Mutant Liver Research Articles

OTC deficiency in females: Phenotype-genotype correlation based on a 130-family cohort.

OTC deficiency, an inherited urea cycle disorder, is caused by mutations in the X-linked OTC gene. Phenotype-genotype correlations are well understood in males but still poorly known in females. Taking advantage of a cohort of 130 families (289 females), we assessed the relative contribution of OTC enzyme activity, X chromosome inactivation, and OTC gene sequencing to genetic counseling in heterozygous females. Twenty two percent of the heterozygous females were clinically affected, with episodic (11%), chronic (7.5%), or neonatal forms of the disease (3.5%). Overall mortality rate was 4%. OTC activity, ranging from 0% to 60%, did not correlate with phenotype at the individual level. Analysis of multiple samples from 4 mutant livers showed intra-hepatic variability of OTC activity and X inactivation profile (range of variability: 30% and 20%, respectively) without correlation between both parameters for 3 of the 4 livers. Ninety disease-causing variants were found, 27 of which were novel. Mutations were classified as "mild" or "severe," based on male phenotypes and/or in silico prediction. In our cohort, a serious disease occurred in 32% of females with a severe mutation, compared to 4% in females with a mild mutation (odds ratio=1.365; P=1.6e-06). These data should help prenatal diagnosis for heterozygous females and genetic counseling after fortuitous findings of OTC variants in pangenomic sequencing.

The Lysosomal Storage Disorder Due to fig4a Mutation Causes Robust Liver Vacuolation in Zebrafish.

The phospholipid phosphatase FIG4/Fig4 is a subunit of PIKFYVE/Pikfyve kinase complex that synthesizes phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a key regulator of endolysosomal trafficking and function. Loss of FIG4/Fig4 leads to intracellular deficiency of PI(3,5)P2 signaling and multiple endolysosomal defects. Previous works were focused on the effects of FIG4/Fig4 mutations in the nervous and musculoskeletal systems in human clinical and animal studies. In this study, we describe a zebrafish recessive mutant cq35 showing robust liver vacuolation and lethality, with a predicted truncating mutation in fig4a gene. The liver vacuolation progress in fig4a mutant was reversible after regaining normal fig4a transcripts. The hepatic vacuolation pathology was identified as abnormal lysosomal storage with numerous accumulated cargoes, including autophagy intermediates, and caused progressive degeneration of bile canaliculi in mutant liver. These hepatic pathological details of fig4a mutant were repeated in zebrafish pikfyve mutant. Thus, zebrafish possess the conserved structural and functional mechanisms in Pikfyve kinase complex, based on which, pikfyve mutant phenotype covered fig4a mutant phenotype in their double mutant. Our findings represent the first description of the in vivo defects caused by FIG4/Fig4 mutation or PI(3,5)P2 deficiency in liver, and reveal the conserved complex mechanisms associated with FIG4/Fig4-deficient disorders in zebrafish.

SIRT3 is required for liver regeneration but not for the beneficial effect of nicotinamide riboside.

Liver regeneration is critical to survival after traumatic injuries, exposure to hepatotoxins, or surgical interventions, yet the underlying signaling and metabolic pathways remain unclear. In this study, we show that hepatocyte-specific loss of the mitochondrial deacetylase SIRT3 drastically impairs regeneration and worsens mitochondrial function after partial hepatectomy. Sirtuins, including SIRT3, require NAD as a cosubstrate. We previously showed that the NAD precursor nicotinamide riboside (NR) promotes liver regeneration, but whether this involves sirtuins has not been tested. Here, we show that despite their NAD dependence and critical roles in regeneration, neither SIRT3 nor its nuclear counterpart SIRT1 is required for NR to enhance liver regeneration. NR improves mitochondrial respiration in regenerating WT or mutant livers and rapidly increases oxygen consumption and glucose output in cultured hepatocytes. Our data support a direct enhancement of mitochondrial redox metabolism as the mechanism mediating improved liver regeneration after NAD supplementation and exclude signaling via SIRT1 and SIRT3. Therefore, we provide the first evidence to our knowledge for an essential role for a mitochondrial sirtuin during liver regeneration and insight into the beneficial effects of NR.

Uhrf1 and dnmt1 Loss Induces an Immune Response in Zebrafish Livers Due to Viral Mimicry by Transposable Elements.

Activation of transposable elements (TEs) can cause cellular damage. Cytoplasmic nucleic acid sensing pathways evolved to detect pathogens, but can also serve to cull cells with inappropriate TE activation as TEs can be viral mimetics. Epigenetic silencing of TEs is mediated in part by DNA methylation, but it is not clear if TE activation or the immune system contribute to the cellular damage caused by loss of DNA methylation. Here, we provide mechanistic insight into the observation of an activated interferon response in the liver of zebrafish larvae with deletion in critical components of the DNA methylation machinery, uhrf1 and dnmt1. We focus on dissecting the relationship between DNA methylation, TE activation and induction of an immune response through cytoplasmic DNA and double stranded RNA sensing pathways and identify tnfa as a mediator of cell death in the liver of these mutants. Integrated RNAseq and methylome analysis identified LTR transposons as the most upregulated in these mutants and also the most methylated in control larvae, indicating a direct role of DNA methylation in suppressing this TE subclass. RNAseq analysis from these same samples revealed expression signatures of a type-I interferon response and of tnfa activation, mimicking the pattern of gene expression in virally infected cells. CRISPR/Cas9 mediated depletion of the cellular antiviral sensors sting and mavs reduced expression of interferon response genes and tnfa depletion dramatically reduced cell death in uhrf1 mutant livers. This suggests that the antiviral response induced by DNA hypomethylation and TE activation in the liver is mediated by the signaling pathways activated by both cytoplasmic double stranded RNA and DNA and that tnfa mediates cell death as a potential mechanism to eliminate these damaged cells.

Flavin Adenine Dinucleotide Depletion Caused by electron transfer flavoprotein subunit alpha Haploinsufficiency Leads to Hepatic Steatosis and Injury in Zebrafish.

The electron transfer flavoprotein (ETF) complex, made up of the ETF alpha subunit (ETFA), ETF beta subunit (ETFB), and ETF dehydrogenase (ETFDH), regulates fatty acid β‐oxidation activity while scavenging leaked electrons through flavin adenine dinucleotide (FAD)/reduced form FAD (FADH2) redox reactions in mitochondria. Here, we hypothesized that ETF dysfunction‐mediated FAD deficiency may result in increased mitochondrial oxidative stress and steatosis and subsequent liver injury. We report that etfa haploinsufficiency caused hyperlipidemia, hypercholesterolemia, and hepatic steatosis and injury in adult zebrafish. Further, etfa+/ − mutant livers had reduced levels of FAD and glutathione and an increase in reactive oxygen species. Because FAD depletion might be critical in the pathogenesis of the liver lesion identified in etfa+/ − mutants, we used riboflavin to elevate FAD levels in the liver and found that riboflavin supplementation significantly suppressed hepatic steatosis and injury in etfa+/ − mutants through suppression of oxidative stress and de novo lipogenesis in the liver. Additionally, we found that adenosine triphosphate‐linked mitochondrial oxygen consumption and mitochondrial membrane potential were reduced in etfa+/ − primary hepatocytes and that riboflavin supplementation corrected these defects. Conclusion: FAD depletion caused by etfa haploinsufficiency plays a key role in hepatic steatosis and oxidative stress‐mediated hepatic injury in adult zebrafish. This raises the possibility that people with ETFA haploinsufficiency have a high risk for developing liver disease.

ASK1 inhibition reduces cell death and hepatic fibrosis in an Nlrp3 mutant liver injury model.

Hepatic inflammasome activation is considered a major contributor to liver fibrosis in NASH. Apoptosis signal-regulating kinase 1 (ASK1) is an apical mitogen-activated protein kinase that activates hepatic JNK and p38 to promote apoptosis, inflammation, and fibrosis. The aim of the current study was to investigate whether pharmacologic inhibition of ASK1 could attenuate hepatic fibrosis driven by inflammasome activation using gain-of-function NOD-like receptor protein 3 (Nlrp3) mutant mice. Tamoxifen-inducible Nlrp3 knock-in (Nlrp3A350V/+CreT-KI) mice and WT mice were administered either control chow diet or diet containing the selective ASK1 inhibitor GS-444217 for 6 weeks. Livers of Nlrp3-KI mice had increased inflammation, cell death, and fibrosis and increased phosphorylation of ASK1, p38, and c-Jun. GS-444217 reduced ASK1 pathway activation, liver cell death, and liver fibrosis. ASK1 inhibition resulted in a significant downregulation of genes involved in collagen production and extracellular matrix deposition, as well as in a reduced hepatic TNF-α expression. ASK1 inhibition also directly reduced LPS-induced gene expression of Collagen 1A1 (Col1a1) in hepatic stellate cells isolated from Nlrp3-KI mice. In conclusion, ASK1 inhibition reduced liver cell death and fibrosis downstream of inflammatory signaling induced by NLRP3. These data provide mechanistic insight into the antifibrotic mechanisms of ASK1 inhibition.

Intraflagellar‐transport A dysfunction causes hyperphagia‐induced systemic insulin resistance in a pre‐obese state

Deletion of murine Thm1, an intraflagellar transport A (IFT-A) component that mediates ciliary protein trafficking, causes hyperphagia, obesity, and metabolic syndrome. The role of Thm1 or IFT-A in adipogenesis and insulin sensitivity is unknown. Here, we report that Thm1 knockdown in 3T3-L1 pre-adipocytes promotes adipogenesis and enhances insulin sensitivity in vitro. Yet, pre-obese Thm1 conditional knockout mice show systemic insulin resistance. While insulin-induced AKT activation in Thm1 mutant adipose depots and skeletal muscle are similar to those of control littermates, an attenuated insulin response arises in the mutant liver. Insulin treatment of control and Thm1 mutant primary hepatocytes results in similar AKT activation. Moreover, pair-feeding Thm1 conditional knockout mice produces a normal insulin response, both in the liver and systemically. Thus, hyperphagia caused by a cilia defect, induces hepatic insulin resistance via a non-cell autonomous mechanism. In turn, hepatic insulin resistance drives systemic insulin resistance prior to an obese phenotype. These data demonstrate that insulin signaling across cell types is regulated differentially, and that the liver is particularly susceptible to hyperphagia-induced insulin resistance and a critical determinant of systemic insulin resistance.

Fasting reveals largely intact systemic lipid mobilization mechanisms in respiratory chain complex III deficient mice

Mice homozygous for the human GRACILE syndrome mutation (Bcs1lc.A232G) display decreased respiratory chain complex III activity, liver dysfunction, hypoglycemia, rapid loss of white adipose tissue and early death. To assess the underlying mechanism of the lipodystrophy in homozygous mice (Bcs1lp.S78G), these and wild-type control mice were subjected to a short 4-hour fast. The homozygotes had low baseline blood glucose values, but a similar decrease in response to fasting as in wild-type mice, resulting in hypoglycemia in the majority. Despite the already depleted glycogen and increased triacylglycerol content in the mutant livers, the mice responded to fasting by further depletion and increase, respectively. Increased plasma free fatty acids (FAs) upon fasting suggested normal capacity for mobilization of lipids from white adipose tissue into circulation. Strikingly, however, serum glycerol concentration was not increased concomitantly with free FAs, suggesting its rapid uptake into the liver and utilization for fuel or gluconeogenesis in the mutants. The mutant hepatocyte mitochondria were capable of responding to fasting by appropriate morphological changes, as analyzed by electron microscopy, and by increasing respiration. Mutants showed increased hepatic gene expression of major metabolic controllers typically associated with fasting response (Ppargc1a, Fgf21, Cd36) already in the fed state, suggesting a chronic starvation-like metabolic condition. Despite this, the mutant mice responded largely normally to fasting by increasing hepatic respiration and switching to FA utilization, indicating that the mechanisms driving these adaptations are not compromised by the CIII dysfunction. Summary statementBcs1l mutant mice with severe CIII deficiency, energy deprivation and post-weaning lipolysis respond to fasting similarly to wild-type mice, suggesting largely normal systemic lipid mobilization and utilization mechanisms.

Inducing and exploiting vulnerabilities for the treatment of liver cancer.

Liver cancer remains difficult to treat due to a paucity of drugs that target critical dependencies1,2 and broad spectrum kinase inhibitors like sorafenib provide only modest benefit to hepatocellular carcinoma (HCC) patients3. Induction of senescence may represent a promising strategy for the treatment of cancer, especially when such pro-senescence therapy is combined with a second drug that selectively eliminates senescent cancer cells (senolysis)4,5. Through a kinome-focused genetic screen, we report here that pharmacological inhibition of the DNA replication kinase CDC7 induces senescence selectively in TP53 mutant liver cancer cells. A follow-up chemical screen identified the anti-depressant sertraline as an agent that kills HCC cells rendered senescent by CDC7 inhibition. Sertraline supressed mTOR signalling, and selective drugs targeting this pathway were highly effective in causing apoptotic cell death of CDC7 inhibitor-treated HCC cells. Mechanistically, we report that the feedback re-activation of mTOR signalling following its inhibition6 is blocked in CDC7-inhibitor treated cells, leading to sustained mTOR inhibition and cell death. Using multiple in vivo liver cancer models, we show that combination of CDC7 and mTOR inhibitors results in dramatic tumour growth inhibition. More generally, our data indicate that exploiting an induced vulnerability could be an effective treatment of liver cancer.

Ankrd45 Is a Novel Ankyrin Repeat Protein Required for Cell Proliferation.

Ankyrin repeats, the most common protein–protein interaction motifs in nature, are widely present in proteins of both eukaryotic and prokaryotic cells. Ankyrin repeat-containing proteins play diverse biological functions. Here, we identified the gene ankrd45, which encodes a novel, two ankyrin repeat-containing protein. Zebrafish ankrd45 displayed a tissue specific expression pattern during early development, with high expression in ciliated tissues, including otic vesicles, Kupffer’s vesicles, pronephric ducts, and floor plates. Surprisingly, zebrafish ankrd45 mutants were viable and developed grossly normal cilia. In contrast, mutant larvae developed enlarged livers when induced with liver specific expression of KrasG12V, one of the common mutations of KRAS that leads to cancer in humans. Further, histological analysis suggested that multiple cysts developed in the mutant liver due to cell apoptosis. Similarly, knockdown of ANKRD45 expression with either siRNA or CRISPR/Cas9 methods induced apoptosis in cultured cells, similar to those in zebrafish ankrd45 mutant livers after induction. Using different cell lines, we show that the distribution of ANKRD45 protein was highly dynamic during mitosis. ANKRD45 is preferably localized to the midbody ring during cytokinesis. Together, our results suggest that ANKRD45 is a novel ankyrin repeat protein with a conserved role during cell proliferation in both zebrafish embryos and mammalian cells.

Functional iron deficiency in toxic milk mutant mice (tx-J) despite high hepatic ferroportin: a critical role of decreased GPI-ceruloplasmin expression in liver macrophages.

Jackson toxic milk mutant mice (tx-J) carrying a missense mutation in the Atp7b gene are animal models of the Wilson disease. In both the Wilson patients and the tx-J mice, mutations in the ATP7B/Atp7b gene lead to disturbances in copper metabolism. The dysfunction of ATP7B/Atp7b leads to a reduction in the incorporation of copper into apoceruloplasmin; this decreases the ferroxidase activity of ceruloplasmin necessary for the efflux of iron from cells and reduces the release of copper from hepatocytes to the bile; this results in a massive hepatic copper accumulation. A decrease in the ferroxidase activity of ceruloplasmin in the tx-J mice emphasises the practicality of this animal model for the exploration of disturbances in iron balance triggered by dysregulation of copper metabolism. We found that 6-month-old tx-J mutants developed mild anaemia caused by functional iron deficiency. The tx-J mutants showed decreased plasma iron levels with concomitant iron accumulation in hepatocytes and liver macrophages. Hepatic iron retention was accompanied by decreased expression of the membrane form of ceruloplasmin in both liver cell types. Interestingly, in the liver of mutants, we found high levels of ferroportin (an iron exporter) on the surface of liver macrophages despite increased hepatic expression of hepcidin, a peptide inducing internalization and degradation of ferroportin. We conclude that even when the ferroportin expression is high, ceruloplasmin remains a limiting factor in the release of iron to the extracellular environment.

A novel GTPCH deficiency mouse model exhibiting tetrahydrobiopterin-related metabolic disturbance and infancy-onset motor impairments

BackgroundGTP cyclohydrolase I (GTPCH) deficiency could impair the synthesis of tetrahydrobiopterin and causes metabolic diseases involving phenylalanine catabolism, neurotransmitter synthesis, nitric oxide production and so on. Though improvements could be achieved by tetrahydrobiopterin and neurotransmitter precursor levodopa supplementation, residual motor and mental deficits remain in some patients. An appropriate GTPCH deficiency animal model with clinical symptoms, especially the motor impairments, is still not available for mechanism and therapy studies yet. Objectives and methodsTo investigate whether the heterozygous GTPCH missense mutation p.Leu117Arg identified from a patient with severe infancy-onset dopa-responsive motor impairments is causative and establish a clinical relevant GTPCH deficiency mouse model, we generated a mouse mutant mimicking this missense mutation using the CRISPR/Cas9 technology. Series of characterization experiments on the heterozygous and homozygous mutants were conducted. ResultsThe expressions of GTPCH were not significantly changed in the mutants, but the enzyme activities were impaired in the homozygous mutants. BH4 reduction and phenylalanine accumulation were observed both in the liver and brain of the homozygous mutants. Severer metabolic disturbance occurred in the brain than in the liver. Significant reduction of neurotransmitter dopamine, norepinephrine and serotonin was observed in the brains of homozygous mutants. Live-born homozygous mutants exhibited infancy-onset motor and vocalization deficits similar to the disease symptoms observed in the patient, while no obvious symptoms were observed in the young heterozygous mutant mice. With benserazide-levodopa treatment, survival of the homozygous mutants was improved but not completely rescued. ConclusionsThe GTPCH p.Leu117Arg missense mutation is deleterious and could cause tetrahydrobiopterin, phenylalanine and neurotransmitter metabolic disturbances and infancy-onset motor dysfunctions recessively. This is the first GTPCH deficiency mouse model which could be live-born and exhibits significant motor impairments. The different extents of BH4 reduction and phenylalanine accumulation observed between liver and brain in response to GTPCH deficiency gives potential new insights into the vulnerability of brain to GTPCH deficiency.

MeCP2 isoform e1 mutant mice recapitulate motor and metabolic phenotypes of Rett syndrome.

Mutations in the X-linked gene MECP2 cause the majority of Rett syndrome (RTT) cases. Two differentially spliced isoforms of exons 1 and 2 (MeCP2-e1 and MeCP2-e2) contribute to the diverse functions of MeCP2, but only mutations in exon 1, not exon 2, are observed in RTT. We previously described an isoform-specific MeCP2-e1-deficient male mouse model of a human RTT mutation that lacks MeCP2-e1 while preserving expression of MeCP2-e2. However, RTT patients are heterozygous females that exhibit delayed and progressive symptom onset beginning in late infancy, including neurologic as well as metabolic, immune, respiratory and gastrointestinal phenotypes. Consequently, we conducted a longitudinal assessment of symptom development in MeCP2-e1 mutant females and males. A delayed and progressive onset of motor impairments was observed in both female and male MeCP2-e1 mutant mice, including hind limb clasping and motor deficits in gait and balance. Because these motor impairments were significantly impacted by age-dependent increases in body weight, we also investigated metabolic phenotypes at an early stage of disease progression. Both male and female MeCP2-e1 mutants exhibited significantly increased body fat compared to sex-matched wild-type littermates prior to weight differences. Mecp2e1-/y males exhibited significant metabolic phenotypes of hypoactivity, decreased energy expenditure, increased respiratory exchange ratio, but decreased food intake compared to wild-type. Untargeted analysis of lipid metabolites demonstrated a distinguishable profile in MeCP2-e1 female mutant liver characterized by increased triglycerides. Together, these results demonstrate that MeCP2-e1 mutation in mice of both sexes recapitulates early and progressive metabolic and motor phenotypes of human RTT.

3026 - Bi-Allelic CEBPA Mutation Combined with a Novel GATA2 Mutation Develops an Acute Erythroid Leukemia Through a Bi-Potent Leukemic Initiating Population

Acute erythroid leukemia (AEL) is a subtype of acute myeloid leukemia (AML) representing <5% of AML cases. There are two subtypes of AEL: pure erythroid leukemia and erythroleukemia (erythroid/myeloid leukemia). Sequencing studies has comprehensively characterized the mutational landscape of AML identifying mutations that are significantly associated such as the bi-allelic CEBPA and GATA2 mutations (Papaemmanuil et al.; 2016). Recently, sequencing AEL patients also found a high frequency of bi-allelic CEBPA and GATA2 co-mutated (Ping et al.; 2016). As there are currently no models of AEL we combined three knock-in mouse strains, a novel Gata2 point mutation in the N-terminal zinc finger crossed with a knock-in of the N-terminal Cebpa mutation, and a C-terminal Cebpa mutation to generate CebpaN/C;Gata2+/+ (NC genotype) and CebpaN/C;Gata2G320D/ + (NCG genotype). To bypass Cebpa mutation perinatal lethality we performed competitive transplantations using mutant fetal liver cells and wild type bone marrow (BM) competitor cells. By analyzing myeloid reconstitution over time in the peripheral blood we found the myeloid differentiation block caused by the N-terminal mutation in NC mice was prevented when co-expressed with Gata2G320D. This correlated with a more aggressive disease in NCG compared to NC transplanted mice. 100% of NC mice developed an AML while 50% of NCG mice developed an AML, and the remaining 50% developed an AEL of the erythroleukemia subtype, identified by the presence of both myeloblasts and erythroblasts in the BM, spleen and peripheral blood. NCG AEL mice had a significantly shorter overall survival than NCG AML mice. Secondary transplant of different sorted BM populations from NCG AML or NCG AEL mice showed that Lin-cKit+FcR+ GMP-like cells could propagate and fully replicate the two diseases, identifying the putative leukemic initiating cell (LIC) population. Together our study provides a novel model of the erythroleukemia subtype of AEL where both the myeloblasts and erythroblasts develop through a bi-potent LIC population.

Mineralization of alpha-1-antitrypsin inclusion bodies in Mmalton alpha-1-antitrypsin deficiency

BackgroundAlpha-1-antitrypsin (AAT) deficiency (AATD) of Z, Mmalton, Siiyama type is associated with liver storage of the mutant proteins and liver disease. The Z variant can be diagnosed on isoelectric focusing (IEF) while Mmalton and Siiyama may be missed or misdiagnosed with this technique. Therefore, molecular analysis is mandatory for their characterization. In particular, that holds true for the Mmalton variant as on IEF profile it resembles the wild M2 subtype.MethodsThis is a retrospective analysis involving review of medical records and of liver biopsy specimens from a series of Mmalton, Z and Siiyama Alpha-1-antitrypsin deficiency patients. The review has been implemented by additional histological stains, electron microscopic observations and 3-D modeling studies of the sites of the mutations.ResultsZ, Mmalton and Siiyama liver specimen contained characteristic intrahepatocytic PAS-D globules. The globules differed in the three variants as only Mmalton cases showed dark basophilic precipitates within the AAT inclusions. The precipitates were visualized in haematoxylin-eosin (H.E.) stained preparations and corresponded to calcium precipitates as demonstrated by von Kossa staining. On immunohistochemistry, ZAAT inclusions were stained by polyclonal as well as monoclonal noncommercial anti-AAT antibody (AZT11), whilst Mmalton and Siiyama inclusion bodies remained negative with the monoclonal anti-Z antibody. 3-D protein analysis allowed to predict more severe misfolding of the Mmalton molecule as compared to Z and Siiyama that could trigger anomalous interaction with endoplasmic reticulum chaperon proteins, namely calcium binding proteins.ConclusionsMmalton AAT inclusion bodies contain calcium precipitates inside them that allow the differential diagnosis with Siiyama and ZAAT inclusions in routine histological sections. The study has confirmed the specificity of the monoclonal AZT11 for the Z mutant. Thus, the combination of these two features is crucial for the distinction between the three variants and for predicting the genotype, whose confirmation would definitely require molecular analysis. Our study provides new data on the pathomorphogenesis of Mmalton inclusion bodies whose mineralization could play a central role in disease pathogenesis of Mmalton that is distinct from the Z and Siiyama variants. Calcium is known to be a major effector of cell death either via the increased intracellular concentration or the alteration of homeostasis.

Hepatocyte-specific expression of constitutively active Alk5 exacerbates thioacetamide-induced liver injury in mice.

While Transforming growth factor-βs (Tgf-βs) have been known to play an important role in liver fibrosis through an activation of Hepatic Stellate Cells (HSC), their fibrotic role on hepatocytes in liver damage has not been addressed thoroughly. To shed more light on the hepatocyte-specific role of Tgf-β signaling during liver fibrosis, we generated transgenic mice expressing constitutively active Tgf-β type I receptor Alk5 under the control of albumin promoter. Uninjured mice with increased Tgf-β/Alk5 signaling in hepatocytes (caAlk5/Alb-Cre mice) did not show characteristics related to hepatocyte death, fibrosis and inflammation. When subjected to thioacetamide (TAA) treatment, caAlk5/Alb-Cre mice exhibited more severe liver injury, when compared to control littermates. After TAA administration for 12 weeks, an increase in pathological changes was evident in caAlk5/Alb-Cre livers, with higher number of infiltrating cells in the portal and periportal area. Immunohistochemistry for F4/80, myeloperoxidase and CD3 showed that there was an increased accumulation of macrophages, neutrophils and T-lymphocytes, respectively, in caAlk5/Alb-Cre livers. Coincidently, we observed an exacerbated liver damage as seen by increases in serum aminotransferase level and number of apoptotic hepatocytes in caAlk5/Alb-Cre mice. Sirius staining of collagen demonstrated that the fibrotic response was worsened in caAlk5/Alb-Cre mice. The enhanced fibrosis in mutant livers was associated with marked production of α-SMA-positive myofibroblast. Hepatic expression of genes indicative of HSC activation was greater in caAlk5/Alb-Cre mice. In conclusion, our data indicated that elevation of Tgf-β signaling via Alk5 in hepatocytes is not sufficient to induce liver pathology but plays an important role in amplifying TAA-induced liver damage.

Fah Knockout Animals as Models for Therapeutic Liver Repopulation.

Several animal models of Fah deficiency have been developed, including mice, pigs and most recently rats. Initially, the murine models were developed with the intent to mirror the human disease for pathophysiologic and therapeutic studies. However, it soon became apparent that Fah-positive hepatocytes have a potent selective growth advantage in mutant liver and can extensively repopulate the diseased organ. For this reason, Fah mutant mice have become a workhorse for liver biology and are widely used in liver stem cell and hepatic gene therapy research. Immune deficient Fah-knockout mice can be repopulated with human hepatocytes, creating "mice with human livers". These chimeric animals have become an important preclinical model for infectious diseases, metabolism and gene therapy. The potent expansion of human hepatocytes in Fah knockout mice has given rise to the concept of using Fah mutants as living bioreactors to produce large quantities of fully mature hepatocytes. As a consequence, larger animal models of Fah deficiency have recently been developed.

Connecting cancer biology and clinical outcomes to imaging in KRAS mutant and wild-type colorectal cancer liver tumors following selective internal radiation therapy with yttrium-90.

To determine whether pathologic colorectal tumor KRAS mutation status is correlated with progression-free survival (PFS) by imaging after selective internal radiation therapy with Yttrium-90 (SIRT Y90) for metastatic colorectal cancer in the liver (mCRC). This was an IRB approved, HIPAA compliant retrospective cohort study. Consecutive patients with unresectable mCRC with documented KRAS mutation status treated at a single center from 2002 to 2013 with SIRT Y90 were investigated. Treatment response was compared between KRAS wild-type (wt) and mutant (mut) using an anatomic tumor response criteria based on RECIST 1.0. Kaplan-Meier estimation and Cox regression analysis were used to measure progression-free survival (PFS) and to assess independent prognostic factors for PFS. 82 of 186 patients met review criteria. 33 (40.2%) patients were identified as KRAS mut. PFS was longer in KRAS wt (median 166days [95% CI 96-258days]) vs. mut (median 91days [95% CI 79-104days], p=0.002). KRAS mut patients were 1.48 times more likely to progress at first follow-up imaging than wt (95% CI 1.06-2.08, p=0.024). Univariate analysis identified high pre-SIRT Y90 INR, KRAS wt, any use of anti-EGFR therapy, and post-SIRT Y90 chemotherapy as prognostic factors for longer PFS. In multivariate analysis, only KRAS wt was an independent prognostic factor for longer PFS (RR: 1.80 [95% CI 1.08-2.99], p=0.024). Longer PFS is associated with KRAS wt vs. mut following SIRT Y90.

Characterization of the Plasmodium falciparum and P. berghei glycerol 3-phosphate acyltransferase involved in FASII fatty acid utilization in the malaria parasite apicoplast.

SummaryMalaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti‐malarial drug target, but surprisingly little is known about its role in lipid metabolism. Here we characterize the apicoplast glycerol 3‐phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei enzymes are confirmed by fusion of the N‐terminal targeting sequence to GFP and 3′ tagging of the full length protein. Activity of the P. falciparum enzyme is demonstrated by complementation in mutant bacteria, and critical residues in the putative active site identified by site‐directed mutagenesis. Genetic disruption of the P. falciparum enzyme demonstrates it is dispensable in blood stage parasites, even in conditions known to induce FASII activity. Disruption of the P. berghei enzyme demonstrates it is dispensable in blood and mosquito stage parasites, and only essential for development in the late liver stage, consistent with the requirement for FASII in rodent malaria models. However, the P. berghei mutant liver stage phenotype is found to only partially phenocopy loss of FASII, suggesting newly made fatty acids can take multiple pathways out of the apicoplast and so giving new insight into the role of FASII and apicoplast glycerol 3‐phosphate acyltransferase in malaria parasites.

Liver-Specific Deletion of SRSF2 Caused Acute Liver Failure and Early Death in Mice.

The liver performs a variety of unique functions critical for metabolic homeostasis. Here, we show that mice lacking the splicing factor SRSF2 but not SRSF1 in hepatocytes have severe liver pathology and biochemical abnormalities. Histological analyses revealed generalized hepatitis with the presence of ballooned hepatocytes and evidence of fibrosis. Molecular analysis demonstrated that SRSF2 governs splicing of multiple genes involved in the stress-induced cell death pathway in the liver. More importantly, SRSF2 also functions as a potent transcription activator, required for efficient expression of transcription factors mainly responsible for energy homeostasis and bile acid metabolism in the liver. Consistent with the effects of SRSF2 in gene regulation, accumulation of total cholesterol and bile acids was prominently observed in the mutant liver, followed by enhanced generation of reactive oxygen species and increased endoplasmic reticulum stress, as revealed by biochemical and ultrastructural analyses. Taking these observations together, inactivation of SRSF2 in liver caused dysregulated splicing events and hepatic metabolic disorders, which trigger endoplasmic reticulum stress, oxidative stress, and finally liver failure.