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Abstract Background Renal cell carcinoma is a common malignancy that often presents as a metastatic-disease for which there are no effective treatments. To gain insights into the mechanism of renal cell carcinogenesis, a number of genome-wide expression profiling studies have been performed. Surprisingly, there is very poor agreement among these studies as to which genes are differentially regulated. To better understand this lack of agreement we profiled renal cell tumor gene expression using genome-wide microarrays (45,000 probe sets) and compare our analysis to previous microarray studies. Methods We hybridized total RNA isolated from renal cell tumors and adjacent normal tissue to Affymetrix U133A and U133B arrays. We removed samples with technical defects and removed probesets that failed to exhibit sequence-specific hybridization in any of the samples. We detected differential gene expression in the resulting dataset with parametric methods and identified keywords that are overrepresented in the differentially expressed genes with the Fisher-exact test. Results We identify 1,234 genes that are more than three-fold changed in renal tumors by t-test, 800 of which have not been previously reported to be altered in renal cell tumors. Of the only 37 genes that have been identified as being differentially expressed in three or more of five previous microarray studies of renal tumor gene expression, our analysis finds 33 of these genes (89%). A key to the sensitivity and power of our analysis is filtering out defective samples and genes that are not reliably detected. Conclusions The widespread use of sample-wise voting schemes for detecting differential expression that do not control for false positives likely account for the poor overlap among previous studies. Among the many genes we identified using parametric methods that were not previously reported as being differentially expressed in renal cell tumors are several oncogenes and tumor suppressor genes that likely play important roles in renal cell carcinogenesis. This highlights the need for rigorous statistical approaches in microarray studies.

Human empathy towards non-human animals (Animal Empathy; AE) has shown a strong gender bias, with women demonstrating higher levels than men. This study aimed to investigate the influence of animal experiences on AE in a male-only sample. It was hypothesised that there would be different levels of AE between men with experiences caring for pets, men with experience in animal agriculture, and men with limited animal experiences. Ninety-one Australian men (18yrs+) completed an online survey evaluating their level of AE using the Animal Empathy Scale (AES). Additionally, they were asked what in their experience they think has influenced their beliefs about how animals think and feel. As expected, AE levels differed significantly between groups, with those in the pet ownership experience group demonstrating higher AE levels than the other two groups. All three groups displayed high endorsement for direct interactions with animals in adulthood as being most influential in shaping their beliefs about how animals think and feel. However, our quantitative results support the idea that not all experiences are worth the same, with the responsibility and sacrifice involved in pet caring appearing to be most influential to the development of AE. These findings have implications for the importance of human-animal interactions in understanding animal sentience and the development of AE in males., Competing Interests: None., (© The Author(s) 2024.)

The Hippo signaling pathway and the Notch signaling pathway are evolutionary conserved signaling cascades that have important roles in embryonic development of many organs. In murine liver, disruption of either pathway impairs intrahepatic bile duct development. Recent studies suggested that the Notch signaling receptor Notch2 is a direct transcriptional target of the Hippo signaling pathway effector YAP, and the Notch signaling is a major mediator of the Hippo signaling in maintaining biliary cell characteristics in adult mice. However, it remains to be determined whether the Hippo signaling pathway functions through the Notch signaling in intrahepatic bile duct development. We found that loss of the Hippo signaling pathway tumor suppressor Nf2 resulted in increased expression levels of the Notch signaling pathway receptor Notch2 in cholangiocytes but not in hepatocytes. When knocking down Notch2 on the background of Nf2 deficiency in mouse livers, the excessive bile duct development induced by Nf2 deficiency was suppressed by heterozygous and homozygous deletion of Notch2 in a dose-dependent manner. These results implicated that Notch signaling is one of the downstream effectors of the Hippo signaling pathway in regulating intrahepatic bile duct development.

Recent studies have found that vasogenic brain edema is present during hepatic encephalopathy following acute liver failure and is dependent on increased matrix metalloproteinase 9 (MMP9) activity and downregulation of tight junction proteins. Furthermore, circulating transforming growth factor β1 (TGFβ1) is increased following liver damage and may promote endothelial cell permeability. This study aimed to assess whether increased circulating TGFβ1 drives changes in tight junction protein expression and MMP9 activity following acute liver failure. Blood-brain barrier permeability was assessed in azoxymethane (AOM)-treated mice at 6, 12, and 18 h post-injection via Evan's blue extravasation. Monolayers of immortalized mouse brain endothelial cells (bEnd.3) were treated with recombinant TGFβ1 (rTGFβ1) and permeability to fluorescein isothiocyanate-dextran (FITC-dextran), MMP9 and claudin-5 expression was assessed. Antagonism of TGFβ1 signaling was performed in vivo to determine its role in blood-brain barrier permeability. Blood-brain barrier permeability was increased in mice at 18 h following AOM injection. Treatment of bEnd.3 cells with rTGFβ1 led to a dose-dependent increase of MMP9 expression as well as a suppression of claudin-5 expression. These effects of rTGFβ1 on MMP9 and claudin-5 expression could be reversed following treatment with a SMAD3 inhibitor. AOM-treated mice injected with neutralizing antibodies against TGFβ demonstrated significantly reduced blood-brain barrier permeability. Blood-brain barrier permeability is induced in AOM mice via a mechanism involving the TGFβ1-driven SMAD3-dependent upregulation of MMP9 expression and decrease of claudin-5 expression. Therefore, treatment modalities aimed at reducing TGFβ1 levels or SMAD3 activity may be beneficial in promoting blood-brain barrier integrity following liver failure.

Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. It has previously been shown that the endocannabinoid anandamide exerts antiproliferative effects on cholangiocarcinoma independent of any known cannabinoid receptors, and by the stabilization of lipid rafts, thereby allowing the recruitment and activation of the Fas death receptor complex. Recently, GPR55 was identified as a putative cannabinoid receptor; therefore, the role of GPR55 in the antiproliferative effects of anandamide was evaluated. GPR55 is expressed in all cholangiocarcinoma cells and liver biopsy samples to a similar level as in non-malignant cholangiocytes. Treatment with either anandamide or the GPR55 agonist, O-1602, reduced cholangiocarcinoma cell proliferation in vitro and in vivo. Furthermore, knocking down the expression of GPR55 prevented the antiproliferative effects of anandamide. Coupled to these effects was an increase in JNK activity. The antiproliferative effects of anandamide could be blocked by pretreatment with a JNK inhibitor and the lipid raft disruptors β-methylcyclodextrin and fillipin III. Activation of GPR55 by anandamide or O-1602 increased the amount of Fas in the lipid raft fractions, which could be blocked by pretreatment with the JNK inhibitor. These data represent the first evidence that GPR55 activation by anandamide can lead to the recruitment and activation of the Fas death receptor complex and that targeting GPR55 activation may be a viable option for the development of therapeutic strategies to treat cholangiocarcinoma.

Endocannabinoids are ubiquitous signalling molecules that exert their effects through a number of specific cannabinoid receptors. Recent studies have indicated that this endocannabinoid system is involved in the pathophysiological processes associated with both acute and chronic liver diseases as well as in the complications that arise from these diseases such as hepatic encephalopathy and cardiac problems. Targeting this signalling system has been useful in ameliorating some of the symptoms and consequences in experimental models of these liver diseases. This review summarises the recent advances into our knowledge and understanding of endocannabinoids in liver diseases and highlights potential novel therapeutic strategies that may prove useful to treat these diseases., (Copyright © 2010 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Cholangiocarcinomas are devastating cancers that are resistant to chemotherapies. Resveratrol, a food-derived polyphenol with antitumorigenic properties, can regulate the expression of cytochrome p450 1b1 (Cyp1b1), which may confer chemoresistance in various cancers. Our aims were to assess the effects of resveratrol on the sensitivity of cholangiocarcinoma cells to chemotherapeutic agents and show an association between Cyp1b1 expression and chemosensitivity. Cholangiocarcinoma cell lines were treated with resveratrol before the addition of 5-fluorouracil (5-FU), gemcitabine, or mitomycin C. Cell proliferation and apoptosis were assessed by MTS assays and Annexin staining. Resveratrol effects on cholangiocarcinoma tumor sensitivity to 5-FU was assessed in an in vivo xenograft model using Mz-ChA-1 cells. After resveratrol treatment, Cyp1b1 expression was assessed by real-time PCR and immunoblotting. Stable-transfected cell lines with Cyp1b1 expression knocked down (Mz-Cyp1b1) were used to assess sensitivity to chemotherapeutic agents by MTS assays and Annexin staining and in a xenograft model using Mz-ChA-1 and Mz-Cyp1b1 cells, respectively. For each chemotherapeutic agent, co-treatment with resveratrol in vitro decreased cell proliferation and increased apoptosis to a greater extent than with the chemotherapeutic agent alone. In vivo, 5-FU+resveratrol decreased tumor size and increased TUNEL staining to a greater extent than 5-FU alone. In parallel, resveratrol decreased Cyp1b1 expression in Mz-ChA-1 cells and in cholangiocarcinoma tumors. Mz-Cyp1b1 cells were more sensitive to chemotherapeutic agents in vitro than mock-transfected cells, and Mz-Cyp1b1-induced tumors were more susceptible to 5-FU treatment. We suggest that resveratrol treatment may be a useful adjunct therapy to improve chemosensitivity in cholangiocarcinoma.

Biogenic amines, such as serotonin and dopamine, regulate a multitude of cellular responses. A great deal of effort has been invested into understanding the effects of these molecules and their corresponding receptor systems on cholangiocyte and cholangiocarcinoma secretion, apoptosis and growth. This review summarizes the results of these efforts and highlights the importance of these regulatory molecules on the physiology and pathophysiology of cholangiocytes. Specifically we have focused on the recent findings into the effects of serotonin and dopamine on cholangiocyte hyperplasia and neoplastic growth.

Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. Symptoms are usually evident after blockage of the bile duct by the tumor, and at this late stage, they are relatively resistant to chemotherapy and radiation therapy. Therefore, it is imperative that alternative treatment options are explored. We have previously shown that serotonin metabolism is dysregulated in cholangiocarcinoma leading to an increased secretion of serotonin, which has growth-promoting effects. Because serotonin and dopamine share the degradation machinery, we evaluated the secretion of dopamine from cholangiocarcinoma and its effects on cell proliferation. Using 4 cholangiocarcinoma cell lines and human biopsy samples, we demonstrated that there was an increase in mRNA and protein expression of the dopamine synthesis enzymes tyrosine hydroxylase and dopa decarboxylase in cholangiocarcinoma. There was increased dopamine secretion from cholangiocarcinoma cell lines compared to H69 and HIBEC cholangiocytes and increased dopamine immunoreactivity in human biopsy samples. Furthermore, administration of dopamine to all cholangiocarcinoma cell lines studied increased proliferation by up to 30%, which could be blocked by the pretreatment of the D2 and D4 dopamine receptor antagonists, whereas blocking dopamine production by alpha-methyldopa administration suppressed growth by up to 25%. Administration of alpha-methyldopa to nude mice also suppressed cholangiocarcinoma tumor growth. The data presented here represent the first evidence that dopamine metabolism is dysregulated in cholangiocarcinoma and that modulation of dopamine synthesis may represent an alternative target for the development of therapeutic strategies.