Figura 1. Esquema del número de pacientes reclutados y que hayan finalizado el estudio.

ABSTRACTS PART 1:

Abstracts PhD Programme in Biosciences

e00001 Role of Lipid Transfer Proteins at ER-Golgi Membrane Contact Sites during Bunyavirus Infection.

e00002 Exploring the regulation of frataxin expression by neurotrophic factors in the mouse cerebellum after physical exercise.

e00003 Undesired sample preparation artifacts can hamper the immunopurification of lysine acetylation in proteomic studies.

e00004 RNA-sequencing analysis identifies downstream genes of TCF4 involved in the development of non-small cell lung cancer.

e00005 NDUFS2 Mitochondrial Complex I deficiency induces adipose tissue degeneration via IIS in a Drosophila melanogaster model.

e00006 Induction of antiviral CD8+ T lymphocyte-mediated protective memory responses.

e00007 A novel role for the Hippo pathway mediator TAZ in thyroid differentiation.

e00008 Neuroprotective strategies against circadian alterations in a glioma model.

e00009 Mechanisms of resistance of basal cell carcinoma to Photodynamic Therapy.

e00010 S100A9 mediates resistance of brain metastasis to radiation therapy.

e00011 NRF2 controls proteostasis through the transcriptional regulation of autophagy.

e00012 The role of p27kip1 in the development, differentiation and maturation of mesencephalic dopaminergic neurons.

e00013 Role of the transcription factor NRF2 in hippocampal neurogenesis and in a mouse model of Alzheimer´s Disease.

e00014 In vitro enhancement of collagen matrix deposition and crosslinking by coupling lysyl oxidase (LOX) with bone morphogenetic protein-1 (BMP-1) and its application in tissue engineering.

e00015 Vesicular trafficking and polarity of ICAM-1 in inflamed liver tissue.

e00016 Lamin A/C enhances APC capacity to stimulate CD4 T cell responses.

e00017 Effectiveness and molecular basis of CDK4/6 inhibition in combination with taxanes in pancreatic cancer.

e00018Relevance of coronavirus viroporins and pdz-binding- motifs in virus replication and virulence.

e00019 The Role of Liver X Receptors in the homeostasis of Splenic Red Pulp Macrophages and Iron Metabolism.

e00020Unravelling transformation of Follicular Lymphoma to Diffuse Large B- Cell Lymphoma.

e00021 Adipose tissue and liver crosstalk: new insights into gender differences in hepatocellular carcinoma incidence.

e00022 Functional characterization of CNS2 DNA regulatory element of the mouse Tyr gene by CRISPR-Cas9 mutagenesis.

e00023 CDCA7 regulates lymphoma cells migration and invasion through reorganization of the tubulin and the actomyosin cytoskeleton.

e00024 Preclinical Safety and Efficacy Evaluation of Lentivirally transduced Hematopoietic Stem Cells for the treatment of Leukocyte Adhesion Deficiency type I.

e00026 Tofacitinib restores the inhibition of reverse cholesterol transport induced by inflammation: understanding the lipid paradox associated with rheumatoid arthritis.

e00027 The role of PKD1 in brain injury: ROS detoxification and neuroprotection.

e00028 Generation and characterization of a reversible HGPS mouse model to design potential future therapies.

e00029 Study of the role of microRNAs in T-cell lymphoblastic lymphoma development through the regulation of expression of FBXW7 gene.

e00030 Protection against Middle East respiratory syndrome coronavirus infection by immunization with genetically engineered liveattenuated viruses.

e00031 Mechanisms controlling the cilia localization of INPP5E, a phosphoinositide 5-phosphatase mutated in MORM and Joubert
syndromes.

e00032 Generation of “mini-brains” from pluripotent stem cells to study brain development.

e00033 Lysyl oxidase-like 3 in melanoma progression.

e00034 Activation of brown adipose tissue (BAT) might play a major and beneficial role against insulin resistance associated to inflammation. Beneficial effects of modulating SIRT1 activity.

e00035 Therapeutic activity of GSE4 peptide in cellular models of idiopathic pulmonary fibrosis.

e00036 Evaluation of the physiopathology of the allan-herndon-dudley syndrome. A characterization of double knock-out mice model of the disease.

e00037 Studying resistance to antivirals in single cells with Correlative Light and Electron microscopy.

e00038 Are the exosomes involved in the response to chemotherapy in cancer?

e00039 FADD phosphorylation is altered in human T-cell lymphoblastic lymphoma.

e00040 Role of cell cycle checkpoint proteins in gastric cancer stem cells.

e00041  Determination of IGFBP-3 methylation levels at ctDNA could be a prognostic biomarker in advanced stages of NSCLC.

e00042  Identification of a novel epigenetic biomarker of early lung cancer detection in COPD patients.

e00043  VMP1 controls lipid trafficking at ER contact sites  Identification of a novel epigenetic biomarker of early lung cancer detection in COPD patients.

e00044 Synaptic T cell contacts prime DC against pathogen infection.

e00045 Computational metabolism modeling predicts drug sensitivity in breast cancer cells.

e00046  The role of Gasdermin B in ulcerative colitis and inflammation.

e00047  “Insulin receptor substrate 2 (IRS2) deficiency reduces inflammatory and fibrogenic responses of the liver to cholestatic injury”.

e00048  Functional analysis of Mastl mutations in cancer.

e00049  Deciphering the RNA-dependent DNA synthesis fidelity of retroviral reverse transcriptases: impact of transcriptional
inaccuracy threshold.

Abstracts PhD Programme in Microbiology

e00086 ISG15 governs mitochondrial function in macrophages following Vaccinia virus infection.

e00087 An undercover bacterium for health applications.

Abstracts PhD Programme in Microbiology:

e00086
ISG15 governs mitochondrial function in macrophages following Vaccinia virus infection.

Manuel Albert1, Martina Bécares1, Rebeca Acín-Pérez2, Sara Baldanta1, Mercedes Fernández-Escobar1, Jesús Vázquez3, José Antonio Enríquez2,
Susana Guerra1*.

*Corresponding author:
Susana Guerra, Dept. Preventive Medicine, Public Health and Microbiology, Autonomous University of Madrid; Madrid, Spain.
E-mail: susana.guerra@uam.es

Details of affiliation

1Dept. Preventive Medicine, Public Health and Microbiology, Autonomous University of Madrid; Madrid, Spain.
2Functional Genetics of the Oxidative Phosphorylation System, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
3Laboratory of Cardiovascular Proteomics, CNIC, and CIBER de Enfermedades Cardiovasculares (CIBER-CV), Madrid, Spain.

Funding

This research is supported by a grant from Spanish Ministry of Economy and Competitiveness (MINECO) to SG (SAF2014-
54623-R), and to JV from MINECO (BIO2015- 67580-P) and from the Carlos III Institute of Health-Fondo de Investigacio´n Sanitaria
(PRB2, IPT13/0001—ISCIII-SGEFI/FEDER, ProteoRed). The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.

Competing Interests:

The authors have declared that no competing interests exist.

Keywords: ISG15, mitochondria, OXPHOS, mitophagy, macrophages, Vaccinia virus

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00086

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as:Manuel Albert, Martina Bécares, Rebeca Acín-Pérez, Sara Baldanta, Mercedes Fernández-Escobar, Jesús Vázquez, José Antonio
Enríquez, Susana Guerra. ISG15 governs mitochondrial function in macrophages following Vaccinia virus infection. IBJ Plus 2018
(S2):e00086 doi: 10.24217/2531-0151.18v1s2.00086.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Protein modification by ubiquitin and ubiquitin-like proteins is a key regulatory process of the innate
and adaptive immune response. In turn, many viruses, including poxviruses, have evolved strategies to antagonize
the antiviral and inflammatory effects of the innate immune response in order to keep infected cells alive, until virus
replication is complete. The interferon (IFN)-stimulated gene 15 (ISG15) encodes the protein ISG15, an ubiquitin-like
modifier. It is one of the most abundant proteins induced by interferon, and its expression is associated with antiviral
immunity.

Material and Methods: To identify protein components implicated in IFN and ISG15 signaling, we compared the
proteomes of murine ISG15-/- and ISG15+/+ bone marrow derived macrophages (BMDM) after Vaccinia virus (VACV)
infection. We focused on the mitochondria, as the levels of several mitochondrial proteins were different. Mitochondria
were isolated from BMDM stimulated or not with IFN, and were treated to determine the localization of ISGylated
mitochondrial proteins by western-blot. Also, using the Seahorse technology, we performed different metabolic assays
to analyze the respiratory metabolism in presence or not of ISG15, and western-blot to evaluate mitophagy-related
proteins. Furthermore, analysis of iNOS production and Arginase-1 activity was performed to explore the different
macrophage polarization patterns.

Results: These analyses revealed that several mitochondrial pathways were altered in ISG15-/- BMDM treated with IFN.
Oxidative phosphorylation (OXPHOS), Adenosine triphosphate (ATP) and reactive oxygen species (ROS) production
was higher in ISG15+/+ BMDM than in ISG15-/-BMDM following IFN treatment, indicating the involvement of ISG15-
dependent mechanisms. An additional consequence of ISG15 depletion was an impaired mitophagy and a significant
change in macrophage polarization. Although infected ISG15-/- macrophages showed a robust proinflammatory cytokine
expression pattern typical of an M1 phenotype, a clear blockade of nitric oxide (NO) production and Arginase-1 activation
was detected. Accordingly, following IFN treatment, NO release was higher in ISG15+/+ BMDM than in ISG15-/- BMDM
concomitant with a decrease in viral titer. Thus, ISG15-/- macrophages were permissive for VACV replication following
IFN treatment.

Conclusion: Our results describe a novel role for ISG15 controlling the dynamic functionality of mitochondria, specifically,
OXPHOS and mitophagy, broadening its physiological role as an antiviral agent. These findings are clinically relevant
since mitochondrial dysfunction is seen in many pathologies, underscoring the importance of the relationship between
cellular metabolism and immune response.

e00087
An undercover bacterium for health applications.

Huseyin Tas1,2, Esteban Martínez-García1, Víctor de Lorenzo1*.

*Corresponding author:
Víctor de Lorenzo, CNB-CSIC, Madrid , Spain. E-mail: vdlorenzo@cnb.csic.es

Details of affiliation

1Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), Calle Darwin 3, Madrid 28049, Country.
2Universidad Autónoma de Madrid (UAM), Ciudad Universitaria de Cantoblanco, Madrid 28049, Spain.

Funding

This project is funded by European Union under RIA (Research and Innovation Action) as H2020-LEIT-BIO-2014-1.

Competing Interests:

The authors disclose any conflict of interest.

Keywords: non-pathogenic, probiotic, chassis, invisible, therapeutic bacteria, orthogonal, biosensors, synthetic biology, P. putida.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00086

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as:Manuel Albert, Martina Bécares, Rebeca Acín-Pérez, Sara Baldanta, Mercedes Fernández-Escobar, Jesús Vázquez, José Antonio
Enríquez, Susana Guerra. ISG15 governs mitochondrial function in macrophages following Vaccinia virus infection. IBJ Plus 2018
(S2):e00086 doi: 10.24217/2531-0151.18v1s2.00086.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Pseudomonas putida KT2440 is a non-pathogenic soil bacterium (GRAS-Generally Regarded As Safe)1,2,3. High stress-tolerating
capacity especially towards organic compounds has put it into the center of attention in biotechnology applications.
It has been exploited from industrial applications i.e. the Pseudomonas 2.011, to environmental applications i.e.
bioindicators of toxics12 in details. Here, our purpose is to refactor that species for health related areas.

Such deep genome reprogramming requires precise genetic tools to use. Moreover, having a model organism that can
intuitively endure harsh conditions and to disturb least the harmony where it is introduced to is an advantage. In this
aspect, the enhanced genomic manipulation methods8,9,10 recently introduced for P. putida in addition to robust central
metabolism together with being orthogonal to the microbiome of humans, we propose to use a new chassis of P. putida
specially engineered for health related applications for the first time to our knowledge.

In this work, we are showing our endeavor and methodology for a proof-of-concept towards using P. putida as a chassis
organism for Human Health fields. The idea is to keep the homeostasis of the host organism and avoid interfering the
microbiome as much possible. As it is shown in many studies, there is a relationship between disrupted microbiome and
certain diseases such as allergies, obesity, cancer, asthma, inflammations and infections4,5,7. Taking this into account, the
platform strain we are developing is designed to make emphasis on non-invasive and non-immune response creating
conditions.

We are in the process of performing the deletions to make this strain invisible to the immune system. To do that, the
genomic manipulation techniques i.e. homologous recombination8, or ssDNA based deletion9 enforced with CRISPR/
Cas9 system10 are used. For the circuitry designs, we are developing an automated platform in P. putida based on the
Cello system6 converted into SEVA format13 (Standard European Vector Architecture). Horseshoe crab immunity testing
platform will be used to assay the immune response of this Invisible Putida chassis.

We have managed to make important deletions from the genome of P. putida, in order to suppress biofilm formation
capacity and avoid immune response in the recipient. These deletions correspond to the operons of flagella, biofilm
formation responsible operons (lapA, lapF) and other genes responsible for creating immune response in the host. The
planned deletions correspond to 131 kb of the genome, and 69 kb of it is already deleted. A deeper understanding may
be possible with upcoming deletions and immune response tests.

Abstracts PhD Programme in Molecular Biosciences:

e00001
Role of Lipid Transfer Proteins at ER-Golgi Membrane Contact
Sites during Bunyavirus Infection.

Alberto Fernández-Oliva1* and Cristina Risco1*.

*Corresponding authors: Fernández-Oliva, Alberto (alberto.fernandez@cnb.csic.es) and Risco, Cristina (crisco@cnb.csic.es).

Details of affiliation

1Cell Structure Laboratory, National Center for Biotechnology, CNB-CSIC, UAM, Campus de Cantoblanco, 28049 Madrid, Spain.

Funding

This work is supported by grant BIO2015-68758-R from the Spanish Ministry of Economy, Industry and Competitiveness
(to Cristina Risco). Alberto Fernández-Oliva is recipient of a FPI contract from the Spanish Ministry of Economy, Industry and
Competitiveness. The founders have no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.

Competing Interests:

The authors declare that no competing interests exit.

Keywords: bunyavirus, viral factories, lipid transfer protein, microscopy

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00001

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as:Fernández-Oliva, A. and Risco, C. Role of Lipid Transfer Proteins at ER-Golgi Membrane Contact Sites during Bunyavirus
Infection. IBJ Plus 2018 (S2):e00001 doi: 10.24217/2531-0151.18v1s2.00001

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction. Bunyavirales order compromises a large group of enveloped, negative-stranded RNA viruses which
includes serious emerging and re-emerging pathogens for animal and plants, and are responsible of encephalitis and
haemorrhagic fevers in humans. However, there are currently no vaccines or antiviral drugs to treat their infection.
Bunyaviruses assemble a complex viral factory involving Golgi membranes where virus particles bud and mature. To
build their membranous scaffolds, virus interfere cellular lipid metabolism and transport. Recent evidence suggests
that viruses usurp lipid transfer proteins (LTPs) from their hosts to provide lipids for their replication complexes. For
that reason, LTPs are expected to be potential targets for developing new therapeutic antiviral drugs. We are studying
bunyavirus factories in HEK293 cells and the role of four different LTPs localised at membrane contact sites between
endoplasmic reticulum and Golgi (LTP1-4) to identify which ones are subverted by bunyaviruses to their replication sites.

Materials and Methods. We have used HEK293 cell lines expressing tagged variants of human LTPs by tetracycline
induction. Cells have been infected with the ATCC Br-87 strain of Bunyamwera virus (BUNV) or a fluorescent recombinant
Gc-GFP-BUNV virus. Intracellular viral protein accumulation, viral titter and percentage of infected cells have been
evaluated by western botting, plaque-based assay and flow cytometry, respectively. LTPs location after infection have
been visualized by confocal microscopy. For high-resolution imaging of bunyavirus factories, cells have been visualized
by electron microscopy.

Results. Confocal and electron microscopy have shown that BUNV induces morphological changes at rough endoplasmic
reticulum. Changes are visualized as condensed and randomly folded membranes covered by ribosomes at the perinuclear
region, which resembles convoluted membranes described in other viral infections. Regarding the role of first selected
LTP1 and LTP2 in viral infection, no significant viral titter differences have been found when overexpressing any of them.
LTP1 has been imaged by confocal microscopy at Golgi-derived viral factories and it increases the intracellular production
of viral proteins during replication. LTP1 inhibition partially reduces BUNV intracellular protein accumulation.

Conclusions. BUNV infection of HEK293 cells modifies the architecture of rough endoplasmic reticulum. Preliminary
results support that LTP1 and LTP2 are involved in bunyavirus infection cycle.

e00002
Exploring the regulation of frataxin expression by neurotrophic
factors in the mouse cerebellum after physical exercise.

Mauro Agrò1, Yurika Katsu-Jiménez2, Paula da Silva3, Jorge Ruas3, Alfredo Giménez-Cassina1, Javier Díaz-Nido1.

*Corresponding author:
Mauro Agrò, Centro de Biología Molecular “Severo Ochoa”/ Universidad Autónoma de Madrid. E-mail: mauro.agro@cbm.csic.es

Details of affiliation

1Centro de Biología Molecular “Severo Ochoa” / Universidad Autónoma de Madrid, Madrid, Spain.
2Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
3Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.

Funding

UAM+CSIC International Excellence and European Molecular Biology Organization (EMBO).

Competing Interests:

We declare no competing interests.

Keywords: Friedreich’s ataxia, frataxin, physical exercise, neurotrophic factors, neurodegeneration.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00002

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as:Mauro Agrò, Yurika Katsu-Jiménez, Paula da Silva, Jorge Ruas, Alfredo Giménez-Cassina, Javier Díaz-Nido. Exploring the
regulation of frataxin expression by neurotrophic factors in the mouse cerebellum after physical exercise. IBJ Plus 2018 (S2):e00002
doi: 10.24217/2531-0151.18v1s2.00002.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Friedreich’s ataxia is a predominantly neurodegenerative disease caused by recessive mutations that ultimately lead
to a deficiency of frataxin (FXN) protein. It mainly affects the spinocerebellar system, thus leading to lack of motor
coordination and loss of balance. However, little is known about the regulation of frataxin gene expression under different
physiopathological situations. Our group has recently shown the prominent role of neurotrophic factors (specifically the
Brain-derived Neurotrophic Factor; BDNF) to elicit neuroprotection against frataxin deficiency both in vitro and in vivo.
Compelling evidence has pointed out a link between physical activity and the expression of neurotrophic factors in
the nervous system through several molecular mechanisms In this work, we aim to explore the link between physical
exercise, neurotrophic factors and frataxin gene expression in the mouse cerebellum. To achieve this, we subjected our
mice to a spontaneous exercise protocol lasting 8 weeks, allowing us to identify, between the “runners” a sub group of
“high runners” in order to check for “activity-amount” specific effects. We tested, through qPCRs, the levels of mRNAs
of the factors we have previously demonstrated are involved in frataxin regulation such as BDNF, Neurotrophin 3 (NT3)
and Sonic Hedgehog (SHH) as well as FXN itself. Our results show an increment in the levels of mRNAs for NT3 and SHH,
while we were not able to detect significant changes in FXN or BDNF levels. Then we checked FXN protein levels by
performing an ELISA assay, showing a significant increment in FXN protein in an amount directly related to the physical
activity performed by the mouse. Similar, but not significant, results for BDNF protein levels were found. To search
for more possible mediators of the effect of physical exercise on FXN protein level, we analyzed the levels of multiple
cytokines using a protein array, which have led to the identification of several potential candidates for FXN upregulation.
In view of these data, we suggest that physical exercise up-regulated FXN protein possibly through a posttranscriptional
mechanism. A more thorough knowledge of the mediators and molecular mechanisms underlying FXN up-regulation
may provide some clues for new therapeutic approaches to curb neurodegeneration in Friedreich’s ataxia.

e00003
Undesired sample preparation artifacts can hamper the immunopurification
of lysine acetylation in proteomic studies.

Ana Martinez-Val1, 2, Fernando Garcia1, 2, Pilar Ximénez-Embún1, Ailyn Martínez Teresa-Calleja1, Nuria Ibarz1, Isabel Ruppen1, Javier Munoz1*.

*Corresponding author: Javier Muñoz, Proteomics Unit, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain. ISCIII-ProteoRed.
E-mail: jmunozpe@cnio.es

Details of affiliation

1 Proteomics Unit, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain. ISCIII-ProteoRed.
2 These authors contribute equally to this work

Funding

The CNIO Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. Part of this work was funded
by SAF2013-45504-R (MINECO). J.M. is also supported by Ramon y Cajal Programme (MINECO) RYC-2012-10651. A.M.-V. is supported
by BES-2014-070098 (MINECO).

Competing Interests:

The authors declare no competing financial interest.

Keywords: Lysine acetylation, carbamylation, immune-purification, urea.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00003

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as:Ana Martinez-Val, Fernando Garcia, Pilar Ximénez-Embún, Ailyn Martínez Teresa-Calleja, Nuria Ibarz, Isabel Ruppen, Javier
Munoz. Undesired sample preparation artifacts can hamper the immuno-purification of lysine acetylation in proteomic studies. IBJ
Plus 2018 (S2):e00003 doi: 10.24217/2531-0151.18v1s2.00003.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Large-scale analysis of post-translational modifications by mass spectrometry relies on highly selective
affinity tools directed against specific chemical moieties. In a set of lysine acetylation (K-ac) immuno-purifications, we
noticed a fraction of high-quality spectra not assigned to peptide sequences. A mass-tolerant search revealed a K-ac
peptide distribution at +42.0103 Da and an unexpected distribution at +43.00543 Da. The presence of CHNO neutral
losses in the spectra unequivocally confirmed the identity of carbamylation.

Methods: We used a mass-tolerant search to identify the chemical modification that was copurified during immunepurification
(IP) of acetylated peptides. Afterwards, we performed the lysis and protein extraction for subsequent IP
under four different conditions: (1) Urea-Hepes and heat, (2) Urea-Hepes and room temperature, (3) Urea-Tris and room
temperature and (4) sodium deoxycholate.

Results: Urea-based buffers typically used in proteomics can artifactually induce carbamylation at the side chains of
lysine (K-cam) and/or peptide N-termini (Nter-cam). Thus, we examined the “basal levels” of carbamylation in the
experimental conditions frequently used in K-ac protocols (i.e. HEPES and 56 ºC reduction). We performed fractionation
of the total proteome and found 5% Nter-cam and 0.8% K-cam. The higher proportion of Nter-cam over K-cam peptides
(1731/286) agrees with previous reports and other published data sets that we re-analysed. Then, we searched again
our immuno-purified samples and found the inverted Nter-cam/K-cam proportion (462/3005). This suggested that the
antibody co-purified, in addition to K-ac, K-cam peptides in a specific manner. This could be explained by their similar
structures which only differ in the methyl (acetylation) and amino (carbamylation) groups.
We devised a strategy to minimize carbamylation in order to improve the selectivity of K-ac immuno-purifications.
Either protein reduction at room temperature or replacement of HEPES for a primary amine Tris buffer decreased
carbamylation levels ~6-fold which produced a ~2-fold improvement of K-ac peptides. However, substitution of urea for
sodium deoxycholate increased K-ac almost 4-fold, with 96% of the modified peptides being acetylated.

Conclusions: We recommend the use of urea-free buffers for lysis and digestion prior purification of K-ac. The use of
an ionic detergent improves the selectivity of the IP. Our findings suggest that the tools used for the enrichment of
modifications in proteins/peptides should be carefully evaluated.
(Project originally published as: Martinez-Val, et al. Urea Artifacts Interfere with Immuno-Purification of Lysine
Acetylation. J. Proteome Res. 2017(16), 1061-1068.)

e00004
RNA-sequencing analysis identifies downstream genes of TCF4
involved in the development of non-small cell lung cancer.

Olga Vera1,2, Alvaro García-Guede1,2, Carlos Rodríguez-Antolín1,2 , Olga Pernía1,2, Javier de Castro1,2, Inmaculada Ibáñez de Cáceres1,2 *

*Corresponding author:
Inmaculada Ibáñez de Cáceres. Cancer Epigenetics Laboratory, INGEMM. Biomarkers and Experimental Therapeutics in Cancer, IdiPAZ. Paseo de la
Castellana 261, 28046 Madrid, Spain. Phone +34-91-2071010-248, Fax +34-91-2071010. E-mail: inma.ibanezca@salud.madrid.org

Details of affiliation

1 Cancer Epigenetics Laboratory, INGEMM, La Paz University Hospital, Madrid, Spain
2 Biomarkers and Experimental Therapeutics in Cancer, IdiPAZ, Madrid, Spain

Funding

Supported by ISCIII PI15/00186 and the Miguel Servet II program (CP08/00068) to I. Ibáñez de Cáceres.

Competing Interests:

The authors have no conflicts of interest to declare.

Keywords: TCF4, Cisplatin (CDDP), cancer

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00004

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as:Olga Vera, Alvaro García-Guede, Carlos Rodríguez-Antolín, Olga Pernía, Javier de Castro, Inmaculada Ibáñez de Cáceres. RNAsequencing
analysis identifies downstream genes of TCF4 involved in the development of non-small cell lung cancer. IBJ Plus 2018
(S2):e00004 doi: 10.24217/2531-0151.18v1s2.00004.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: The standard treatment for non-small cell lung cancer (NSCLC) and ovarian cancer is a Platinum-based
chemotherapy, although the main clinical problem associated is the progression of the disease to a platinum-resistant
state. This fact has limited its efficacy in these tumor types, which is one of the first causes of cancer deaths in developed
countries. Thus, it is of great interest to identify predictive molecular biomarkers that could help in the patient treatment
selection.

Material and methods: In this study we used array-CGH to analyze the cytogenetic alterations that arise in NSCLC
after cisplatin treatment, by using four paired sensitive (S) and resistant (R) cell lines: H23S/R and H460S/R, A2780S/R
and OVCAR3S/R. Validation of the results was performed through qRT-PCR in the same cells. Finally, our translational
approach included the analysis of the expression in a total of 22 lung primary tumors and 7 adjacent non-tumor tissues,
followed by the RNA-sequencing of a group of these samples and the validation by RT-PCR to confirm the results.

Results: Our experimental approach revealed the presence of a common deletion of the gene TCF4 in a mosaic manner
in at least 50% of the resistant cells in both tumor types, while a decrease in TCF4 expression was confirmed through qRTPCR
in the same cells. As TCF4 is a transcription factor of Wnt signaling, we analyzed the expression of the downstream
effector DKK1 that is involved in the Wnt pathway. The translational analysis of the tumor and control samples showed
that TCF4 expression is frequently downregulated in these tumor types. Moreover, RNA-sequencing and Wnt-pathway
analysis in a subgroup of these patients allowed us to identify four genes with potential role in the development of this
malignancy.

Conclusions: Our results indicate that TCF4 is regulating downstream effectors of the Wnt-Signaling pathway that can
be involved in the tumor establishment and progression in NSCLC. Altogether, we present a novel role for Wnt signaling
pathway in the response to CDDP-based chemotherapy, which could be use as a potential therapeutic target for lung
cancer.

e00005
NDUFS2 Mitochondrial Complex I deficiency induces adipose
tissue degeneration via IIS in a Drosophila melanogaster model.

José María Becedas1, Alba Rocío Tornero1, Roberto Serna1, Sara Laine-Menéndez1, Sonia Rodríguez1, Rafael Garesse1, Margarita Cervera1, Juan
José Arredondo1*

*Corresponding author:
Juan José Arredondo, UAM- CSIC, Madrid, Spain. E-mail: juan.arredondo@uam.es

Details of affiliation

1Departamento de Bioquímica (Facultad de Medicina-UAM) & Instituto de Investigaciones Biomédicas Alberto Sols (CSIC), Madrid, Spain.

Funding

Fondo de Investigaciones Sanitarias (IP: Rafael Garasse).

Competing Interests:

No conflict of interests exists.

Keywords: mitochondria, Drosophila, NDUFS2, cardiomyopathy.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00005

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: José María Becedas, Alba Rocío Tornero, Roberto Serna, Sara Laine-Menéndez, Sonia Rodríguez, Rafael Garesse, Margarita
Cervera, Juan José Arredondo. NDUFS2 Mitochondrial Complex I deficiency induces adipose

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Cardiac diseases are the foremost cause of death due to health problems in the western society. Amongst the causes of
cardiac disease, those caused by metabolic defects, especially mitochondrial inborn errors, have difficult treatment and
prognosis. This organelle provides energy trough oxidative phosphorylation, abbreviated as OXPHOS, and participates in
apoptotic pathways. Permanent OXPHOS metabolism decay due to deficiencies in electron transport chain genes activates
cell-signalling routes, leading to tissue wasting and disease. To study these diseases, we decide to develop a Drosophila
melanogaster model in which nuclear encoded respiratory chain gene NDUFS2 has been specifically interfered in indirect
flight skeletal-like muscle using UAS-GAL4 system. NDUFS2 is a structural subunit of the complex I core reported to cause
hypertrophic cardiomyopathy among other symptoms. NDUFS2 IFM specific interference causes muscle and fat bodies
degeneration, although the last ones are not NDUFS2 interfered and present healthy mitochondria. Gene expression
assays indicate that the phenotype of wasted skeletal muscle leads to the increased expression of Impl2 gene in this
tissue. Impl2 is an inhibitor of insulin/ insulin-like growth factor signalling. We suggest Impl2 overexpression induces lipid
and glucose mobilization, and therefore, the observed fat bodies reduction leading to a phenotype reminiscent to insulin
resistance and lipodystrophy in mammals. These results highlight the importance of knowing the routes triggered by
mitochondrial malfunction and, thereafter, the tissue metabolic interdependence involved.

e00006
Induction of antiviral CD8+ T lymphocyte-mediated protective
memory responses.

Andrea C. Méndez1, Tihana Tršan2, Astrid Krmpotić2, Stipan Jonjić2, Margarita del Val1,*.

*Corresponding author:
Margarita del Val. Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain. E-mail: mdval@cbm.csic.es

Details of affiliation

1Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain.
2Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Croatia

Funding

Supported by grants SAF2010-18917 and SAF2013-48754-C2-1-R to MDV and cofinanced by the European Regional
Development Fund. ACM is supported by “Ayudas para contratos predoctorales para la formación de doctores 2014” from Ministerio
de Economía y Competitividad.

Competing Interests:

The authors declare no competing financial interests.

Keywords: memory CD8+ T lymphocytes, murine cytomegalovirus, vaccines.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00006

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Andrea C. Méndez, Tihana Tršan, Astrid Krmpotić, Stipan Jonjić, Margarita del Val. Induction of antiviral CD8+ T lymphocytemediated
protective memory responses. IBJ Plus 2018 (S2):e00006 doi: 10.24217/2531-0151.18v1s2.00006.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

CD8+ T lymphocyte-based vaccines are necessary to provide effective protection against pathogens that cannot be
controlled by humoral immunity. For this purpose, cytomegaloviruses (CMVs) are one of the most attractive vaccine
vector candidates due to their induction of a strong and long-lasting CD8+ T lymphocyte response. Previously in our
team we have characterized the role of Nras in the generation of protective memory CD8+ T lymphocytes. We found that
Nras-deficient mice have impaired memory CD8+ T lymphocyte responses caused by a marked deficiency of the antigenmediated
early induction of T-box transcription factor Eomesodermin (Eomes) (Iborra et al, J Exp Med 2013).
We took advantage of a murine CMV (MCMV) vector developed by Dr S. Jonjić that conferred strong and long-lasting CD8+
T-lymphocyte-based protection against bacterial challenge in mice (Tršan et al., PNAS 2013), and analyzed the capacity
of MCMV vectors to improve CD8+ T lymphocyte memory responses in Nras-deficient mice. By doing this, we aimed to
assess the potential of MCMV to overcome situations of deficient immune memory, providing helpful information for
further vaccine design by describing the detailed mechanisms underlying the superior protective capacity of MCMV
vaccine vectors.

e00007
A novel role for the Hippo pathway mediator TAZ in thyroid differentiation.

Celia Fernández-Méndez1, Pilar Santisteban1, 2.

Pilar Santisteban, Instituto de Investigaciones Biomédicas “Alberto Sols”; Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma
de Madrid (UAM), Madrid, Spain. Centro de Investigación Biomédica en Red de Cáncer (CIBERONC) Instituto de Salud Carlos III (ISCIII), Madrid, Spain.
E-mail: psantisteban@iib.uam.es

Details of affiliation

1 Instituto de Investigaciones Biomédicas “Alberto Sols”; Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid
(UAM), Madrid, Spain.
2 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC) Instituto de Salud Carlos III (ISCIII), Madrid, Spain.

Funding

SAF2016-75531-R

Competing Interests:

There are no conflicts of interest.

Keywords: Hippo pathway, TAZ, thyroid, NIS.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00007

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Celia Fernández-Méndez, Pilar Santisteban. A novel role for the Hippo pathway mediator TAZ in thyroid differentiation. IBJ Plus
2018 (S2):e00007 doi: 10.24217/2531-0151.18v1s2.00007.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction. The Hippo signalling pathway plays a key role in the control of cell proliferation and its dysregulation has
been associated with tumourigenesis. However, little is known about the regulatory mechanisms of the Hippo pathway
in the thyroid gland. TAZ, a transcriptional coactivator in Hippo signalling, has been described to interact with Pax8, a
main driver of thyroid differentiation, increasing thyroglobulin transcription. Pax8 is the main regulator of sodium iodide
symporter (NIS), an important protein not only for the correct function of the thyroid gland, but also for radioiodide
treatment of thyroid cancer.
The aim of this work was to study the role of the Hippo pathway and its mediator TAZ in NIS regulation, and hence in
thyroid differentiation.

Material and Methods. We assessed the levels and the location of Hippo pathway components in thyroid follicular cells
by RT-qPCR, Western Blotting and Immunofluorescence. The involvement of TAZ in NIS regulation was evaluated using
chromatin immunoprecipitation (ChIP) and luciferase reporter assays. Cell-surface biotinylation and radioiodide uptake
assays were used to analyse NIS membrane location and function.
Results. TSH the main inductor of NIS expression activates the Hippo pathway, thereby increasing pYAP and promoting
TAZ translocation to the cytoplasm where it is marked for degradation by the proteasome machinery. On the other hand,
a known negative regulator of NIS function such as TGFβ increases TAZ nuclear levels. On the contrary to what has been
described for the thyroglobulin gene, TAZ is a negative regulator of NIS expression. This effect is due to a decreased Pax8
binding to the NIS enhancer. Accordingly, TAZ silencing partially impairs TGFβ-induced NIS repression and allows NIS
membrane location, improving iodine uptake.

Conclusion. Altogether these data establish a novel role of the Hippo pathway, and particularly the cofactor TAZ,
in the regulation of NIS expression in thyroid cells by the crosstalk with the main pathways playing a role in thyroid
differentiation.

e00008
Neuroprotective strategies against circadian alterations in a glioma model.

Patricia Jarabo, Francisco A. Martín, Sergio Casas-Tintó*

*Corresponding author scasas@cajal.csic.es

Details of affiliation

Instituto Cajal; Molecular, Cellular and Developmental Neurobiology Department, Madrid

Funding

This work was support by Plan Nacional grant BFU2015-65685-P (S.C-T.) from the Spanish Ministry of Economy and Consejo
Superior de Investigaciones Científicas.

Competing Interests:

No potential conflict of interest was reported by the authors.

Keywords: Glioma, neurodegeneration, circadian rhythms.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00008

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Patricia Jarabo, Francisco A. Martín, Sergio Casas-Tintó. Neuroprotective strategies against circadian alterations in a glioma
model. IBJ Plus 2018 (S2):e00008 doi: 10.24217/2531-0151.18v1s2.00008.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Glioblastoma multiforme (GBM) is the most common and lethal tumor from the central nervous system,
and it is resistant to all current treatments. GMB causes neurological symptoms in patients such as sleep disturbances
and memory loss. We propose that neurodegeneration plays a central role in GBM progression and death. Our results
indicate that GBM induces synapse loss in the neuromuscular junction in a Drosophila melanogaster model of glioma,
one of the first events leading to neurodegeneration. Our goal is to study the signals from the glioma that cause
degeneration in neighbouring neurons.

Materials and methods: Using RT-qPCR, the results show that glioma brains upregulate ImpL2, an antagonist of the
insulin pathway. The downregulation of ImpL2 using RNAi meanwhile a glioma is developed is able to rescue the synapse
number in neuromuscular junctions and the glial cell number.
Currently, we focus our study on the effect of GMB in circadian rhythms analysing the circadian locomotor activity in
adult flies. Preliminary results show that, as GBM advances, circadian rhythmicity is progressively disturbed. Moreover,
we have shown that, in the absence of GBM, a genetically driven decrease of the number of synapses in clock neurons
is sufficient to cause circadian disruptions. Using neuroprotective strategies like the overexpression of Rheb, a target of
insulin pathway, we can improve the circadian performance, and increase the lifespan.

Results and conclusions: The upregulation of ImpL2 in glioma brains leads the neurodegenerative process disrupting the
insulin pathway in neurons. One of the functional effects is the circadian activity distribution. However, the protection
of this pathway allows us to increase the quality of life and lifespan. This may open an opportunity to develop a novel
therapy based on the treatment of the neurological symptoms in glioma patients.

e00009
Mechanisms of resistance of basal cell carcinoma to Photodynamic Therapy.

Silvia Rocío Lucena1*, Ángeles Juarranz1.

*Corresponding author:
Silvia Rocío Lucena, Faculty of Sciences, Autonomous University of Madrid, Spain. E-mail: silvialucenablas@gmail.com

Details of affiliation

1Biology Department, Faculty of Sciences, Autonomous University of Madrid, Spain.

Funding

FPI-UAM and Ministerio de Economía y Competitividad (PI12/01253 and PI15/00974).

Competing Interests:

Competing interest explanation.

Keywords: skin cancer, photodynamic therapy, resistance

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00009

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Silvia Rocío Lucena, Ángeles Juarranz. Mechanisms of resistance of basal cell carcinoma to Photodynamic Therapy. IBJ Plus
2018 (S2):e00009 doi: 10.24217/2531-0151.18v1s2.00009.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Basal cell carcinoma is the most common cancer worldwide. One of the non-invasive treatments for BCC is Photodynamic
Therapy that, like many other anticancer therapies, in occasions, may cause recurrences. So, in this work, there were
evaluated possible PDT- resistance mechanisms in three murine basal cell carcinoma cells lines (ASZ, BSZ and CSZ),
comparing original populations with cells resistant to 10 cycles of PDT (10thG). Also, after the generation of 10thG
populations, they were inoculated in mice; and then, developed tumors were cultured by explant to isolate two new
populations (P T and 10thG T). In general, resistant populations acquired fusiform morphologies, diminished their
proliferation capacity and their size and presented higher tumorigenic capacity than original cells. Besides, their
resistance depend on location or production of protoporphyrin IX, the photosensitizer employed for PDT. Finally, it
was observed that p53 determined the characteristics acquired by resistant populations: resistant cell line with p53
expression presented less proliferation rate than those without p53, and inhibition of Wnt/β-catenin pathway by
increasing Gsk3β and increased expression of N-cadherin compared to original cells and. On the other hand, resistant
cells without p53 had their Wnt/β-catenin signaling pathway activated by diminishing Gsk3β and presented less adhesion
capacity by decreasing E-cadherin and β-catenin expression on the membrane. All these results put light on the different
mechanisms of basal cell carcinoma resistance to PDT, what may help to improve its use, for example, by combining it
with other co-adjuvant treatments.

e00010
S100A9 mediates resistance of brain metastasis to radiation therapy.

Cátia Monteiro1, Coral Fustero2, Aurelio Hernández-Laín3, Riccardo Soffietti4 and Manuel Valiente1*.

*Corresponding author: E-mail: mvaliente@cnio.es

Details of affiliation

1Brain Metastasis Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.
2Bioinformatics Unit, CNIO, Spanish National Cancer Research Center (CNIO), Madrid, Spain.
3Neuropathology Unit, Hospital Universitario 12 de Octubre Research Institute, Madrid, Spain.
4Neuro-oncology Department, University and City of Health and Science University Hospital of Turin, Turin, Italy.

Funding

This work is supported by FCT- Fundação para a Ciência e Tecnologia, Portugal.

Competing Interests:

There are no conflicts of interest related to the work presented

Keywords: Brain Metastasis/ S100A9/ Radiation therapy

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00010

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Cátia Monteiro, Coral Fustero, Aurelio Hernández-Laín, Riccardo Soffietti, Manuel Valiente. S100A9 mediates resistance of
brain metastasis to radiation therapy. IBJ Plus 2018 (S2):e00010 doi: 10.24217/2531-0151.18v1s2.00010.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Background: Brain metastasis affects 10-30% of cancer patients. Standard of care is still based on palliative treatments
including surgery and radiation. In order to understand why current therapies fail and whether there are ways to improve
them we have developed an experimental model that incorporates whole brain radiation therapy (WBRT). With this
new model we aim to identify critical mediators of radiation resistance to explore novel ways to radiosensitize brain
metastasis.

Material and methods: We analyzed the effects of radiation in different brain metastasis models using in vitro, ex
vivo and in vivo experimental approaches. In vivo WBRT was applied to experimental mouse models harboring
established brain metastases using several hypofractionated protocols mimicking those given in the clinic. Additionally,
we performed RNAseq comparing experimental conditions that correlate with different sensitivities to radiation to
identify potential mediators of radiation resistance.

Results and discussion: We found no impairment in the growth rate and thus no impact in overall survival when three
different WBRT protocols were applied to mice harboring brain metastasis. However, in vitro, brain tropic cell lines from
lung and breast cancer with different oncogenomic profiles were highly sensitive to radiation. Interestingly, when we
used culture methods that promote metastasis initiation capabilities or incorporate the brain microenvironment, we
observed significantly decreased sensitivity to radiation. We then compared gene expression between BrM cell lines
under conditions inducing resistance versus sensitivity to radiation, and identified S100A9 as a top candidate. Although
absence of S100A9 does not influence the incidence of brain metastasis, when combined with WBRT a dramatic
decrease in brain colonization is evident by histology. There exists a blood-brain barrier-permeable inhibitor targeting
potential receptors of S100A9-mediated activation of NFκB. We have shown that this inhibitor is effective at impairing
radiation resistance in organotypic brain cultures, and we are currently testing its potential as novel radiosensitizer in
vivo. Analysis of S100A9 levels in human brain metastasis samples reflects inter-patient heterogeneity, suggesting that
this molecule could be used as a biomarker to rationalize the use of WBRT.

Conclusions: Our results support that in spite of not affecting metastatic colonization per se, targeting S100A9 in BrM
models, combined with WBRT, reduces tumor burden ex vivo and in vivo. Further, we have identified an inhibitor blocking
S100A9 mediated activation of NFkb capable of reproducing this result, suggesting a potential therapeutic application
of our findings.

e00011
NRF2 controls proteostasis through the transcriptional regulation of autophagy.

Marta Pajares 1,2,3, Ana I Rojo 1,2,3 and Antonio Cuadrado 1,2,3.

*Corresponding author:
Ana I Rojo: airojo@iib.uam.es, Antonio Cuadrado: antonio.cuadrado@uam.es.
Centro de Investigación en red en enfermedades Neurodegenerativas (Ciberned). Instituto de Investigación Sanitaria La Paz (IdiPaz). Instituto de
investigaciones Biomédicas “Albertos Sols”. Departamento de Bioquímica de la Facultad de Medicina de la Universidad Autónoma de Madrid, (UAM).
Madrid, Spain.

Details of affiliation

1Centro de Investigación en red en enfermedades Neurodegenerativas (Ciberned).
2 Instituto de Investigación Sanitaria La Paz (IdiPaz).
3Instituto de investigaciones Biomédicas “Albertos Sols”. Departamento de Bioquímica de la Facultad de Medicina de la Universidad Autónoma de
Madrid, (UAM).

Funding

FPI UAM, SAF2016-76520-R.

Competing Interests:

The authors disclose no conflicts of interest.

Keywords: NRF2, autophagy, oxidative stress.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00011

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Marta Pajares, Ana I Rojo, Antonio Cuadrado. NRF2 controls proteostasis through the transcriptional regulation of autophagy.
IBJ Plus 2018 (S2):e00011 doi: 10.24217/2531-0151.18v1s2.00011.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Cells control the abundance and quality of the proteome through a wide network that integrates signaling
pathways, gene expression and protein degradation systems. Degradation of cytosolic components inside lysosomes is
carried out by specific types of autophagy in mammals, including macroautophagy and chaperone mediated autophagy
(CMA). Considering autophagy as a proteostatic and defensive mechanism, we sought to determine if this process
could be regulated by the transcription factor NRF2, classically considered the master regulator of the antioxidant cell
response.

Material and methods: We performed a bioinformatics analysis to identify putative NRF2 binding sequences, termed
antioxidant response elements (AREs) among autophagy related genes. Some of them were further validated by ChIP
analysis. Messenger RNA and protein levels of cells under basal levels or submitted to different treatments were
assessed by quantitative PCR and immunoblot. CMA activity in intact cells was determined with a photoswitchable
reporter. The impact of NRF2 deficiency in vivo was determined by immunofluorescence and immunoblot.

Results: The bioinformatics analysis allowed us to identify putative NRF2 binding sequences, termed antioxidant
response elements (AREs), in many genes whose products participate in macroautophagy and CMA. Several were
further validated as NRF2-regulated genes by ChIP assays and quantitative PCR in Nrf2-deficient cells. Interestingly,
Nrf2-knockout cells exhibited impaired macroautophagy flux in response to oxidative stress. Nrf2-knockout cells also
showed reduced LAMP2A lysosomal levels (the limiting step for CMA) and, consequently, impaired CMA activity.
Treatment with pro-oxidants up-regulated CMA through the transcriptional induction of Lamp2a, but to a lesser
extent in Nrf2-deficient cells. Interestingly, pharmacological activation of NRF2 led to increased expression of many
of these autophagy-related genes. This novel role of NRF2 in the regulation of autophagy may have an impact on
proteinopathies, such as Alzheimer’s Disease (AD). Indeed, NRF2 deficiency results in reduced neuronal expression of
some of these markers as well as increased intracellular APP/Aβ and insoluble TAU in a mouse model of AD.

Conclusion: Our results point to a novel role of NRF2 in the regulation of autophagy and suggest a new strategy to
combat proteinopathies.

e00012
The role of p27kip1 in the development, differentiation and maturation of mesencephalic dopaminergic neurons.

Charlotte Palmer1, Adela Bernabeu-Zornoza1, Raquel Coronel1, Maria Lachgar1, Laura Silva1, Nerea Jiménez-Téllez1, Cris Gil1, Manuel Serrano2, Isabel
Liste1*.

*Corresponding author: Isabel Liste, PhD
Unidad Funcional de Investigación de Enfermedades Crónicas (UFIEC), Instituto de Salud Carlos III (ISCIII), Majadahonda, Madrid, Spain. E-mail:
iliste@isciii.es

Details of affiliation

1Unidad Funcional de Investigación de Enfermedades Crónicas (UFIEC), Instituto de Salud Carlos III (ISCIII), Majadahonda, Madrid, Spain.
2Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain.

Funding

This study was supported by the MICINN-ISCIII (grants MPY1412/09 and PI10/00291), Comunidad de Madrid (NEUROSTEM
consortium; S2010/BMD-2336) and MINECO-Retos SAF2015-71140-R.

Competing Interests:

The authors declare no competing interest.

Keywords: Parkinson’s, DAn, DAn development, iPSCs, p27Kip1.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00012

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Charlotte Palmer, Adela Bernabeu-Zornoza, Raquel Coronel, Maria Lachgar, Laura Silva, Nerea Jiménez-Téllez, Cris Gil, Manuel
Serrano, Isabel Liste. The role of p27kip1 in the development, differentiation and maturation of mesencephalic dopaminergic neurons.
IBJ Plus 2018 (S2):e00012 doi: 10.24217/2531-0151.18v1s2.00012.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Parkinson’s Disease (PD) is one of the most common neurodegenerative disorders, generally characterized by the loss
of specific dopaminergic neurons (DAn) in the midbrain. Current treatment options are available to help relieve primary
motor symptoms, but their long-term effectiveness is limited. For this reason, alternative treatment options are being
sought in the form of stem cell replacement therapies. Pluripotent stem cells, known for their ability to self-renew
and differentiate into any cell lineage of the three germ layers, are a popular source of cells for the differentiation
of DAn. In our lab, we are investigating the role of the protein p27Kip1 (p27) in the differentiation process of these types
of neurons. p27 is a cyclin/cyclin dependent kinase inhibitor (CKI) belonging to the Cip/Kip family of proteins, best
known for its function in the cell cycle. In this work, we investigate and analyze the effects of p27 on the development,
differentiation and maturation of dopaminergic neurons in vivo and in vitro, using standard techniques of immunocytoand
histochemistry, western blot and quantitative-PCR. Our preliminary results show that mouse induced pluripotent
stem cells that lack the protein p27 has led to a significant decrease in the production of TH+ (tyrosine hydroxylase,
the limiting enzyme in dopamine production) cells, while the opposite effect was seen in mouse embryonic stem cells
nucleofected to overexpress p27. In vivo, we have seen that at embryonic age E13.5, the production of TH in p27 knockout
mice was greatly reduced compared to wild type controls. We are currently doing a deeper analysis of other markers
important for proper dopaminergic neuron development to decipher the mechanistic effects of p27. This would allow
us to better apply the use of this protein to improve current differentiation protocols of dopaminergic neurons for stem
cell replacement therapies in Parkinson’s Disease.

e00013
Role of the transcription factor NRF2 in hippocampal neurogenesis
and in a mouse model of Alzheimer´s Disease.

Natalia Robledinos-Antón1, Ana Isabel Rojo1, Elisabete Ferreiro2, Ángel Núñez3, Karl-Heinz Krause4, Vincent Jaquet4, and Antonio Cuadrado1*

*Corresponding author:
Antonio Cuadrado, Madrid, Spain. E-mail: antonio.cuadrado@uam.es

Details of affiliation

1Instituto de Investigaciones Biomédicas “Alberto Sols”, Faculty of Medicine, Autonomous University of Madrid (UAM), Centro de Investigación
Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain.
2Center for Neuroscience and Cell Biology, Institute for Interdisciplinary Research (IIIUC), University of Coimbra, Portugal.
3Department of Anatomy Histology and Neuroscience, Autonomous University of Madrid, Madrid, Spain.
4Department of Pathology and Immunology, University of Geneva Medical School, 1 rue Michel Servet, 1211 Geneva, Switzerland.

Funding

This work was funded by Grant SAF2016-76520-R of the Spanish Ministry of Economy and Competitiveness. NRA is
recipient of a FPU contract of Spanish Ministry of Education Culture and Sports. EF is a recipient a postdoctoral fellowship: SFRH/
BPD/86551/2012 (Financiado por Fundos FEDER através do Programa Operacional Factores de Competitividade – COMPETE 2020
e por Fundos Nacionais através da FCT –Fundação para a Ciência e a Tecnologia no âmbito do projecto Estratégico com referência
atribuida pelo COMPETE: POCI-01- 0145-FEDER-007440). EF enjoyed a short term stay visit at AC’s laboratory founded by COST action
BM1402 MouseAge.

Competing Interests:

The authors declare that they have no conflict of interest relating to the publication of this manuscript.

Keywords: neurogenesis, NRF2, NSPCs.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00013

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Natalia Robledinos-Antón, Ana Isabel Rojo, Elisabete Ferreiro, Ángel Núñez, Karl-Heinz Krause, Vincent Jaquet, Antonio
Cuadrado. Role of the transcription factor NRF2 in hippocampal neurogenesis and in a mouse model of Alzheimer´s Disease. IBJ Plus
2018 (S2):e00013 doi: 10.24217/2531-0151.18v1s2.00013.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: During adulthood, new hippocampal granule neurons are generated in the hippocampus by differentiation
of neural stem/progenitor cells (NSPCs) in the subgranular zone (SGZ). The implication of hippocampal neurogenesis in
learning and memory functions point it as a therapeutic strategy to face the cognitive deficits related with aging and
neurodegenerative diseases. Hippocampal neurogenesis can be modulated by oxidative stress, neuroinflammation and
proteinopathy. Here, we hypothesized that Nuclear Factor-Erythroid 2-Related Factor 2 (NRF2), as a master regulator of
cellular homeostasis, might modulate the fate of NSPCs at the hippocampus.

Material and methods: immunohistochemistry analysis of hippocampal coronal sections of WT, NRF2-/-, APP/TAU/WT
and APP/TAU/NRF2-/- mice at indicated age points. Long term potentiation (LTP) and Morris water maze test assays in 6
month- old mice of indicated genotypes. Immunocytochemistry of primary cultures of NSPCs of mice in postnatal day 0
to 4 (P0-P4) and 3 months of age in proliferation and differentiation conditions and lentiviral silence and overexpression
of NRF2.

Results: NRF2-/- mice showed an impairment in LTP, correlating with an exacerbated reduction in hippocampal
NSPCs from birth to adulthood. In vitro analysis using neurosphere assay corroborated this data, showing a reduced
proliferative capacity of SGZ-derived NSPCs from newborn and 3-month-old NRF2-/- mice. Differentiation analysis
pointed that NRF2-deficiency alters proper differentiation profile, favouring an abnormal rate between glial and
neuronal differentiation. Ectopic expression of NRF2 in Nrf2-deficiency NSPCs attenuated the impact in their clonogenic,
proliferative and differentiating capacity. Furthermore, when we performed the knockdown of the NRF2 gene in wild
type NSPCs, data showed the alterations described previously for NRF2-/- NSPCs. Subsequently, to further analyse NRF2
implication in pathology, we used mice that express human mutated forms of TAU(P301L) and the amyloid protein
precursor APP(V717I), in the presence or absence of NRF2. We report cognitive deficits in APP/TAU/NRF2-deficient
mice considering the registered decrease in hippocampal LTP and poor performance in the Morris water maze test.
Immunohistochemistry analysis of SGZ evidenced the detriment of NSPCs pool and neuronal differentiation in APP/TAU/
NRF2-deficient mice at different age points.

Conclusions: The data support that NRF2 is important in the maintenance of proper proliferation and differentiation
rates of hippocampal NSPCs. Our findings highlight the importance of NRF2 pharmacological upregulation to preserve
the neurogenic functionality of the hippocampus and improve cognitive functions in AD.

e00014
In vitro enhancement of collagen matrix deposition and crosslinking
by coupling lysyl oxidase (LOX) with bone morphogenetic
protein-1 (BMP-1) and its application in tissue engineering.

Rosell-García T1, Rodriguez-Pascual F1*.

Corresponding author: Dr. Fernando Rodríguez-Pascual
Centro de Biología Molecular “Severo Ochoa”
Consejo Superior de Investigaciones Científicas (C.S.I.C)/Universidad Autónoma de Madrid (U.A.M.)
Nicolás Cabrera, 1 E-28049,
Madrid, Spain
Phone: 34 91 196 4505
Fax: 34 91 196 4420
Email: frodriguez@cbm.csic.es

Details of affiliation

1 Centro de Biología Molecular “Severo Ochoa” Consejo Superior de Investigaciones Científicas (C.S.I.C)/Universidad Autónoma de Madrid (Madrid),
Madrid, Spain.

Funding

This work was supported by grants from Ministerio de Economía y Competitividad (Plan Nacional de I + D + I : SAF 2012 –
34916 , and SAF 2015 – 65679 – Rto F. R – P).

Competing Interests:

The authors declare no competing interest.

Keywords: Extracellular matrix, collagen deposition, lysyl oxidase, bone morphogenetic protein-1, tissue engineering.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00014

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Rosell-García T, Rodriguez-Pascual F. In vitro enhancement of collagen matrix deposition and cross-linking by coupling lysyl
oxidase (LOX) with bone morphogenetic protein-1 (BMP-1) and its application in tissue engineering. IBJ Plus 2018 (S2):e00014 doi:
10.24217/2531-0151.18v1s2.00014.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Tissue engineering is emerging as a powerful therapeutic strategy to treat injured or degenerated tissues by implanting
natural, synthetic, or semisynthetic tissue and organ mimics. The use of native ECM substrates is favored, as, theoretically,
they should provide the structural and functional cues required for better preservation and growing conditions. However,
standard cell culture conditions are far from ideal given the fact that the diluted microenvironment does not favor
the production of ECM components. This is particularly true for collagen, the most important structural biomolecule,
as its synthesis and deposition onto matrix is enzymatically rate-limited. An incomplete conversion of procollagen by
C-proteinase/bone morphogenetic protein-1 (BMP-1) has been proposed to severely limit in vitro collagen deposition.
BMP-1 also catalyzes the proteolytic activation of the precursor of the collagen cross-linking enzyme, lysyl oxidase (LOX)
to yield the active form, suggesting a deficit in cross-linking activity under standard conditions. We hypothesized that the
addition of LOX and BMP-1 may represent a strategy to boost in vitro deposition of collagen.

For that purpose we have generated HEK293-based cell systems that produced supernatants enriched with LOX and
BMP-1 enzymes which, when combined together, recapitulated in vitro the proteolytic activation of LOX. Then we
have implemented fibroblast cultures with these supernatants enriched in LOX and BMP-1 that strongly increased the
deposition of collagen onto the insoluble matrix at the expense of the soluble fraction in the extracellular medium.
Using decellularization protocols, we also provide evidence that fibroblast-derived matrices are able to regulate the
adipogenic and osteogenic differentiation of human mesenchymal stem cells (MSC), a powerful cell tool in regenerative
medicine, and these actions were modulated by LOX/BMP-1-modified matrices. These results demonstrate that we have
developed a convenient protocol to enhance the capacity of in vitro cell cultures to deposit collagen in the ECM, and that
this technology represents a promising approach for application in tissue engineering.

e00015
Vesicular trafficking and polarity of ICAM-1 in inflamed liver tissue.

Cristina Cacho-Navas1, Natalia Reglero-Real2, Susana Barroso1, Jaime Millán1*.

*Corresponding author: Jaime Millán, Centro de Biología Molecular Severo Ochoa-CSIC, Calle Nicolás Cabrera 1, Madrid, Spain.
E-mail: jmillan@cbm.csic.es

Details of affiliation

1Centro de Biología Molecular Severo Ochoa-CSIC, Calle Nicolás Cabrera 1, 28049 Madrid, Spain.
2William Harvey Research Institute, Queen Mary University of London, United Kingdom.

Funding

This work was supported by grants SAF2014-57950-R and SAF2017-88187-R from the Ministerio de Economía, Industria y
Competitividad; grant B2017/BMD-3817 from Comunidad de Madrid; Proyectos Endocórnea (convenio colaboración) from Fundación
Jiménez Díaz.

Competing Interests:

None of the authors declared a conflict of interest.

Keywords: Hepatocyte, apicobasal polarity, ICAM-1.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00015

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Cristina Cacho-Navas, Natalia Reglero-Real, Susana Barroso, Jaime Millán. Vesicular trafficking and polarity of ICAM-1 in
inflamed liver tissue. IBJ Plus 2018 (S2):e00015 doi: 10.24217/2531-0151.18v1s2.00015.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: During the inflammatory response, cellular barriers are transiently disrupted to let immune cells reach
the inflammatory focus. The liver regulates immune tolerance by regulating leukocyte trafficking and exposure to
immune receptors. Hepatocytes are polarized epithelial cells with an apical domain facing the bile canaliculus (BC)
and a basolateral domain in contact with the liver parenchyma. Polarized hepatocytes confine ICAM-1, the ligand of
leukocyte β2 integrins, in the BC so it cannot access infiltrated immune cells. Upon loss of polarity, ICAM-1 is exposed
and mediates leukocyte adhesion to dysfunctional cells. We study mechanisms of intracellular trafficking that determine
ICAM-1 polarization in hepatic cells and how inflammation modulates it. We are also generating an in vitro co-culture
system based on adult liver 3D organoids in which we will mimic liver parenchyma to study the crosstalk between
lymphocytes, endothelial and parenchymal cell barriers.

Material and methods: Cells and Hepatic Organoids. HepG2 human liver hepatocellular carcinoma cell lines were
culture in DMEM supplemented with 10% fetal bovine serum. Human liver organoids from EpCAM+ ductal cells have
been generated in collaboration with Dr. M. Huch, at the Gurdon Institute (Broutier et al., 2016). Intracellular trafficking
assays and confocal microscopy. Transcytosis assays, cell line and liver organoid immunostaining were performed as
described (Reglero-Real et al., 2014, Broutier et al., 2016). Mass spectrometry and validation of the interactome of
ICAM-1-BirA*. HepG2 cells stably expressing ICAM-1-BirA* were grown for 24 h in 50 μm biotin supplemented medium
and pull down assays were performed. Extracts were analyzed by quantitative proteomics.

Results and Conclusions: Hepatic basolateral ICAM-1 relocates to the tight junctions domains where is accumulated and
probably mediates the recruitment of vesicular trafficking that translocate it to the microvilli at the BC. The basolateral
translocation of ICAM-1 is partially prevented by endocytosis inhibitors. Taking advance of BioID assays, we performed
mass spectrometry analysis of ICAM-1-BirA* cells and proteomics revealed trafficking machinery in proximity of ICAM-1.
We validated by immunoprecipitation and colocalization assays the direct interaction of ICAM-1 with some members of
the SNARE and MAL family, both implicated in the regulation of apicobasal polarity in epithelial cells. Giving the limitations
of culturing primary hepatocytes, we have introduce the new technology for growing adult human liver organoids and
our aim is to co-culture them with endothelial cells to analyze the mechanisms mediating ICAM-1 trafficking and its
relevant function in hepatic cells.

e00016
Lamin A/C enhances APC capacity to stimulate CD4 T cell responses

Beatriz Herrero-Fernandez1+, Virginia Zorita2+, Raquel Fernandez-Toribio2, Jose M Gonzalez-Granado1*.

+Authors with equal contribution

*Corresponding author:
Jose M Gonzalez-Granado, Instituto de Investigación Hospital 12 Octubre, Madrid, Spain. E-mail: jmgonzalez.imas12@h12o.es

Details of affiliation

1 LamImSys Lab. Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12), Av. Cordoba, s/n, 28041 Madrid, Spain.
2 LamImSys Lab. Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Calle de Melchor Fernandez Almagro, 3, 28029 Madrid, Spain.

Funding

ISCIII (CPII16/00022, CP11/00145, PI14/00526 and PI17/01395), Fundación Ramón Areces and imas12.

Competing Interests:

No potential conflicts of interest were disclosed.

Keywords: Lamin A/C, CD4 T cell, APC, adaptive immunity.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00016

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Beatriz Herrero-Fernandez, Virginia Zorita, Raquel Fernandez-Toribio, Jose M Gonzalez-Granado. Lamin A/C enhances APC capacity to stimulate CD4 T cell responses.. IBJ Plus 2018 (S2):e00016 doi: 10.24217/2531-0151.18v1s2.00016.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction:
Antigen presentation by dendritic cells (DCs) stimulates naïve CD4+ T cells, triggering T cell activation and adaptive
immune responses. Several pathological processes are linked to abnormal Antigen Presenting Cells (APC) antigen
presentation that alters the subsequence T cell response. Nuclear envelope lamin A/C controls chromatin organization,
gene transcription, DNA replication and damage responses, cell differentiation and polarization during migration.
Moreover, lamin A/C, when expressed in T cells, enhances CD4+ T cell responses. However lamin A/C role in APC function
has not been determined. Our objective is to analyze the role of lamin A/C in APC capacity to trigger adaptive immune
responses.

Material and methods:
We compared by flow cytometry and live imaging microscopy, the capacity of WT and lamin A/C deficient (Lmna-/-) DCs
to promote activation, proliferation and differentiation of naïve CD4+ T cells in both in vitro and in vivo experiments. DCs
were obtained from LysMcre+/+ x LmnaFl/Fl and their control mice.

Results:
Comparing Lmna-/- GM-CSF bone marrow derived-DCs with WT DCs in in vitro experiments, we observed a reduced
capacity of Lmna-/- DCs to form conjugates with naïve CD4+ T cells and to promote CD4+ T cells activation and proliferation.
We also observed a significant decreased production of IFNγ+ CD4+ T cells after T Cell Receptor (TCR) stimulation
accompanied by less induction of the T helper 1 (Th1)-specific transcription factor T-bet. Lamin A/C deficiency in DCs
also facilitates a greater differentiation towards a T regulatory (Treg) phenotype measured by the expression of Foxp3
and CD25. This effect was also corroborated in vivo since Lmna-/- mice in myeloid LysM-expressing cells exhibited less
differentiation towards a Th1 phenotype and increased Treg phenotype upon vaccinia virus infection.

Conclusion:
Lamin A/C in APCs is a critical regulator of the activation, proliferation and differentiation of naïve CD4+ T cells.

e00017
Effectiveness and molecular basis of CDK4/6 inhibition in
combination with taxanes in pancreatic cancer.

Beatriz Salvador1, Mónica Álvarez1, Camino Menéndez1, Pedro P. López-Casas1, David Shields2,3, Manuel Hidalgo4,*, Marcos Malumbres1,*

*Corresponding author:
Manuel Hidalgo, Beth Israel Deaconess Medical Center, Boston, MA, USA. E-mail: mhidalgo@bidmc.harvard.edu
Marcos Malumbres, CNIO, Madrid, Spain. E-mail: mmm@cnio.es

Details of affiliation

1CNIO, Madrid, Spain.
2Pfizer, Inc., Pearl River, NY, USA.
3Pfizer, Inc., Boston, MA, USA.
4Beth Israel Deaconess Medical Center, Boston, MA, USA.

Funding

Pfizer has found all the costs of the project.

Competing Interests:

This project is founded by Pfizer.

Keywords: CDK4/6 inhibitor, taxol, Pancreatic Ductal Adenocarcinoma

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00017

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Beatriz Salvador, Mónica Álvarez, Camino Menéndez, Pedro P. López-Casas, David Shields, Manuel Hidalgo, Marcos Malumbres.
Effectiveness and molecular basis of CDK4/6 inhibition in combination with taxanes in pancreatic cancer. IBJ Plus 2018 (S2):e00017
doi: 10.24217/2531-0151.18v1s2.00017.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Pancreatic Ductal Adenocarcinoma (PDAC) is among the deadliest human cancers with a 5-year survival rate
of less than 5% using the standard of care gemcitabine/nab-paclitaxel. A very frequently disrupted gene in PDACs is CDKN2A
(>90%), which encodes the cyclin-dependent kinase (CDK)4/6 inhibitor p16. Recently, CDK4/6 inhibitors have been approved
for breast cancer treatment, and preclinical assays for PDAC are giving promising results.

Material and methods: PDAC Patient-Derived Xenografts (PDX) models and PDX-derived cell lines were used for in vivo and in
vitro studies, respectively. Cellular studies were performed using proliferation and cell cycle assays in combination with flow
cytometry, immunoblotting, fluorescence microscopy and live cell imaging techniques. Drug treatments were performed with
the CDK4/6 inhibitor PD-0332991 (Palbociclib®), and with Paclitaxel (Taxol®) or Nab-Paclitaxel (Abraxane®) for in vitro and in
vivo studies, respectively.

Results: Treatment of different PDX-derived cell lines with the combination of taxanes and CDK4/6 inhibitors resulted in
a higher anti-proliferative effect than both drugs used as single agent. Cell cycle studies showed that inhibition of CDK4/6
prevented recovery from treatment with taxol. At the molecular level we found that the combined treatment induced a
clear interruption in retinoblastoma pathway, even higher than CDK4/6 inhibition in monotherapy. Gene expression profiles
comparing single versus combined treatment are currently being performed to further understand the molecular basis
underlying the effectiveness of this type of treatment. Moreover, to assess the efficacy of this new combined treatment in
vivo, we treated nine PDAC PDX models with PD-0332991 and nab-paclitaxel, following the same schedule. Importantly, eight
of them presented an increased tumor growth inhibition in the combination with respect to the monotherapies.

Conclusions: Although the molecular mechanism underlying the effectiveness of this treatment is not completely understood
yet, our data suggest a good therapeutic value for the combination of CDK4/6 inhibitors and taxanes in PDAC treatment.

e00018
Relevance of coronavirus viroporins and pdz-binding- motifs in virus replication and virulence.

Carlos Castaño-Rodriguez1, Jose M. Honrubia1, Javier Gutierrez-Alvarez1, Raul Fernandez-Delgado1, Carmina Verdia-Baguena2, Maria Queralt-
Martin2, Vicente Aguilella2, Eric Bailly3, Pascale Zimmermann3, Jean-Paul Borg3, Stanley Perlman4, Isabel Sola1, Luis Enjuanes1

Corresponding author: Luis Enjuanes, L.Enjuanes@cnb.csic.es

Details of affiliation

1Department of Molecular and Cell Biology, National Center of Biotechnology (CNB-CSIC), Madrid, Spain.
2Department of Physics, Laboratory of Molecular Biophysics. Universitat Jaume I, Castellón, Spain.
3Centre de Recherche en Cancérologie de Marseille (CRCM), Inserm, CNRS, Aix-Marseille University, Institut Paoli-Calmettes, France.
4Department of Microbiology, University of Iowa, IA 52242, United States.

Funding

PhD Fellowship La Caixa.
SARS-CoV host cell interactions and vaccine development (NIH 2P01AI060699-06A1, W000306844)

Competing Interests:

Keywords: Coronavirus, SARS-CoV, viroporins, PDZ, PBM.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00018

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Carlos Castaño-Rodriguez, Jose M. Honrubia, Javier Gutierrez-Alvarez, Raul Fernandez-Delgado, Carmina Verdia-Baguena,
Maria Queralt-Martin, Vicente Aguilella, Eric Bailly, Pascale Zimmermann, Jean-Paul Borg, Stanley Perlman, Isabel Sola, Luis Enjuanes.
Relevance of coronavirus viroporins and pdz-binding- motifs in virus replication and virulence. IBJ Plus 2018 (S2):e00018 doi:
10.24217/2531-0151.18v1s2.00018.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Coronaviruses (CoVs) are pathogens responsible for a wide range of existing and emerging diseases in
humans and domestic and companion animals. A CoV causing the severe acute respiratory syndrome (SARS-CoV) was
identified in Southeast China in 2002, infecting more than 8,000 people, with mortality in approximately 10% of cases
Viroporins are viral proteins with ion channel (IC) activity that play an important role in several processes including virus
replication and pathogenesis. SARS-CoV encodes three: proteins 3a, E and 8a. Additionally, 3a and E proteins have a PDZbinding
motif (PBM), which may bind over 400 cellular proteins containing a PDZ domain, making them relevant for the
control of cell function. In the present work, a comparative study of the functional motifs included within the SARS-CoV
viroporins was performed focusing on the roles of the IC and PBM of E and 3a proteins.

Material and methods: Mutant viruses deleting each of the viroporins or affecting E and 3a proteins IC activities and
PBMs were engineered from a mouse adapted infectious cDNA clone. Virus replication and virulence was assessed in
vivo in BALB/c mice. 3a protein gene and its mutant variants were introduced in a BaculoVirus vector, produced in insect
cells and purified by affinity chromatography. Then, their IC activity was measured in planar lipid bilayers. The interaction
between CoVs PBMs and each of the cellular PDZ domains was studied using yeast-two hybrid.

Results and Discussion: Our results showed that both the full-length E and 3a proteins were required for optimum SARSCoV
replication and virulence whereas viroporin 8a had a minor impact on these activities. However, IC and PBM activities
of E, but not 3a protein, were necessary for virulence in mice. Interdependence between E and 3a was identified. A virus
missing both proteins was not viable, whereas the presence of either protein with a functional PBM restored virus
viability, indicating functional redundancy between the PBMs of these proteins. Given the relevance of SARS-CoV PBMs,
the presence of these motifs was studied in MERS-CoV, another highly pathogenic human CoV which includes a PBM
in both E and 5 proteins. The interaction between the 256 known human PDZ domains and the PBMs of SARS-CoV and
MERS-CoV was studied and more than 20 cellular proteins mostly involved in virus-cell and cell-cell interaction were
identified. Some of these proteins are involved in immune response or in the infectivity of other viruses. Collectively,
these results demonstrate redundant roles for the IC and PBMs for optimal virus replication and pathogenesis suggesting
that they are potential targets for antiviral therapy.

e00019
The Role of Liver X Receptors in the homeostasis of Splenic Red Pulp Macrophages and Iron Metabolism.

M. C. Orizaola1, A. Sánchez1, J. Vladimir de la Rosa2, A. Hidalgo3, S. Alemany1, A. Castrillo1,2*

*Corresponding author:
Antonio Castrillo, E-mail: acastrillo@iib.uam.es

Details of affiliation

1Dpt. Metabolismo y señalización celular, Universidad Autónoma de Madrid (UAM)- Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM),
Madrid Spain
2Unidad de Biomedicina IIBM-ULPGC (Unidad Asociada al CSIC), Instituto Universitario de Investigaciones Biomédicas y Sanitarias (IUIBS) ULPGC, Las
Palmas, Spain.
3Fundación Centro Nacional de Investigaciones Cardiovasculares (CNIC-CSIC), Madrid, Spain

Funding

This work was supported by grants from MINECO SAF56819-R and SAF2015-71878-REDT, and PhD fellowship BES-2015-
075339.

Competing Interests:

There are no competing interests.

Keywords: LXR, RPM, iron, CD163, Hemoglobin.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00019

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: M. C. Orizaola, A. Sánchez, J. Vladimir de la Rosa, A. Hidalgo, S. Alemany, A. Castrillo. The Role of Liver X Receptors in the
homeostasis of Splenic Red Pulp Macrophages and Iron Metabolism. IBJ Plus 2018 (S2):e00019 doi: 10.24217/2531-0151.18v1s2.00019.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: The liver X receptors (LXRα and LXRβ) are members of the nuclear receptor superfamily of transcription
factors. In macrophages, LXRs play essential roles in the coordination of both metabolic and immune responses, such
as the transcriptional control of lipid metabolism or the modulation of innate and adaptive immune responses. Tissue
resident macrophages are professional phagocytes that orchestrate innate immune responses and have considerable
phenotypic diversity at different anatomical locations. In the spleen, there are different macrophage subpopulations,
including red pulp and marginal zone macrophages, which play specific roles in homeostasis and disease. Red pulp
macrophages (RPMs, identified as CD45+F4/80hiCD11blo by flow cytometry) are specialized cells that are important for
the maintenance of Red Blood Cells (RBC) homeostasis, by actively phagocytosing injured and senescent erythrocytes,
and thus being critical for the recycling of hemoglobin iron. We have previously reported that LXRs are crucial for the
differentiation of splenic marginal zone (MZ) macrophages. Here we now show these nuclear receptors importance in
the correct functioning of the red pulp of the spleen.

Material and methods: We used C57Bl/6 LXRαβ-/-, LXRα-/- and wild type mice, to compare the RPM and monocyte
subpopulations both in the red pulp of the spleen and bone marrow using Flow Cytometry. Cell Sorting technique
allowed us to perform transcriptional profiling, and quantitative PCR to monitor specific gene expression in these cell
populations. Iron and hemoglobin concentration was analyzed through nephelometry, and iron distribution in the spleen
by Prussian Blue histological staining.

Results: LXRαβ null mice present marked defects in splenic RPM subpopulation, despite elevated proportion of
monocytes in the spleen. Presumably as a result of these alterations, iron handling is impaired in LXRαβ-deficient
mice, that accumulate excessive iron in the splenic red pulp. In addition, LXRαβ-deficient mice also present defects
in F4/80hiCD11blo iron-recycling bone-marrow resident macrophages. Strickingly, transcriptional analysis of RPM
population in LXR-null mice showed defective expression of CD163, the hemoglobin scavenger receptor, which results in
increased hemoglobin concentrations in the tissue.
Discussion: These results indicate a new role for LXR nuclear receptors in the regulation of iron homeostasis, possibly in
part through the generation of an appropriate splenic RPM compartment.

e00020
Unravelling transformation of Follicular Lymphoma to Diffuse Large B- Cell Lymphoma.

Julia González-Rincón1,2, Miriam Méndez1,3, Sagrario Gómez1, Juan F García4, Paloma Martín2, Socorro M Rodríguez-Pinilla2,6, David Pérez-Callejo1,3,
Miguel A. Piris2,6, Lucia Pedrosa1, Ivo Kwee8, Francesco Bertoni8, Manuela Mollejo9, Mariano Provencio³, Margarita Sánchez-Beato1*

*Corresponding author: Margarita Sánchez-Beato Gómez, IIS Puerta de Hierro, Madrid (España). E-mail: msbeato@idiphim.org

Details of affiliation

¹Lymphoma Research Group, Medical Oncology Department, Instituto de Investigación Sanitaria Puerta de Hierro-Segovia de Arana, Madrid, Spain
²Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Spain
³Medical Oncology Department, Hospital Universitario Puerta de Hierro, Madrid, Spain
⁴Pathology Department, Hospital MD Anderson Cancer Center, Madrid, Spain
⁵Pathology Department, Hospital Universitario Puerta de Hierro, Madrid, Spain
⁶Pathology Department, Fundación Jiménez Díaz, Madrid, Spain
⁷Pathology Department/Translational Hematology Group, Hospital Universitario Marqués de
Valdecilla/IDIVAL, Santander, Spain
⁸Institute of Oncology Research (IOR), Belinzona, Switzerland
⁹Pathology Department, Hospital Virgen de la Salud, Toledo, Spain

Funding

ISCIIII- AES-FEDER (IFI14/00003, CPII16/00024, PI16/01294, CIBERONC CB16/12/00291, DTS17/00039)

Competing Interests:

The authors declare no potential conflict of interests.

Keywords:

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00020

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Julia González-Rincón, Miriam Méndez, Sagrario Gómez, Juan F García, Paloma Martín, Socorro M Rodríguez-Pinilla, David
Pérez-Callejo, Miguel A. Piris, Lucia Pedrosa, Ivo Kwee, Francesco Bertoni, Manuela Mollejo, Mariano Provencio, Margarita Sánchez-
Beato. Unravelling transformation of Follicular Lymphoma to Diffuse Large B- Cell Lymphoma. IBJ Plus 2018 (S2):e00020 doi:
10.24217/2531-0151.18v1s2.00020.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Follicular lymphoma (FL) is an indolent but typically incurable disease with a long natural history. Disease
outcome is variable: some patients do not need treatment, while others follow a more aggressive course characterized
by interspersed episodes of remission and relapse, associated with lower sensitivity to therapy . The reported frequency
of histological transformation (HT) to more aggressive lymphomas (transformed FL, tFL), most commonly to diffuse large
B-cell lymphoma (DLBCL), varies markedly, from 30% to 60% of patients, is associated with poor prognosis.

Material and methods: We conducted targeted massive parallel sequencing of 22 FL/transformed diffuse large B-cell
lymphoma (tFL) pairs and 20 FL samples from non-transformed patients.

Results: We have identified genes more frequently mutated in tFL than in matched FL samples. The most relevant were
SOCS1 (FL=4.5% vs. tFL=22.7%), GNA13 (9.1% / 22.7%), B2M (9.1% / 22.7%), POU2AF1 (18.2% / 31.8%) and LRP1B
(18.2% / 27.2%). The POU2AF1 gene was found recurrently mutated in tFL sampels (31.8%). All the mutations were in
the 3 bp exon 1 splice donor site. Additionally, two of the POU2AF1-mutated tFL cases had mutations in the POU2F2
gene. We examined whether there was any difference in FL diagnosis samples from patients who transformed compared
with those from non-transformed (ntFL) patients. We sequenced 20 samples from ntFL patients (without evidence of
transformation after a follow-up of at least 5 years) with the same sequencing panel. When comparing FL and ntFL,
although a similar number of genes were recurrently mutated in ntFL as in FL from patients who transformed (5 in
ntFL, 6 in FL), the heterogeneity in the patterns of mutations was greater in FLs of transformed patients: 48 genes were
recurrently mutated in FL biopsies from transformed patients while only 23 genes were recurrently mutated in ntFL
samples. Finally, we looked for genes that were more frequently mutated in FL than in ntFL samples. We found a group
of eight such genes: UBE2A, POU2F2, DSP, TAGAP, PCLO, LRP1B, NOTCH2 and CSMD. We found that a mutation in any of
these genes was strongly associated with transformation (Fisher exact test P=0.0005 ).

Conclusions: We identified recurrently mutated genes that may be involved in transformation, the most relevant of
them (POU2AF1, GNA13 and LRP1B) having roles in B-cell differentiation, GC architecture and migration. We also found
genetic alterations in FL samples that differ in patients who did or did not transform, and so could be used to predict
transformation at the time of FL diagnosis. This information might be useful for following up patients at higher risk of
transformation.

e00021
Adipose tissue and liver crosstalk: new insights into gender differences in hepatocellular carcinoma incidence.

Elisa Manieri1,2#, Leticia Herrera-Melle1#, Alfonso Mora¹, Antonia Tomás-Loba¹, Luis Leiva-Vega¹, Delia Irene Fernández¹, María Elena Rodríguez¹,
Lourdes Hernández-Cosido³, Jorge Luis Torres⁴, Luisa María Seoane³, Miguel Marcos⁴, Guadalupe Sabio1*

*Corresponding author:
Guadalupe Sabio, Spanish National Center for Cardiovascular Research Carlos III (CNIC), 28029 Madrid, Spain. E-mail: guadalupe.sabio@cnic.es
#These authors contributed equally to this work.

Details of affiliation

¹Spanish National Center for Cardiovascular Research Carlos III (CNIC), C/Melchor Fernández Almagro 3, 28029 Madrid, Spain.
²Spanish National Center for Biotechnology (CNB/CSIC), C/Darwin 3, 28049 Madrid, Spain.
³Department of Physiology, CIMUS, University of Santiago de Compostela-Instituto de Investigación Sanitaria, Avda. Barcelona, 15782 Santiago de
Compostela, Spain.
⁴Department of Internal Medicine, University Hospital of Salamanca-IBSAL, Paseo de San Vicente 58-182, 37007 Salamanca, Spain.

Funding

G.S. is an investigator of the Ramón y Cajal Program. E.M. is a La Caixa Foundation fellow. L.H-M. is a Ministry of Education,
Culture and Sport (MECD) fellow. This study was funded by the following grants to G.S: ERC 260464, EFSD 2030, MICINN /SAF1305
Comunidad de Madrid S2010/BMD-2326 and 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation. The CNIC
is supported by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) and the Pro CNIC Foundation, and is a Severo
Ochoa Center of Excellence (MEIC award SEV-2015-0505).

Competing Interests:

The authors report no conflict of interest.

Keywords: hepatocellular carcinoma, liver, adipose tissue, adipokines, gender.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00021

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Elisa Manieri, Leticia Herrera-Melle, Alfonso Mora, Antonia Tomás-Loba, Luis Leiva-Vega, Delia Irene Fernández, María
Elena Rodríguez, Lourdes Hernández-Cosido, Jorge Luis Torres, Luisa María Seoane, Miguel Marcos, Guadalupe Sabio. Adipose
tissue and liver crosstalk: new insights into gender differences in hepatocellular carcinoma incidence. IBJ Plus 2018 (S2):e00021 doi:
10.24217/2531-0151.18v1s2.00021.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Hepatocellular carcinoma (HCC) is the third most common cancer type and the second leading cause of cancer-related
death. The incidence of HCC is rising worldwide due to the increased prevalence of obesity. Therapeutic options for this
malignancy are limited, being survival after diagnosis very poor. Better preventive, diagnostic and therapeutic tools are
therefore urgently needed, particularly in view of the important contribution of obesity to HCC incidence worldwide.
Obesity multiplies the risk of developing liver cancer, probably because it can result in fatty liver disease, inflammation
and cirrhosis, a well-known predisposing factor for this malignancy. In addition, epidemiological studies have shown a
higher incidence of HCC in men than in women, a dimorphism also observed in mouse models. However, the specific
mechanisms underlying the correlation between obesity, gender and HCC are unknown.

Adipose tissue is one of the most important contributors to the adaptation to obesity, through the regulation of
fuel metabolism storage, the release of nutrients and, indirectly, through the production of circulating adipokines.
Importantly, this organ shows evident gender disparities in the production of these secreted factors, but their role in
HCC is controversial and requires further investigation.
Here, we used allograft experiments and diethylnitrosamine (DEN)-induced HCC mouse models to understand the
connection between adipokine´s secretion and liver cancer regarding gender differences. We demonstrate that decreased
levels of an adipokine account for the increased hepatic cancer risk in males. We found that higher activation of a protein
kinase is responsible for the inhibition of white adipose tissue adipokine secretion in males, therefore promoting tumor
development. This increased hepatic cancer cells proliferation is a consequence of the reduced activation of relevant
signaling pathways in this organ, which exert a protective effect against tumor development in females.

This study shows that gender differences in adipocytes are important players in HCC development and can contribute to
the increased incidence of HCC in males. Our results unravel a clear adipose tissue and liver crosstalk, clarifying a new
mechanism underlying gender disparity in liver cancer development, and suggest its potential to guide new strategies
for cancer therapeutics.

e00022
Functional characterization of CNS2 DNA regulatory element of
the mouse Tyr gene by CRISPR-Cas9 mutagenesis.

Santiago Josa1,2,, Almudena Fernández1,2, Marta Cantero1,2, Julia Fernández1,2, Lluis Montoliu1,2,*.

*Corresponding author:
montoliu@cnb.csic.es

Details of affiliation

¹National Centre for Biotechnology (CNB-CSIC), Madrid, Spain.
²CIBERER-ISCIII, Madrid, Spain.

Funding

PhD fellowship from MINECO (FPI-2013). Grant to LM from MINECO: BIO2015-70978-R

Competing Interests:

There is not conflict of interest.

Keywords: CRISPR/Cas9, albinism, tyrosinase.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00022

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Santiago Josa, Almudena Fernández, Marta Cantero, Julia Fernández, Lluis Montoliu. Functional characterization of CNS2
DNA regulatory element of the mouse Tyr gene by CRISPR-Cas9 mutagenesis. IBJ Plus 2018 (S2):e00022 doi: 10.24217/2531-
0151.18v1s2.00022.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Tyrosinase (Tyr) is the main enzyme of the melanin biosynthetic pathway. The Tyr gene is only expressed
in pigment cells, of which there are two different known types: melanocytes from neural crest and retinal pigment epithelium
(RPE) cells from optic cup. Tyr mutations cause cell hypopigmentation or albino (null) phenotype, with severe
associated visual deficits. In human, mutations in TYR are responsible of a rare disease called Oculocutaneous Albinism
Type 1 (OCA1). Previous works using standard and artificial chromosome-type BACs determined that promoter and
proximal regulatory elements where sufficient to drive Tyr expression. Later on, a major regulatory region located at
-15kb was found, containing enhancer and boundary elements, which our lab has been able to evaluate in melanocytes
using CRISPR/Cas9 technique. However, Tyr expression in RPE cells was not driven by this element. Some years ago, an
element called CNS2 (Conserved Non-coding Sequence) was proposed to be responsible of Tyr expression in RPE. We
have analyzed this region to assess its relevance and functional implications.

Methods: Using CRISPR/Cas9 technique, we have designed sgRNAs at each side of the CNS2 element to produce both
Double Strand Breaks (DSB) aiming to achieve a deletion. This has allowed us to obtain several alleles, among which is
our desired deletion. Once the deletion was well established in a mouse colony, its function has been assessed by histology
analysis of skin and eye, expression of Tyr and adjacent genes by RT-qPCR, and melanin content.

Results: We have been able to obtain 34 mice out of 81 (42%) with a mutation in any of the DSB, of which 9 out of 81
had a deletion (11%). This includes deletions of the desired region of interest. We have also obtained alleles with point
mutations and an inversion. We decided to maintain four of the mouse lines: one with the CNS2 complete deletion, two
with different partial deletions and an inversion. When analyzed by histological techniques or melanin content assay, we
have not observed differences between any of our mice and wild type. However, when Tyr gene expression was evaluated
in eye, we observed a decrease of expression in CNS2 complete deletion mice versus wild type animals

Conclusions: In this project, we have been able to achieve a 42% of mice with a mutation, and 11% with a deletion,
which demonstrate the robustness and efficiency of CRISPR/Cas9 system. When CNS2 relevance has been evaluated, we
have observed that this element regulates Tyr expression in RPE but it is not completely necessary for its proper function,
probably because of the presence of other regulatory elements in Tyr locus.

e00023
CDCA7 regulates lymphoma cells migration and invasion through
reorganization of the tubulin and the actomyosin cytoskeleton.

Carla Martín-Cortázar1, Raúl Jiménez-Pérez¹, Yuri Chiodo¹, María L. Cayuela², Teresa Iglesias³, and Miguel R. Campanero1*.

*Corresponding author:
Miguel R. Campanero, Cancer Biology Department, Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM. C/ Arturo Duperier, 4; Madrid
28029, Spain. mcampanero@iib.uam.es

Details of affiliation

¹Department of Cancer Biology and ³Department of Endocrine and Nervous Systems Pathophysiology; Instituto de Investigaciones Biomédicas
Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid 28029, Spain.
²Unidad de Cirugía Experimental, Hospital Universitario Virgen de la Arrixaca, El Palmar 30120, Murcia, Spain.

Funding

This work was supported by the Spanish Ministerio de Economía, Industria y Competitividad (MINECO) grant to M.R.C.
(SAF2013-45258P).

Competing Interests:

The authors have no conflict of interest to declare.

Keywords: CDCA7, lymphoma, invasion, migration.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00023

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Carla Martín-Cortázar, Raúl Jiménez-Pérez, Yuri Chiodo, María L. Cayuela, Teresa Iglesias, Miguel R. Campanero. CDCA7
regulates lymphoma cells migration and invasion through reorganization of the tubulin and the actomyosin cytoskeleton. IBJ Plus
2018 (S2):e00023 doi: 10.24217/2531-0151.18v1s2.00023.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Cancer cells accumulate numerous molecular alterations relative to their normal counterparts that confer
them tumor formation capacities including indefinite proliferation activity (immortality), cell death resistance, or the
capacity to invade the surrounding tissue. Great efforts have been made to uncover genes involved specifically in tumor
formation, but most of the identified genes participate in processes related with cell proliferation. Accordingly, therapies
targeting these genes also impair the proliferation of normal cells. Although immortality is a characteristic of tumor
cells, it is not sufficient for tumor formation. Indeed, some non-tumor cells are capable to divide indefinitely, such as
Lymphoblastoid B-Cell Lines (LCLs). To identify genes involved in tumor traits independent of limitless proliferation, we
compared gene expression profiles of B-cell lymphomas with those of LCLs and identified >1,600 differentially expressed
genes. One of the most significantly up-regulated genes was CDCA7, whose knockdown in lymphoma cells decreased
their capacity to form tumors in immunodeficient mice (manuscript under revision).

Methods and Results: Histological examination of xenotransplanted lymphoma tumors showed that while CDCA7-
silenced lymphoma cells did not infiltrate the surrounding muscle and adipose tissues, CDCA7-competent lymphoma
cells massively infiltrated them, suggesting that CDCA7 might be a critical mediator of lymphoma invasiveness. Lentivirus
encoding a control shRNA or various CDCA7-specific shRNAs were used to knockdown (KD) CDCA7 in DG-75, BL-2 and
Toledo lymphoma cells and assess the invasion and migration capacity of these cells in vitro and in vivo and the molecular
mechanisms involved. We found that CDCA7 KD sharply decreased invasion and migration of DG-75, BL-2 and Toledo
cells in matrigel- and fibronectin-coated transwells, respectively. Moreover, CDCA7 KD also impaired invasion/migration
of DG-75 cells in zebrafish embryos. CDCA7 KD did not affect cell adhesion to fibronectin or fibronectin receptors
expression, but markedly altered the actomyosin and the tubulin cytoskeleton activation and polarization.

Conclusions: Our results strongly suggest that CDCA7 is a critical mediator of lymphoma cells invasion through its
capacity to regulate the dynamics of both the tubulin and the actomyosin cytoskeleton.

e00024
Preclinical Safety and Efficacy Evaluation of Lentivirally transduced
Hematopoietic Stem Cells for the treatment of Leukocyte Adhesion
Deficiency type I.

Mesa Núñez C1, 2, Damián C1, 2, León-Rico D1, 2, Aldea M1, 2, Carrascoso-Rubio C1, 2, Lozano ML1, 2, Guenechea G1, 2, Campo B3, Santilli G4, Kohn DB3,
Thrasher AJ4, Bueren JA1,2*, Almarza E1,2*

*Corresponding authors: Elena Almarza, PhD elena.almarza@ciemat.es and Juan A Bueren, PhD juan.bueren@ciemat.es. Division of Hematopoietic
Innovative Therapies, CIEMAT/CIBERER/IIS-FJD, Avenida Complutense, 40; 28040; Madrid (Spain).

Details of affiliation

1Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT) and Centro de
Investigación Biomédica en Red de Enfermedades Raras (CIBERER-ISCIII). Madrid, 28040, Spain
2Advanced Therapies Unit, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD/UAM). Madrid, 28040, Spain.
3Department of Microbiology, Immunology and Molecular Genetics and Department of Pediatrics University of California, Los Angeles (UCLA), Los
Ángeles CA 90095–7364, California
4Molecular Immunology Unit, Institute of Child Health – University College London (ICH-UCL), London, WC1N1EH, United Kingdom.

Funding

Partially supported with a grant from Rocket Pharma.

Competing Interests:

The ChimhCD18-LV has been licensed to Rocket Pharma. Juan A Bueren is scientific advisor for Rocket Pharma.

Keywords: Primary immunodeficiencies; LAD-I, hCD18, Integrins, Gene Therapy.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00024

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Mesa Núñez C, Damián C, León-Rico D, Aldea M, Carrascoso-Rubio C, Lozano ML, Guenechea G, Campo B, Santilli G, Kohn
DB, Thrasher AJ, Bueren JA, Almarza, E. Preclinical Safety and Efficacy Evaluation of Lentivirally transduced Hematopoietic Stem Cells
for the treatment of Leukocyte Adhesion Deficiency type I. IBJ Plus 2018 (S2):e00024 doi: 10.24217/2531-0151.18v1s2.00024.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Leukocyte Adhesion Deficiency Type I (LAD-I) is a primary immunodeficiency characterized by recurrent
and life-threatening bacterial and fungal infections. It is caused by mutations in the ITGB2 gene, encoding the CD18
subunit of β2 integrins. These mutations lead to defective or absent expression of β2 integrins on the leukocytes’ surface,
limiting their adhesion to the endothelium and therefore their extravasation to infection sites. As it is the case with other
monogenic immunodeficiencies, LAD-I is a good candidate for ex vivo gene therapy. In a previous work, a therapeutic
Chim.hCD18 lentiviral vector (LV) was generated in which the human CD18 protein was expressed under the control of a
chimeric promoter preferentially active in myeloid cells. This vector obtained the Orphan Drug Designation by the EMA
(EU/3/16/1753) and FDA (DRU-2016-5430) Agencies.

Materials and Methods: We have tested the efficacy of the LV.Chim.hCD18 vector in mouse CD18 KO cells that resemble
the severe phenotype of the disease. Bio-distribution studies have been performed in wild type mice transplanted with
LV.Chim.hCD18 transduced Lin- BM cells. The regulation of the hCD18 expression was also evaluated by transducing
healthy donor CD34+ cells from cord blood with the lentiviral vector.

Results: Ex vivo gene therapy experiments performed in CD18 KO mice have shown that even transductions leading to
low vector copy numbers in the transduced cells are able to show restoration of the membrane expression of leukocyte’s
integrins. Bio-distribution studies demonstrated that the presence of the LV was restricted to the hematopoietic tissue
and that no changes in the hematopoietic reconstitution were observed. Also, neither histopathology alterations in the
organs of these mice, nor unexpected deaths or weight changes were observed in these mice, indicating the absence of
toxicity of the gene therapy approach. Moreover, studies conducted with human hematopoietic cells showed that the
integration of the therapeutic LV.Chim.hCD18, even when used at high multiplicities of infection, did not result in supraphysiological
expression levels of CD18 in the membrane of transduced cells.

Conclusions: Altogether, these results evidence the preclinical efficacy and safety of the proposed LAD-I gene therapy
approach.

e00026
Tofacitinib restores the inhibition of reverse cholesterol
transport induced by inflammation: understanding the lipid
paradox associated with rheumatoid arthritis.

Pérez-Baos Sandra¹, Barrasa Juan Ignacio1, 2, Gratal Paula¹, Larrañaga-Vera Ane¹, Prieto-Potin Iván¹, Herrero-Beaumont Gabriel1*, Largo Raquel¹

*Corresponding author:
Professor Gabriel Herrero-Beaumont, Bone and Joint Research Unit, Rheumatology Department, IIS-Fundación Jiménez Díaz UAM, Reyes Católicos,
28040 Madrid, Spain. E-mail: gherrero@fjd.es

Details of affiliation

1Affiliations: Bone and Joint Research Unit, Rheumatology Dept, IIS-Fundación Jiménez Díaz UAM, Madrid. Spain.
²Current affiliation: Department of Molecular Biology, Umeå University, Umeå, Sweden.

Funding

This work has been partially supported by a PFIS Fellowship (ISCIII), a Conchita Rábago de Jiménez Díaz Felowship (Fundación
Conchita Rábago, IIS-FJD), a Pfizer Investigator Initiated Research (IIR) Competitive Grant (ASPIRE, #XZJ-IIR-01-1) and grants from the
Instituto de Salud Carlos III (PI13/00570; PI15/00340, PI16/00065 and RETICEF RD12/0043/0008), co-funded by Fondo Europeo de
Desarrollo Regional (FEDER).a Pfizer Investigator Initiated Research (IIR) Competitive Grant (ASPIRE, #XZJ-IIR-01-1) and grants from
the Instituto de Salud Carlos III (PI13/00570; PI15/00340, PI16/00065 and RETICEF RD12/0043/0008), co-funded by Fondo Europeo
de Desarrollo Regional (FEDER).

Competing Interests:

The authors declare no conflicts of interest.

Keywords: Rheumatoid Arthritis, Inflammation, Cholesterol, Macrophages, ABCA1

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00026

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Pérez-Baos Sandra, Barrasa Juan Ignacio, Gratal Paula, Larrañaga-Vera Ane, Prieto-Potin Iván, Herrero-Beaumont Gabriel,
Largo Raquel. Tofacitinib restores the inhibition of reverse cholesterol transport induced by inflammation: understanding the lipid
paradox associated with rheumatoid arthritis. IBJ Plus 2018 (S2):e00026 doi: 10.24217/2531-0151.18v1s2.00026.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Rheumatoid arthritis (RA) patients have increased cardiovascular mortality, paradoxically associated
with reduced serum lipid levels. In fact, an inverse relationship between C-reactive protein (CRP) and circulating lipid
levels has been observed. The JAK inhibitor tofacitinib (TOFA) ameliorates RA systemic and joint inflammation with a
concomitant increase in serum lipids. Our aim was to analyze the effect of TOFA on the lipid and inflammatory profile
of hyperlipidemic rabbits with chronic arthritis (CA), and on the regulation of reverse cholesterol transport (RCT) during
chronic inflammation.

Methods: CA was induced in 18 high fat diet (HFD)-fed rabbits. Four weekly intra-articular injections of ovalbumin were
given to previously immunized animals. Nine CA rabbits were treated with TOFA (10mg/kg/day) for two weeks. Six
healthy HFD-fed rabbits were used as controls. Fully differentiated THP-1 cells were exposed to HFD rabbit serum or
ox-LDL to become foam cells. Thereafter, cells were stimulated with IFNγ in presence or absence of TOFA for 24 hours.
Protein was collected for western blot studies and cholesterol accumulation was assessed by an Oil Red-O staining.

Results: CA rabbits showed lower levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) than
controls (p=0.001 and p=0.012). C-reactive protein (CRP) levels were dramatically increased in all CA animals, although
a reduction was observed with the treatment (p=0.006). We also found an inverse correlation between serum TC and
CRP (R=-0.454, p=0.029). TOFA tended to increase serum TC and LCL-C and was able to reduce the lipid content within
synovial macrophages up to a 58% (p=0.041), without modifying synovial macrophage density. In foam macrophages
in culture, IFNγ further stimulated the intracellular lipid accumulation (p=0.041) along with a decrease in the protein
levels of the nuclear factor liver X receptor alfa (LXRα) and the cholesterol transporter ATP-binding cassette transporter
1 (ABCA1) (p=0.002 and p=0.0047, respectively). TOFA prevented the lipid accumulation within macrophages (p=0.029)
by increasing LXRα and ABCA1 synthesis in a JAK/STAT-dependent manner (p=0.047 and p=0.004, respectively), while it
was unable to reduce lipid accumulation in ABCA1 silenced macrophages.

Conclusions: Our results suggest that active inflammation could be favouring lipid accumulation within tissue
macrophages, thus inducing a decrease in serum lipid levels. TOFA may prevent this phenomenon, at least partially, by
restoring ABCA1-mediated cholesterol efflux in macrophages. These findings further explain how serum lipid levels are
diminished in RA and partially justify the effect of TOFA on the lipid profile of RA patients.

e00027
The role of PKD1 in brain injury: ROS detoxification and
neuroprotection.

J Pose-Utrilla1,2, L García-Guerra1,2, A Del Puerto1,2, A Martín-Muñóz4, J Jurado-Arjona3,2 , N S De León-Reyes1, A Gamir-Morralla1,2, A Sebastián-
Serrano1,2,J Fielitz5,6, I Ferrer7,2, F Hernández3,2, J Ávila3,2, M R Campanero1,8, T Iglesias1,2*

*Corresponding author:
Teresa Iglesias. Instituto de Investigaciones Biomédicas “Alberto Sols”, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de
Madrid (CSIC-UAM), Madrid, Spain
CIBERNED, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Madrid, Spain
E-mail: tiglesias@iib.uam.es

Details of affiliation

1Instituto de Investigaciones Biomédicas “Alberto Sols”, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM),
Madrid, Spain
2CIBERNED, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Madrid,Spain
3Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Madrid, Spain
4Experimental Molecular Imaging, (Molecular Imaging Unit), CIC biomaGUNE,San Sebastian, Spain.
5Experimental and Clinical Research Center (ECRC), Charité-Universitätsmedizin, Max-Delbrück-Center (MDC) for Molecular Medicine in the Helmholtz
Association, Berlin, Germany.
6Department of Cardiology, Heart Center Brandenburg and Medical University Brandenburg (MHB), Brandenburg, Germany
7Instituto de Neuropatología, Hospital Universitario de Bellvitge, Hospitalet de Llobregat, Barcelona, Spain
8CIBERCV, Centro de InvestigaciónBiomédica en Red de EnfermedadesCardiovasculares, Instituto de Salud Carlos III, Madrid, Spain

Funding

Competing Interests:

Keywords: Excitotoxicity, oxidative stress, neuronal death, stroke, PKD1

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00027

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: J Pose-Utrilla, L García-Guerra, A Del Puerto, A Martín-Muñóz, J Jurado-Arjona, N S De León-Reyes, A Gamir-Morralla, A
Sebastián-Serrano, J Fielitz, I Ferrer, F Hernández, J Ávila MR. Campanero, T Iglesias. The role of PKD1 in brain injury: ROS detoxification
and neuroprotection. IBJ Plus 2018 (S2):e00027 doi: 10.24217/2531-0151.18v1s2.00027.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Oxidative stress is a major pathophysiological mediator of degenerative processes in many
neurodegenerative diseases. It is an early event produced during excitotoxicity and is one of the main causes of neuronal
damage. It has been described that Protein Kinase D1 (PKD1) is activated by oxidative stress and regulate detoxification
of free radicals in tumor cells. However, the role of PKD1 in brain injury associated with excitotoxicity and oxidative stress
damage has not yet been explored.

Methods: We analyze the activity of PKD1 using in vitro and in vivo models of neuronal damage, as well as human
ischemic stroke samples. In addition, and through the use of pharmacological inhibitors, lentiviral silencing and neuronal
conditional knockout mice we study the role of this kinase in the pro-survival oxidative stress detoxification pathway and
the regulation of their activity in these processes.

Results: We find that excitotoxicity provokes an inactivation of neuronal PKD1 and as a consequence there is a decline
of IKK/NF-κB survival cascade and an increase in reactive oxygen species. We identify the first molecular mechanism
involved in PKD1 inactivation, caused in excitotoxicity by the action of phosphatases not identified so far. Consistent
with these results, we demonstrate that the elimination of PKD1 in murine models increases neuronal damage after
ischemic stroke and the neurospecific expression of dephosphorylation-resistant active PKD1 prevents kainic acidinduced
neuronal death in vitro and in vivo.

Conclusions: Our data support that the loss of neuronal PKD1 activity is detrimental for neuronal survival and ROSdetoxification
and suggest that this kinase could be a promising target for treatment of excitotoxic brain damage
associated to acute and chronic neurodegeneration.

e00028
Generation and characterization of a reversible HGPS mouse
model to design potential future therapies.

Amanda Sánchez-López1,2, Álvaro Macías1,2, Victor Fanjul1,2,3, Mª Jesús Andrés-Manzano1,2, Cristina González1,2, Lara del Campo1,2, Vicente Andrés1,2.

*Corresponding author:
Vicente Andrés: vandres@cnic.es
Author E-mail: asanchez@cnic.es

Details of affiliation

1Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain.
2CIBER de Enfermedades Cardiovasculares, Madrid, Spain.
3Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Spain & Centro de
Investigación Biomédica en Red de Cáncer (CIBERONC), Spain.

Funding

This study was supported by research grants from the Spanish Ministry of Economy, Industry and Competitiveness (MEIC)
and the Instituto de Salud Carlos III with cofounding from Fondo Europeo de Desarrollo Regional (FEDER) (grants SAF2016-79490-R,
and AC17/00067). The CNIC is supported by the MEIC and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence
(award SEV-2015-0505). The MEIC supported A.S.L (FPI Severo Ochoa 2014 predoctoral contract), Fundación La Caixa supported V.F.
(predoctoral fellowship), and the Red de Investigación Cardiovascular (RETIC Program, Instituto de Salud Carlos III) supported L.d.C.
(Jordi Soler postdoctoral contract).

Competing Interests:

The authors declare no conflict of interest.

Keywords: progeria, mouse model, reversion

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00028

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Amanda Sánchez-López, Álvaro Macías, Victor Fanjul, Mª Jesús Andrés-Manzano, Cristina González, Lara del Campo, Vicente
Andrés. Generation and characterization of a reversible HGPS mouse model to design potential future therapies. IBJ Plus 2018
(S2):e00028 doi: 10.24217/2531-0151.18v1s2.00028.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder characterized by premature aging and early
death (average life span: 14.6 years). Classical HGPS is caused by a de novo dominant point mutation in the LMNA gene
(encoding mainly lamin A and C). This LMNA mutation (typically c.1824 C>T, p.G608G) gives rise to a lamin A variant
called progerin, a mutant protein that remains permanently farnesylated due to incomplete maturation and that exerts
a dominant-negative effect.

HGPS patients appear healthy at birth but develop several clinical features during the first and second year of life. Taking
into account that there is still no definitive cure for HGPS and that HGPS cannot be diagnosed right after birth, it is
critically important to know if this disease is reversible or if, at least, it is possible to stop its progression. In addition, it is
necessary to investigate what is the contribution of tissue-specific and systemic factors to the development of the HGPS
phenotype in order to ascertain whether a therapy delivered in a tissue-specific manner would be enough to restore
normal body condition in HGPS patients.

Using the CRISPR-Cas9 technology, we have generated for the first time a “reversible” transgenic mouse model called
LmnaHGPSrev which expresses progerin ubiquitously and recapitulates the HGPS phenotype. Importantly, our design
allows the elimination of progerin expression and the restoration of lamin A expression upon activation of the Crerecombinase.
We have demonstrated that LmnaHGPSrev mice recapitule HGPS features, and that it is possible to remove progerin and
restore lamin A expression after tamoxifen-inducible Cre expression. This HGPS model will allow us to control progerin
suppression and lamin A restoration at different times during disease progression, either ubiquitously or in a tissuespecific
manner. We therefore plan to investigate the potential reversibility of the damage caused by progerin, and
assess the effectiveness of possible future therapies designed to suppress progerin expression in specific tissues.

As shown in the figure, our new mouse model expresses only progerin and lamin C but not lamin A. However, after
tamoxifen induction to activate Cre, progerin expression is progressively suppressed while lamin A protein expression is
restored.

e00029
Study of the role of microRNAs in T-cell lymphoblastic lymphoma
development through the regulation of expression of FBXW7
gene.

Irene Vázquez-Domínguez1,2*, Laura González-Sánchez1,2,3, Pilar López-Nieva1,2,3, María Villa-Morales1,2,3, María Ángeles Cobos-Fernández1,2, Pablo
Fernández-Navarro4,5,6, Isabel Sastre1, Agustín F. Fernández7, Marcos Malumbres8, Javier Santos1,2,3, José Fernández-Piqueras 1,2,3

*Corresponding author:
Irene Vázquez-Domínguez1, 2. Madrid, Spain. E-mail: ivazquez@cbm.csic.es or irene.vazquez@uam.es
José Fernández-Piqueras 1,2,3. Madrid, Spain. E-mail: jfpiqueras@cbm.csic.es

Details of affiliation

1Centro de Biología Molecular Severo Ochoa (CBMSO), Consejo Superior de Investigaciones Científicas- Universidad Autónoma de Madrid (CSICUAM),
Madrid, Spain.
2IIS Fundación Jiménez Díaz, Madrid, Spain.
3Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), ISCIII, Spain
4Unidad de Epidemiología Ambiental y Cáncer, Centro Nacional de Epidemiología, Instituto de Salud Carlos III, Madrid, Spain.
5Consorcio de Investigación Biomédica de Epidemiología y Salud Pública (CIBERESP), Madrid, Spain.
6IIS Puerta de Hierro, Majadahonda, Spain.
7Unidad de Epigenética del Cáncer, IUOPA-CSIC, Hospital Universitario Central de Asturias (HUCA), Oviedo, Spain.
8Spanish National Cancer Research Centre (CNIO), Madrid, Spain

Funding

The authors would like to thank the Spanish Ministry of Economy and Competitiveness (SAF2015-70561-R; MINECO/FEDER,
EU) and the Autonomous Community of Madrid, Spain (B2017/BMD-3778; LINFOMAS-CM) for funding this work. Institutional grants
from the Fundación Ramón Areces and Banco de Santander are also acknowledged. IVD is the recipient of a predoctoral fellowship
from the Spanish Ministry of Economy and Competitiveness (FPI: BES-2013-065740).

Competing Interests:

The authors declare no competing financial interests.

Keywords: T-LBL development, Regulation of FBXW7 expression by microRNAs, FBXW7-isoform specific functions.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00029

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Irene Vázquez-Domínguez, Laura González-Sánchez, Pilar López-Nieva, María Villa-Morales, María Ángeles Cobos-Fernández,
Pablo Fernández-Navarro, Isabel Sastre, Agustín F. Fernández, Marcos Malumbres, Javier Santos, José Fernández-Piqueras. Study of
the role of microRNAs in T-cell lymphoblastic lymphoma development through the regulation of expression of FBXW7 gene. IBJ Plus
2018 (S2):e00029 doi: 10.24217/2531-0151.18v1s2.00029.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: T-cell lymphoblastic leukaemia/lymphoma (T-ALL/T-LBL) are agressive hematological cancers prevalent in
the childhood. Different genes have been related with the development of this disease including the tumor suppressor
gene FBXW7, which plays an important role acting through the proteasome degradation of key oncoproteins. In this
work, we showed that up-regulation of a pool of microRNAs contributes significantly to the down-regulation of FBXW7
in T-LBL development.

Material and methods: In order to elucidate the mutational status of FBXW7, we performed targeted-deep-sequencing
analyses in a sample series of human T-LBL. Massive sequencing by RNA-Seq was accomplished to study the expression
levels of FBXW7 isoforms and to select a pool of miRNAs candidates to be involved in a FBXW7 regulation: hsa-miR-
223-3p, hsa-miR-195-5p and has-miR-101-3p. Transfecting SUP-T1 cell line with miRVana-miRNA mimics and inhibitors
assessed the experimental validation of the effect of miRNA up-regulation over FBXW7 levels and its specific targets. We
completed the studies of mRNA and protein expression with proliferation assays. Finally, we performed rescue experiments
in order to define the role of each isoform in the T-LBL development.

Results: Despite we did not observe any mutation in FBXW7, we noticed a significant reduction in the amount of mRNA
of two of the three FBXW7 isoforms (α and β isoforms), indicating that down-regulation of FBXW7 is a common feature
in the pathogenesis of these diseases. Two of the selected miRNAs hsa-miR-101-3p and has-miR-195-5p are able to
cause a significant reduction of FBXW7 protein levels acting alone or in combination, respectively. This drop in FBXW7
levels is also related with a reduction of cell survival and proliferation mainly due to the up-regulation of c-MYC and
CCNE1. According to our data, this relationship between FBXW7 and its targets are dependent on the reduction of α and
β isoforms reduction.

Conclusions: In our disease context, up-regulation of a pool of microRNAs portrays a highly dynamic landscape in the
regulation of FBXW7 expression. Besides this, each FBXW7 isoforms has a different role on its targets regulation and
consequently in the T-LBL development.

e00030
Protection against Middle East respiratory syndrome coronavirus infection by immunization with genetically engineered liveattenuated viruses

Francisco Javier Gutiérrez-Álvarez1*, Raúl Fernandez-Delgado1, Carlos Castaño-Rodríguez1, José M. Honrubia1, Sonia Zuñiga1, Paul McCray2, Stanley Perlman2, Isabel Sola1, Luis Enjuanes1

*Corresponding author:
Francisco Javier Gutiérrez-Álvarez, Department of Molecular and Cell Biology, National Center for Biotechnology (CNB-CSIC), Madrid, Spain,
fjgutierrez@cnb.csic.es

Details of affiliation

1Department of Molecular and Cell Biology, National Center for Biotechnology (CNB-CSIC), Madrid, Spain
2Department of Microbiology, University of Iowa, USA

Funding

This study was financed by a grant from the Zoonotic Anticipation and Preparedness Initiative (ZAPI project; IMI Grant
Agreement no. 115760) and a grant from the Ministry of Science and Innovation of Spain (Bio2016-75549-R AEI/FEDER, UE)

Competing Interests:

The authors did not declare any competing interests.

Keywords: MERS, coronavirus, vaccine, live-attenuated, Envelope

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00030

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Francisco Javier Gutiérrez-Álvarez, Raúl Fernandez-Delgado, Carlos Castaño-Rodríguez, José M. Honrubia, Sonia Zuñiga, Paul
McCray, Stanley Perlman, Isabel Sola, Luis Enjuanes. Protection against Middle East respiratory syndrome coronavirus infection by
immunization with genetically engineered live-attenuated viruses. IBJ Plus 2018 (S2):e00030 doi: 10.24217/2531-0151.18v1s2.00030.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Middle East respiratory syndrome (MERS) coronavirus (CoV) is a life threatening human CoV, against which
there are no approved treatments or vaccines. The development of vaccines against MERS-CoV is mainly based on
the Spike (S), which is highly immunogenic. Nonetheless, vectored or subunit vaccines against MERS-CoV have been
demonstrated to carry out side effects. Despite the concerns about it safety, live-attenuated vaccines (LAV) have proven
it potential to induce strong and lasting immune responses. Our laboratory demonstrated that the Envelope (E) protein of
severe acute respiratory syndrome CoV (SARS-CoV) is a virulent factor. Lack of E gene attenuated the virus and provided
full protection against virulent SARS-CoV challenge.

Material and methods: Two types of LAV have been engineered using a MERS-CoV reverse genetic system. The first
one is based on the full deletion of the envelope (E) protein (rMERS-CoV-ΔE), which resulted in a replication-competent
and propagation-defective one-cycle virus. The second type, a replication-competent and propagation-competent virus,
initially consisted of a collection of six rMERS-CoVs carrying partial deletions of 9-11 amino acids spanning the C-terminal
domain of the E protein (rMERS-CoV-E*); of them, three deletion mutants (E*-Δ1, E*-Δ2, and E*-mutPBM) were selected
for further analysis. E*-Δ1 and E*-mutPBM mutants were viable, while E*-Δ2 was stabilized by introducing five amino
acids within the engineered deletion (E*-Δ2ins). In order to produce ΔE virus, wild type (wt) E protein was provided in
trans. Since E protein induced cell death, the complementation was performed using an inducible system optimized for
transient expression.

Results: In the presence of E protein provided in trans, titers of the ΔE virus were raised ten-fold (>105 pfu/ml), whereas
titers of E* and wt viruses were not significantly increased. Attenuation of rMERS ΔE, E*-Δ1, E*-Δ2ins, and E*-mutPBM
viruses was evaluated in vivo in a transgenic mouse model susceptible to MERS-CoV infection. All E*-Δ1 infected animals
lost weight and died after 8 days post-infection (dpi). Interestingly, ΔE, E*-Δ2ins, and E*-mutPBM infected animals
recovered from the infection and survived, indicating that these viruses were attenuated. ΔE, E*-Δ2ins, and E*-mutPBM
immunized animals were then challenged with virulent MERS-CoV. All the animals survived and no weight loses were
observed.

Conclusions: Three LAV candidates have been obtained against MERS-CoV infection. ΔE seems to be the most promising
due to its capacity to replicate but not to propagate. However, a lot of work is still to be done concerning the evaluation
of the immune response, the genetic stability, and the safety.

e00031
Mechanisms controlling the cilia localization of INPP5E, a phosphoinositide 5-phosphatase mutated in MORM and Joubert syndromes.

Sierra Rodero MB1, Cilleros Rodriguez D1, Martin-Morales R1, Barbeito P1, Garcia-Gonzalo FR1*.

*Corresponding author:
Garcia-Gonzalo FR. E-mail: francesc.garcia@uam.es

Details of affiliation

1Alberto Sols Biomedical Research Institute UAM-CSIC (IIBM), La Paz University Hospital Research Institute (IdiPAZ) & Department of Biochemistry,
School of Medicine, Autonomous University of Madrid. 28029 Madrid, Spain.

Funding

Work in the Garcia-Gonzalo lab is funded by a MINECO/FEDER project from the Spanish government (SAF2015-66568-R).
MBSR has been the recipient of a one-year predoctoral contract from the Youth Employment Initiative (YEI) of FEDER and the Madrid regional government (CAM).

Competing Interests:

The authors declare no competing interests.

Keywords: INPP5E, Joubert syndrome, MORM, ARL13B, cilia, phosphorylation.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00031

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Sierra Rodero MB, Cilleros Rodriguez D, Martin-Morales R, Barbeito P, Garcia-Gonzalo FR. Mechanisms controlling the cilia localization of INPP5E, a phosphoinositide 5-phosphatase mutated in MORM and Joubert syndromes. IBJ Plus 2018 (S2):e00031 doi:
10.24217/2531-0151.18v1s2.00031.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: INPP5E is a ciliary phosphoinositide 5-phosphatase whose mutations are the cause of two human
ciliopathies, Joubert (JBTS) and MORM syndromes, and whose activity supports Hedgehog-dependent tumor progression.
We previously showed that INPP5E is a critical regulator of ciliary phosphoinositide levels, which in turn control ciliary
protein composition and Hedgehog signaling (Garcia-Gonzalo et al. 2015 Dev Cell). However, the mechanisms controlling
INPP5E ciliary localization remain unclear.

Material and methods: We have used site-directed mutagenesis to examine how INPP5E cilia localization is controlled.

Results: We find that INPP5E cilia localization depends on two separate regions located at the beginning and end of
its catalytic domain. These two regions, albeit separated by circa 300 residues, come together as a concave surface on
the folded catalytic domain. Several residues on this surface are needed for INPP5E to bind ARL13B, a JBTS-causative
ciliary protein known to be required for INPP5E to localize to cilia. Since INPP5E is reported to leave cilia in response
to mitogenic signals, we also studied whether INPP5E ciliary targeting is regulated by phosphorylation. We find that
INPP5E undergoes tyrosine phosphorylation, and that a mutant mimicking phosphorylation of a tyrosine on the abovementioned
surface prevents INPP5E cilia localization.

Conclusions: We conclude that INPP5E cilia localization is controlled by a ciliary targeting signal (CTS) present in its folded
catalytic domain. This CTS acts, at least partly, by allowing INPP5E to interact with ARL13B. Moreover, our preliminary
data suggest that CTS phosphorylation can interfere with INPP5E cilia localization.

e00032
Generation of “mini-brains” from pluripotent stem cells to study brain development.

Adela Bernabeu-Zornoza1*, Charlotte Palmer1, Raquel Coronel1, María Lachgar1, Laura Silva1, Alberto Zambrano1, Isabel Liste1*.

*Corresponding author:
Isabel Liste Ph.D.,Unidad de Regeneración Neural, Unidad Funcional de Investigación de Enfermedades Crónicas. Instituto de Salud Carlos III (ISCIII)
28220 Majadahonda, Madrid, Spain. E-mail: Iliste@isciii.es / adela.bernabeu@gmail.com

Details of affiliation

1Unidad Funcional de Investigación de Enfermedades Crónicas (UFIEC). Instituto de Salud Carlos III (ISCIII), Majadahonda, Madrid, Spain.

Funding

This study was supported by the MICINN-ISCIII (grants MPY1412/09 and PI10/00291), Comunidad de Madrid (NEUROSTEMCM consortium; S2010/BMD-2336), and MINECO- Retos SAF 2015- 71140-R.

Competing Interests:

The authors indicate that they have no potential conflict of interest with this work.

Keywords: Embryonic stem cells, pluripotency, organoids.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00032

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Adela Bernabeu-Zornoza, Charlotte Palmer, Raquel Coronel, María Lachgar, Laura Silva, Alberto Zambrano, Isabel Liste.
Generation of “mini-brains” from pluripotent stem cells to study brain development. IBJ Plus 2018 (S2):e00032 doi: 10.24217/2531-0151.18v1s2.00032.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Due to the complexity of the human brain, it is difficult to study many brain disorders in model organisms. The results that
we can obtain nowadays are all made in monolayer cell cultures (2D), and, although highly valuable, those methods are
devoid of a tridimensional component necessary for normal organ development. Therefore, the ability to model human
brain development in vitro represents an important step in our study of developmental processes and neurological
disorders.

It has recently been described that pluripotent stem cells (Embryonic Stem Cells (ES) or induced Pluripotent Stem Cells
(iPS)) in a suitable environment are capable of generating three-dimensional (3D) structures called “cerebral organoids
or mini-brains”. They recapitulate different stages of human cortical development, generating a variety of regional
identities organized in discrete domains able to connect with each other.

We are setting up a human and mouse ES cells three dimensional organoids culture system. The differentiation in
organoids (3D) is being carried out according to the protocol recently published by Lancaster and Knoblich (2014) Nature
Protocols 9(10):2329-40. It is based on a first phase of generation of floating embryoid bodies, followed by a second
phase of transferring these embryoid bodies to plates for their neural induction and subsequent differentiation. The
characterization of cultures and identification of different neural structures and phenotypes are being performed at
cellular and molecular level by immunocytochemistry and quantitative-RT-PCR.

Together, these studies would indicate that three-dimensional organoids can recapitulate human or mouse
neurodevelopment and it can be useful to study the pathogenesis of neurological diseases.

e00033
Lysyl oxidase-like 3 in melanoma progression.

José Bustos-Tauler1, Alberto Vázquez-Naharro1, Lourdes Yuste1, Amparo Cano1 and Patricia G. Santamaría1*.

*Corresponding author:
Patricia G. Santamaría, Departamento de Bioquímica Universidad Autónoma de Madrid, Instituto de Investigaciones Biomédicas “Alberto Sols” CSICUAM,
IdiPAZ, CIBERONC, Madrid, Spain. E-mail: pgsantamaria@iib.uam.es

Details of affiliation

1Departamento de Bioquímica Universidad Autónoma de Madrid, Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-UAM, IdiPAZ, CIBERONC,
Madrid, Spain.

Funding

SAF2013-44739R, SAF2016-76504-R Ministerio de Economía y Competitividad (MINECO), CB16/12/00295 CIBERONC, 16-
0295 Worldwide Cancer Research (WWCR), Red de Cáncer RETIC-RD12/0036/0007 (Instituto de Salud Carlos III).

Competing Interests:

The authors indicate that they have no potential conflict of interest with this work.

Keywords: LOXL3, BRAFV600E, PTEN, melanoma.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00033

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: José Bustos-Tauler, Alberto Vázquez-Naharro, Lourdes Yuste, Amparo Cano, Patricia G. Santamaría. Lysyl oxidase-like 3 in melanoma progression. IBJ Plus 2018 (S2):e00033 doi: 10.24217/2531-0151.18v1s2.00033.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Malignant melanoma is the skin cancer with highest mortality rate, characterized by its heterogeneity, aggressiveness
and resistance to treatment. Melanoma is characterized by a high mutation burden. The common BRAFV600E oncogenic
mutation is already present in nevi, and further genetic alterations, both in vitro and in animal models, are required for
malignant transformation.

LOXL3 has been described as one of the most highly expressed proteins in melanoma cell lines compared to normal
melanocytes. Our previous results indicate that LOXL3 is overexpressed in human melanoma cell lines and primary and
metastatic human melanoma samples, whereas in vitro studies suggest that LOXL3 downregulation is detrimental for
their proliferation and survival but the specific role played by LOXL3 in vivo in melanomagenesis is still ill defined.
We thus decided to characterize the involvement of LOXL3 in melanoma initiation, progression and/or dissemination
and the underlying molecular mechanisms involved using a combination of in vitro and in vivo approaches.
For that purpose, we have depleted Loxl3 expression in B16 F1 and F10 mouse melanoma cell lines using lentiviral shRNAs
to analyze the effects of Loxl3 silencing on cell proliferation and survival. We have also performed in vivo tumorigenesis
and metastasis assays to study the role of Loxl3 after orthotopical or tail vein injection of Loxl3-silenced cells. Our initial
results suggest that Loxl3 depletion negatively affects melanoma cell proliferation.

Besides, we have generated a genetically engineered melanoma mouse model with Loxl3 conditional loss-of-function.
In these conditional mice, upon 4-hydroxytamoxifen (4-HT) treatment, the Tyr::CreERT2 transgene allows melanocyte
specific Cre expression promoting constitutive active mutant BRAFV600E expression and PTEN loss and concomitant Loxl3
deletion. Topical application of 4-HT results in the development of pigmented skin lesions which progress to malignant
melanoma whereas metastasis is detected in lymph nodes and lungs. In order to evaluate the contribution of Loxl3 to
melanoma initiation and/or progression we have performed a pilot experiment using the generated BRAFV600E/+/
PTENlox/lox/Loxl3loxl/lox and corresponding controls BRAFV600E/+/PTENlox/lox/Loxl3+/+. Upon evaluating the timing
and incidence of nevi and melanomas as well as overall survival after 4-HT treatment, our preliminary results show that,
in the absence of Loxl3, the onset of pigmented lesions is delayed while overall survival is increased compared to control
animals. Altogether, the present data support a key role for Loxl3 in melanomagenesis.

e00034
Activation of brown adipose tissue (BAT) might play a major and beneficial role against insulin resistance associated to inflammation. Beneficial effects of modulating SIRT1 activity.

Carmen Escalona Garrido1,2, Patricia Vázquez1,2, Ester García-Casarrubios1, MJ Obregón1, AM Valverde1,2*.

*Corresponding author:
Email: avalverde@iib.uam.es

Details of affiliation

1Instituto de Investigaciones Biomédicas “Alberto Sols” (IIB, CSIC-UAM), Madrid, Spain.
2Affiliations two, address, city, country. CIBER de Diabetes y Enfermedades Metabólicas (CIBERDEM), ISCIII, Madrid, Spain.

Funding

This study was supported by funding from project SAF 2015-65267-R.

Competing Interests:

The authors declare no conflict of interest.

Keywords: BAT, SIRT1, INFFLAMATION.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00034

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Carmen Escalona Garrido, Patricia Vázquez, Ester García-Casarrubios, MJ Obregón, AM Valverde. Activation of brown adipose tissue (BAT) might play a major and beneficial role against insulin resistance associated to inflammation. Beneficial effects of modulating SIRT1 activity. IBJ Plus 2018 (S2):e00034 doi: 10.24217/2531-0151.18v1s2.00034.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Activation of brown adipose tissue (BAT) plays a promising role against metabolic diseases such as obesity or
type 2 diabetes mellitus (T2DM). These conditions are associated with chronic low-grade systemic inflammation, which is
considered a critical underlying factor in the development of insulin resistance.
Sirtuin 1 (SIRT1), a NAD+-dependent protein deacetylase, has emerged as a key metabolic sensor in various metabolic tissues
that modulates a variety of cellular processes like energy metabolism or stress response. Although SIRT1 overexpression is
protective against diverse metabolic complications, little is known about the etiology of these benefits.

Material and methods: To identify the mechanisms implicated in the potential therapeutic benefit of targeting SIRT1 in BAT
to ameliorate inflammation-mediated insulin resistance we have used an in vivo model of lean mice with or without moderate
SIRT1 overexpression. We performed an in vitro model of differentiated brown adipocytes obtained from these mice in
order to study the role of this protein in thermogenesis and insulin signaling. The impact of SIRT1 in BAT inflammation was
studied by an acute treatment with bacterial lipopolysaccharide (LPS). All these processes were analyzed by western blot
and RT-PCR.

Results: Our results indicated that SIRT1 overexpression enhanced insulin sensitivity in BAT, and after an acute treatment
with LPS, the induction of the proinflammatory cascades were attenuated in these cells.
Conclusions: Our results suggest that activation of SIRT1 in brown adipocytes might play a major and beneficial role against
insulin resistance associated to inflammation.

e00035
Therapeutic activity of GSE4 peptide in cellular models of idiopathic pulmonary fibrosis.

Beatriz Fernández-Varas1, Javier Rodríguez-Centeno1,2, Laura Pintado-Berninches1,3, Cristina Manguán-García1,2, Leandro Sastre1,2, Rosario Perona1,2*.

*Corresponding author:
E-mail: rperona@iib.uam.es

Details of affiliation

1Institute for Biomedical Research CSIC/UAM, c/ Arturo Duperier 4, 28029 Madrid (Spain)
2Spanish Network on Rare Diseases (CIBERER), Av. Monforte de Lemos 3-5, 28029 Madrid (Spain)
3Advanced Medical Projects, 28029 Madrid (Spain)

Funding

R.P laboratory is funded by grant PI14-01495 and PI17-01401 (Fondo de Investigaciones Sanitarias, Instituto de Salud
Carlos III, Spain, supported by FEDER funds) and CIBER 576/805_ER16PE06P2016.

Competing Interests:

The authors declare that they have no conflict of interest relating to the publication of this manuscript.

Keywords: idiopathic pulmonary fibrosis, telomeropathy, GSE4, TERT, TERC.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00035

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Fernández-Varas B, Rodríguez-Centeno J, Pintado-Berninches L, Manguán-García C, Sastre L, Perona, R. Therapeutic activity of GSE4 peptide in cellular model of idiopathic pulmonary fibrosis. IBJ Plus 2018 (S2):e00035 doi: 10.24217/2531-0151.18v1s2.00035.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Idiopathic pulmonary fibrosis (IPF) is considered a rare disease but also the most common manifestation within
telomeropathies, mainly in adulthood. Although 80% of families with IPF have not been genetically characterized, some
mutations have been described in hTERT, hTERC and other genes of the telomerase complex, causing telomerase activity
failure, telomeric shortening, DNA damage, senescence, inflammation and oxidative stress. GSE4-peptide, corresponding
to an internal domain of dyskerin, has proved to reverse these effects in dyskeratosis congenita (DC) cells, another
telomeropathy. Taking into account the therapeutic role of this peptide in DC, the objective of this work was to verify if
GSE4 is also effective reversing some of these parameters in different cellular models of IPF.

In the VA13 cell line, lung fibroblasts deficient in telomerase, we compared by TRAP assay the differences that occur in
telomerase activity by transiently expressing the hTERT/hTERC WT genes or different mutations associated with IPF in
these genes. In addition, we expressed GSE4 in these cells to check if the peptide corrects the deficiency in telomerase
activity generated by these mutations. In the same way, we expressed these mutations in a human fibroblast cell line
called FN1iib to compare the differences that occur in DNA damage, senescence and inflammation, and to check if GSE4
reduces some of these parameters delivering it by nanoparticles. DNA damage was analyzed by detection of γ-H2A.X
expression by western blot, and senescence and inflammation by measuring CDKN1A/CDKN2A and IL6 expression,
respectively, by q-RT-PCR. Moreover, we treated a rat alveolar cell line called RLE-6TN with bleomycin to induce fibrosis,
and we expressed GSE4 on it to check if the peptide reduces DNA damage, senescence and inflammation and if it
corrects the deficiency in telomerase activity.

We observed that all mutants show a deficiency in telomerase activity, being more severe in a hTERC mutant. In addition,
GSE4 manages to correct this deficiency in the most severe mutations, suggesting that it acts when telomerase activity
is lower. We verified that all mutants exhibit DNA damage and senescence, being higher in the hTERC mutants. Besides,
GSE4 is able to reduce DNA damage in all the mutants. These results agree with the fact that hTERC plays a main role in
the maintenance of the structure and function of the telomerase complex since mutations in this gene produce the most
severe alterations. Finally, in the rat cells model we saw that GSE4 reduces the levels of cellular DNA damage, senescence
and inflammation and increases telomerase activity, suggesting that this peptide could be a good therapeutic tool in IPF.

e00036
Evaluation of the physiopathology of the allan-herndon-dudley syndrome. A characterization of double knock-out mice model of
the disease.

García-Aldea A1, Guillen-Yunta M1, Grijota-Martínez C1, Rausell E1, Guadaño-Ferraz A1.

*Corresponding author:
Ana Guadaño Ferraz. E-mail: aguadano@iib.uam.es

Details of affiliation

1Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM),
Madrid, Spain

Funding

Competing Interests:

Keywords: Allan-Herndon-Dudley syndrome, hypothyroidism, synaptogenesis, microglia, astrocyte.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00036

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: García-Aldea A, Guillen-Yunta M, Grijota-Martínez C, Rausell E, Guadaño-Ferraz A. Evaluation of the physiopathology of the
allan-herndon-dudley syndrome. A characterization of double knock-out mice model of the disease. IBJ Plus 2018 (S2):e00036 doi:
10.24217/2531-0151.18v1s2.00036.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Thyroid Hormones (THs, T4 and T3) are essential in the development of the brain, regulating processes such as
differentiation of neural cells and synaptogenesis. THs are secreted into the blood from the thyroid gland, mainly as
T4, which is converted into T3, the nuclear active form, by the enzyme deiodinase 2 (DIO2), to exert genomic actions.
Allan-Herndon-Dudley Syndrome (AHDS) is associated to a X-linked condition caused by mutations in monocarboxilate
transporter 8 (MCT8), a transmembrane transporter highly specific for THs. AHDS is characterized by an endocrine and
a neurological syndrome with congenital hypotonia that progresses to spasticity with severe psychomotor impairment.
Evidences strongly suggest that the neurological syndrome in MCT8 deficiency is mainly due to a brain hypothyroidism,
since the access of THs across brain barriers is impaired.

We analyzed the expression of synaptic scaffold proteins and the expression of different proteins in different populations
of nerve cells in MCT8-deficient brain samples from an 11-year-old subject, in comparison to a control subject of the
same age. In parallel, we studied the synaptogenesis markers distribution in neurons and the expression of different
proteins in nerve cells in double knock-out mice for Mct8 and Dio2 brains, as a model for the disease. Protein expression
was analyzed by immunohistochemistry. Our results showed a decreased expression of most of the synaptic proteins
in the MCT8-deficient human brain in comparison to the control brain. To characterize glial cells, we evaluated the
expression of GFAP, and we observed an increase in the number of astrocytes. Additionally, to evaluate microglial cells,
we analyzed the expression of Iba-1. Our results revealed an increase in the number of Iba-1 immunopositive cells in the
MCT8-deficient human brain. We also observed an increase in the number of GFAP immunopositive astrocytes in the
cerebral cortex of Mct8/Dio2 knock-out mice. Regarding microglial cells, our results revealed an increase in the number
of Iba-1 positive cells, in the cerebral cortex and hippocampus of Mct8/Dio2 knock-out mice. To differentiate resting and
reactive microglial cells, we studied CD68 expression, a membrane protein much more abundant in reactive microglia
cells. Our results showed a significant increase in the expression of this protein in the brain of the double knock-out in
comparison to the wild-type mice. Our results suggest that synaptic transmission could be altered in patients with MCT8
deficiency, and the characterization of nerve cells suggests an inflammatory response due to MCT8 deficiency, what
could be related to the psychomotor delay.

e00037
Studying resistance to antivirals in single cells with Correlative Light and Electron microscopy.

Moisés García-Serradilla1* and Cristina Risco1*.

*Corresponding authors:
García-Serradilla, Moisés moises.garcia@cnb.csic.es, Risco, Cristina crisco@cnb.csic.es.

Details of affiliation

1Cell Structure Laboratory, National Center for Biotechnology, CNB-CSIC, UAM, Campus de Cantoblanco, 28049 Madrid, Spain.

Funding

This work is supported by grant BIO2015-68758-R from the Spanish Ministry of Economy, Industry and Competitiveness (to
Cristina Risco).

Competing Interests:

The authors declare that no competing interests exit.

Keywords: Bunyavirus, Ribavirin, Correlative Light and Electron Microscopy (CLEM), Antivirals, Drug Resistance.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00037

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: García-Serradilla M, Risco C. Studing resistance to antivirals in single cells with Correlative Light and Electron microscopy. IBJ
Plus 2018 (S2):e00037 doi: 10.24217/2531-0151.18v1s2.00037.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Development of highly effective, broad-spectrum antivirals is the major objective shared by the fields of
virology and pharmaceutics. High throughput screening of molecules has identified promising candidates for developing
optimal broad-spectrum antiviral agents. In our lab we study the replication factories of several pathogenic RNA viruses.
In the last few years we have developed new imaging techniques to study viruses in cells and applied these methods
to study the factories assembled by bunyaviruses. Several bunyaviruses are important pathogens for humans. Climate
change together with increased global trade and travel is causing a rapid dissemination of the vectors of bunyaviruses
and some other important arboviruses such as Dengue, Zika and Chikungunya. These viruses constitute a global threat
because there are currently no vaccines or specific antiviral drugs for many of them.

Material and methods: We have recently started to study viral infections at the level of single cells. With fluorescent
recombinant bunyaviruses and Correlative Light and Electron Microscopy (CLEM), we have studied the heterogeneous
response of cell populations to bunyavirus infection and to treatment with antivirals. The response of cells to the
antiviral treatment was studied by live cell video microscopy and confocal microscopy. Production of infectious viruses
was quantified by plaque assay and western blot.

Results: Vero cells infected with a fluorescent bunyavirus (Gc-GFP-BUNV) at different MOI (1, 5 and 10 PFU/cell) and
times post infection (0, 4, 6, 10, 15, 24 and 36 hp.i.) were treated with different concentrations of Ribavirin (10, 20, 32,
50 and 150 μg/mL). As expected, Ribavirin was non-toxic and efficient at 20, 32 and 50 μg/mL. Infection was blocked in
~ 90% of cells when the antiviral was added at 0 h p.i. Those cells where the virus escaped from the antiviral treatment
were selected and processed by CLEM and ultrastructural analysis. A small percentage of cells (~3%) presented a peculiar
phenotype. These cells expressed fluorescent viral proteins but did not exhibit the characteristic morphological changes
associated to infection, such as Golgi fragmentation.

Conclusions: The principal target site of Ribavirin in Vero cells infected by BUNV seems to reside in the RNA-dependent
RNA-polymerase (RdRp). Nucleus and nucleolus are shown as good markers of the infective status of cells. Further
studies to identify the role of the nucleolus in bunyavirus infection are in progress. Ribavirin inhibits BUNV replication
and cytopathic effect in Vero cells. CLEM in combination with cell sorting and biochemical analysis is revealing how
antiviral resistance is generated in cells.

e00038
Are the exosomes involved in the response to chemotherapy in cancer?.

Jiménez J1,2*, Rodríguez-Antolín C1,2, Rodríguez C3, Pernía O1,2, Rosas R1,2, De Castro Carpeño J2, Ibáñez de Cáceres I1,2.

*Corresponding author:
Julia Jiménez Hernandez. Cancer Epigenetics Laboratory (INGEMM). Experimental Therapies and Biomarkers in Cancer (IdiPAZ). University Hospital La
Paz. Paseo La Castellana 261. Edificio Bloque Quirúrgico Planta -2 28046 Madrid, Spain. e-mail: julia.jimenez.hdez@gmail.com

Details of affiliation

1Cancer Epigenetics Laboratory, INGEMM, La Paz University Hospital. Madrid, Spain.
2Biomarkers and Experimental Therapies in Cancer. IdiPAZ. Madrid, Spain.
3Metabolic diseases Laboratory, INGEMM, La Paz University Hospital. Madrid, Spain.

Funding

PI15/0186, Roche Farma and Fondos FEDER “Una Manera de hacer Europa”

Competing Interests:

Authors declare no conflict of interest.

Keywords: Exosomes, Chemoresistance, Non-Small Cell Lung Cancer (NSCLC).

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00038

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Jiménez J, Rodríguez-Antolín C, Rodríguez C, Pernía O, Rosas R, De Castro Carpeño J, Ibáñez de Cáceres I. Are the exosomes
involved in the response to chemotherapy in cancer? IBJ Plus 2018 (S2):e00038 doi: 10.24217/2531-0151.18v1s2.00038.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Non-small cell lung cancer accounts for the highest number of cancer-related deaths worldwide,
so does ovarian cancer when referring to gynecological tumors. This mortality rate is due to the advanced stage of
both diseases at diagnosis and its resistance to the standard therapy based on platinum-derived drugs. This makes it
essential to identify predictive biomarkers of response to these compounds, improving the use of available treatments
for each patient. Exosomes are small double-membrane vesicles released from most types of cells that are involved
in intercellular communication through transferring proteins, mRNAs and miRNAs. In the recent decades it has been
described that tumor-derived exosomes are involved in tumorigenesis and hence its potential as a source of cancer
biomarkers. However their role in the response to anti-tumor drugs is still poorly understood.

Objective: This project is focused on the study of the role of exosomes in response to anticancer treatment and the
epigenetic characterization of their content. That could allow identifying cisplatin-resistance biomarkers in NSCLC and
ovarian cancer exosomes to be used for liquid biopsies.
Material y Methods: Exosomes from cell culture medium, were isolated by ultracentrifugation. Cell viability curves
to cisplatin were developed incubating sensitive cells with resistant exosomes. Transcriptomic and proteomic
characterization of exosome content of three paired cell lines, sensitive and resistant, of lung and ovarian cancer were
carried out by small-RNAseq and LC-MS/MS respectively. Functional validation of the miRNA candidates was performed
overexpressing the specific mimics in sensitive cells followed by the treatment with increasing CDDP doses and analysis
of cell viability. Exosomal RNA from 60 NSCLC plasma samples was isolated using ExoRNeasy serum/plasma kit and the
analysis of the expression with TaqMan Advanced Assays by qRT-PCR is undergone.

Results: Firstly, our results showed a time of incubation-dependent acquisition of resistance to cisplatin of the sensitive
cells seeded with resistant exosomes. Secondly, the global comparison of protein and microRNA content of “resistant vs
sensitive” exosomes allowed us to identify 8 miRNA and 20 peptides highly represented in resistant exosomes compared
with sensitive ones. Four of those miRNAs were validated by qRT-PCR and currently we are testing their functional role
in the acquisition of resistance to cisplatin.

Conclusions: Exosomal miRNAs are involved, at least in part, in the acquisition of resistance to cisplatin in cancer and
therefore may be used as potential biomarker of response in liquid biopsy.

e00039
FADD phosphorylation is altered in human T-cell lymphoblastic lymphoma.

Marín-Rubio JL1,2,3, Barrios-Donoso C3, Cobos-Fernández MA1,2,3, Sastre I1, Fernández-Piqueras J1,2,3,4, Villa-Morales M1,2,3,4.

*Corresponding author:
María Villa-Morales, Madrid, Spain. E-mail: mvilla@cbm.csic.es
José Fernández-Piqueras, Madrid, Spain. E-mail: jfpiqueras@cbm.csic.es

Details of affiliation

1Centro de Biología Molecular Severo Ochoa (CBMSO), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSICUAM),
Madrid, Spain.
2IIS-Fundación Jiménez Díaz, Madrid, Spain.
3Universidad Autónoma de Madrid, Departamento de Biología, Madrid, Spain.
4Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Valencia, Spain.

Funding

This word was supported by grants from Spanish Ministry of Economy and Competitiveness (SAF2015-70561-R and SAF2012-
36566). Predoctoral fellowship from Spanish Ministry of Education, Culture and Sports (FPU13/00338).

Competing Interests:

The authors have declared that no competing interests exist.

Keywords: T-cell lymphoblastic lymphomas (T-LBL), FADD, phosphorylation, prognostic market.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00039

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Marín-Rubio JL, Barrios-Donoso C, Cobos-Fernández MA, Sastre I, Fernández-Piqueras J, Villa-Morales M. FADD phosphorylation
is altered in human T-cell lymphoblastic lymphoma. IBJ Plus 2018 (S2):e00039 doi: 10.24217/2531-0151.18v1s2.00039.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: T-cell lymphoblastic lymphoma (T-LBL) is an aggressive tumour type derived from immature thymocytes
in various differentiation stages. Although FADD canonical function is as a principal adaptor in apoptotic signalling, it has
become evident that FADD has a non-apoptotic role related to proliferation and cell cycle control which seems to depend
on its phosphorylation status. Moreover, aberrant phosphorylation of FADD in T-LBL samples inversely correlates with
the proliferation capacity and tumour aggressiveness.

Material and methods: FADD and S194-FADD protein levels were detected by immunohistochemistry in human T-LBL
biopsies which were provided by Biobanks integrated in the Spanish Hospital Biobanks Network. We generated four
stable cell lines by lentiviral transduction of the FADD-deficient cell line JURKAT I 2.1: a cell line expressing an empty
vector, thus FADD-deficient (NEG), a cell line expressing wildtype FADD (FADD), a cell line expressing a phosphomimetic
FADD mutant (S194D) and a cell line expressing a non-phosphorylatable FADD mutant (S194A). Cell proliferation were
analyzed using XTT assay. Cell cycle were performed by flow cytometry with propidium iodide. For protein stability
assays, the stable cell lines were treated with cycloheximide or anisomycin.

Results: 1) FADD presence rendered the cells significantly more proliferative, in comparison with FADD absence. 2)
The FADD-deficient and non-phosphorylatable mutant FADD cell lines are less sensitive to the G2/M-arrest induced by
mitotic inhibitors, strongly suggesting that both the presence and the status of FADD phosphorylation are involved in the
progression along the cell cycle. 3) The phosphomimetic S194D mutant FADD protein is remarkably more stable than
wild-type FADD or the non-phosphorylatable S194A mutant FADD, suggesting that phosphorylation at Ser194 positively
affects FADD protein stability.

Conclusions: FADD reduction would impair apoptosis in tumour cells, but we have demonstrated that the phosphorylation
status of FADD did not affect such apoptotic role. However, proliferation and cell cycle seem to be affected both by level
and phosphorylation of FADD protein. Thus, FADD phosphorylation can be used as a biomarker with prognostic value in
human T-LBL, to improve the clinical management of this rare disease and, in turn, to improve cancer survival.

e00040
Role of cell cycle checkpoint proteins in gastric cancer stem cells.

Pajuelo-Lozano N1, Perona R3,4,5, Sanchez-Perez I1,2,3,5.

*Corresponding author:
Natalia Pajuelo Lozano, Dpto. Bioquímica. Facultad Medicina. Instituto de Investigaciones Biomédicas CSIC-UAM; Madrid, Spain.
E-mail: npajuelo@iib.uam.es

Details of affiliation

1Dpto. Bioquímica. Facultad Medicina. Instituto de Investigaciones Biomédicas CSIC-UAM; Madrid, Spain.
2Unidad Asociada de Biomedicina UCLM-CSIC; Albacete, Spain.
3CIBER for Rare Diseases (CIBERER); Valencia, Spain.
4Instituto de Investigaciones Biomédicas CSIC/UAM; Madrid, Spain.
5Biomarkers and Experimental Therapeutics Group; IdiPAZ; University Hospital La Paz; Madrid, Spain.

Funding

This work was supported by PI1401495 (supported by FEDER funds) from Fondo de Investigaciones Sanitarias, Instituto de
Salud Carlos III, Spain

Competing Interests:

None declared

Keywords: Gastric cancer stem cells; tumorigenesis; Mad2.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00040

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Pajuelo-Lozano N, Perona R, Sanchez-Perez I. Role of cell cycle checkpoint proteins in gastric cancer stem cells. IBJ Plus 2018
(S2):e00040 doi: 10.24217/2531-0151.18v1s2.00040.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Gastric cancer (GC) is the fourth type of tumor more common and one of the first causes of death related with cancer
worldwide. The cancer stem cells (CSCs) hypothesis believes that few cells in cancer tissues fuel tumor growth. The
characteristics of these cells (self-regenerate, proliferation and multiple differentiation potentials) are responsible for
tumor maintenance, recurrence and resistance to therapy. In GC tissues, CSC populations (GCSCs) have been identified.
In addition to the known stem cell markers in CSCs, some cell cycle checkpoint proteins have been implicated in the
generation and maintenance of GCSCs, such as, Chk1, Mad2 and BubR1. In this study, we aimed to analyze the role of
these proteins in GCSCs and their relationship with tumorigenesis and therapy response. First, we isolated GCSCs from
different stablished GC cell lines (MKN45, ST2957 and SNU638), by sphere formation assay. RT-qPCR and Western Blot
analysis showed that Nanog is increased in all GCSCs, but OCT4 and SOX2 depend on the cell line. Furthermore, flow
cytometry demonstrated the increased of CXCR4 in GCSCs. Our data indicated that the expression levels of Mad2 are
increased in GCSCs and this situation exacerbates in differentiated cells. The interference of Mad2 decreased the capacity
of migration and invasion of GCSCs, indicating the involvement of Mad2 in metastasis. Surprisingly, downregulation of
Mad2 increased the generation of tumourospheres. Finally, GCSCs were more resistance to CDDP, BLM and PXL compared
with their parental cells; but downregulation of Mad2 do not clearly contributed to therapy response. As a conclusion,
our data demonstrated the high inter- and intra-tumor heterogeneity and that Mad2 could be a good prognostic marker.
More studies are needed to clarify the behavior of GCSCs, in order to identify easily those populations of cells and use
them in programs of discovery of new therapeutic agents.

e00041
Determination of IGFBP-3 methylation levels at ctDNA could be a prognostic biomarker in advanced stages of NSCLC.

Olga Pernía1,2*, Rocio Rosas1,2, Julia Jiménez1,2, Olga Vera1,2, Isabel Esteban3, Ana M. Rodríguez García3, Patricia Cruz-Castellanos4, Darío Sánchez-
Cabrero4, Javier de Castro2,4, Inmaculada Ibáñez de Caceres1,2.

*Corresponding author:
Olga Pernía Arias. Cancer Epigenetics Laboratory, INGEMM, Biomarkers and Experimental Therapeutics in Cancer, IdiPAZ, Paseo de la Castellana
261, Madrid, 28046, Spain Phone 34-91-2071010 ext 248 E-mail: olgacarpe@gmail.com.

Details of affiliation

1Cancer Epigenetics Laboratory, INGEMM, La Paz University Hospital, Madrid, Spain;
2Biomarkers and Experimental Therapeutics in Cancer, IdiPAZ, Madrid, Spain;
3Department of Pathology, La Paz University Hospital, Madrid, Spain;
4Department of Oncology, La Paz University Hospital, Madrid, Spain.

Funding

Supported by PI15/00186, RTC2016-5314-1 and Fondos FEDER, “una manera de hacer Europa”.

Competing Interests:

The authors declare not conflict of interest.

Keywords: Non-Small Cell Lung Cancer (NSCLC), Liquid Biopsy, Methylation, IGFBP3

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00041

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Olga Pernía, Rocio Rosas, Julia Jiménez, Olga Vera, Isabel Esteban, Ana M. Rodríguez García, Patricia Cruz-Castellanos, Darío
Sánchez-Cabrero, Javier de Castro, Inmaculada Ibáñez de Caceres. Determination of IGFBP-3 methylation levels at ctDNA could be a
prognostic biomarker in advanced stages of NSCLC. IBJ Plus 2018 (S2):e00041 doi: 10.24217/2531-0151.18v1s2.00041.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Non-small cell lung cancer (NSCLC) is the 80-85% of the lung cancer. Cisplatin-based chemotherapy is the
paradigm of NSCLC treatment; however, it also induces de novo DNA hyper methylation that is associated with gene
expression regulation. We have previously reported that the loss of IGFBP-3 expression by promoter hypermethylation
results in reduced tumor cell sensitivity to cisplatin in NSCLC. Liquid biopsies have gained increasing attention in recent
years, as they are a convenient and minimally invasive means of interrogating tumor DNA (ctDNA). Many studies have
shown concordant genetic and epigenetic alterations between ctDNA and corresponding tumor tissue DNA.

Objective: To study the correlation between the IGFBP-3 gene promoter methylation levels in both ctDNA and paired
tumor tissues from NSCLC patients with advanced stages.
Methodology: 52 FFPE/plasma paired samples from stages III-IV NSCLC patients have been collected in a prospective
study at Hospital La Paz together with the associated clinical history. ctDNA and tumor tissue DNA was isolated and
bisulfite modified. We then measured the methylation of the IGFBP3-promoter by qMSP. To obtain the percentage of
methylation for each sample we use the following equation: Cmeth = 100/ [1+2(CTCG– CTTG)]

Results: When comparing DNA methylation levels from tumor and ctDNA, 69% (33 out of 48) of the patients followed
the same methylation pattern. To determine whether IGFBP-3 methylation data obtained from ctDNA correlates with
overall survival (OS) we analyzed 34 NSCLC from our prospective cohort from which we had already collected clinical data
associated with progression or decease. We also included a small group of 4 control samples, to set the percentage of
baseline methylation levels at circulating DNA, that was 10%. The NSCLC samples were separated into 2 groups based on
their IGFBP-3 methylation levels compared with the control group. The survival functions were plotted using the Kaplan-
Meier estimator. We found a clear tendency for a better survival in those patients with lower IGFBP-3 methylation
levels at ctDNA in advanced stages NSCLC patients. Twenty three out of 34 patients (67%), harbored an unmethylated
promoter and 11 out of 34 a methylated promoter, these last showed an evident less survival.

Conclusion: DNA methylation status was determined in 48 out of 52 paired samples showing a 92% sensitivity and a
positive correlation between tumor/ctDNA from the same patient.
These results indicate that the determination of IGFBP-3 methylation levels at ctDNA could be an independent prognostic
marker, in terms of Overall survival, in advanced stages NSCLC patients that could potentially be used in liquid biopsy.

e00042
Identification of a novel epigenetic biomarker of early lung cancer detection in COPD patients.

C Rodríguez-Antolín1,2*, R Rosas1,2*, R Galera3*, J J Sánchez-Pascuala1,2, R Casitas3*, L Felguera, O Pernía1,2, O Vera1,2, J De Castro1,2, F García-Rio
3,5#, I Ibáñez de Caceres1,2.

*Corresponding author:
Carlos Rodriguez Antolín. Cancer Epigenetics Laboratory, INGEMM, Biomarkers and Experimental Therapeutics in Cancer, IdiPAZ, Paseo de la
Castellana 261, Madrid, 28046, Spain Phone 34-91-2071010 ext 248 E-mail: rodriguez.antolin.c@gmail.com.

Details of affiliation

1Cancer Epigenetics Laboratory, INGEMM, La Paz University Hospital, Madrid, Spain;
2Biomarkers and Experimental Therapeutics in Cancer, IdiPAZ, Madrid, Spain;
3Department of Respiratory Diseases, La Paz University Hospital, IdiPAZ, Madrid, Spain;
4Autonomous University of Madrid, Madrid, Spain

Funding

Supported by PI15/00186

Competing Interests:

The authors declare not conflict of interest.

Keywords: COPD, lung cancer, miR-X, Methylation

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00042

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: C Rodríguez-Antolín, R Rosas, R Galera, J J Sánchez-Pascuala, R Casitas, L Felguera, O Pernía, O Vera, J De Castro, F García- Rio, I Ibáñez de Caceres. Identification of a novel epigenetic biomarker of early lung cancer detection in COPD patients. IBJ Plus 2018
(S2):e00042 doi: 10.24217/2531-0151.18v1s2.00042.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Chronic obstructive pulmonary disease (COPD) is a health problem with high prevalence. Currently, it is the fourth
leading cause of death in the world, with an increased incidence, which placed it in the coming years in third place globally. Among
the leading causes of death in these patients, lung cancer represents the 14-26,5% of all deaths. These data, together with the high
prevalence of COPD in lung cancer patients, have shown that COPD is an independent risk for lung cancer factor, increasing in 2-5 times
its incidence rates with respect to smoker subjects without COPD. Changes at the DNA methylation profile from non-neoplastic tissues
from lung cancer patients could be used to identify potential individuals with higher risk of cancer development. Previous reported
studies from our group showed that the methylation of miR-X is an event related to tumor progression in ovarian and lung cancer cells.

Objective: The status of the epigenetic mark involving DNA-methylation of miR-X regulatory region could play a role as new biomarker
for clinical use to identify patients at high risk of developing lung cancer among the smoking population with chronic respiratory diseases.

Methodology: We collected the DNA from buccal epithelial samples from 124 stable COPD patients by using buccal swaps at the
clinical consultation. DNA was isolated and bisulfite modified. We evaluated the methylation status of the mir-X by qMSP
by using double probes. To obtain the percentage of methylation for each sample we use the following equation: Cmeth
= 100/ [1+2(CTCG– CTTG)]

Results: The miR-X levels of buccal epithelial samples between the several phenotypes of COPD were: 19.4±9.9 in exacerbator
phenotype, 27.1±10.2 in emphysema, 17.3±9.0 in chronic bronchitis and 16.0±7.2 in asthma-COPD overlap. miR-X levels
were significantly higher in emphysema phenotype ( p<0.05 vs ACO and p<0.01 vs exacerbator and chronic bronchitis). In addition,
univariate linear regression models of predictors of miR-X methylation in COPD patients showed a significant Pearson’s
correlation with static hyperinflation (functional residual capacity (FRC)/ total lung capacity (TLC)) (p<0.001) and diffusion capacity
for carbon monoxide (DLCO) (p<0.001). Both variables are related to emphysema, which is the phenotype associated with
higher risk of lung cancer development.

Conclusion: The presence of high rates of miR-X methylation levels allows identifying a subgroup of patients with chronic
airflow limitation that need to be closely followed with image techniques in order to identify early stages of lung cancer tumor. The early
diagnosis in lung cancer patients is associated with a better outcome in terms of overall survival and therapeutic response.

e00043
VMP1 controls lipid trafficking at ER contact sites.

Tábara LC.1*, Vincent O.1, Escalante R.1.

*Corresponding author:
Email: lctabara@iib.uam.es

Details of affiliation

1Instituto de Investigaciones Biomédicas Alberto Sols; C.S.I.C./U.A.M.; 28029-Madrid, Spain

Funding

This work was supported by grants BFU2012-32536, BFU2015-64440-P (MINECO/FEDER). Luis C. Tábara was recipient of a
FPU fellowship from the Spanish Ministerio de Educación, Cultura y Deporte.

Competing Interests:

The authors declare no conflict of interest.

Keywords: VMP1, Endoplasmic Reticulum, Membrane Contact Sites.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00043

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Tábara LC, Vincent O, Escalante R. VMP1 controls lipid trafficking at ER contact sites. IBJ Plus 2018 (S2):e00043 doi:
10.24217/2531-0151.18v1s2.00043.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: VMP1 is an endoplasmic reticulum (ER) protein of unknown function which is highly conserved
among metazoans but it is absent in yeast. VMP1 has been considered an autophagic protein since around 60% of
autophagosomes originates from VMP-enriched ER. However, only a little proportion of VMP1 puncta (5%) are engaged
with autophagy suggesting that VMP1 could have additional functions beside autophagosome biogenesis.

Material and methods: Detailed confocal miscroscopy was used to study VMP1 dynamics in mammalian cell culture.
Results: VMP1 is in close proximity to various organelles besides autophagosome at ER-organelles contact sites. Moreover,
VMP1 is enriched at ER subdomains where phospholipid synthesizing enzymes are accumulated. Thus, VMP1-enriched
ER might function as a platform for phospholipid synthesis and lipid trafficking between organelles. Interestingly, VMP1
regulates the length of Membrane Contact Sites (MCS) between ER and endosomes and is required for proper lipid
trafficking between both organelles.

Conclusion: Our results demonstrate for the first time a function for VMP1 in endosome maturation in mammalian cells
and suggest that its role could be in maintaining lipid homeostasis in the interface between ER and other organelles.

e00044
Synaptic T cell contacts prime DC against pathogen infection.

Daniel Torralba1,2, Francesc Baixauli1,3, Carolina Villarroya-Beltri1,2, Irene Fernández-Delgado1,2, Jose Antonio Enríquez4, Maria Mittelbrunn5,
Francisco Sánchez-Madrid1,2*.

*Corresponding author:
Francisco Sánchez-Madrid, 1Vascular Pathophysiology Research Area, Centro Nacional Investigaciones Cardiovasculares (CNIC), Madrid, Spain. Servicio de
Inmunología, Instituto Investigación Sanitaria Princesa, Universidad Autónoma de Madrid, Madrid, Spain.
E-mail: fsmadrid@salud.madrid.org

Details of affiliation

1Vascular Pathophysiology Research Area, Centro Nacional Investigaciones Cardiovasculares (CNIC), Madrid, Spain.
2Servicio de Inmunología, Instituto Investigación Sanitaria Princesa, Universidad Autónoma de Madrid, Madrid, Spain.
3Immunometabolism Department, Max Planck Institute for Immunobiology and Epigenetics, Freiburg, Germany.
4Myocardial Pathophysiology Research Area, Centro Nacional Investigaciones Cardiovasculares (CNIC), Madrid, Spain
5Centro de Biología Molecular, UAM-CSIC, Departamento de Biología Celular e Inflamación, Madrid, Spain

Funding

This study was supported by grants SAF2017/82886-R from the Spanish Ministry of Economy and Competitiveness, CAM
S2017/BMD-3671 from the Comunidad de Madrid, CIBER Cardiovascular (Fondo de Investigación Sanitaria del Instituto de Salud
Carlos III and co-funding by Fondo Europeo de Desarrollo Regional FEDER), ERC-2011-AdG 294340-GENTRIS and COST-Action BM1202
to F.S.-M.; D.T is funded by La Caixa Foundation.; grant SAF2015-65633-R from the Spanish Ministry of Economy and Competitiveness
to J.A.E. M.M. is supported by MS14/00219 from Instituto de Salud Carlos III. Centro Nacional de Investigaciones Cardiovasculares
(CNIC) is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation, and is a Severo
Ochoa Center of Excellence (MINECO award SEV-2015-0505).

Competing Interests:

No Competing interest.

Keywords: immune intercellular contacts, extracellular vesicles, exosomes,

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00044

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Daniel Torralba, Francesc Baixauli, Carolina Villarroya-Beltri, Irene Fernández-Delgado, Jose Antonio Enríquez, Maria
Mittelbrunn, Francisco Sánchez-Madrid. Synaptic T cell contacts prime DC against pathogen infection. IBJ Plus 2018 (S2):e00044 doi:
10.24217/2531-0151.18v1s2.00044.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Diverse quality control mechanisms ensure mitochondrial functioning during stress conditions. Mitochondrial dysfunction
enhances exosome secretion dependent on reactive oxygen species (ROS) signaling alleviating its consequences. We
demonstrate that mitochondrial components are transferred through exosomes between immune cells during cognate
immune interactions. It has been deeply studied how T cell contacts with antigen-bearing dendritic cells (DCs) result in T
cell activation. However, whether this interaction has physiological consequences on DC function is largely unexplored.

Using proteomics, deep RNA sequencing, flow cytometry and high resolution microscopy techniques we describe a new
mechanism of intercellular communication between immune cells.
In this study we show that antigen-dependent T cell contacts with DCs trigger anti-pathogenic programs in the latter,
priming them against pathogen infection. Information is transmitted through exosomes from the T cell to the DC. T
cell transfer of exosomes to DCs triggers transcellular antiviral responses. These events confer resistance to DCs to
subsequent viral infections. Together, our results reveal that T cells prime DCs, supporting a specific role for antigendependent
contacts in conferring protection to DCs against pathogen infection.

e00045
Computational metabolism modeling predicts drug sensitivity in breast cancer cells.

Lucía Trilla-Fuertes1,2, Mariana Díaz-Almirón3, Angelo Gámez-Pozo1,2, Guillermo Prado-Vázquez1, Andrea Zapater-Moros1, Sara Llorente-
Armijo1,Francisco Gayá Moreno3, Rosa Aras-Lopez4, Enrique Espinosa5, Juan Ángel Fresno Vara1.

*Corresponding author:
Lucía Trilla-Fuertes, Hospital Universitario La Paz, Biomedica Molecular Medicine SL. , Madrid, Spain. E-mail: lucia.trilla@biomedicamm.com

Details of affiliation

1Molecular Oncology & Pathology Lab, Instituto de Genética Médica y Molecular-INGEMM, Hospital Universitario La Paz-IdiPAZ, Madrid, Spain.
2Biomedica Molecular Medicine SL, Madrid, Spain.
3Biostatistics Unit, Hospital Universitario La Paz, Madrid, Spain
4Congenital Malformations Lab, Instituto de Genética Médica y Molecular-INGEMM – Hospital Universitario La Paz -IdiPAZ, Madrid, Spain,
5Medical Oncology Service, Hospital Universitario La Paz -IdiPAZ, Madrid, Spain.

 

NOT AVAILABLE AT THIS MOMENT

e00046
The role of Gasdermin B in ulcerative colitis and inflammation.

María Pérez-López1, Lidia Martínez1, Eva Díaz-Martín3, Alejandro Rojo-Sebastián2,3, David Sarrió1,3*, Gema Moreno-Bueno1,2,3*.

*Correspondings authors: Gema Moreno-Bueno & David Sarrió, UAM-IIBm, Madrid. E-mail: gmoreno@iib.uam.es

Details of affiliation

1Universidad Autónoma de Madrid & Instituto de Investigaciones Biomédicas (IIBm-UAM), c/ Arzobispo Morcillo 2, 28029 Madrid-Spain.
2Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Monforte de Lemos 3-5, 28029 Madrid, Spain.
3Traslational research lab &Pathology Department; MD Anderson Cancer Center, C/ Arturo Soria 270, 28033 Madrid-Spain

 

NOT AVAILABLE AT THIS MOMENT

e00047
“Insulin receptor substrate 2 (IRS2) deficiency reduces inflammatory and fibrogenic responses of the liver to cholestatic injury”.

Andrea Villar-Lorenzo1, Patricia Rada1, Carmelo García-Monzón2, Ángela M Valverde1, Águeda González-Rodríguez2.

*Corresponding author:
Águeda González-Rodríguez, Unidad de Investigación, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria Princesa, CIBERehd,
Madrid, Spain. E-mail. aguedagr.phd@gmail.com

Details of affiliation

1Instituto de Investigaciones Biomédicas “Alberto Sols” (CSIC), CIBERdem, Madrid, Spain.
2Unidad de Investigación, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria Princesa, CIBERehd, Madrid, Spain.

Funding

Competing Interests:

Keywords: Liver, Cholestasis, IRS2

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00047

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Andrea Villar-Lorenzo, Patricia Rada, Carmelo García-Monzón, Ángela M Valverde, Águeda González-Rodríguez. “Insulin
receptor substrate 2 (IRS2) deficiency reduces inflammatory and fibrogenic responses of the liver to cholestatic injury”. IBJ Plus 2018
(S2):e00047 doi: 10.24217/2531-0151.18v1s2.00047.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Hepatocellular injury is the major triggering event of the wound healing response that leads to liver fibrosis.
As insulin receptor substrate 2 (IRS2) is one of the key downstream mediators of insulin signaling pathway, which play
major roles in liver disease, we investigated whether IRS2 influences the hepatocellular stress response in the liver.
Methods: For that goal, cholestatic liver injury was induced by bile duct ligation (BDL) in wild-type (WT) and IRS2-
deficient (IRS2KO) mice. Histological analysis, inflammatory and fibrogenic responses were evaluated in livers from these
mice 3, 7 and 28 days following BDL.

Results: Although no differences between genotypes were found at the end of the experiment (28 days), IRS2KO
mice displayed less BDL-induced liver histological alterations, including hepatocyte damage and excess deposition
of extracellular matrix components compared to WT mice after 3 and 7 days. Moreover, hepatic expression levels of
collagen 1 alpha, transforming growth factor 1 and smooth muscle actin were all lower in IRS2KO mice than in WT
animals after 3 and 7 days. In parallel, mRNA expression of pro-inflammatory cytokines such us tumor necrosis factor
alpha and interleukin 6 was also reduced in livers from IRS2KO mice at these time points. Interestingly, hemeoxygenase
1 expression, used as a maker of oxidative stress, was also decreased in livers lacking IRS2.

Conclusions: Taken together, our results indicate that IRS2 contributes to the progression of cholestatic liver injury since
its deficiency reduced inflammatory and fibrogenic responses induced by BDL. Modulation of this protein represents a
potential therapeutic strategy for cholestatic liver diseases.

e00048
Functional analysis of Mastl mutations in cancer.

M Maroto1, M Trakala2, B Hurtado1, M Malumbres1.

*Corresponding author:
M. Malumbres, Spanish National Cancer Research Centre (CNIO), Madrid, Spain. E-mail: mmm@cnio.es

Details of affiliation

1Spanish National Cancer Research Centre (CNIO), Madrid, Spain
2Massachusetts Institute of Technology (MIT) Koch institute for Integrative Cancer Research, Cambridge, Massachussets

Funding

FPI, MINECO

Competing Interests:

No Competing interest.

Keywords: Mastl, frameshift mutation, MSI, CRC.

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00048

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: M Maroto, M Trakala, B Hurtado, M Malumbres. Functional analysis of Mastl mutations in cancer. IBJ Plus 2018 (S2):e00048
doi: 10.24217/2531-0151.18v1s2.00048.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Mastl, also known as Greatwall, is a protein kinase essential for proper chromosome condensation and
progression through mitosis and meiosis. It belongs to the AGC kinase family and, particularly, presents a non-conserved
insertion of 550 aa at the corresponding T-activation loop site in the C-lobe (usually 20-30 aa). This non-conserved
middle region (NCMR) is not considered to have an essential role for Mastl activity but its function in unknown. Mastl
is involved in the inhibition of protein phosphatase 2A (PP2A)-B55 complexes to maintain the mitotic state. By using a
conditional knockout in mouse generated in our lab, it was shown that mammalian Mastl is essential for mouse embryonic
development and cell cycle progression. Mastl was initially found in humans as a gene mutated in thrombocytopenia
and preliminary data suggests its overexpression in tumors. However, very little is known about this protein in human
disease.

Material and methods: Genomic data from repositories of cancer somatic mutations include MASTL NCMR indel
mutations, leading to the generation of a truncated protein. Exome sequencing studies in Mastl in gastric cancers show
that mutant tumors present microsatellite instability (MSI). We have studied the prevalence of these mutations by
sequencing MASTL in a subset of samples from colon and stomach patients.
To evaluate the therapeutic relevance of this kinase, functional assays have been performed, as complementation
studies and kinase assays.
To mimic the cancer mutations we have generated a new mouse model using CRISPR/Cas9 technology. We are currently
performing several assays such as focus assays, scratch assays, soft agar and colony formation on cells derived from
this model. In addition, we are using a chemical-induced colorectal carcinoma model to study the role of these mutant
kinases in cancer.

Results: A heterozygous exonic indel mutation has been found in an MSI+ CRC from the 21 patient samples sequenced.
Functional assays with the mutant enzyme resulted in a partial rescue in DNA segregation observed in Mastl-null cells.
Mastl mutant forms resulted in embryonic lethality in homozygosis. Therefore, our carcinogenesis models are performed
in heterozygous mice, thus mimicking cancer mutations.
Conclusions: Mastl indel mutations in the NCMR region lead to the expression of truncated shorter forms. These Mastl
mutated forms are not able to fully accomplish the role of Mastl in mitosis. Mutant heterozygous mice, mimicking
MASTL cancer mutations, are viable and fertile.

e00049
Deciphering the RNA-dependent DNA synthesis fidelity of retroviral reverse transcriptases: impact of transcriptional inaccuracy threshold.

Alba Sebastián-Martín1, Verónica Barrioluengo1,2 and Luis Menéndez-Arias1,*

*Corresponding author:
Luis Menéndez-Arias, Centro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Científicas & Universidad Autónoma de
Madrid). C/ Nicolás Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain. E-mail: lmenendez@cbm.csic.es

Details of affiliation

1Centro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid). C/ Nicolás
Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain
2Present address: DiaSorin Iberia S.A., Avenida de la Vega 1, 28108, Alcobendas (Madrid), Spain.

Funding

This work was supported by grants of the Spanish Ministry of Economy and Competitiveness (BIO2013–48788-C2-1-R and
BIO2016-76716-R (AEI/FEDER, UE)), and an institutional grant (to CBMSO) of the Fundación Ramón Areces. A. S.-M. is a recipient of a
predoctoral fellowship of the Spanish Ministry of Education, Culture and Sport (FPU2013-00693).

Competing Interests:

The authors declare that they have no competing interests.

Keywords: reverse transcriptase, fidelity, retrovirus, RNA polymerase, HIV, transcription

Published May 18, 2018.

DOI: 10.24217/2531-0151.18v1s2.00049

Copyright: © 2018 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Alba Sebastián-Martín, Verónica Barrioluengo, Luis Menéndez-Arias. Deciphering the RNA-dependent DNA synthesis fidelity
of retroviral reverse transcriptases: impact of transcriptional inaccuracy threshold. IBJ Plus 2018 (S2):e00049 doi: 10.24217/2531-
0151.18v1s2.00049.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

In retrovirus, the reverse transcriptase (RT) is the enzyme responsible for the replication of the viral genome. RTs are widely
used in biotechnology for their ability to synthesize complementary DNA using RNA templates. Despite its importance,
the fidelity of RNA-dependent DNA polymerization catalyzed by RTs has not been properly determined, although there
are many studies reporting on the accuracy of the enzyme using DNA templates. In M13mp2 lacZα forward mutation
assays measuring intrinsic fidelity of DNA-dependent DNA synthesis, wild-type human immunodeficiency virus type 1
(HIV-1) RTs of group M/subtype B previously showed >10-fold higher error rates than murine leukaemia virus (MLV) and
avian myeloblastosis virus (AMV) RTs.

Here, we provide error rates and mutant frequencies in RNA-dependent DNA synthesis reactions for several RTs, including
wild-type HIV-1BH10, HIV-1ESP49, AMV and MLV RTs, and the high-fidelity mutants of HIV-1ESP49 RT K65R and K65R/V75I, by
using an adapted version of the forward mutation assay. In order to analyze the transcriptional inaccuracy threshold of
the RNA template, kinetics of T7 RNA polymerase were also studied by using single-nucleotide incorporation assays with
correct and incorrect nucleotides using different transcription conditions (pH and Mg2+ concentration).
Our results showed that there were less than two-fold differences in fidelity between the studied RTs, with error rates
ranging from 2.5×10-5 to 3.5×10-5. These results were consistent with the existence of a transcriptional inaccuracy
threshold, generated by the RNA polymerase while synthesizing the RNA template used in the assay. A modest but
consistent reduction of the inaccuracy threshold was achieved by lowering the pH and Mg2+ concentration of the
transcription reaction.

Despite assay limitations, we conclude that HIV-1BH10 and HIV-1ESP49 RTs are less accurate when copying DNA templates
than RNA templates. Analysis of the RNA-dependent mutational spectra revealed a higher tendency to introduce large
deletions at the initiation of reverse transcription by all HIV-1 RTs except the double-mutant K65R/V75I. With RNA
templates (as well as with DNA), the HIV-1BH10 RT remained as the least accurate enzyme, while the K65R/V75I mutant
was one of the most faithful RTs. A better assessment of reverse transcription fidelity is expected to be helpful to improve
next-generation sequence platforms that require retroviral RTs for RNA sequencing.