Centre de recherche en cancérologie - Lyon

Hepatitis B and C viruses determine chronic infections of the liver in about 600 million people worldwide, and foster hepatic injuries such as steatosis and fibrosis that can lead to cirrhosis and hepatocarcinoma (HCC). The team's main aims consist in a better understanding of molecular mechanisms that allow establishment and maintenance of HBV and HCV infections, in the hope of developing novel antiviral strategies (either directly targeting the virus or through modulation of immune functions modulation). Our global purpose is to contribute to the eradication of hepatic viral infections, the prevention of liver cancer and the regression of its precursor pathologies such as fibrosis and cirrhosis.

Biology of cccDNA and novel biomarkers for in chronic HBV infections

Context: The goals of current HBV therapy are to prevent the development of cirrhosis, hepatic decompensation, hepatocellular carcinoma (HCC) and death from HBV-related liver disease, which represents the 3^rd mortality cause worldwide. This, associated to suboptimal vaccination coverage in highly endemic areas, make CHB a worldwide major health burden, with more than 240 million people being chronic carriers of the infection.

Current first-choice treatments for chronic hepatitis B (CHB) are able to efficiently induce viral suppression in the majority of patients, but life-long therapy is needed to maintain infection under control due to their inability to eliminate the virus from infected hepatocytes. Indeed, the hepatitis B virus (HBV) minichromosome, the so-called covalently closed circular (ccc)DNA, is wrapped around nucleosomes to form a stable, chromatinized structure that is regulated by epigenetic mechanisms and is responsible for viral persistence in the nucleus of infected cells. Moreover, the ability of HBV to integrate into the host genome hampers a complete sterilizing cure.

The residual viral replication and antigen production in most patients under treatment substantially contributes to the residual risk of hepatocarcinogenesis.

New therapeutic approaches are needed to overcome HBV persistence in the infected cells, or at least, to control its transcriptional and replicative activity.

Concomitantly, the advent of new combinatorial therapies requires the need of defining new endpoints for the assessment of therapeutic efficiency and of serum standardized assays representing surrogate markers intrahepatic viral activity.

Objectives:

1°) Generate new knowledge on cccDNA biology, in particular on the key steps leading to its formation and to its transcriptional regulation once the pool is established, in vitro and in vivo

2°) Investigate Gene editing approaches to induce cccDNA degradation

3°) Investigate new serum surrogate markers for intrahepatic cccDNA amount and/or activity; assess their correlation with intrahepatic HBV activity and evaluate their prognostic value in CHB patients cohorts

4°) Investigate the impact of viral/host genome genetic variability on cccDNA epigenetic regulation and on new antiviral treatments efficiency

5°) Analyze HBV integration in the host genome and its role in liver pathogenesis

Using original and relevant systems such as primary liver cells, 3D cultures, as well as mouse models, our research programs aim at generating knowledges on the life-threatening HBV and HDV (co-)infections in order to develop innovative therapeutic strategies, including immune therapeutics. The specific aims of the group are to:

1°) Determine the role of liver non-parenchymal cells in the establishment and maintenance of HBV/HDV (co)infections.

2°) Analyze the early hepatic intrinsic responses against incoming viral genomes in the nucleus. In particular our studies aim at characterizing the early DNA damage response (DDR) induced by the release of the HBV rcDNA in the nucleus and its impact on cccDNA formation.

3°) Identify HBc nuclear regulatory functions and its impact on the regulation of intrinsic cell responses Our approach relies on the proteomic identification of HBc interacting partners in the hepatocyte nucleus.

4°) Identify host (i.e. innate sensors) and viral factors influencing HBV/HDV infections outcome and response to IFN alpha treatment.

5°) Understand how HDV super-infection accelerate and increase the liver diseases originally caused by HBV persistence

6°) Test and develop innovative therapeutic strategies based on either the use of PRR agonists, the targeting of HBc with specific inhibitors (i.e., DAA), or the targeting of host-factors (i.e., HTA) required for both HBV and HDV replications.

Staff: 3 tenured researchers, 1 tenured Engineer (IE INSERM), 1 AI (CDD), 4 Post-docs, 6 PhD students;

Fundings: ANRS, FINOVI, Infect-ERA, Janssen, Gilead, Novira Pharma., Arbutus Biopharma, Roche, Assembly Biosciences;

Recent publications: 1. Diab et al., Hepatology, 2017; 2. Alfaiate et al., Antiviral Res. 2016; 3. Durantel et al., J Hepatol. 2016 ; 4. Lucifora et al., J Hepatol. 2016 ;  5. Zannetti et al., J Immunol. 2016; 6. Isorce et al., Antiviral Res. 2016; 7. Luangsay et al., J Hepatol. 2015a; 8. Luangsay et al., J Hepatol. 2015b; 9. Lucifora et al., Science. 2014 ; 10. Jammart et al., J Virol. 2013.  

Tumor cell-like metabolic adaptations in the pathophysiology of chronic viral hepatitis

Chronic infection with hepatitis B and C and D viruses (HBV/ HCV/HDV) is one of the main etiologies of hepatocellular carcinoma (HCC), the most common form of liver cancer. A major feature of chronic viral hepatitis is the frequent occurrence of oxidative stress and metabolic alterations, which play a major role in liver fibrosis and disease progression towards liver cancer.  At the interface between virology, cell biology and biochemistry, our laboratory is analyzing metabolic alterations and associated intracellular oxidative stress induced by hepatitis viruses and their respective roles in the development of liver fibrosis. We have found for example evidence for the installation of a tumor cell-like metabolism in HCV-infected cells, characterized by a special dependence on glutamine utilization. Importantly, altered glutamine fluxes impact mitochondria and mitochondrial functions and may alter cellular resistance to apoptosis, inflammatory processes, metabolic and redox homeostasis in infected cells and is thus potentially strongly pro-carcinogenic.

Our particular aims are:

·      To  show how hepatitis viruses modulate metabolic fluxes using biochemistry, molecular biology and metabolomics

·      To investigate how hepatitis viruses impact and alter mitochondrial structure and functions, via direct binding to these organelles or by altered function of mitochondrial or metabolic enzymes

·      Investigate how hepatitis virus-induced metabolic changes are linked to increased oxidative stress frequently observed in infection in vitro and in patients

·      To investigate how the above events drive fibrosis progression and hepatocarcinogenesis in infected patients

To find answers to these questions, we use a mixture of molecular and cellular biology, biochemistry, metabolomics, imaging and state of the art infection assays (P3 biosafety level). In addition, we have access to clinical cohorts and samples.

These studies will on the long term lead to the development of biomarkers and optimization of treatment modalities for fibrosis progression and prevention of hepatocarcinogenesis in viral hepatitis.

Selected publications from 2015-2017:

·      AV Ivanov, O Khomich, B Bartosch. Oxidative Stress in Hepatitis C infection; Bookchapter in  Liver Oxidative Stress and Dietary Antioxidants, edited by Vinood Patel. Elsevier. In press

·      O. Smirnova, T. Keinanen, O. Ivanova M. Hyvonen, A. Khomutov, S. Kochetkov, B. Bartosch, and A. Ivanov  Hepatitis C virus Alters metabolism of biogenic polyamines by affecting expression of key enzymes of their metabolism 2017 BBRC

·      AV Ivanov, VT Valuev-Elliston, DA Tyurina, ON Ivanova, SN Kochetkov, B Bartosch, MG Isaguliants Oxidative stress, a trigger of hepatitis C and B virus-induced liver carcinogenesis. 2017 Oncotarget

·      P.L. Lévy, S. Duponchel, H. Eischeid, M. Michelet, J. Molle, HP. Dienes, HM. Steffen, M. Odenthal, F. Zoulim, B. Bartosch. Hepatitis C virus infection triggers a tumor-like glutamine metabolism. 2017 Hepatology

·      A V. Ivanov, V T. Valuev-Elliston, O Ivanova, S N. Kochetkov, B Bartosch and M G. Isaguliants. Oxidative stress during HIV infection: mechanisms and consequences. 2016 Oxid Med Cell Longev

·      J  Rieusset ,  J  Fauconnier,  M  Paillard ,  E  Belaidi ,  E  Tubbs ,   M-A  Chauvin,  A  Durand ,  A  Bravard ,  G  Teixeira ,  B  Bartosch ,  M Michelet, P Theurey , G Vial , M Demion, E Blond , F Zoulim , L Gomez , H Vidal , A Lacampagne, M Ovize.  Disruption of calcium transfer from ER to mitochondria links alterations of mitochondria-associated endoplasmic reticulum membranes (MAM) integrity to hepatic insulin resistance. 2016 Diabetologia

·      Charlène Brault, Pierre Lévy, Sarah Duponchel, Maud Michelet, Aurèlie Sallé, Eve-Isabelle Pecheur, Marie-Laure Plissonnier, Romain Parent, Evelyne Véricel, Alexander V Ivanov, Münevver Demir, Hans-Michael Steffen, Margarete Odenthal, Fabien Zoulim, Birke Bartosch. Glutathione peroxidase 4 is reversibly induced by HCV to control lipid peroxidation and to increase virion infectivity. 2016 Gut

·      Pierre Lévy, Birke Bartosch, Metabolic reprogramming: a hallmark of viral oncogenesis. 2015 Oncogene

·      Bartosch B. Piecing together the key players of fibrosis in chronic hepatitis C: what roles do non-hepatic liver resident cell types play? 2015 Gut

The netrin-1/UNC5 axis in chronic liver disease associated with liver cancer

Netrin-1 is a secreted molecule implicated in neural development. It has also been shown to be involved in the pathogenesis and treatment resistance of a substantial range of cancer types.

However, no study has been adressing the role of netrin-1 in the liver field for years. We have previously implemented several studies aiming at defining the role of the netrin-1  / UNC5A axis in chronic liver disease, using the HCV and the UPR models.

We have shown that:

-  Netrin-1 and HCV are reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions, in an EGFR-dependent manner. This functional association involving a cancer-related virus and netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.

- The UNC5A dependence receptor counteracts HCV persistence through regulation of autophagy in a DAPK-dependent manner, and is dramatically decreased in all instances in HCC samples, and specifically by HCV in cirrhosis.

- Unlike several structurally related oncogenic transcripts (l-myc, c-myc, c-myb), netrin-1 messenger RNA was selected for translation during UPR both in human hepatocytes and in mice livers. Depletion of netrin-1 during UPR induces apoptosis, leading to cell death through an uncoordinated phenotype-5A/C-mediated involvement of protein phosphatase 2A and death-associated protein kinase 1 in vitro and in netrin transgenic mice. IRES-driven netrin-1 translation leads to the inhibition of UNC5A/DAPK1-mediated apoptosis in the hepatic context during UPR, a hallmark of chronic liver disease.

Our objectives are to define:

- The implication of netrin-1 in liver inflammation (current Labex funded PhD student)

- The implication of netrin-1 in HCC onset (current ANRS funded program involving also a research assistant)

Since netrin-1 upregulation spans a wide spectrum of liver diseases (Plos Biol 2016), such programs represent an important part of the Pathology program of the Zoulim Team, which lies besides the HBV basic and the HBV immunopathology program of the team.

Selected publications

1: Plissonnier ML, Lahlali T, Raab M, Michelet M, Romero-López C, Rivoire M, Strebhardt K, Durantel D, Levrero M, Mehlen P, Zoulim F, Parent R. Reciprocal antagonism between the netrin-1 receptor uncoordinated-phenotype-5A (UNC5A) and the hepatitis C virus. Oncogene. 2017 Aug 7. doi: 10.1038/onc.2017.271. [Epub ahead of print] PubMed PMID: 28783179.

2: Lahlali T, Plissonnier ML, Romero-López C, Michelet M, Ducarouge B, Berzal-Herranz A, Zoulim F, Mehlen P, Parent R. Netrin-1 Protects Hepatocytes Against Cell Death Through Sustained Translation During the Unfolded Protein Response. Cell Mol Gastroenterol Hepatol. 2016 Jan 9;2(3):281-301.e9. doi: 10.1016/j.jcmgh.2015.12.011. eCollection 2016 May. PubMed PMID: 28174720; PubMed  Central PMCID: PMC5042567.

3: Plissonnier ML, Lahlali T, Michelet M, Lebossé F, Cottarel J, Beer M, Neveu G, Durantel D, Bartosch B, Accardi R, Clément S, Paradisi A, Devouassoux-Shisheboran

M, Einav S, Mehlen P, Zoulim F, Parent R. Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus. PLoS Biol. 2016 Mar 31;14(3):e1002421. doi: 10.1371/journal.pbio.1002421.  eCollection 2016 Mar. PubMed PMID: 27031829; PubMed Central PMCID: PMC4816328.

4: Plissonnier ML, Lahlali T, Mehlen P, Parent R. [Hepatitis C, EGFR, cirrhosis and netrin-1: potential implications for HCC onset]. Med Sci (Paris). 2016 Jun-Jul;32(6-7):566-8. doi: 10.1051/medsci/20163206013. Epub 2016 Jul 12. French. PubMed PMID: 27406760.

There is mounting evidence that virion-bound proteins are prone to be involved either at the replication, budding/egress or entry/release steps of the viral cycle. HCV particles acquire cellular proteins via budding and encapsidation. This is even more so regarding HCV virions, known to be composed of a high percentage of cellular material, and replicating in the hepatocyte, a cell type displaying constitutively high protein secretion activity. We have shown that in vitro-grown virions bear at least 46 HCV virions-bound proteins including HCS70. Identifying such targets in clinical samples may yield ideal candidates to gain insight on the dependence of HCV upon a restricted subset of host proteins, therefore providing refined sets of genetically stable targets for therapy. The clinical interest has been confirmed by the identification of an anti-HCV compound targeting HSC70. The project goals are therefore to set up adequate conditions for robust and reproducible purification of HCV virions in clinical samples obtained from an ongoing clinical study in our Hepatology department. These clinical virions that display distinct biophysical properties as compared to their in vitro-grown counterparts will undergo MS-based identification of their HCV-bound host proteins and the characterization of their functions. Proteomics profiling of HCV particles purified from clinical samples will be overlaid with proteins identified and characterized in cell culture-grown HCV particles. Targets identified in both samples sets will be subjected to in vitro investigations using HCV-virions producing cells. Conventional biochemical and imaging methods will be used in order to: (i) ascertain their physical association with HCV virions; (ii) define the features of their interaction with HCV proteins; (iii) decipher the topology and subcellular localization of their association with HCV proteins and virions; (iv) quantitatively assess their functional involvement in particle budding, egress or secretion and infectivity. A candidate that yield satisfactory results in these experiments will be further investigated at the level of structural biology in the hope of eventually interfering with its virally hijacked function.

The second phase of this project aims at increasing as much as possible the pathophysiological relevance of this study by implementing the purification of HCV particles of clinical origin (by recruiting 15 to 20 nonresponders as well as 3 to 5 responding patients as controls) through the identification by mass spectrometry of virion-associated proteins. Comparison of plasma-derived virions-associated proteins and virions-associated proteins previously identified in in vitro HCV culture systems will enable development of research programs on targets of maximum pathophysiological relevance but also on proteins whose function may be mechanistically adressed in vitro. By this study, we hope to contribute to the identification of novel viral and cellular interaction partners, potentially applicable to therapeutic perspectives.

Research Theme #2 : Netrin and Hepatitis C (Collaboration between P. Mehlen and F. Zoulim teams)

HCV elevates transcripts of netrin-1. Huh7.5 cells were infected with HCV (JFH1 strain containing three adaptive mutations) and netrin-1 transcript was monitored by quantitative PCR for 10 days.

One of the most prominent advances in the fields of developmental, cell biology, and oncology lies in the dependence receptor (DR) concept. Unlike conventional receptors, DRs are active and induce cell death through apoptosis when unbound. These receptors bind the neural guidance cue netrin-1 as their canonical ligand. Netrin-1 is implicated in neural development, but also inflammation, and tumorigenesis. Netrin-1 is involved in at least five types of carcinomas (breast, glioma, lung, pancreas, colon) and three sites of inflammatory illnesses (kidney, respiratory epithelium, and colon). Inflammation and carcinoma are associated with HCV infection. No link has been evidenced so far between human oncoviruses and DRs. Preliminary results recently obtained in our group showing clear interplay between netrin-1 and HCV represented solid impetus to spur a research program at the intersection of HCV infection and netrin-1. This project aims at studying (i) netrin-1's influence on the HCV life cycle and vice versa, (ii) netrin-1's modulation of leucocytic and hepatocytic innate immunity, and (iii) netrin-1-associated alteration of the sinusoidal endothelial phenotype. Results will be validated on HCV patients' plasmas and biopsies cohorts through our Inserm / HCL-certified biobanks.


The biological world according to Wassily Kandinsky (Composition IX, Paris, 1936)

Heparans sulfates proteoglycans of the hepatic microenvironment in the infection and early carcinogenesis induced by the hepatotropic viruses B, C and delta.

Chronic viral hepatitis is responsible for 75 % of hepatocellular carcinomas (HCC), the 2nd most lethal cancer after lung cancer and the 5th in terms of world incidence. No adequate therapy is currently effective against HCC, the only option at that stage is liver transplantation.

The hepatitis C virus (HCV) chronically infects 180 million individuals, and although effective antiviral treatments are available, there remains difficult-to-treat patients, at great risk of developing HCC. Hepatitis B and delta affect 350 and 20 million people respectively. Due to the hepatitis B (HBV) and delta (HDV) viruses, they are major public health problems because of the diversity of viral genotypes, their fast dissemination, the severity of hepatic damages, the emergence of viral resistant variants, the insufficient pharmacological control with current therapies and the variable access to expensive treatments.

In this context, a better understanding of the infection cycles and of the early stages of viral oncogenesis will allow to propose new therapeutic strategies. In a first axis of research centered on HCV, we shall study the hepatocyte micro-environment during infection, and the changes it undergoes under the influence of oxidative stress generated by viral replication. These fundamental results will be used in a second axis to pave new therapeutic tracks, in particular against HBV and HDV.

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First Axis

In the liver, HCV infects only hepatocytes in a productive way (1), while the space of Disse in which they are located also contain stellate cells, macrophages (or Kupffer cells) and endothelial cells lining the sinusoid capillaries (cf Figure). From this observation, we hypothesized that the same molecule, present near or on the surface of hepatocytes, could play a role in the onset of infection (at the stage of viral entry into the hepatocyte) and in the process of carcinogenesis. We concentrated our efforts on a molecule of the family of heparans sulfates proteoglycans (HSPG), syndecan-1, present in the hepatocyte micro-environment or glycocalyx, because: (i) it is a transmembrane glycoprotein capable of signal transduction and a lipoprotein receptor, playing an essential role in the liver lipid metabolism, diverted and disrupted by the virus; (ii) it plays a pivotal role via its contacts with proteins of the adhesion and cellular communication, and finally (iii) it is a tumoral biomarker of certain leukaemia.

Our recent results demonstrate the implication of syndecan-1 in HCV infection, at the early virus entry stage, but also at later stages where the virus replicates in association with intracellular membrane compartments (2-4). Syndecan-1 acts in close structural and functional cooperation with CD81, the first receptor of HCV discovered. From the first days of infection, the expression and subcellular localization of syndecan-1 are modified in infected cells by comparison with uninfected cells. In parallel, the composition of the hepatocyte glycocalyx is altered, due to perturbations of the activity of key enzymes of the biosynthesis and metabolism of HSPG. Also HCV infection leads to : (i) an accumulation of intermediate metabolites of glycolysis, which triggers an intracellular oxidative stress (5), (ii) an overexpression of some metalloproteinases, reshuffling the hepatic micro-environment and extracellular matrix (ECM), and (iii) an alteration of the expression and subcellular localization of cell junction proteins such as E-cadherin. This suggests a close intricacy between infection, changes of protein glycosylation profile (in particular HSPG of the glycocalyx), and dysregulation of cell/cell and cell/ECM communications, an early hallmark of carcinogenesis.

From these observations, our project will develop in 3 acts:

- Identification of the syndecan-1-dependent gene pathways dysregulated during infection, by transcriptomic and CRISPR-Cas9 approaches;

- Thorough study of how HCV reshuffles the hepatocyte micro-environment, stricto sensu (glycocalyx) and lato sensu (space of Disse);

- Dissection of the link between oxidative stress, changes of the hepatocyte micro-environment and HCV infection, by specifically studying enzymes and key proteins involved in or responsible for these phenomena (heparanases, matrix-metalloproteinases, superoxide dismutase, glutathione peroxydase, syndecan-1, glypican-3…), in cell cultures and biopsies of infected patients.

Second Axis

Understanding the fundamental mechanisms of a viral infection allows advances on the front of antiviral strategies adapted to the management of a chronic disease, evolving by stages (acute infection, fibrosis, cirrhosis, HCC). For several months, we have been exploring the antiviral properties of two molecules already administered in human medicine for other indications: arbidol and silibinin. We demonstrated their inhibitory activity of HCV entry into hepatocytes (6-9), but also of other stages of the viral cycle such as replication and assembly of neo-formed particles (10). Arbidol also exhibits an anti-HBV activity (11).

Both molecules directly act on the virus and also on cellular targets ; they are therefore broad-spectrum antivirals and host-targeting agents, and have a double therapeutic interest: they are usable in acute (inhibition of viral entry; 8,12) and chronic infection phases. Our recent results indicate on one hand that silibinin inhibits HBV and HDV entry into the hepatocyte, and on the other hand that it down-regulates the expression of HSPG. Since HSPG are attachment and entry factors of HBV and HDV (13,14), this down-regulation phenomenon could thus contribute to explain silibinin’s mechanism of antiviral action. The antiviral potential of these molecules will thus be explored in depth, while aiming at the highest physio-pathological relevance. Indeed, this potential will be estimated during infection of human primary hepatocytes by sera from infected patients, and in infected animals. Moreover silibinin demonstrated an anti-tumoral effect in cellulo; we shall therefore study its potential on viro-induced HCC cell lines or not, and in vivo.

References

1 - Perrault M, Pécheur EI. Biochem J. 2009 Nov;423:303-314. Invited Review.

2 - http://viewer.zmags.com/publication/03efcb6d#/03efcb6d/34 : poster highlight, EASL 2014 - International Liver Congress (London, 9-13 april 2014).

3 - Grigorov B, Gentil dit Maurin A, Varbanov M, Blaising J, Michelet M, Ruggiero F, Pécheur EI. P199. J Hepatol. 2014;60(1) Supplement S132.

4 - Grigorov B, Reungoat E *, Gentil dit Maurin A *, Varbanov M *, Blaising J, Michelet M, Manuel R, Parent R, Bartosch B, Zoulim F, Ruggiero F, Pécheur EI. Submitted.

5 - Brault C, Lévy P, Duponchel S, Michelet M, Sallé A, Pécheur EI, Plissonnier ML, Parent R, Véricel E, Ivanov AV, Demir M, Steffen HM, Odenthal M, Zoulim F, Bartosch B. Gut. 2016 Jan;65(1):144-154.

6 - Blaising J, Lévy PL, Gondeau C, Phelip C, Varbanov M, Teissier E, Ruggiero F, Polyak SJ, Oberlies NH, Ivanovic T, Boulant S, Pécheur EI. Cell Microbiol. 2013 Nov;15(11):1866-82.

7 - Blaising J *, Lévy PL *, Polyak SJ, Stanifer M, Boulant S, Pécheur EI. Antiviral Res. 2013 Oct;100(1):215-9.

8 - Blaising J, Polyak SJ, Pécheur EI. Antiviral Res. 2014 Jul;107C:84-94. Invited Review.

9 - Teissier E, Zandomeneghi G, Loquet A, Lavillette D, Lavergne JP, Montserret R, Cosset FL, Böckmann A, Meier BH, Penin F, Pécheur EI. PLoS One. 2011 Jan 25;6(1):e15874.

10 - Wagoner J, Negash A, Kane OJ, Martinez LE, Nahmias Y, Bourne N, Owen DM, Grove J, Brimacombe C, McKeating JA, Pécheur EI, Graf TN, Oberlies NH, Lohmann V, Cao F, Tavis JE, Polyak SJ. Hepatology. 2010 Jun;51(6):1912-21.

11 - Pécheur EI, Borisevich V, Halfmann P, Morrey JD, Smee DF, Prichard M, Mire CE, Kawaoka Y, Geisbert TW, Polyak SJ. J Virol. In revision.

12 - Pécheur EI. Gut. 2014 Jul;63(7):1035-7. Invited Editorial.

13 - Schulze A, Gripon P, Urban S. Hepatology. 2007 Dec;46(6):1759-68.

14 - Lamas Longarela O, Schmidt TT, Schöneweis K, Romeo R, Wedemeyer H, Urban S, Schulze A. PLoS One. 2013;8(3):e58340.

Ideas are like fish. If you want to catch little fish, you can stay in the shallow water. But if you want to catch the big fish, you’ve got to go deeper.

David Lynch.