Zucker Lean Rats With Hepatic Steatosis Recapitulate Asymptomatic Metabolic Syndrome and Exhibit Greater Sensitivity to Drug-Induced Liver Injury Compared With Standard Nonclinical Sprague-Dawley Rat Model.

Fatty liver disease is a potential risk factor for drug-induced liver injury (DILI). Despite advances in nonclinical in vitro and in vivo models to assess liver injury during drug development, the pharmaceutical industry is still plagued by idiosyncratic DILI. Here, we tested the hypothesis that certain features of asymptomatic metabolic syndrome (namely hepatic steatosis) increase the risk for DILI in certain phenotypes of the human population. Comparison of the Zucker Lean (ZL) and Zucker Fatty rats fed a high fat diet (HFD) revealed that HFD-fed ZL rats developed mild hepatic steatosis with compensatory hyperinsulinemia without increases in liver enzymes. We then challenged steatotic HFD-fed ZL rats and Sprague-Dawley (SD) rats fed normal chow, a nonclinical model widely used in the pharmaceutical industry, with acetaminophen overdose to induce liver injury. Observations in HFD-fed ZL rats included increased liver injury enzymes and greater incidence and severity of hepatic necrosis compared with similarly treated SD rats. The HFD-fed ZL rats also had disproportionately higher hepatic drug accumulation, which was linked with abnormal hepatocellular efflux transporter distribution. Here, we identify ZL rats with HFD-induced hepatic steatosis as a more sensitive nonclinical in vivo test system for modeling DILI compared with SD rats fed normal chow.

Trunk mutational events present minimal intra- and inter-tumoral heterogeneity in hepatocellular carcinoma.

BACKGROUND & AIMS: According to the clonal model of tumor evolution, trunk alterations arise at early stages and are ubiquitous. Through the characterization of early stages of hepatocarcinogenesis, we aimed to identify trunk alterations in hepatocellular carcinoma (HCC) and study their intra- and inter-tumor distribution in advanced lesions. METHODS: A total of 151 samples representing the multistep process of hepatocarcinogenesis were analyzed by targeted-sequencing and a single nucleotide polymorphism array. Genes altered in early lesions (31 dysplastic nodules [DNs] and 38 small HCCs [sHCC]) were defined as trunk. Their distribution was explored in: a) different regions of large tumors (43 regions, 21 tumors), and b) different nodules of the same patient (39 tumors, 17 patients). Multinodular lesions were classified as intrahepatic metastases (IMs) or synchronous tumors based on chromosomal aberrations. RESULTS: TERT promoter mutations (10.5%) and broad copy-number aberrations in chromosomes 1 and 8 (3-7%) were identified as trunk gatekeepers in DNs and were maintained in sHCCs. Trunk drivers identified in sHCCs included TP53 (23%) and CTNNB1 (11%) mutations, and focal amplifications or deletions in known drivers (6%). Overall, TERT, TP53 and CTNNB1 mutations were the most frequent trunk events and at least one was present in 51% of sHCCs. Around 90% of mutations in these genes were ubiquitous among different regions of large tumors. In multinodular HCCs, 35% of patients harbored IMs; 85% of mutations in TERT, TP53 and/or CTNNB1 were retained in primary and metastatic tumors. CONCLUSIONS: Trunk events in early stages (TERT, TP53, CTNNB1 mutations) were ubiquitous across different regions of the same tumor and between primary and metastatic nodules in >85% of cases. This concept supports the knowledge that single biopsies would suffice to capture trunk mutations in HCC. LAY SUMMARY: Trunk alterations arise at early stages of cancer and are shared among all malignant cells of the tumor. In order to identify trunk alterations in HCC, we characterized early stages of hepatocarcinogenesis represented by dysplastic nodules and small lesions. Mutations in TERT, TP53 and CTNNB1 genes were the most frequent. Analyses in more advanced lesions showed that mutations in these same genes were shared between different regions of the same tumor and between primary and metastatic tumors, suggesting their trunk role in this disease.

Drosophila melanogaster retrotransposon and inverted repeat-derived endogenous siRNAs are differentially processed in distinct cellular locations.

BACKGROUND: Endogenous small interfering (esi)RNAs repress mRNA levels and retrotransposon mobility in Drosophila somatic cells by poorly understood mechanisms. 21 nucleotide esiRNAs are primarily generated from retrotransposons and two inverted repeat (hairpin) loci in Drosophila culture cells in a Dicer2 dependent manner. Additionally, proteins involved in 3' end processing, such as Symplekin, CPSF73 and CPSR100, have been recently implicated in the esiRNA pathway. RESULTS: Here we present evidence of overlap between two essential RNA metabolic pathways: esiRNA biogenesis and mRNA 3' end processing. We have identified a nucleus-specific interaction between the essential esiRNA cleavage enzyme Dicer2 (Dcr2) and Symplekin, a component of the core cleavage complex (CCC) required for 3' end processing of all eukaryotic mRNAs. This interaction is mediated by the N-terminal 271 amino acids of Symplekin; CCC factors CPSF73 and CPSF100 do not contact Dcr2. While Dcr2 binds the CCC, Dcr2 knockdown does not affect mRNA 3' end formation. RNAi-depletion of CCC components Symplekin and CPSF73 causes perturbations in esiRNA abundance that correlate with fluctuations in retrotransposon and hairpin esiRNA precursor levels. We also discovered that esiRNAs generated from retrotransposons and hairpins have distinct physical characteristics including a higher predominance of 22 nucleotide hairpin-derived esiRNAs and differences in 3' and 5' base preference. Additionally, retrotransposon precursors and derived esiRNAs are highly enriched in the nucleus while hairpins and hairpin derived esiRNAs are predominantly cytoplasmic similar to canonical mRNAs. RNAi-depletion of either CPSF73 or Symplekin results in nuclear retention of both hairpin and retrotransposon precursors suggesting that polyadenylation indirectly affects cellular localization of Dcr2 substrates. CONCLUSIONS: Together, these observations support a novel mechanism in which differences in localization of esiRNA precursors impacts esiRNA biogenesis. Hairpin-derived esiRNAs are generated in the cytoplasm independent of Dcr2-Symplekin interactions, while retrotransposons are processed in the nucleus.

Bioinformatic analyses of sense and antisense expression from terminal inverted repeat transposons in Drosophila somatic cells.

Understanding regulation of transposon movement in somatic cells is important as mobile elements can cause detrimental genomic rearrangements. Generally, transposons move via one of 2 mechanisms; retrotransposons utilize an RNA intermediate, therefore copying themselves and amplifying throughout the genome, while terminal inverted repeat transposons (TIR Tns) excise DNA sequences from the genome and integrate into a new location. Our recently published work indicates that retrotransposons in Drosophila tissue culture cells are actively transcribed in the antisense direction. Our data support a model in which convergent transcription of retrotransposons from intra element transcription start sites results in complementary RNAs that hybridize to form substrates for Dicer-2, the endogenous small interfering (esi)RNA generating enzyme. Here, we extend our previous analysis to TIR Tns. In contrast to retrotransposons, our data show that antisense TIR Tn RNAs result from transcription of intronic TIR Tns oriented antisense to their host genes. Also, disproportionately less esiRNAs are generated from TIR transcripts than from retrotransposons and transcription of very few individual TIR Tns could be confirmed. Collectively, these data support a model in which TIR Tns are regulated at the level of Transposase production while retrotransposons are regulated with esiRNA post-transcriptional mechanisms in Drosophila somatic cells.

Antisense Transcription of Retrotransposons in Drosophila: An Origin of Endogenous Small Interfering RNA Precursors.

Movement of transposons causes insertions, deletions, and chromosomal rearrangements potentially leading to premature lethality in Drosophila melanogaster. To repress these elements and combat genomic instability, eukaryotes have evolved several small RNA-mediated defense mechanisms. Specifically, in Drosophila somatic cells, endogenous small interfering (esi)RNAs suppress retrotransposon mobility. EsiRNAs are produced by Dicer-2 processing of double-stranded RNA precursors, yet the origins of these precursors are unknown. We show that most transposon families are transcribed in both the sense (S) and antisense (AS) direction in Dmel-2 cells. LTR retrotransposons Dm297, mdg1, and blood, and non-LTR retrotransposons juan and jockey transcripts, are generated from intraelement transcription start sites with canonical RNA polymerase II promoters. We also determined that retrotransposon antisense transcripts are less polyadenylated than sense. RNA-seq and small RNA-seq revealed that Dicer-2 RNA interference (RNAi) depletion causes a decrease in the number of esiRNAs mapping to retrotransposons and an increase in expression of both S and AS retrotransposon transcripts. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and processed by Dicer-2 into esiRNAs that silence both sense and antisense retrotransposon transcripts. Reduction of sense retrotransposon transcripts potentially lowers element-specific protein levels to prevent transposition. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active.

Progenitor cell markers predict outcome of patients with hepatocellular carcinoma beyond Milan criteria undergoing liver transplantation.

BACKGROUND & AIMS: In patients with hepatocellular carcinoma (HCC), liver transplantation (LT) is an excellent therapy if tumor characteristics are within the Milan criteria. We aimed to define genomic features enabling to identify HCC patients beyond Milan criteria who have acceptable transplant outcomes. METHODS: Among 770 consecutive HCC patients transplanted between 1990 and 2013, 132 had tumors exceeding Milan criteria on pathology and were enrolled in the study; 44% of the patients satisfied the 'up-to-7 rule' [7=sum of the size of the largest tumor and the number of tumors]. Explant tumors were assessed for genomic signatures and immunohistochemical markers associated with poor outcome. RESULTS: At a median follow-up of 88months, 64 patients had died and 45 recurred; the 5-year overall survival (OS) and recurrence rates were 57% and 35%, respectively. Cytokeratin 19 (CK19) gene signature was independently associated with recurrence [Hazard ratio (HR)=2.95, p<0.001], along with tumor size (HR=3.37, p=0.023) and presence of satellites (HR=2.98, p=0.001). S2 subclass signature was independently associated with poor OS (HR=3.18, p=0.001), along with tumor size (HR=5.06, p<0.001) and up-to-7 rule (HR=2.50, p=0.002). Using the presence of progenitor cell markers (either CK19 or S2 signatures) patients were classified into poor prognosis (n=58; 5-year recurrence 53%, survival 45%) and good prognosis (n=74; 5-year recurrence 19%, survival 67%) (HR=3.16, p<0.001 for recurrence, and HR=1.72, p=0.04 for OS). CONCLUSIONS: HCC patients transplanted beyond Milan criteria without gene signatures of progenitor markers (CK19 and S2) achieved survival rates similar as those within Milan criteria. Once prospectively validated, these markers may support a limited expansion of LT indications.

Genome-wide methylation analysis and epigenetic unmasking identify tumor suppressor genes in hepatocellular carcinoma.

BACKGROUND & AIMS: Epigenetic silencing of tumor suppressor genes contributes to the pathogenesis of hepatocellular carcinoma (HCC). To identify clinically relevant tumor suppressor genes silenced by DNA methylation in HCC, we integrated DNA methylation data from human primary HCC samples with data on up-regulation of gene expression after epigenetic unmasking. METHODS: We performed genome-wide methylation analysis of 71 human HCC samples using the Illumina HumanBeadchip27K array; data were combined with those from microarray analysis of gene re-expression in 4 liver cancer cell lines after their exposure to reagents that reverse DNA methylation (epigenetic unmasking). RESULTS: Based on DNA methylation in primary HCC and gene re-expression in cell lines after epigenetic unmasking, we identified 13 candidate tumor suppressor genes. Subsequent validation led us to focus on functionally characterizing 2 candidates, sphingomyelin phosphodiesterase 3 (SMPD3) and neurofilament, heavy polypeptide (NEFH), which we found to behave as tumor suppressor genes in HCC. Overexpression of SMPD3 and NEFH by stable transfection of inducible constructs into an HCC cell line reduced cell proliferation by 50% and 20%, respectively (SMPD3, P = .003 and NEFH, P = .003). Conversely, knocking down expression of these genes with small hairpin RNA promoted cell invasion and migration in vitro (SMPD3, P = .0001 and NEFH, P = .022), and increased their ability to form tumors after subcutaneous injection or orthotopic transplantation into mice, confirming their role as tumor suppressor genes in HCC. Low levels of SMPD3 were associated with early recurrence of HCC after curative surgery in an independent patient cohort (P = .001; hazard ratio = 3.22; 95% confidence interval: 1.6-6.5 in multivariate analysis). CONCLUSIONS: Integrative genomic analysis identified SMPD3 and NEFH as tumor suppressor genes in HCC. We provide evidence that SMPD3 is a potent tumor suppressor gene that could affect tumor aggressiveness; a reduced level of SMPD3 is an independent prognostic factor for early recurrence of HCC.

Serum markers for predicting abdominal surgery outcomes in patients with cirrhosis.

BACKGROUND: Determinants of adverse events for cirrhotic patients undergoing abdominal surgery have not been adequately assessed. Child-Turcotte-Pugh (CTP) and Model for End-Stage Liver Disease (MELD) have estimated perioperative outcomes with inconsistent results. Our study sought to combine novel serum markers with CTP and MELD to improve prognostication of 30-day postoperative mortality or liver transplant in cirrhotic patients undergoing abdominal surgery. METHODS: A review was performed on 120 cirrhotic patients undergoing nonhepatic abdominal surgeries at Mount Sinai Medical Center from 2001-2011. Preoperative serum markers were evaluated by logistic regression and receiver-operator characteristics. Prognostic ability of scoring systems was assessed using Youden's J statistic (J). RESULTS: Albumin and hematocrit were independently predictive of 30-day mortality or transplant with optimal cutoff values of albumin at <3.05 mg/dl and hematocrit at <35.55 %. Adding these criteria to CTP>A, CTP>B, MELD ≥ 10, MELD ≥ 15, and MELD ≥ 20 improved sensitivity and specificity by an average of 6.1 and 32.1 %, respectively. The highest J values resulted from combining novel criteria with CTP>A (sensitivity, 80 %; specificity, 82 %; p < 0.01; J, 0.63) and MELD ≥ 10 (sensitivity, 63 %; specificity, 90 %; p < 0.01; J, 0.53). CONCLUSION: Augmenting CTP and MELD with albumin and hematocrit significantly improved the identification of cirrhotic patients at risk of 30-day mortality or transplantation following nonhepatic abdominal surgery.

The impact of native competitors on an alien invasive: temporal niche shifts to avoid interspecific aggression?

The American mink, Neovison vison, is an established, alien invasive species in the United Kingdom that originally colonized the country at a time when two native mustelids (otters, Lutra lutra, and polecats, Mustela putorius) were largely absent. Both of these species are now recovering their populations nationally. We compared the relative abundance and the behavior of mink in the 1990s and in the 2000s in an area of southern England where both otters and polecats were absent in the 1990s but reappeared in the intervening years. We found that mink were still abundant in the 2000s in the presence of otters and polecats, but that they appeared to have altered some aspects of their behavior. In accordance with previous studies, we found that mink consumed fewer fish in the presence of otters. We also found that mink were predominantly nocturnal in the 1990s (in the absence of competitors) but were predominantly diurnal in the 2000s (in the presence of competitors). We hypothesize that this temporal shift may be an avoidance mechanism allowing the coexistence of mink with the otter and the polecat, although we are unable to attribute the shift to one or the other species. We also found that mink in the presence of competitors weighed less but remained the same size, suggesting the possibility of a competitor-mediated decline in overall body condition. This is one of very few field studies demonstrating a complete temporal shift in apparent response to competitors. The implications of this study are that recovering otter populations may not lead to significant and long-term reductions in the number of invasive mink in the United Kingdom as has been suggested in the media, although we cannot exclude the possibility of a decline in mink in the longer-term.

Recording the free-living behaviour of small-bodied, shallow-diving animals with data loggers.

1. Time-depth data recorders (TDRs) have been widely used to explore the behaviour of relatively large, deep divers. However, little is known about the dive behaviour of small, shallow divers such as semi-aquatic mammals. 2. We used high-resolution TDRs to record the diving behaviour of American mink Mustela vison (weight of individuals 580-1275 g) in rivers in Oxfordshire (UK) between December 2005 and March 2006. 3. Dives to > 0.2 m were measured in all individuals (n = 6). Modal dive depth and duration were 0.3 m and 10 s, respectively, although dives up to 3 m and 60 s in duration were recorded. Dive duration increased with dive depth. 4. Temperature data recorded by TDRs covaried with diving behaviour: they were relatively cold (modal temperature 4-6 degrees C across individuals) when mink were diving and relatively warm (modal temperature 24-36 degrees C across individuals) when mink were not diving. 5. Individuals differed hugely in their use of rivers, reflecting foraging plasticity across both terrestrial and aquatic environments. For some individuals there was < 1 dive per day while for others there was > 100 dives per day. 6. We have shown it is now possible to record the diving behaviour of small free-living animals that only dive a few tens of centimetres, opening up the way for a new range of TDR studies on shallow diving species.

Hallucinogen-like actions of 2,5-dimethoxy-4-(n)-propylthiophenethylamine (2C-T-7) in mice and rats.

RATIONALE: Few studies have examined the effects of 2,5-dimethoxy-4-(n)-propylthiophenethylamine (2C-T-7) in vivo. OBJECTIVES: 2C-T-7 was tested in a drug-elicited head twitch assay in mice and in several drug discrimination assays in rats; 2C-T-7 was compared to the phenylisopropylamine hallucinogen R(-)-1-(2,5-dimethoxy-4-methylphenyl)-2aminopropane (DOM) in both assays, with or without pretreatment with the selective 5-HT2A antagonist (+)-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol (M100907). Finally, the affinity of 2C-T-7 for three distinct 5-HT receptors was determined in rat brain. METHODS: Drug-elicited head twitches were quantified for 10 min following administration of various doses of either 2C-T-7 or R(-)-DOM, with and without pretreatments of 0.01 mg/kg M100907. In rats trained to discriminate lysergic acid diethylamide (LSD), 2C-T-7 and R(-)-DOM were tested for generalization. In further studies, rats were trained to discriminate 2C-T-7 from saline, then challenged with 0.05 mg/kg M100907. In competition binding studies, the affinity of 2C-T-7 was assessed at 5-HT2A receptors, 5-HT1A receptors, and 5-HT2C receptors. RESULTS: 2C-T-7 and R(-)-DOM induced similar head twitch responses in the mouse that were antagonized by M100907. In the rat, 2C-T-7 produced an intermediate degree of generalization (75%) to the LSD cue and served as a discriminative stimulus; these interoceptive effects were attenuated by M100907. Finally, 2C-T-7 had nanomolar affinity for 5-HT2A and 5-HT2C receptors and lower affinity for 5-HT1A receptors. CONCLUSIONS: 2C-T-7 is effective in two rodent models of 5-HT2 agonist activity and has affinity at receptors relevant to hallucinogen effects. The effectiveness with which M100907 antagonizes the behavioral actions of 2C-T-7 strongly suggests that the 5-HT2A receptor is an important site of action for this compound.