Fostering responsible research with genome editing technologies: a European perspective

In this consensus paper resulting from a meeting that involved representatives from more than 20 European partners, we recommend the foundation of an expert group (European Steering Committee) to assess the potential benefits and draw-backs of genome editing (off-targets, mosaicisms, etc.), and to design risk matrices and scenarios for a responsible use of this promising technology. In addition, this European steering committee will contribute in promoting an open debate on societal aspects prior to a translation into national and international legislation.     

Effects of increasing the number of players and memory size in the iterated Prisoner's Dilemma: a numerical approach

The Prisoner''s Dilemma has become a paradigm for the evolution of altruistic behaviour. Here we present results of numerical simulations of the infinitely iterated stochastic simultaneous Prisoner''s Dilemma considering players with longer memory, encounters of more than two players as well as different pay-off values. This provides us with a better foundation to compare theoretical results to experimental data. We show that the success of the strategy Pavlov, regardless of its simplicity, is far more general by having an outstanding role in the iterated N-player N-memory Prisoner''s Dilemma. Besides, we study influences of increased memory sizes in the iterated two-player Prisoner''s Dilemma, and present comparisons to results of experiments with first-year students.     

Isozyme differentiation of aldolase and pyruvate kinase in fetal,regenerating, preneoplastic,and malignant rat hepatocytes during culture

Summary Aldolase and pyruvate kinase isozymes were investigated in cultured hepatocytes from fetal, regenerating, and 2-acetyl-aminofluorene-fed rat liver as well as in some epithelial liver cell lines. Our results show that: (a) cell proliferation and prolonged expression of specific isozymes were found only in cultured hepatocytes from 17-day old fetuses; (b) the fetal type of pyruvate kinase expressed in regenerating and carcinogen-treated liver was temporarily lost only in cultured hepatocytes from regenerating liver; (c) the adult type of aldolase and pyruvate kinase was absent in one epithelial cell line derived from a carcinogen-treated liver and in the hepatoma tissue cell (HTC) line but was found in the Faza clone of the Reuber H₃₅ cell line during the 50 first passages in vitro; and (d) the isozyme pattern of pyruvate kinase was always more strongly shifted than that of aldolase. The observations suggest that: (a) hepatocytes from carcinogen-treated liver exhibit the same lack of ability to proliferate in primary culture as normal adult hepatocytes; (b) adult hepatocytes can produce fetal isozymes without prior cell division; (c) pyruvate kinase is a stronger marker of dedifferentiation (retrodifferentiation) than aldolase; and (d) regulatory processes of isozyme expression are different during ontogenesis, regeneration, and hepatocarcinogenesis. This work was supported by the “Institut National de la Santé et de la Recherche Médicale” and the “Fondation pour la Recherche Medicale Fran?aise”     

M-CSF stimulated differentiation requires persistent MEK activity and MAPK phosphorylation independent of Grb2-Sos association and phosphatidylinositol 3-kinase activity

Macrophage colony-stimulating factor (M-CSF) is a physiological regulator of monocyte-macrophage lineage. Ectopic expression of the M-CSF receptor (M-CSFR, or Fms) in murine myeloid cell line FDC-P1 (FD/Fms cells) results in M-CSF-dependent macrophage differentiation. Previously, we observed that M-CSF induces two temporally distinct phases of mitogen-activated protein kinase (MAPK) phosphorylation. Here we show that levels of phosphorylated MAPK kinase MEK1 follow the same kinetics as MAPK phosphorylation, characterized by an early and transient phase (the first 30 min of M-CSF stimulation) and a late and persistent phase from 4 h of stimulation. The MEK inhibitor U0126 strongly inhibited both phases of MAPK phosphorylation as well as FD/Fms cell differentiation, indicating that MAPK may relay M-CSF differentiation signaling downstream of M-CSFR. Treatment of FD/Fms cells with U0126 during the first hour of M-CSF stimulation reversibly blocked the early phase of MAPK phosphorylation but did not affect differentiation. In contrast, U0126 still inhibited FD/Fms cell differentiation when its addition was delayed by 24 h. This demonstrated that late and persistent MEK activity is specifically required for macrophage differentiation to occur. Furthermore, disrupting Grb2-Sos complexes with a specific blocking peptide did not prevent FD/Fms cells differentiation in response to M-CSF, nor did it abolish MAPK phosphorylation. The role of phosphatidylinositol 3-kinase (PI 3-kinase), another potential regulator of the MAPK pathway, was examined using the specific inhibitor LY294002. This compound could not impede FD/Fms cell commitment to macrophage differentiation and did not significantly affect MAPK phosphorylation in response to M-CSF. Therefore, M-CSF differentiation signaling in myeloid progenitor cells is mediated through persistent MEK activity but it is not strictly dependent upon Grb2-Sos interaction or PI 3-kinase activity.     

Characterization of Determinants Important for Hepatitis C Virus p7 Function in Morphogenesis by Using trans-Complementation

Hepatitis C virus (HCV) p7 is an integral membrane protein that forms ion channels in vitro and that is crucial for the efficient assembly and release of infectious virions. Due to these properties, p7 was included in the family of viroporins that comprises proteins like influenza A virus M2 and human immunodeficiency virus type 1 (HIV-1) vpu, which alter membrane permeability and facilitate the release of infectious viruses. p7 from different HCV isolates sustains virus production with variable efficiency. Moreover, p7 determinants modulate processing at the E2/p7 and the p7/NS2 signal peptidase cleavage sites, and E2/p7 cleavage is incomplete. Consequently, it was unclear if a differential ability to sustain virus production was due to variable ion channel activity or due to alternate processing at these sites. Therefore, we developed a trans-complementation assay permitting the analysis of p7 outside of the HCV polyprotein and thus independently of processing. The rescue of p7-defective HCV genomes was accomplished by providing E2, p7, and NS2, or, in some cases, by p7 alone both in a transient complementation assay as well as in stable cell lines. In contrast, neither influenza A virus M2 nor HIV-1 vpu compensated for defective p7 in HCV morphogenesis. Thus, p7 is absolutely essential for the production of infectious HCV particles. Moreover, our data indicate that p7 can operate independently of an upstream signal sequence, and that a tyrosine residue close to the conserved dibasic motif of p7 is important for optimal virus production in the context of genotype 2a viruses. The experimental system described here should be helpful to investigate further key determinants of p7 that are essential for its structure and function in the absence of secondary effects caused by altered polyprotein processing.Hepatitis C virus (HCV) is a highly variable enveloped virus. It is the sole member of the genus Hepacivirus within the family Flaviviridae (36). Based on sequence homology, patient isolates are classified into seven genotypes and more than 100 subtypes (17, 52).The genome of HCV is a single-stranded RNA molecule of positive polarity with a size of ∼9.6 kb. It encodes a polyprotein of ca. 3,000 amino acids and contains nontranslated regions (NTRs) at both the 5′ and 3′ termini that are required for translation and RNA replication (33). Cellular and two viral proteases, NS2-3 and NS3-4A, liberate the individual viral proteins. The N-terminal portion of the polyprotein contains the structural proteins core and envelope glycoproteins 1 and 2 (E1, E2), which constitute the virus particle. These proteins are cleaved from the polyprotein by the host cell signal peptidase (18, 24). In the case of the core protein, an additional cleavage step mediated by the signal peptide peptidase liberates its mature C terminus (41). Further downstream of the structural proteins the polyprotein harbors p7, a short membrane-associated polypeptide required for virus assembly and release (27, 55), and the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Proteins NS3 to NS5B are the minimal components of the membrane-bound replication complexes that catalyze RNA replication (16, 38).Using the novel JFH1-based HCV infection model (35, 61, 65), it has been demonstrated recently that besides the canonical structural proteins core, E1, and E2, NS5A, p7, NS3, and NS2 also are crucial for the production of infectious HCV particles (1, 26, 27, 39, 40, 55, 57). These data highlight that HCV assembly and release is a coordinated process involving both structural and nonstructural proteins. However, how the aforementioned proteins contribute to the production of infectious virus particles remains poorly understood.HCV p7 comprises two helical domains connected by a polar loop. Studies with epitope-tagged p7 variants indicate that both termini of the protein are resident in the lumen of the endoplasmic reticulum (ER) (4) or that, in addition, a second alternative topology with the C terminus exposed to the cytoplasm can be adopted (25). Using such constructs for fluorescent microscopy, a complex localization of p7 was revealed. While most prominent staining generally was observed at the ER (4, 19, 23), pools of p7 also were detected at mitochondria (19) and at the plasma membrane (4). These data suggest that p7 influences virus replication at various sites within infected cells, and that the function and/or localization of p7 is regulated by different trafficking signals that could be exposed in a topology-dependent manner. However, caution is warranted since, due to the lack of antibodies, epitope-tagged p7 variants had to be employed for most analyses, and since localization studies of virus-producing cells with functional p7 still are lacking.One hallmark of p7 is its ability to form cation-selective channels in artificial membranes (20, 46, 49), a property that likely depends on the oligomerization of the protein (7, 21). There are intriguing correlations that link p7''s function as an ion channel protein in vitro to its role in the assembly and release of infectious HCV particles in tissue culture. First, the mutation of the conserved dibasic motif in the polar loop of p7 abrogates ion channel activity and interferes with virus production in tissue culture (20, 27, 55). Second, iminosugars coupled to long alkyl chains like N-nonyl deoxygalactonojirimycin (NN-DGJ) not only interfere with ion channel activity but also repress the release of infectious particles from transfected Huh-7 cells (46, 56). Taken together, these data suggest that the ion channel activity of p7 is crucial for its role in the late steps of the HCV replication cycle, and that this function is amenable to the development of selective inhibitors for antiviral therapy. However, presently it is unknown how mechanistically p7, as an ion channel protein, facilitates HCV assembly and release or if p7 also is a component of virus particles and participates in entry.Besides its function as an ion channel, p7 harbors a signal-like sequence in its C-terminal domain that directs the insertion of the N terminus of NS2 into the lumen of the ER (4). Strikingly, due to structural determinants within the C terminus of E2, p7, and the N terminus of NS2, signalase cleavages at the E2/p7 and the p7/NS2 sites are incomplete, thus yielding E2-p7-NS2 and E2-p7 precursor proteins (3, 18, 34, 42). Although these precursors are not absolutely essential for the production of infectious HCV particles (26, 27), a defined ratio between mature and precursor proteins might play a role to orchestrate optimal virus assembly. Given these circumstances, genetic studies of p7 function are complicated, since mutations may, on the one hand, affect ion channel activity, and on the other hand influence processing at the E2-p7 and p7-NS2 junctions.To circumvent this problem, in this study we developed a complementation system that permits the rescue of genomes with defects in p7 by the ectopic expression of p7 in trans. This enabled us to directly assess the function of p7 in the absence of secondary effects caused by aberrant polyprotein cleavage. Using this approach, we analyzed the role of the native signal sequence of p7 and p7-containing precursor proteins. In addition, we investigated key determinants that are essential for the optimal function of p7 in the course of HCV infectious particle production.     

Antiviral Activity of a Small Molecule Deubiquitinase Inhibitor Occurs via Induction of the Unfolded Protein Response

Ubiquitin (Ub) is a vital regulatory component in various cellular processes, including cellular responses to viral infection. As obligate intracellular pathogens, viruses have the capacity to manipulate the ubiquitin (Ub) cycle to their advantage by encoding Ub-modifying proteins including deubiquitinases (DUBs). However, how cellular DUBs modulate specific viral infections, such as norovirus, is poorly understood. To examine the role of DUBs during norovirus infection, we used WP1130, a small molecule inhibitor of a subset of cellular DUBs. Replication of murine norovirus in murine macrophages and the human norovirus Norwalk virus in a replicon system were significantly inhibited by WP1130. Chemical proteomics identified the cellular DUB USP14 as a target of WP1130 in murine macrophages, and pharmacologic inhibition or siRNA-mediated knockdown of USP14 inhibited murine norovirus infection. USP14 is a proteasome-associated DUB that also binds to inositol-requiring enzyme 1 (IRE1), a critical mediator of the unfolded protein response (UPR). WP1130 treatment of murine macrophages did not alter proteasome activity but activated the X-box binding protein-1 (XBP-1) through an IRE1-dependent mechanism. In addition, WP1130 treatment or induction of the UPR also reduced infection of other RNA viruses including encephalomyocarditis virus, Sindbis virus, and La Crosse virus but not vesicular stomatitis virus. Pharmacologic inhibition of the IRE1 endonuclease activity partially rescued the antiviral effect of WP1130. Taken together, our studies support a model whereby induction of the UPR through cellular DUB inhibition blocks specific viral infections, and suggest that cellular DUBs and the UPR represent novel targets for future development of broad spectrum antiviral therapies.     

Discovery and optimization of a potent and selective triazolopyridinone series of c-Met inhibitors

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of a structurally diverse series of carbon-linked quinoline triazolopyridinones, which demonstrates nanomolar inhibition of c-Met kinase activity. This novel series of inhibitors exhibits favorable pharmacokinetics as well as potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.