinfectious source and caused obvious pathological injury of gastrointestinal mucosa, which play a very important role in developing abdominal distension, toxic intestinal expansion, alimentary tract haemorrhage and endotoxemia in patients with hepatic failure.