Oncogenic context shapes the fitness landscape of tumor suppression.

Tumors acquire alterations in oncogenes and tumor suppressor genes in an adaptive walk through the fitness landscape of tumorigenesis. However, the interactions between oncogenes and tumor suppressor genes that shape this landscape remain poorly resolved and cannot be revealed by human cancer genomics alone. Here, we use a multiplexed, autochthonous mouse platform to model and quantify the initiation and growth of more than one hundred genotypes of lung tumors across four oncogenic contexts: KRAS G12D, KRAS G12C, BRAF V600E, and EGFR L858R. We show that the fitness landscape is rugged-the effect of tumor suppressor inactivation often switches between beneficial and deleterious depending on the oncogenic context-and shows no evidence of diminishing-returns epistasis within variants of the same oncogene. These findings argue against a simple linear signaling relationship amongst these three oncogenes and imply a critical role for off-axis signaling in determining the fitness effects of inactivating tumor suppressors.

MYC Overexpression Drives Immune Evasion in Hepatocellular Carcinoma That Is Reversible through Restoration of Proinflammatory Macrophages.

Cancers evade immune surveillance, which can be reversed through immune-checkpoint therapy in a small subset of cases. Here, we report that the MYC oncogene suppresses innate immune surveillance and drives resistance to immunotherapy. In 33 different human cancers, MYC genomic amplification and overexpression increased immune-checkpoint expression, predicted nonresponsiveness to immune-checkpoint blockade, and was associated with both Th2-like immune profile and reduced CD8 T-cell infiltration. MYC transcriptionally suppressed innate immunity and MHCI-mediated antigen presentation, which in turn impeded T-cell response. Combined, but not individual, blockade of PDL1 and CTLA4 could reverse MYC-driven immune suppression by leading to the recruitment of proinflammatory antigen-presenting macrophages with increased CD40 and MHCII expression. Depletion of macrophages abrogated the antineoplastic effects of PDL1 and CTLA4 blockade in MYC-driven hepatocellular carcinoma (HCC). Hence, MYC is a predictor of immune-checkpoint responsiveness and a key driver of immune evasion through the suppression of proinflammatory macrophages. The immune evasion induced by MYC in HCC can be overcome by combined PDL1 and CTLA4 blockade. SIGNIFICANCE: Macrophage-mediated immune evasion is a therapeutic vulnerability of MYC-driven cancers, which has implications for prioritizing MYC-driven hepatocellular carcinoma for combination immunotherapy.

Inducible lncRNA transgenic mice reveal continual role of HOTAIR in promoting breast cancer metastasis.

HOTAIR is a 2.2-kb long noncoding RNA (lncRNA) whose dysregulation has been linked to oncogenesis, defects in pattern formation during early development, and irregularities during the process of epithelial-to-mesenchymal transition (EMT). However, the oncogenic transformation determined by HOTAIR in vivo and its impact on chromatin dynamics are incompletely understood. Here, we generate a transgenic mouse model with doxycycline-inducible expression of human HOTAIR in the context of the MMTV-PyMT breast cancer-prone background to systematically interrogate the cellular mechanisms by which human HOTAIR lncRNA acts to promote breast cancer progression. We show that sustained high levels of HOTAIR over time increased breast metastatic capacity and invasiveness in breast cancer cells, promoting migration and subsequent metastasis to the lung. Subsequent withdrawal of HOTAIR overexpression reverted the metastatic phenotype, indicating oncogenic lncRNA addiction. Furthermore, HOTAIR overexpression altered both the cellular transcriptome and chromatin accessibility landscape of multiple metastasis-associated genes and promoted EMT. These alterations are abrogated within several cell cycles after HOTAIR expression is reverted to basal levels, indicating an erasable lncRNA-associated epigenetic memory. These results suggest that a continual role for HOTAIR in programming a metastatic gene regulatory program. Targeting HOTAIR lncRNA may potentially serve as a therapeutic strategy to ameliorate breast cancer progression.

Metabolic convergence on lipogenesis in RAS, BCR-ABL, and MYC-driven lymphoid malignancies.

BACKGROUND: Metabolic reprogramming is a central feature in many cancer subtypes and a hallmark of cancer. Many therapeutic strategies attempt to exploit this feature, often having unintended side effects on normal metabolic programs and limited efficacy due to integrative nature of metabolic substrate sourcing. Although the initiating oncogenic lesion may vary, tumor cells in lymphoid malignancies often share similar environments and potentially similar metabolic profiles. We examined cells from mouse models of MYC-, RAS-, and BCR-ABL-driven lymphoid malignancies and find a convergence on de novo lipogenesis. We explore the potential role of MYC in mediating lipogenesis by 13C glucose tracing and untargeted metabolic profiling. Inhibition of lipogenesis leads to cell death both in vitro and in vivo and does not induce cell death of normal splenocytes. METHODS: We analyzed RNA-seq data sets for common metabolic convergence in lymphoma and leukemia. Using in vitro cell lines derived in from conditional MYC, RAS, and BCR-ABL transgenic murine models and oncogene-driven human cell lines, we determined gene regulation, metabolic profiles, and sensitivity to inhibition of lipogenesis in lymphoid malignancies. We utilize preclinical murine models and transgenic primary model of T-ALL to determine the effect of lipogenesis blockade across BCR-ABL-, RAS-, and c-MYC-driven lymphoid malignancies. Statistical significance was calculated using unpaired t-tests and one-way ANOVA. RESULTS: This study illustrates that de novo lipid biogenesis is a shared feature of several lymphoma subtypes. Using cell lines derived from conditional MYC, RAS, and BCR-ABL transgenic murine models, we demonstrate shared responses to inhibition of lipogenesis by the acetyl-coA carboxylase inhibitor 5-(tetradecloxy)-2-furic acid (TOFA), and other lipogenesis inhibitors. We performed metabolic tracing studies to confirm the influence of c-MYC and TOFA on lipogenesis. We identify specific cell death responses to TOFA in vitro and in vivo and demonstrate delayed engraftment and progression in vivo in transplanted lymphoma cell lines. We also observe delayed progression of T-ALL in a primary transgenic mouse model upon TOFA administration. In a panel of human cell lines, we demonstrate sensitivity to TOFA treatment as a metabolic liability due to the general convergence on de novo lipogenesis in lymphoid malignancies driven by MYC, RAS, or BCR-ABL. Importantly, cell death was not significantly observed in non-malignant cells in vivo. CONCLUSIONS: These studies suggest that de novo lipogenesis may be a common survival strategy for many lymphoid malignancies and may be a clinically exploitable metabolic liability. TRIAL REGISTRATION: This study does not include any clinical interventions on human subjects.

Conducting Concurrent Electroencephalography and Functional Near-Infrared Spectroscopy Recordings with a Flanker Task.

Concurrent EEG and fNIRS recordings offer an excellent opportunity to gain a full understanding of the neural mechanism of cognitive processing by inspecting the relationship between the neural and hemodynamic signals. EEG is an electrophysiological technology that can measure the rapid neuronal activity of the cortex, whereas fNIRS relies on the hemodynamic responses to infer brain activation. The combination of EEG and fNIRS neuroimaging techniques can identify more features and reveal more information associated with the functioning of the brain. In this protocol, fused EEG-fNIRS measurements were performed for concurrent recordings of evoked-electrical potentials and hemodynamic responses during a Flanker task. In addition, the critical steps for setting up the hardware and software system as well as the procedures for data acquisition and analysis were provided and discussed in detail. It is expected that the present protocol can pave a new avenue for improving the understanding of the neural mechanisms underlying various cognitive processes by using the EEG and fNIRS signals.

MYC and Twist1 cooperate to drive metastasis by eliciting crosstalk between cancer and innate immunity.

Metastasis is a major cause of cancer mortality. We generated an autochthonous transgenic mouse model whereby conditional expression of MYC and Twist1 enables hepatocellular carcinoma (HCC) to metastasize in >90% of mice. MYC and Twist1 cooperate and their sustained expression is required to elicit a transcriptional program associated with the activation of innate immunity, through secretion of a cytokinome that elicits recruitment and polarization of tumor associated macrophages (TAMs). Systemic treatment with Ccl2 and Il13 induced MYC-HCCs to metastasize; whereas, blockade of Ccl2 and Il13 abrogated MYC/Twist1-HCC metastasis. Further, in 33 human cancers (n = 9502) MYC and TWIST1 predict poor survival (p=4.3×10-10), CCL2/IL13 expression (p<10-109) and TAM infiltration (p<10-96). Finally, in the plasma of patients with HCC (n = 25) but not cirrhosis (n = 10), CCL2 and IL13 were increased and IL13 predicted invasive tumors. Therefore, MYC and TWIST1 generally appear to cooperate in human cancer to elicit a cytokinome that enables metastasis through crosstalk between cancer and immune microenvironment.

Periodontitis links to exacerbation of myocardial dysfunction in subjects with type 2 diabetes.

BACKGROUND AND OBJECTIVE: Subjects with diabetes and periodontitis are at high risk of cardiovascular events, while the subclinical alterations of cardiac function in this cohort remain unclear. This cross-sectional study investigated the association of periodontitis with left ventricle (LV) structural and functional abnormalities in subjects with type 2 diabetes mellitus (T2DM). MATERIAL AND METHODS: A total of 115 subjects with T2DM were divided into Control group (n = 32) with no or mild periodontitis, and the rest with moderate to severe chronic periodontitis (CP) were further categorized into CP-1 (n = 41) and CP-2 (n = 42) based on disease severity. Echocardiography was performed to precisely assess (a) LV hypertrophy by LV mass index (LVMi); (b) LV diastolic function by tissue Doppler imaging index E/e' ratio; and (c) LV systolic function by speckle tracking derived global longitudinal strain (GLS). RESULTS: Overall, a linear trend in LVMi, E/e', and GLS existed among the Control, CP-1, and CP-2 groups, respectively (P < 0.05). After adjustments of multiple confounders, CP-2 subjects showed significantly higher E/e' (log scale, 2.22 ± 0.05 vs 2.07 ± 0.06, P < 0.01) and GLS (-17.42 ± 0.46% vs -18.95 ± 0.54%, P < 0.05) than the Controls. Multivariate analysis revealed that sites% with probing depth ≥4 mm and sites% with clinical attachment loss ≥5 mm were independent indicators for E/e' (ß = 0.005 and ß = 0.002, P < 0.01) and GLS (ß = 0.03 and ß = 0.02, P < 0.05) , respectively. Moreover, the number of missing teeth was significantly associated with LVMi (ß = 0.01, P < 0.01). CONCLUSION: This study provides the first evidence that severe periodontitis is significantly associated with the exacerbation of LV diastolic and systolic dysfunction in subjects with T2DM.

Lipid nanoparticles that deliver IL-12 messenger RNA suppress tumorigenesis in MYC oncogene-driven hepatocellular carcinoma.

Interleukin-12 (IL-12) is a promising candidate for cancer immunotherapy because of its ability to activate a number of host immune subsets that recognize and destroy cancer cells. We found that human hepatocellular carcinoma (HCC) patients with higher than median levels of IL-12 have significantly favorable clinical outcomes. Here, we report that a messenger RNA (mRNA) lipid nanoparticle delivering IL-12 (IL-12-LNP) slows down the progression of MYC oncogene-driven HCC. IL-12-LNP was well distributed within the HCC tumor and was not associated with significant animal toxicity. Treatment with IL-12-LNP significantly reduced liver tumor burden measured by dynamic magnetic resonance imaging (MRI), and increased survival of MYC-induced HCC transgenic mice in comparison to control mice. Importantly, IL-12-LNP exhibited no effect on transgenic MYC levels confirming that its therapeutic efficacy was not related to the downregulation of a driver oncogene. IL-12-LNP elicited marked infiltration of activated CD44+ CD3+ CD4+ T helper cells into the tumor, and increased the production of Interferon γ (IFNγ). Collectively, our findings suggest that IL-12-LNP administration may be an effective immunotherapy against HCC.

Anti-miR-17 therapy delays tumorigenesis in MYC-driven hepatocellular carcinoma (HCC).

Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options. Genomic amplification and/or overexpression of the MYC oncogene is a common molecular event in HCC, thus making it an attractive target for drug therapy. Unfortunately, currently there are no direct drug therapies against MYC. As an alternative strategy, microRNAs regulated by MYC may be downstream targets for therapeutic blockade. MiR-17 family is a microRNA family transcriptionally regulated by MYC and it is commonly overexpressed in human HCCs. In this study, we performed systemic delivery of a novel lipid nanoparticle (LNP) encapsulating an anti-miR-17 oligonucleotide in a conditional transgenic mouse model of MYC driven HCC. Treatment with anti-miR-17 in vivo, but not with a control anti-miRNA, resulted in significant de-repression of direct targets of miR-17, robust apoptosis, decreased proliferation and led to delayed tumorigenesis in MYC-driven HCCs. Global gene expression profiling revealed engagement of miR-17 target genes and inhibition of key transcriptional programs of MYC, including cell cycle progression and proliferation. Hence, anti-miR-17 is an effective therapy for MYC-driven HCC.

Prognostic factors of short-term outcomes of intravitreal ranibizumab in diabetic macular edema.

AIM: To evaluate the prognostic factors for short-term visual and anatomical improvement of intravitreal ranibizumab (IVR) for diabetic macular edema (DME). METHODS: Fifty-one eyes from 35 patients that received three consecutive monthly IVR for DME with moderate visual loss were retrospectively recruited; all cases had their baseline best-corrected visual acuity (BCVA) between 20/400 and 20/40. BCVA and central subfield thickness (CST) at baseline and month 3 were collected. Linear mixed models were used to evaluate the prognostic factors for visual and anatomical improvement at month 3. RESULTS: Younger age, poorer baseline BCVA and proliferative diabetic retinopathy (PDR) were correlated with better visual improvement at month 3 (P=0.002, 0.0001 and 0.007, respectively). Thicker CST and the presence of subretinal fluid at baseline were correlated with a greater reduction in CST (P<0.0001 and P=0.018, respectively). The presence of epiretinal membrane or previous posterior subtenon injection of triamcinolone acetonide (PSTA) were associated with a smaller reduction in CST (P=0.029 and 0.018, respectively), but had no significant effects in visual improvement at month 3 (P>0.05 for both). CONCLUSION: For eyes with DME and moderate visual loss, those with younger age, poorer baseline BCVA or PDR tend to have better visual improvement after three consecutive monthly IVR. Epiretinal membrane or previous PSTA result in less resolution of CST, but do not significantly affect visual improvement.

Dietary antioxidants (selenium and N-acetylcysteine) modulate paraoxonase 1 (PON1) in PCB 126-exposed rats.

Environmental pollutants polychlorinated biphenyls (PCBs), especially dioxin-like PCBs, cause oxidative stress and associated toxic effects, including cancer and possibly atherosclerosis. We previously reported that PCB 126, the most potent dioxin-like PCB congener, not only decreases antioxidants such as hepatic selenium (Se), Se-dependent glutathione peroxidase, and glutathione (GSH) but also increases levels of the antiatherosclerosis enzyme paraoxonase 1 (PON1) in liver and serum. To probe the interconnection of these three antioxidant systems, Se, GSH, and PON1, we examined the influence of varying levels of dietary Se and N-acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS) and precursor for GSH synthesis, on PON1 in the absence and presence of PCB 126 exposure. Male Sprague-Dawley rats, fed diets with differing Se levels (0.02, 0.2, or 2 ppm) or NAC (1%), were treated with a single intraperitoneal injection of corn oil or various doses of PCB 126 and euthanized 2 weeks later. PCB 126 significantly increased liver PON1 mRNA, protein level and activity, and serum PON1 activity in all dietary groups but did not consistently increase thiobarbituric acid levels (thiobarbituric acid reactive substances, TBARS), an indicator of lipid oxidation and oxidative stress, in liver or serum. Inadequate (high or low) dietary Se decreased baseline and PCB 126-induced aryl hydrocarbon receptor (AhR) expression but further increased PCB 126-induced cytochrome P450 1A1 (CYP1A1) expression, the enzyme believed to be the cause for PCB 126-induced oxidative stress. In addition, a significant inverse relationship was observed not only between dietary Se levels and PON1 mRNA and PON1 activity but also with TBARS levels in the liver, suggesting significant antioxidant protection from dietary Se. NAC lowered serum baseline TBARS levels in controls and increased serum PON1 activity but lowered liver PON1 activities in animals treated with 1 µmol/kg PCB 126, suggesting antioxidant activity by NAC primarily in serum. These results also show an unexpected predominantly inverse relationship between Se or NAC and PON1 during control and PCB 126 exposure conditions. These interactions should be further explored in the development of dietary protection regimens.

Does dietary copper supplementation enhance or diminish PCB126 toxicity in the rodent liver?

Copper is essential for the function of the mitochondrial electron transport chain and several antioxidant proteins. However, in its free form copper can participate in Fenton-like reactions that produce reactive hydroxyl radicals. Aryl-hydrocarbon receptor (AhR) agonists, including the most potent polychlorinated biphenyl (PCB) congener, 3,3',4,4',5-pentachlorobiphenyl (PCB126), increase copper levels in rodent livers. This is accompanied by biochemical and toxic changes. To assess the involvement of copper in PCB toxicity, male Sprague-Dawley rats were fed an AIN-93G diet with differing dietary copper levels: low (2 ppm), adequate (6 ppm), and high (10 ppm). After three weeks, rats from each group were given a single ip injection of corn oil (control), 1, or 5 µmol/kg body weight PCB126. Two weeks following injections, biochemical and morphological markers of hepatic toxicity, trace metal status, and hepatic gene expression of metalloproteins were evaluated. Increasing dietary copper was associated with elevated tissue levels of copper and ceruloplasmin. In the livers of PCB126-treated rats, the hallmark signs of AhR activation were present, including increased cytochrome P450 and lipid levels and decreased glutathione. In addition, a doubling of hepatic copper levels was seen, and overall metal homeostasis was disturbed, resulting in decreased hepatic selenium, manganese, zinc, and iron. Expression of key metalloproteins was either decreased (cytochrome c oxidase), unchanged (ceruloplasmin and CuZnSOD), or increased (tyrosinase and metallothioneins 1 and 2) with exposure to PCB126. Increases in metallothionein may contribute/reflect the increased copper seen. Alterations in dietary copper did not amplify or abrogate the hepatic toxicity of PCB126. PCB126 toxicity, i.e., oxidative stress and steatosis, is clearly associated with disturbed metal homeostasis. Understanding the mechanisms of this disturbance may provide tools to prevent liver toxicity by other AhR agonists.

N-acetylcysteine (NAC) diminishes the severity of PCB 126-induced fatty liver in male rodents.

Potent aryl hydrocarbon receptor agonists like PCB 126 (3,3',4,4',5-pentachlorobiphenyl) cause oxidative stress and liver pathology, including fatty liver. Our question was whether dietary supplementation with N-acetylcysteine (NAC), an antioxidant, can prevent these adverse changes. Male Sprague-Dawley rats were fed a standard AIN-93G diet (sufficient in cysteine) or a modified diet supplemented with 1.0% NAC. After one week, rats on each diet were exposed to 0, 1, or 5µmol/kg body weight PCB 126 by i.p. injection (6 rats per group) and euthanized two weeks later. PCB-treatment caused a dose-dependent reduction in growth, feed consumption, relative thymus weight, total glutathione and glutathione disulfide (GSSG), while relative liver weight, glutathione transferase activity and hepatic lipid content were dose-dependently increased with PCB dose. Histologic examination of liver tissue showed PCB 126-induced hepatocellular steatosis with dose dependent increase in lipid deposition and distribution. Dietary NAC resulted in a reduction in hepatocellular lipid in both PCB groups. This effect was confirmed by gravimetric analysis of extracted lipids. Expression of CD36, a scavenger receptor involved in regulating hepatic fatty acid uptake, was reduced with high dose PCB treatment but unaltered in PCB-treated rats on NAC-supplemented diet. These results demonstrate that NAC has a protective effect against hepatic lipid accumulation in rats exposed to PCB 126. The mechanism of this protective effect appears to be independent of NAC as a source of cysteine/precursor of glutathione.

Dietary selenium as a modulator of PCB 126-induced hepatotoxicity in male Sprague-Dawley rats.

Homeostasis of selenium (Se), a critical antioxidant incorporated into amino acids and enzymes, is disrupted by exposure to aryl hydrocarbon receptor (AhR) agonists. Here we examined the importance of dietary Se in preventing the toxicity of the most toxic polychlorinated biphenyl congener, 3,3',4,4',5-pentachlorobiphenyl (PCB 126), a potent AhR agonist. Male Sprague-Dawley rats were fed a modified AIN-93 diet with differing dietary Se levels (0.02, 0.2, and 2 ppm). Following 3 weeks of acclimatization, rats from each dietary group were given a single ip injection of corn oil (vehicle), 0.2, 1, or 5 µmol/kg body weight PCB 126, followed 2 weeks later by euthanasia. PCB exposure caused dose-dependent increases in liver weight and at the highest PCB 126 dose decreases in whole body weight gains. Hepatic cytochrome P-450 (CYP1A1) activity was significantly increased even at the lowest dose of PCB 126, indicating potent AhR activation. PCB exposure diminished hepatic Se levels in a dose-dependent manner, and this was accompanied by diminished Se-dependent glutathione peroxidase activity. Both these effects were partially mitigated by Se supplementation. Conversely, thioredoxin (Trx) reductase activity and Trx oxidation state, although significantly diminished in the lowest dietary Se groups, were not affected by PCB exposure. In addition, PCB 126-induced changes in hepatic copper, iron, manganese, and zinc were observed. These results demonstrate that supplemental dietary Se was not able to completely prevent the toxicity caused by PCB 126 but was able to increase moderately the levels of several key antioxidants, thereby maintaining them roughly at normal levels.

Oral health of Chinese people with systemic sclerosis.

The aim was to study oral health status, salivary function, and oral features of Chinese people with Systemic Sclerosis (SSc). Chinese people with SSc attending a university specialist clinic were invited for a questionnaire survey and a clinical examination. Ethics approval was sought (UW 08-305). Gender- and age-matched individuals without SSc who attended a university dental hospital were recruited for comparison. Forty-two SSc patients with a mean age of 54.0 ± 12.2 were examined. This study found no Chinese people with systemic sclerosis were periodontally healthy and many (76%) had periodontal pockets despite most of them (93%) practiced daily tooth-brushing. They all had caries experience (DMFT = 10.5) and many (65%) had untreated decay. Mucosal telangiectasia was a common oral feature (80%). They had lower resting salivary flow rates (0.18 ± 0.17 ml/min vs. 0.31 ± 0.21 ml/min; p = 0.003) and pH values (6.90 ± 0.40 vs. 7.28 ± 0.31; p < 0.001) and reduced maximal mouth opening (40.1 ± 6.5 mm vs. 43.6 ± 7.0 mm) than people without SSc.

Acute toxicity of 3,3',4,4',5-pentachlorobiphenyl (PCB 126) in male Sprague-Dawley rats: effects on hepatic oxidative stress, glutathione and metals status.

Although polychlorinated biphenyl (PCBs) production, and new uses for PCBs, was halted in the 1970s in the United States, PCBs continue to be used in closed systems and persist in the environment, accumulating in fatty tissues. PCBs are efficacious inducers of drug metabolism and may increase oxidative events and alter many other biochemical and morphologic parameters within cells and tissues. The goal of the present study was to evaluate the effects of a single, very low dose of PCB 126 (3,3',4,4',5-pentachlorobiphenyl), a coplanar, dioxin-like PCB congener and aryl hydrocarbon receptor (AhR) agonist, on redox status, metals homeostasis, antioxidant enzymes, and cellular morphology. To examine these parameters, male Sprague-Dawley rats were fed a purified AIN-93 basal diet containing 0.2 ppm selenium for two weeks, then administered a single i.p. injection of corn oil (5 ml/kg body weight) or 1µmol PCB 126/kg body weight (326µg/kg body weight) in corn oil. Rats were maintained on the diet for an additional two weeks before being euthanized. This dose of PCB 126 did not alter feed intake or growth, but significantly increased liver weight (42%) and hepatic microsomal cytochrome P-450 (CYP1A) enzyme activities (10-40-fold increase). Hepatic zinc, selenium, and glutathione levels were significantly decreased 15%, 30%, and 20%, respectively, by PCB 126. These changes were accompanied by a 60% decrease in selenium-dependent glutathione peroxidase activity. In contrast, hepatic copper levels were increased 40% by PCB 126. PCB 126-induced pathology was characterized by hepatocellular hypertrophy and mild steatosis in the liver and a mild decrease in cortical T-cells in the thymus. This controlled study in rats fed a purified diet shows that even a single, very low dose of PCB 126 that did not alter feed intake or growth, significantly perturbed redox and metals homeostasis and antioxidant and enzyme levels in rodent liver.