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1.  European Journal of Medical Research reviewer acknowledgement 2013 
Contributing reviewers
The editor of European Journal of Medical Research would like to thank all our reviewers who have contributed to the journal in Volume 18 (2013).
PMCID: PMC3913951
2.  Prototype Foamy Virus Bet Impairs the Dimerization and Cytosolic Solubility of Human APOBEC3G 
Journal of Virology  2013;87(16):9030-9040.
Cellular cytidine deaminases from the APOBEC3 family are potent restriction factors that are able to block the replication of retroviruses. Consequently, retroviruses have evolved a variety of different mechanisms to counteract inhibition by APOBEC3 proteins. Lentiviruses such as human immunodeficiency virus (HIV) express Vif, which interferes with APOBEC3 proteins by targeting these restriction factors for proteasomal degradation, hence blocking their ability to access the reverse transcriptase complex in the virions. Other retroviruses use less-well-characterized mechanisms to escape the APOBEC3-mediated cellular defense. Here we show that the prototype foamy virus Bet protein can protect foamy viruses and an unrelated simian immunodeficiency virus against human APOBEC3G (A3G). In our system, Bet binds to A3G and prevents its encapsidation without inducing its degradation. Bet failed to coimmunoprecipitate with A3G mutants unable to form homodimers and dramatically reduced the recovery of A3G proteins from soluble cytoplasmic cell fractions. The Bet-A3G interaction is probably a direct binding interaction and seems to be independent of RNA. Together, these data suggest a novel model whereby Bet uses two possibly complementary mechanisms to counteract A3G: (i) Bet prevents encapsidation of A3G by blocking A3G dimerization, and (ii) Bet sequesters A3G in immobile complexes, impairing its ability to interact with nascent virions.
PMCID: PMC3754047  PMID: 23760237
3.  Hepatic stem cell niches 
The Journal of Clinical Investigation  2013;123(5):1874-1880.
Stem cell niches are special microenvironments that maintain stem cells and control their behavior to ensure tissue homeostasis and regeneration throughout life. The liver has a high regenerative capacity that involves stem/progenitor cells when the proliferation of hepatocytes is impaired. In recent years progress has been made in the identification of potential hepatic stem cell niches. There is evidence that hepatic progenitor cells can originate from niches in the canals of Hering; in addition, the space of Disse may also serve as a stem cell niche during fetal hematopoiesis and constitute a niche for stellate cells in adults.
PMCID: PMC3638908  PMID: 23635785
4.  Usp18 Driven Enforced Viral Replication in Dendritic Cells Contributes to Break of Immunological Tolerance in Autoimmune Diabetes 
PLoS Pathogens  2013;9(10):e1003650.
Infection with viruses carrying cross-reactive antigens is associated with break of immunological tolerance and induction of autoimmune disease. Dendritic cells play an important role in this process. However, it remains unclear why autoimmune-tolerance is broken during virus infection, but usually not during exposure to non-replicating cross-reactive antigens. Here we show that antigen derived from replicating virus but not from non-replicating sources undergoes a multiplication process in dendritic cells in spleen and lymph nodes. This enforced viral replication was dependent on Usp18 and was essential for expansion of autoreactive CD8+ T cells. Preventing enforced virus replication by depletion of CD11c+ cells, genetically deleting Usp18, or pharmacologically inhibiting of viral replication blunted the expansion of autoreactive CD8+ T cells and prevented autoimmune diabetes. In conclusion, Usp18-driven enforced viral replication in dendritic cells can break immunological tolerance and critically influences induction of autoimmunity.
Author Summary
Autoimmune diabetes in humans is linked to infection with viruses, which carry cross-reactive antigens. Virus derived cross-reactive antigens break immunological tolerance to pancreatic islets, which initiates disease. Several other non-viral sources of cross-reactive antigens are known, however they usually fail to induce diabetes. Here we found that viral antigen underwent an Usp18 dependent replication in dendritic cells. This mechanism was essential to generate sufficient amounts of cross-reactive antigen and to expand autoreactive CD8+ T cells. Blocking of virus replication by either depletion of dendritic cells, genetic depletion of Usp18 or pharmacological inhibition of replication blunted expansion of autoreactive CD8+ T cells and prevented diabetes. In conclusion we found that enforced virus replication broke the tolerance to self-antigen, which partially explains the strong association of autoimmune diseases with virus infections.
PMCID: PMC3812017  PMID: 24204252
5.  Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME 
Godoy, Patricio | Hewitt, Nicola J. | Albrecht, Ute | Andersen, Melvin E. | Ansari, Nariman | Bhattacharya, Sudin | Bode, Johannes Georg | Bolleyn, Jennifer | Borner, Christoph | Böttger, Jan | Braeuning, Albert | Budinsky, Robert A. | Burkhardt, Britta | Cameron, Neil R. | Camussi, Giovanni | Cho, Chong-Su | Choi, Yun-Jaie | Craig Rowlands, J. | Dahmen, Uta | Damm, Georg | Dirsch, Olaf | Donato, María Teresa | Dong, Jian | Dooley, Steven | Drasdo, Dirk | Eakins, Rowena | Ferreira, Karine Sá | Fonsato, Valentina | Fraczek, Joanna | Gebhardt, Rolf | Gibson, Andrew | Glanemann, Matthias | Goldring, Chris E. P. | Gómez-Lechón, María José | Groothuis, Geny M. M. | Gustavsson, Lena | Guyot, Christelle | Hallifax, David | Hammad, Seddik | Hayward, Adam | Häussinger, Dieter | Hellerbrand, Claus | Hewitt, Philip | Hoehme, Stefan | Holzhütter, Hermann-Georg | Houston, J. Brian | Hrach, Jens | Ito, Kiyomi | Jaeschke, Hartmut | Keitel, Verena | Kelm, Jens M. | Kevin Park, B. | Kordes, Claus | Kullak-Ublick, Gerd A. | LeCluyse, Edward L. | Lu, Peng | Luebke-Wheeler, Jennifer | Lutz, Anna | Maltman, Daniel J. | Matz-Soja, Madlen | McMullen, Patrick | Merfort, Irmgard | Messner, Simon | Meyer, Christoph | Mwinyi, Jessica | Naisbitt, Dean J. | Nussler, Andreas K. | Olinga, Peter | Pampaloni, Francesco | Pi, Jingbo | Pluta, Linda | Przyborski, Stefan A. | Ramachandran, Anup | Rogiers, Vera | Rowe, Cliff | Schelcher, Celine | Schmich, Kathrin | Schwarz, Michael | Singh, Bijay | Stelzer, Ernst H. K. | Stieger, Bruno | Stöber, Regina | Sugiyama, Yuichi | Tetta, Ciro | Thasler, Wolfgang E. | Vanhaecke, Tamara | Vinken, Mathieu | Weiss, Thomas S. | Widera, Agata | Woods, Courtney G. | Xu, Jinghai James | Yarborough, Kathy M. | Hengstler, Jan G.
Archives of Toxicology  2013;87:1315-1530.
This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.
Electronic supplementary material
The online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.
PMCID: PMC3753504  PMID: 23974980
Non-parenchymal cells; Mechanisms of gene regulation; DILI; 3D Models; Cryopreservation; Clearance; Mathematical modeling
6.  Reduced type I interferon production by dendritic cells and weakened antiviral immunity in patients with Wiskott-Aldrich syndrome protein deficiency 
Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency caused by absence of Wiskott-Aldrich syndrome protein (WASP) expression, resulting in defective function of many immune cell lineages and susceptibility to severe bacterial, viral, and fungal infections. Despite a significant proportion of patients with WAS having recurrent viral infections, surprisingly little is known about the effects of WASP deficiency on antiviral immunity.
We sought to evaluate the antiviral immune response in patients with WASP deficiency in vivo.
Viral clearance and associated immunopathology were measured after infection of WASP-deficient (WAS KO) mice with lymphocytic choriomeningitis virus (LCMV). Induction of antiviral CD8+ T-cell immunity and cytotoxicity was documented in WAS KO mice by means of temporal enumeration of total and antigen-specific T-cell numbers. Type I interferon (IFN-I) production was measured in serum in response to LCMV challenge and characterized in vivo by using IFN-I reporter mice crossed with WAS KO mice.
WAS KO mice showed reduced viral clearance and enhanced immunopathology during LCMV infection. This was attributed to both an intrinsic CD8+ T-cell defect and defective priming of CD8+ T cells by dendritic cells (DCs). IFN-I production by WAS KO DCs was reduced both in vivo and in vitro.
These studies use a well-characterized model of persistence-prone viral infection to reveal a critical deficiency of CD8+ T-cell responses in murine WASP deficiency, in which abrogated production of IFN-I by DCs might play an important contributory role. These findings might help us to understand the immunodeficiency of WAS.
PMCID: PMC3757164  PMID: 23141740
Type I interferon; dendritic cells; CD8 T cells; virus; Wiskott-Aldrich syndrome protein; Wiskott-Aldrich syndrome; diabetes; cDC, Conventional dendritic cell; DC, Dendritic cell; FACS, Fluorescence-activated cell sorting; IFN-I, Type I interferon; IL-7R, IL-7 receptor; LCMV, Lymphocytic choriomeningitis virus; NK, Natural killer; pDC, Plasmacytoid dendritic cell; Poly(I:C), Polyinosine-polycytidylic acid; pfu, Plaque-forming units; TLR, Toll-like receptor; VSV, Vesicular stomatitis virus; WAS, Wiskott-Aldrich syndrome; WAS KO, WASP knockout; WASP, Wiskott-Aldrich syndrome protein; YFP, Yellow fluorescent protein
7.  Stellate Cells from Rat Pancreas Are Stem Cells and Can Contribute to Liver Regeneration 
PLoS ONE  2012;7(12):e51878.
The identity of pancreatic stem/progenitor cells is still under discussion. They were suggested to derive from the pancreatic ductal epithelium and/or islets. Here we report that rat pancreatic stellate cells (PSC), which are thought to contribute to pancreatic fibrosis, have stem cell characteristics. PSC reside in islets and between acini and display a gene expression pattern similar to umbilical cord blood stem cells and mesenchymal stem cells. Cytokine treatment of isolated PSC induced the expression of typical hepatocyte markers. The PSC-derived hepatocyte-like cells expressed endodermal proteins such as bile salt export pump along with the mesodermal protein vimentin. The transplantation of culture-activated PSC from enhanced green fluorescent protein-expressing rats into wild type rats after partial hepatectomy in the presence of 2-acetylaminofluorene revealed that PSC were able to reconstitute large areas of the host liver through differentiation into hepatocytes and cholangiocytes. This developmental fate of transplanted PSC was confirmed by fluorescence in situ hybridization of chromosome Y after gender-mismatched transplantation of male PSC into female rats. Transplanted PSC displayed long-lasting survival, whereas muscle fibroblasts were unable to integrate into the host liver. The differentiation potential of PSC was further verified by the transplantation of clonally expanded PSC. PSC clones maintained the expression of stellate cell and stem cell markers and preserved their differentiation potential, which indicated self-renewal potential of PSC. These findings demonstrate that PSC have stem cell characteristics and can contribute to the regeneration of injured organs through differentiation across tissue boundaries.
PMCID: PMC3521726  PMID: 23272184
8.  Running Loose or Getting Lost: How HIV-1 Counters and Capitalizes on APOBEC3-Induced Mutagenesis through Its Vif Protein 
Viruses  2012;4(11):3132-3161.
Human immunodeficiency virus-1 (HIV-1) dynamics reflect an intricate balance within the viruses’ host. The virus relies on host replication factors, but must escape or counter its host’s antiviral restriction factors. The interaction between the HIV-1 protein Vif and many cellular restriction factors from the APOBEC3 protein family is a prominent example of this evolutionary arms race. The viral infectivity factor (Vif) protein largely neutralizes APOBEC3 proteins, which can induce in vivo hypermutations in HIV-1 to the extent of lethal mutagenesis, and ensures the production of viable virus particles. HIV-1 also uses the APOBEC3-Vif interaction to modulate its own mutation rate in harsh or variable environments, and it is a model of adaptation in a coevolutionary setting. Both experimental evidence and the substantiation of the underlying dynamics through coevolutionary models are presented as complementary views of a coevolutionary arms race.
PMCID: PMC3509687  PMID: 23202519
HIV-1; APOBEC3; Vif; coevolution; quasispecies; population genetics; modeling; drug resistance; escape mutant
9.  Investigating Cell-ECM Contact Changes in Response to Hypoosmotic Stimulation of Hepatocytes In Vivo with DW-RICM 
PLoS ONE  2012;7(10):e48100.
Hepatocyte volume regulation has been shown to play an important role in cellular metabolism, proliferation, viability and especially in hepatic functions such as bile formation and proteolysis. Recent studies on liver explants led to the assumption that cell volume changes present a trigger for outside-in signaling via integrins, a protein family involved in mediating cellular response to binding to the extracellular matrix (ECM). However, it remains elusive how these volume change related signaling events are transducted on a single cell level and how these events are influenced and controlled by ECM interactions. One could speculate that an increase in cell volume leads to an increase in integrin/ECM contacts which causes activation of integrins, which act as mechano-sensors. In order to test this idea, it was an important issue to quantify the cell volume-dependence of the contact areas between the cell and the surrounding ECM. In this study we used two wavelength reflection interference contrast microscopy (DW-RICM) to directly observe the dynamics of cell-substrate contacts, mimicking cell-ECM interactions, in response to a controlled and well-defined volume change induced by hypoosmotic stimulation. This is the first time a non-invasive, label-free method is used to uncover a volume change related response of in vitro hepatocytes in real time. The cell cluster analysis we present here agrees well with previous studies on ex vivo whole liver explants. Moreover, we show that the increase in contact area after cell swelling is a reversible process, while the reorganisation of contacts depends on the type of ECM molecules presented to the cells. As our method complements common whole liver studies providing additional insight on a cell cluster level, we expect this technique to be particular suitable for further detailed studies of osmotic stimulation not only in hepatocytes, but also other cell types.
PMCID: PMC3482193  PMID: 23110181
10.  Oxidative Stress Markers in the Brain of Patients with Cirrhosis and Hepatic Encephalopathy 
Hepatology (Baltimore, Md.)  2010;52(1):256-265.
Cell culture studies and animal models point to an important role of oxidative/nitrosative stress in the pathogenesis of cerebral ammonia toxicity. However, it is unknown whether oxidative/nitrosative stress in the brain is also characteristic of hepatic encephalopathy (HE) in humans. We therefore analyzed post mortem cortical brain tissue samples from patients with cirrhosis dying with or without HE in comparison with brains from patients without liver disease. Significantly elevated levels of protein tyrosine-nitrated proteins, heat shock protein-27, and 8-hydroxyguanosine as a marker for RNA oxidation were found in the cerebral cortex of HE patients, but not of patients with cirrhosis but without HE. Glutamine synthetase (GS) activity was significantly decreased, whereas GS protein expression was not significantly affected. Protein expression of the glutamate/aspartate cotransporter was up-regulated in HE, whereas protein expression of neuronal and inducible nitric oxide synthases, manganese-dependent and copper/zinc-dependent superoxide dismutase, and glial glutamate transporter-1 were not significantly increased.
These data indicate that HE in patients with cirrhosis is associated with oxidative/nitrosative stress, protein tyrosine nitration, and RNA oxidation, suggesting a role of oxidative stress in the pathogenesis of HE in patients with cirrhosis.
PMCID: PMC3395472  PMID: 20583283
11.  Evaluation of acoustic radiation force impulse imaging for determination of liver stiffness using transient elastography as a reference 
AIM: To evaluate cut-off values and performance of acoustic radiation force impulse imaging (ARFI) using transient elastography [FibroScan© (FS)] as a reference.
METHODS: Six hundred and six patients were enrolled in this study. All patients underwent liver stiffness measurement with FS (FS-LS) and ARFI (with shear wave velocity quantification; ARFI-SWV) and the performance of ARFI in comparison to FS was determined. Sixty-eight patients underwent liver biopsy.
RESULTS: Significantly higher success rates for the determination of liver stiffness were found using ARFI as compared to FS [604/606 (99.7%) vs 482/606 (79.5%), P < 0.001]. ARFI-SWV correlated significantly with FS-LS (r = 0.920, P < 0.001). ARFI-SWV increased significantly with the stage of fibrosis (1.09 ± 0.13 m/s for patients with no significant fibrosis (FS-LS < 7.6 kPa); 1.46 ± 0.27 m/s for patients with significant liver fibrosis (7.6 < FS-LS ≤ 13.0 kPa); and 2.55 ± 0.77 m/s for patients with liver cirrhosis (FS-LS > 13.0 kPa)). ARFI-SWV cut-off values were identified for no significant fibrosis (1.29 m/s; sensitivity 91.4% and specificity 92.6%) and for liver cirrhosis (1.60 m/s; sensitivity 92.3% and specificity 96.5%). The optimal cut-off value for predicting liver fibrosis (F ≥ 2) was 1.32 m/s (sensitivity 87.0% and specificity 80.0%) and for liver cirrhosis (F4) 1.62 m/s (sensitivity 100% and specificity 85.7%), for patients who underwent liver biopsy. An excellent inter-and intraobserver reproducibility was observed for ARFI-SWV determinations.
CONCLUSION: An ARFI-SWV cut-off value of 1.29 m/s seems to be optimal for patients with no significant liver fibrosis and 1.60 m/s for patients with liver cirrhosis.
PMCID: PMC3296981  PMID: 22416182
Acoustic radiation force impulse imaging; Elastography; Fibroscan; Liver
12.  Onwards and upwards: European Journal of Medical Research continues as an open access publication 
The well-established European Journal of Medical Research has joined BioMed Central's portfolio of journals in January 2012, converting to the open access publishing model. Since its launch in 1995 the journal has been a print-only publication; from now on, it continues as an open access, online-only journal. The conversion to open access opens up the potential for the journal to become a leading, globally visible title in the field of general medicine over the coming years.
PMCID: PMC3337540  PMID: 22472279
13.  FibroSURE™ and FibroScan® in relation to treatment response in chronic hepatitis C virus 
AIM: To compare histological endpoint assessment using noninvasive alternatives to biopsy during treatment in a chronic hepatitis C virus (HCV) cohort.
METHODS: Patients with chronic HCV were randomized to receive interferon-based therapy for 24 (genotypes 2/3) or 48 (genotype 1) wk. FibroSURE™ (FS) was assessed at baseline and at week-12 post-treatment follow-up. Baseline biopsy for METAVIR was assessed by a single pathologist. FibroScan® transient elastography (TE) was performed during treatment in a patient subset.
RESULTS: Two thousand and sixty patients (n = 253 in Asia) were classified as METAVIR F0-1 (n = 1682) or F2-4 (n = 378). For F2-4, FS (n = 2055) had sensitivity and specificity of 0.87 and 0.61, respectively, with area under the receiver-operating curve of 0.82; corresponding values for TE (n = 214) and combined FS/TE (n = 209) were 0.77, 0.88 and 0.88, and 0.93, 0.68 and 0.88. Overall FS/TE agreement for F2-4 was 71% (κ = 0.41) and higher in Asians vs non-Asians (κ = 0.86 vs 0.35; P < 0.001). Combined FS/TE had 97% accuracy in Asians (n = 33). Baseline FS (0.38 vs 0.51, P < 0.001) and TE (8.0 kPa vs 11.9 kPa, P = 0.006) scores were lower in patients with sustained virological response than in nonresponders, and were maintained through follow-up.
CONCLUSION: FS and TE may reliably differentiate mild from moderate-advanced disease, with a potential for high diagnostic accuracy in Asians with chronic HCV.
PMCID: PMC3225094  PMID: 22147963
Albinterferon alfa-2b; FibroScan; FibroSURE; Hepatitis C virus; Interferon; Sustained virological response; Transient elastography
14.  Toponomics method for the automated quantification of membrane protein translocation 
BMC Bioinformatics  2011;12:370.
Intra-cellular and inter-cellular protein translocation can be observed by microscopic imaging of tissue sections prepared immunohistochemically. A manual densitometric analysis is time-consuming, subjective and error-prone. An automated quantification is faster, more reproducible, and should yield results comparable to manual evaluation. The automated method presented here was developed on rat liver tissue sections to study the translocation of bile salt transport proteins in hepatocytes. For validation, the cholestatic liver state was compared to the normal biological state.
An automated quantification method was developed to analyze the translocation of membrane proteins and evaluated in comparison to an established manual method. Firstly, regions of interest (membrane fragments) are identified in confocal microscopy images. Further, densitometric intensity profiles are extracted orthogonally to membrane fragments, following the direction from the plasma membrane to cytoplasm. Finally, several different quantitative descriptors were derived from the densitometric profiles and were compared regarding their statistical significance with respect to the transport protein distribution. Stable performance, robustness and reproducibility were tested using several independent experimental datasets. A fully automated workflow for the information extraction and statistical evaluation has been developed and produces robust results.
New descriptors for the intensity distribution profiles were found to be more discriminative, i.e. more significant, than those used in previous research publications for the translocation quantification. The slow manual calculation can be substituted by the fast and unbiased automated method.
PMCID: PMC3230911  PMID: 21929784
15.  Vorinostat and sorafenib increase CD95 activation in gastrointestinal tumor cells through a Ca2+ - de novo ceramide - PP2A - ROS dependent signaling pathway 
Cancer research  2010;70(15):6313-6324.
The targeted therapeutics sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I evaluation. In this study we determined how CD95 is activated by treatment with this drug combination. Low doses of sorafenib and vorinostat but not the individual drugs rapidly increased ROS, Ca2+ and ceramide levels in GI tumor cells. The production of ROS was reduced in Rho zero cells. Quenching ROS blocked drug-induced CD95 surface localization and apoptosis. ROS generation, CD95 activation and cell killing was also blocked by quenching of induced Ca2+ levels or by inhibition of PP2A. Inhibition of acidic sphingomyelinase or de novo ceramide generation blocked the induction of ROS however combined inhibition of both acidic sphingomyelinase and de novo ceramide generation was required to block the induction of Ca2+. Quenching of ROS did not impact on drug-induced ceramide/dihydro-ceramide levels whereas quenching of Ca2+ reduced the ceramide increase. Sorafenib and vorinostat treatment radiosensitized liver and pancreatic cancer cells, an effect that was suppressed by quenching ROS or knock down of LASS6. Further, sorafenib and vorinostat treatment suppressed the growth of pancreatic tumors in vivo. Our findings demonstrate that induction of cytosolic Ca2+ by sorafenib and vorinostat is a primary event that elevates dihydroceramide levels, each essential steps in ROS generation that promotes CD95 activation.
PMCID: PMC2918282  PMID: 20631069
16.  Sorafenib activates CD95 and promotes autophagy and cell death via Src family kinases in GI tumor cells 
Molecular cancer therapeutics  2010;9(8):2220-2231.
Sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and the present studies have determined individually how sorafenib and vorinostat contribute to CD95 activation. Sorafenib (3-6 μM) promoted a dose-dependent increase in Src Y416, ERBB1 Y845 and CD95 Y232/Y291 phosphorylation, and Src Y527 dephosphorylation. Low levels of sorafenib (3 μM) –induced CD95 tyrosine phosphorylation did not promote surface localization whereas sorafenib (6 μM), or sorafenib (3 μM) and vorinostat (500 nM) treatment promoted higher levels of CD95 phosphorylation that correlated with DISC formation, receptor surface localization and autophagy. CD95 (Y232F, Y291F) was not tyrosine phosphorylated and was unable to plasma membrane localize or induce autophagy. Knock down / knock out of Src family kinases abolished sorafenib –induced: CD95 tyrosine phosphorylation; DISC formation; and the induction of cell death and autophagy. Knock down of PDGFRβ enhanced Src Y416 and CD95 tyrosine phosphorylation that correlated with elevated CD95 plasma membrane levels and autophagy, and with a reduced ability of sorafenib to promote CD95 membrane localization. Vorinostat increased ROS levels; and in a delayed NFκB-dependent fashion, those of FAS ligand and CD95. Neutralization of FAS-L did not alter the initial rapid drug-induced activation of CD95 however, neutralization of FAS-L reduced sorafenib + vorinostat toxicity by ~50%. Thus sorafenib contributes to CD95 activation by promoting receptor tyrosine phosphorylation whereas vorinostat contributes to CD95 activation via initial facilitation of ROS generation and subsequently of FAS-L expression.
PMCID: PMC2933415  PMID: 20682655
Vorinostat; Sorafenib; CD95; c-FLIP-s; FAS-L; cell death; autophagy
17.  Heterologous Overexpression and Mutagenesis of the Human Bile Salt Export Pump (ABCB11) Using DREAM (Directed REcombination-Assisted Mutagenesis) 
PLoS ONE  2011;6(5):e20562.
Homologous recombination in Saccharomyces cerevisiae is a well-studied process. Here, we describe a yeast-recombination-based approach to construct and mutate plasmids containing the cDNA of the human bile salt export pump (BSEP) that has been shown to be unstable in E. coli. Using this approach, we constructed the necessary plasmids for a heterologous overexpression of BSEP in the yeast Pichia pastoris. We then applied a new site-directed mutagenesis method, DREAM (Directed REcombination-Assisted Mutagenesis) that completely bypasses E. coli by using S. cerevisiae as the plasmid host with high mutagenesis efficiency. Finally, we show how to apply this strategy to unstable non-yeast plasmids by rapidly turning an existing mammalian BSEP expression construct into a S. cerevisiae-compatible plasmid and analyzing the impact of a BSEP mutation in several mammalian cell lines.
PMCID: PMC3105083  PMID: 21655228
18.  17AAG and MEK1/2 inhibitors kill GI tumor cells via Ca2+-dependent suppression of GRP78/BiP and induction of ceramide and ROS 
Molecular cancer therapeutics  2010;9(5):1378-1395.
The present studies determined in greater detail the molecular mechanisms upstream of the CD95 death receptor by which geldanamycin HSP90 inhibitors and MEK1/2 inhibitors interact to kill carcinoma cells. MEK1/2 inhibition enhanced 17AAG toxicity that was suppressed in cells deleted for mutant active RAS which were non-tumorigenic but was magnified in isogenic tumorigenic cells expressing H-RAS V12 or K-RAS D13. MEK1/2 inhibitor and 17AAG treatment increased intracellular Ca2+ levels and reduced GRP78/BiP expression in a Ca2+ -dependent manner. GRP78/BiP over-expression, however, also suppressed drug-induced intracellular Ca2+ levels. MEK1/2 inhibitor and 17AAG treatment increased ROS levels that were blocked by quenching Ca2+ or over-expression of GRP78/BiP. MEK1/2 inhibitor and 17AAG treatment activated CD95 and inhibition of ceramide synthesis; ROS or Ca2+ quenching blocked CD95 activation. In SW620 cells that are patient matched to SW480 cells, MEK1/2 inhibitor and 17AAG toxicity was significantly reduced that correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Over-expression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell killing. Inhibition of ceramide signaling abolished drug-induced ROS generation but not drug-induced cytosolic Ca2+ levels. Thus treatment of tumor cells with MEK1/2 inhibitor and 17AAG induces cytosolic Ca2+ and loss of GRP78/BiP function, leading to de novo ceramide synthesis pathway activation that plays a key role in ROS generation and CD95 activation.
PMCID: PMC2868106  PMID: 20442308
Geldanamycin; 17AAG; MEK1/2 inhibitor; CD95; c-FLIP-s; GRP78/BiP; autophagy; cell death; ASMase; de novo
19.  Specific Targeting of Hepatitis C Virus Core Protein by an Intracellular Single-Chain Antibody of Human Origin 
Hepatology (Baltimore, Md.)  2008;48(3):702-712.
The hepatitis C virus (HCV) core protein is essential for viral genome encapsidation and plays an important role in steatosis, immune evasion, and hepatocellular carcinoma. It may thus represent a promising therapeutic target to interfere with the HCV life-cycle and related pathogenesis. In this study, we used phage display to generate single-chain variable domain antibody fragments (scFv) to the core protein from bone marrow plasma cells of patients with chronic hepatitis C. An antibody with high-affinity binding (scFv42C) was thus identified, and the binding site was mapped to the PLXG motif (residues 84–87) of the core protein conserved among different genotypes. Whereas scFv42C displayed diffuse cytoplasmic fluorescence when expressed alone in the Huh7 human hepatoma cell line, cotransfection with the core gene shifted its subcellular distribution into that of core protein. The intracellular association of scFv42C with its target core protein was independently demonstrated by the fluorescence resonance energy transfer technique. Interestingly, expression of the single-chain antibody reduced core protein levels intracellularly, particularly in the context of full HCV replication. Moreover, cell proliferation as induced by the core protein could be reversed by scFv4C coexpression. Therefore, scFv42C may represent a novel anti-HCV agent, which acts by sequestering core protein and attenuating core protein–mediated pathogenesis.
PMCID: PMC3080105  PMID: 18697213
20.  Loss of Ability To Self-Heal Malaria upon Taurine Transporter Deletion▿ †  
Infection and Immunity  2010;78(4):1642-1649.
Deletion of the taurine transporter gene (taut) results in lowered levels of taurine, the most abundant amino acid in mammals. Here, we show that taut−/− mice have lost their ability to self-heal blood-stage infections with Plasmodium chabaudi malaria. All taut−/− mice succumb to infections during crisis, while about 90% of the control taut+/+ mice survive. The latter retain unchanged taurine levels even at peak parasitemia. Deletion of taut, however, results in the lowering of circulating taurine levels from 540 to 264 μmol/liter, and infections cause additional lowering to 192 μmol/liter. Peak parasitemia levels in taut−/− mice are approximately 60% higher than those in taut+/+ mice, an elevation that is associated with increased systemic tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) levels, as well as with liver injuries. The latter manifest as increased systemic ammonia levels, a perturbed capacity to entrap injected particles, and increased expression of genes encoding TNF-α, IL-1β, IL-6, inducible nitric oxide synthase (iNOS), NF-κB, and vitamin D receptor (VDR). Autopsy reveals multiorgan failure as the cause of death for malaria-infected taut−/− mice. Our data indicate that taut-controlled taurine homeostasis is essential for resistance to P. chabaudi malaria. Taurine deficiency due to taut deletion, however, impairs the eryptosis of P. chabaudi-parasitized erythrocytes and expedites increases in systemic TNF-α, IL-1β, and ammonia levels, presumably contributing to multiorgan failure in P. chabaudi-infected taut−/− mice.
PMCID: PMC2849432  PMID: 20100858
21.  Mutational Characterization of the Bile Acid Receptor TGR5 in Primary Sclerosing Cholangitis 
PLoS ONE  2010;5(8):e12403.
TGR5, the G protein-coupled bile acid receptor 1 (GPBAR1), has been linked to inflammatory pathways as well as bile homeostasis, and could therefore be involved in primary sclerosing cholangitis (PSC) a chronic inflammatory bile duct disease. We aimed to extensively investigate TGR5 sequence variation in PSC, as well as functionally characterize detected variants.
Methodology/Principal Findings
Complete resequencing of TGR5 was performed in 267 PSC patients and 274 healthy controls. Six nonsynonymous mutations were identified in addition to 16 other novel single-nucleotide polymorphisms. To investigate the impact from the nonsynonymous variants on TGR5, we created a receptor model, and introduced mutated TGR5 constructs into human epithelial cell lines. By using confocal microscopy, flow cytometry and a cAMP-sensitive luciferase assay, five of the nonsynonymous mutations (W83R, V178M, A217P, S272G and Q296X) were found to reduce or abolish TGR5 function. Fine-mapping of the previously reported PSC and UC associated locus at chromosome 2q35 in large patient panels revealed an overall association between the TGR5 single-nucleotide polymorphism rs11554825 and PSC (odds ratio  = 1.14, 95% confidence interval: 1.03–1.26, p = 0.010) and UC (odds ratio  = 1.19, 95% confidence interval 1.11–1.27, p = 8.5×10−7), but strong linkage disequilibrium precluded demarcation of TGR5 from neighboring genes.
Resequencing of TGR5 along with functional investigations of novel variants provided unique insight into an important candidate gene for several inflammatory and metabolic conditions. While significant TGR5 associations were detected in both UC and PSC, further studies are needed to conclusively define the role of TGR5 variation in these diseases.
PMCID: PMC2928275  PMID: 20811628
22.  Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes 
Klionsky, Daniel J. | Abeliovich, Hagai | Agostinis, Patrizia | Agrawal, Devendra K. | Aliev, Gjumrakch | Askew, David S. | Baba, Misuzu | Baehrecke, Eric H. | Bahr, Ben A. | Ballabio, Andrea | Bamber, Bruce A. | Bassham, Diane C. | Bergamini, Ettore | Bi, Xiaoning | Biard-Piechaczyk, Martine | Blum, Janice S. | Bredesen, Dale E. | Brodsky, Jeffrey L. | Brumell, John H. | Brunk, Ulf T. | Bursch, Wilfried | Camougrand, Nadine | Cebollero, Eduardo | Cecconi, Francesco | Chen, Yingyu | Chin, Lih-Shen | Choi, Augustine | Chu, Charleen T. | Chung, Jongkyeong | Clarke, Peter G.H. | Clark, Robert S.B. | Clarke, Steven G. | Clavé, Corinne | Cleveland, John L. | Codogno, Patrice | Colombo, María I. | Coto-Montes, Ana | Cregg, James M. | Cuervo, Ana Maria | Debnath, Jayanta | Demarchi, Francesca | Dennis, Patrick B. | Dennis, Phillip A. | Deretic, Vojo | Devenish, Rodney J. | Di Sano, Federica | Dice, J. Fred | DiFiglia, Marian | Dinesh-Kumar, Savithramma | Distelhorst, Clark W. | Djavaheri-Mergny, Mojgan | Dorsey, Frank C. | Dröge, Wulf | Dron, Michel | Dunn, William A. | Duszenko, Michael | Eissa, N. Tony | Elazar, Zvulun | Esclatine, Audrey | Eskelinen, Eeva-Liisa | Fésüs, László | Finley, Kim D. | Fuentes, José M. | Fueyo, Juan | Fujisaki, Kozo | Galliot, Brigitte | Gao, Fen-Biao | Gewirtz, David A. | Gibson, Spencer B. | Gohla, Antje | Goldberg, Alfred L. | Gonzalez, Ramon | González-Estévez, Cristina | Gorski, Sharon | Gottlieb, Roberta A. | Häussinger, Dieter | He, You-Wen | Heidenreich, Kim | Hill, Joseph A. | Høyer-Hansen, Maria | Hu, Xun | Huang, Wei-Pang | Iwasaki, Akiko | Jäättelä, Marja | Jackson, William T. | Jiang, Xuejun | Jin, Shengkan | Johansen, Terje | Jung, Jae U. | Kadowaki, Motoni | Kang, Chanhee | Kelekar, Ameeta | Kessel, David H. | Kiel, Jan A.K.W. | Kim, Hong Pyo | Kimchi, Adi | Kinsella, Timothy J. | Kiselyov, Kirill | Kitamoto, Katsuhiko | Knecht, Erwin | Komatsu, Masaaki | Kominami, Eiki | Kondo, Seiji | Kovács, Attila L. | Kroemer, Guido | Kuan, Chia-Yi | Kumar, Rakesh | Kundu, Mondira | Landry, Jacques | Laporte, Marianne | Le, Weidong | Lei, Huan-Yao | Lenardo, Michael J. | Levine, Beth | Lieberman, Andrew | Lim, Kah-Leong | Lin, Fu-Cheng | Liou, Willisa | Liu, Leroy F. | Lopez-Berestein, Gabriel | López-Otín, Carlos | Lu, Bo | Macleod, Kay F. | Malorni, Walter | Martinet, Wim | Matsuoka, Ken | Mautner, Josef | Meijer, Alfred J. | Meléndez, Alicia | Michels, Paul | Miotto, Giovanni | Mistiaen, Wilhelm P. | Mizushima, Noboru | Mograbi, Baharia | Monastyrska, Iryna | Moore, Michael N. | Moreira, Paula I. | Moriyasu, Yuji | Motyl, Tomasz | Münz, Christian | Murphy, Leon O. | Naqvi, Naweed I. | Neufeld, Thomas P. | Nishino, Ichizo | Nixon, Ralph A. | Noda, Takeshi | Nürnberg, Bernd | Ogawa, Michinaga | Oleinick, Nancy L. | Olsen, Laura J. | Ozpolat, Bulent | Paglin, Shoshana | Palmer, Glen E. | Papassideri, Issidora | Parkes, Miles | Perlmutter, David H. | Perry, George | Piacentini, Mauro | Pinkas-Kramarski, Ronit | Prescott, Mark | Proikas-Cezanne, Tassula | Raben, Nina | Rami, Abdelhaq | Reggiori, Fulvio | Rohrer, Bärbel | Rubinsztein, David C. | Ryan, Kevin M. | Sadoshima, Junichi | Sakagami, Hiroshi | Sakai, Yasuyoshi | Sandri, Marco | Sasakawa, Chihiro | Sass, Miklós | Schneider, Claudio | Seglen, Per O. | Seleverstov, Oleksandr | Settleman, Jeffrey | Shacka, John J. | Shapiro, Irving M. | Sibirny, Andrei | Silva-Zacarin, Elaine C.M. | Simon, Hans-Uwe | Simone, Cristiano | Simonsen, Anne | Smith, Mark A. | Spanel-Borowski, Katharina | Srinivas, Vickram | Steeves, Meredith | Stenmark, Harald | Stromhaug, Per E. | Subauste, Carlos S. | Sugimoto, Seiichiro | Sulzer, David | Suzuki, Toshihiko | Swanson, Michele S. | Tabas, Ira | Takeshita, Fumihiko | Talbot, Nicholas J. | Tallóczy, Zsolt | Tanaka, Keiji | Tanaka, Kozo | Tanida, Isei | Taylor, Graham S. | Taylor, J. Paul | Terman, Alexei | Tettamanti, Gianluca | Thompson, Craig B. | Thumm, Michael | Tolkovsky, Aviva M. | Tooze, Sharon A. | Truant, Ray | Tumanovska, Lesya V. | Uchiyama, Yasuo | Ueno, Takashi | Uzcátegui, Néstor L. | van der Klei, Ida | Vaquero, Eva C. | Vellai, Tibor | Vogel, Michael W. | Wang, Hong-Gang | Webster, Paul | Wiley, John W. | Xi, Zhijun | Xiao, Gutian | Yahalom, Joachim | Yang, Jin-Ming | Yap, George | Yin, Xiao-Ming | Yoshimori, Tamotsu | Yu, Li | Yue, Zhenyu | Yuzaki, Michisuke | Zabirnyk, Olga | Zheng, Xiaoxiang | Zhu, Xiongwei | Deter, Russell L.
Autophagy  2007;4(2):151-175.
Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
PMCID: PMC2654259  PMID: 18188003
autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole
23.  Endocrine and paracrine role of bile acids 
Bile acids are not only important for the absorption of dietary lipids and fat soluble vitamins but are signalling molecules with diverse endocrine and paracrine functions. Bile acids regulate bile acid, lipid and glucose metabolism and modulate temperature and energy homeostasis. Furthermore, bile acids can not only promote cell proliferation and liver regeneration but can also induce programmed cell death. Bile acid functions are mediated through different pathways which comprise the activation of nuclear hormone receptors, of intracellular kinases and of the plasma membrane-bound, G-protein coupled bile acid receptor TGR5/Gpbar-1.
PMCID: PMC2748195  PMID: 18837077
Bile acids; Farnesoid X receptor; TGR5; Glucose metabolism; Lipid metabolism
24.  An uncommon cause of gastro-duodenal ulceration 
Gastrointestinal ulcers occur frequently and are mainly caused by H pylori infection. In this report, we present a rare case of gastro-duodenal ulcer following selective internal radiation therapy (SIRT). SIRT is a palliative treatment for unresectable liver tumours. During SIRT, 90Y-microspheres are infused into the hepatic artery. Pre-treatment evaluation for the presence of arterial shunts to neighbouring organs should be determined in order to avoid complications of SIRT.
PMCID: PMC2708377  PMID: 18442213
Selective internal radiation therapy; Duodenal ulcer; Colon carcinoma; Hepatic metastases; Gastroscopy

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