Model of fibrolamellar hepatocellular carcinomas reveals striking enrichment in cancer stem cells

The aetiology of human fibrolamellar hepatocellular carcinomas (hFL-HCCs), cancers occurring increasingly in children to young adults, is poorly understood. We present a transplantable tumour line, maintained in immune-compromised mice, and validate it as a bona fide model of hFL-HCCs by multiple methods. RNA-seq analysis confirms the presence of a fusion transcript (DNAJB1-PRKACA) characteristic of hFL-HCC tumours. The hFL-HCC tumour line is highly enriched for cancer stem cells as indicated by limited dilution tumourigenicity assays, spheroid formation and flow cytometry. Immunohistochemistry on the hFL-HCC model, with parallel studies on 27 primary hFL-HCC tumours, provides robust evidence for expression of endodermal stem cell traits. Transcriptomic analyses of the tumour line and of multiple, normal hepatic lineage stages reveal a gene signature for hFL-HCCs closely resembling that of biliary tree stem cells-newly discovered precursors for liver and pancreas. This model offers unprecedented opportunities to investigate mechanisms underlying hFL-HCCs pathogenesis and potential therapies

Assessing the utilization of high-resolution 2-field HLA typing in solid organ transplantation.

HLA typing in solid organ transplantation (SOT) is necessary for determining HLA-matching status between donor-recipient pairs and assessing patients\u27 anti-HLA antibody profiles. Histocompatibility has traditionally been evaluated based on serologically defined HLA antigens. The evolution of HLA typing and antibody identification technologies, however, has revealed many limitations with using serologic equivalents for assessing compatibility in SOT. The significant improvements to HLA typing introduced by next-generation sequencing (NGS) require an assessment of the impact of this technology on SOT. We have assessed the role of high-resolution 2-field HLA typing (HR-2F) in SOT by retrospectively evaluating NGS-typed pre- and post-SOT cases. HR-2F typing was highly instructive or necessary in 41% (156/385) of the cases. Several pre- and posttransplant scenarios were identified as being better served by HR-2F typing. Five different categories are presented with specific case examples. The experience of another center (Temple University Hospital) is also included, whereby 21% of the cases required HR-2F typing by Sanger sequencing, as supported by other legacy methods, to properly address posttransplant anti-HLA antibody issues

Comprehensive analysis of The Cancer Genome Atlas reveals a unique gene and non-coding RNA signature of fibrolamellar carcinoma

Fibrolamellar carcinoma (FLC) is a unique liver cancer primarily affecting young adults and characterized by a fusion event between DNAJB1 and PRKACA. By analyzing RNA-sequencing data from The Cancer Genome Atlas (TCGA) for >9,100 tumors across ~30 cancer types, we show that the DNAJB1-PRKACA fusion is specific to FLCs. We demonstrate that FLC tumors (n = 6) exhibit distinct messenger RNA (mRNA) and long intergenic non-coding RNA (lincRNA) profiles compared to hepatocellular carcinoma (n = 263) and cholangiocarcinoma (n = 36), the two most common liver cancers. We also identify a set of mRNAs (n = 16) and lincRNAs (n = 4), including LINC00473, that distinguish FLC from ~25 other liver and non-liver cancer types. We confirm this unique FLC signature by analysis of two independent FLC cohorts (n = 20 and 34). Lastly, we validate the overexpression of one specific gene in the FLC signature, carbonic anhydrase XII (CA12), at the protein level by western blot and immunohistochemistry. Both the mRNA and lincRNA signatures support a major role for protein kinase A (PKA) signaling in shaping the FLC gene expression landscape, and present novel candidate FLC oncogenes that merit further investigation

Hepatocyte ABCA1 Deletion Impairs Liver Insulin Signaling and Lipogenesis

Plasma membrane (PM) free cholesterol (FC) is emerging as an important modulator of signal transduction. Here, we show that hepatocyte-specific knockout (HSKO) of the cellular FC exporter, ATPbinding cassette transporter A1 (ABCA1), leads to decreased PM FC content and defective trafficking of lysosomal FC to the PM. Compared with controls, chow-fed HSKO mice had reduced hepatic (1) insulin- stimulated Akt phosphorylation, (2) activation of the lipogenic transcription factor Sterol Regulatory Element Binding Protein (SREBP)-1c, and (3) lipogenic gene expression. Consequently, Westerntype diet-fed HSKO mice were protected from steatosis. Surprisingly, HSKO mice had intact glucose metabolism; they showed normal gluconeogenic gene suppression in response to re-feeding and normal glucose and insulin tolerance. We conclude that: (1) ABCA1 maintains optimal hepatocyte PM FC, through intracellular FC trafficking, for efficient insulin signaling; and (2) hepatocyte ABCA1 deletion produces a form of selective insulin resistance so that lipogenesis is suppressed but glucose metabolism remains normal

No Evidence of Avian Influenza A H5N1 among Returning US Travelers

We reviewed reports to the Centers for Disease Control and Prevention of US travelers suspected of having avian influenza A H5N1 virus infection from February 2003 through May 2006. Among the 59 reported patients, no evidence of H5N1 virus infection was found; none had had direct contact with poultry, but 42% had evidence of human influenza A

CSF from Parkinson disease Patients Differentially Affects Cultured Microglia and Astrocytes

<p>Abstract</p> <p>Background</p> <p>Excessive and abnormal accumulation of alpha-synuclein (α-synuclein) is a factor contributing to pathogenic cell death in Parkinson's disease. The purpose of this study, based on earlier observations of Parkinson's disease cerebrospinal fluid (PD-CSF) initiated cell death, was to determine the effects of CSF from PD patients on the functionally different microglia and astrocyte glial cell lines. Microglia cells from human glioblastoma and astrocytes from fetal brain tissue were cultured, grown to confluence, treated with fixed concentrations of PD-CSF, non-PD disease control CSF, or control no-CSF medium, then photographed and fluorescently probed for α-synuclein content by deconvolution fluorescence microscopy. Outcome measures included manually counted cell growth patterns from day 1-8; α-synuclein density and distribution by antibody tagged 3D model stacked deconvoluted fluorescent imaging.</p> <p>Results</p> <p>After PD-CSF treatment, microglia growth was reduced extensively, and a non-confluent pattern with morphological changes developed, that was not evident in disease control CSF and no-CSF treated cultures. Astrocyte growth rates were similarly reduced by exposure to PD-CSF, but morphological changes were not consistently noted. PD-CSF treated microglia showed a significant increase in α-synuclein content by day 4 compared to other treatments (p ≤ 0.02). In microglia only, α-synuclein aggregated and redistributed to peri-nuclear locations.</p> <p>Conclusions</p> <p>Cultured microglia and astrocytes are differentially affected by PD-CSF exposure compared to non-PD-CSF controls. PD-CSF dramatically impacts microglia cell growth, morphology, and α-synuclein deposition compared to astrocytes, supporting the hypothesis of cell specific susceptibility to PD-CSF toxicity.</p

Transcriptomic Analysis of Chronic Hepatitis B and C and Liver Cancer Reveals MicroRNA-Mediated Control of Cholesterol Synthesis Programs

ABSTRACT Chronic hepatitis B (CHB), chronic hepatitis C (CHC), and associated hepatocellular carcinoma (HCC) are characterized by cholesterol imbalance and dyslipidemia; however, the key regulatory drivers of these phenotypes are incompletely understood. Using gene expression microarrays and high-throughput sequencing of small RNAs, we performed integrative analysis of microRNA (miRNA) and gene expression in nonmalignant and matched cancer tissue samples from human subjects with CHB or CHC and HCC. We also carried out follow-up functional studies of specific miRNAs in a cell-based system. These studies led to four major findings. First, pathways affecting cholesterol homeostasis were among the most significantly overrepresented among genes dysregulated in chronic viral hepatitis and especially in tumor tissue. Second, for each disease state, specific miRNA signatures that included miRNAs not previously associated with chronic viral hepatitis, such as miR-1307 in CHC, were identified. Notably, a few miRNAs, including miR-27 and miR-224, were components of the miRNA signatures of all four disease states: CHB, CHC, CHB-associated HCC, and CHC-associated HCC. Third, using a statistical simulation method (miRHub) applied to the gene expression data, we identified candidate master miRNA regulators of pathways controlling cholesterol homeostasis in chronic viral hepatitis and HCC, including miR-21, miR-27, and miR-33. Last, we validated in human hepatoma cells that both miR-21 and miR-27 significantly repress cholesterol synthesis and that miR-27 does so in part through regulation of the gene that codes for the rate-limiting enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase ( HMGCR ). IMPORTANCE Hepatitis B virus (HBV) and hepatitis C virus (HCV) are phylogenetically unrelated hepatotropic viruses that persistently infect hundreds of millions of people world-wide, often leading to chronic liver disease and hepatocellular carcinoma (HCC). Chronic hepatitis B (CHB), chronic hepatitis C (CHC), and associated HCC often lead to cholesterol imbalance and dyslipidemia. However, the regulatory mechanisms underlying the dysregulation of lipid pathways in these disease states are incompletely understood. MicroRNAs (miRNAs) have emerged as critical modulators of lipid homeostasis. Here we use a blend of genomic, molecular, and biochemical strategies to identify key miRNAs that drive the lipid phenotypes of chronic viral hepatitis and HCC. These findings provide a panoramic view of the miRNA landscape in chronic viral hepatitis, which could contribute to the development of novel and more-effective miRNA-based therapeutic strategies

Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development.

BACKGROUND: We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development. RESULTS: The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements. CONCLUSIONS: Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution

Model of fibrolamellar hepatocellular carcinomas reveals striking enrichment in cancer stem cells

The aetiology of human fibrolamellar hepatocellular carcinomas (hFL-HCCs), cancers occurring increasingly in children to young adults, is poorly understood. We present a transplantable tumour line, maintained in immune-compromised mice, and validate it as a bona fide model of hFL-HCCs by multiple methods. RNA-seq analysis confirms the presence of a fusion transcript (DNAJB1-PRKACA) characteristic of hFL-HCC tumours. The hFL-HCC tumour line is highly enriched for cancer stem cells as indicated by limited dilution tumourigenicity assays, spheroid formation and flow cytometry. Immunohistochemistry on the hFL-HCC model, with parallel studies on 27 primary hFL-HCC tumours, provides robust evidence for expression of endodermal stem cell traits. Transcriptomic analyses of the tumour line and of multiple, normal hepatic lineage stages reveal a gene signature for hFL-HCCs closely resembling that of biliary tree stem cells—newly discovered precursors for liver and pancreas. This model offers unprecedented opportunities to investigate mechanisms underlying hFL-HCCs pathogenesis and potential therapies

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts