Abstracts of 3rd Phd Research Symposium in Health Sciences and Biomedicine

e00001
Role of CD39 in COVID-19 Severity: Dysregulation of Purinergic Signaling and Thromboinflammation.

Elena Díaz-García 1 2, Sara García-Tovar 1, Enrique Alfaro 1, Ester Zamarrón 1 2, Alberto Mangas 1, Raúl Galera 1 2, José Juan RuízHernández 3, Jordi Solé-Violán 2 4, Carlos Rodríguez-Gallego 5 6, Ana Van-Den-Rym 7 8, Rebeca Pérez-de-Diego 7 8, Kapil NanwaniNanwani 9, Eduardo López- Collazo 10, Francisco García-Rio 1 2 11, Carolina Cubillos-Zapata 1 2 *.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00001

Copyright: © 2022 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: Díaz-García E., García-Tovar S., Alfaro E., Zamarrón E., Mangas A., Galera R., Ruíz-Hernández J.J., Sole-Violán J., Rodríguez-Gallego C., VanDen-Rym A., Pérez-de-Diego R., Nanwani-Nanwani K., López-Collazo E., García-Río F. and Cubillos-Zapata C. Role of CD39 in COVID-19 Severity: Dysregulation of Purinergic Signaling and Thromboinflammatio. IBJ Plus 2022 1(s5) e00001. DOI: 10.24217/2531-0151.22v1s5.00001

Edited: Madrid, España.

Abstract

CD39/NTPDase1 has emerged as an important molecule that contributes to maintain inflammatory and coagulatory homeostasis. Various studies have hypothesized the possible role of CD39 in COVID-19 pathophysiology since no confirmatory data shed light in this regard. Therefore, we aimed to quantify CD39 expression on COVID-19 patients exploring its association with severity clinical parameters and ICU admission, while unraveling the role of purinergic signaling on thromboinflammation in COVID-19 patients. We selected a prospective cohort of patients hospitalized due to severe COVID-19 pneumonia (n=75), a historical cohort of Influenza A pneumonia patients (n=18) and sex/age-matched healthy controls (n=30). CD39 was overexpressed in COVID-19 patients’ plasma and immune cell subsets and related to hypoxemia. Plasma soluble form of CD39 (sCD39) was related to length of hospital stay and independently associated with intensive care unit admission (adjusted odds ratio 1.04, 95%CI 1.0-1.08, p=0.038), with a net reclassification index of 0.229 (0.118-0.287; p=0.036). COVID-19 patients showed extracellular accumulation of adenosine nucleotides (ATP and ADP), resulting in systemic inflammation and pro-coagulant state, as a consequence of purinergic pathway dysregulation. Interestingly, we found that COVID-19 plasma caused platelet activation, which was successfully blocked by the P2Y12 receptor inhibitor, ticagrelor. Therefore, sCD39 is suggested as a promising biomarker for COVID-19 severity. As a conclusion, our study indicates that CD39 overexpression in COVID-19 patients could be indicating purinergic signaling dysregulation, which might be at the basis of COVID-19 thromboinflammation disorder

e00002
Obesity-associated renal alterations are mediated by Microsomal Prostaglandin E Synthase-1 (mPGES-1) in a sex-dependent manner.

Ballesteros-Martinez C1, Rodrigues-Diez R1,2,3, Salaices M1,2,3, Briones AM1,2,3.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00002

Copyright: © 2022 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: Ballesteros-Martinez C., Rodrigues-Diez R., Salaices M. and Briones AM. Obesity-associated renal alterations are mediated by Microsomal Prostaglandin E Synthase-1 (mPGES-1) in a sex-dependent manner. IBJ Plus 2022 1(s5) e00002. DOI: 10.24217/2531-0151.22v1s5.00002

Edited: Madrid, España.

Abstract

Introduction: Obesity is characterized by a chronic-low grade inflammation that causes pathophysiological alterations in different target organs, contributing, among others, to the development of renal damage. Microsomal Prostaglandin E Synthase 1 (mPGES-1) is an inducible isomerase responsible for the overexpression of prostaglandin E2 (PGE2) under inflammatory conditions, including obesity. Inhibition of PGE2 production by non-steroidal anti-inflammatory drugs, that block cyclooxygenase-2 (COX-2) cascade, limits inflammatory processes. However, it can be associated with side- effects as nephrotoxicity due to the inhibition of other prostaglandins production. New drugs targeting mPGES-1 have shown promising results in different experimental models. Nevertheless, to our knowledge, efficacy of inhibition downstream COXs in the renal impact of obesity remains unexplored.

Objective: To elucidate whether mPGES-1 depletion could prevent the development of renal alterations in an experimental model of obesity.

Methods: We have developed a model of HFD-induced obesity (60% fat, 13 weeks) in males and females mPGES-1-/- and their control littermates mPGES-1+/+ mice. Renal structural alterations and changes in mRNA expression were studied by histological analysis and q-RT-PCR, respectively.

Results: IHFD decreased renal mPGES-1 gene expression in females but not in males. On the contrary, mPGES-1 expression was upregulated in perirenal adipose tissue (PRAT) of both sexes. In females, HFD induced kidney and PRAT hypertrophy that was prevented by the absence of mPGES-1; whereas in males, HFD did not modify kidney weight, while increasing PRAT weight significantly more in mPGES-1+/+ than in mPGES-1-/- mice. Rt-qPCR studies revealed that HFD induced renal damage, inflammation and fibrosis in mPGES-1+/+ male mice, which was not observed in mPGES-1 deficient mice. In agreement, histological studies showed that HFD induced glomerular hypertrophy and renal fibrosis only in mPGES-1+/+ HFD males. These alterations were not likely mediated by oxidative stress. Of note, females were protected against renal inflammatory and fibrotic alterations induced by HFD. Additionally, mPGES-1 deficiency prevented HFD- induced PRAT inflammation, fibrosis and hypoxia in both males and females. HFD did not modify serum proteinuria in any sex.

Conclusion: mPGES-1 participates in the renal alterations associated with obesity in a sex-dependent manner.

e00003
Early use of corticosteroids and sarilumab in
SARS-Cov-2 pneumonia.

Caballero-Bermejo AF1, Sancho-López A1, Ussetti-Gil P2, Fernández-Cruz A3, Avendaño-Solá C1, Ruiz-Antorán B1.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00003

Copyright: © 2022 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: Caballero-Bermejo AF., Sancho-López A., Ussetti-Gil P., Fernández-Cruz A., Avendaño-Solá C. and Ruiz-Antorán B. Early use
of corticosteroids and sarilumab in SARS-Cov-2 pneumonia. IBJ Plus 2022 1(s5) e00003. DOI: 10.24217/2531-0151.22v1s5.00003.

Edited: Madrid, España.

Abstract

The cytokine storm underlying the inflammatory phase of COVID19 has been suggested since the beginning of
the pandemic as a target for treatment of the disease. The identification of the presence of elevated IL6 levels in
patients with severe SARS-CoV-2 infection led to the hypothesis that the use of already marketed drugs whose
mechanism of action is the inhibition of IL6 could result in clinical benefit for these patients. We hypothesised
that the use of glucocorticoid-associated sarilumab in early stages of the proinflammatory phase of COVID19
could prevent progression to respiratory distress syndrome and death.
Hospitalised patients with SARS-CoV2 pneumonia, treated with nasal cannula and with analytical parameters of
inflammation, were randomised (1:1 ratio) to receive sarilumab + standard therapy (sarilumab group) or standard
therapy (control group). In all cases, standard therapy included glucocorticoid treatment for at least 3 days at
a dose equivalent to methylprednisolone mg/kg/day. The primary endpoint was the percentage of patients
who progressed to severe respiratory failure (defined as a BRESCIA-COVID score ≥3), intensive care unit (ICU)
admission or death at any time up to day 15.
A total of 201 patients were included, of whom 99 were randomised to the sarilumab group and 102 to the
control group. The percentage of patients who progressed to severe respiratory failure (BRESCIA≥3), ICU
admission or death at any time up to day 15 was 16.6% in the sarilumab group and 15.69% in the control group
(RR 1.03; 95% CI, 0.48 to 2.20). No differences in the safety profile between the two groups were detected.
Mortality at day 28 was 2.02% in the sarilumab group vs. 1.96% in the control group (RR 1.03; 95% CI 0.14 – 7.46).
In conclusion, an early intervention with sarilumab and glucocorticoids did not demonstrate clinical benefit in our
study population compared to standard therapy.

e00004
Clinical and Immunometabolic Patterns Determining Efficacy of DCtreatment
reinvigorating HIV-1-specific CD8+ T cells in PLWH.

Marta Calvet-Mirabent1,2, Ildefonso Sánchez-Cerrillo1, 2, Noa Martín-Cófreces1,2,3, Hortensia de la Fuente1,3, Ilya Tsukalov2, Cristina Delgado-
Arévalo1,2, María José Calzada2, Ignacio de los Santos4,7, Jesús Sanz4,7, Lucio García-Fraile4,7, Francisco Sánchez-Madrid1,2,3, Arantzazu Alfranca1,
María Ángeles Muñoz-Fernández5, Maria J. Buzón6, Enrique Martín-Gayo1,2,7.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00004

Copyright: © 2022 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: Calvet-Mirabent M., Sánchez-Cerrillo I., Martín-Cófreces N., De la Fuente H., Tsukalov I., Delgado-
Arévalo C., Calzada MJ., De los Santos I., Sanz J., García-Fraile L., Sánchez-Madrid F., Alfranca A.,
Muñoz-Fernández MA., Buzón M.J. and Martín-Gayo E. Clinical and Immunometabolic Patterns Determining Efficacy of DC-treatment reinvigorating
HIV-1-specific CD8+ T cells in PLWH IBJ Plus 2022 1(s5) e00004. DOI: 10.24217/2531-0151.22v1s5.00004.

Edited: Madrid, España.

Abstract

Introduction: Heterogeneous dysfunctional states of CD8+ T cells in people living with HIV-1 (PWLH) has limited
the efficacy of dendritic cell (DC)-based immunotherapies. Here, we studied associations between improved
functional response to Gag-loaded adjuvant-primed DCs of CD8 T cells from PLWH with ART duration, memory
subset distribution and exhaustion and metabolic profiles in these cells.

Methods: A cohort of n=49 PLWH on ART with undetectable plasma viremia and CD4+ T counts above 400cells/
ml were recruited. Monocyte-derived DC were activated with Poly I:C and 2´3´cdiAM(PS)2 adjuvants in the
presence of a pool of HIV-1 Gag peptides and co-cultured with autologous CD8+ T cells. Induction and polyfunctionality
of HIV- 1 specific CD8+ T responses was evaluated by IFNγ and CD107a expression by FACS. Functionality
of DC- stimulated CD8+ T cells was evaluated by co-culture with autologous CD4+ T cells and the ability to reduce
proportions of p24+ CD4+ T cells. Individual or combined anti-PD1, TIGIT, TIM3 antibodies and Metformin
were used in some functional assays. Characterization of CD8+ T cell memory subset and exhaustion markers was
analyzed by FACS. Metabolic profiles of CD8+ T cells were analyzed by Seahorse.

Results: Polyfunctionality and functional capacities to eliminate p24+ CD4+ T cells of HIV-1 specific CD8+ T cell
responses from PLWH on ART for more than 10 years (LT-ARTp) significantly improved after activation with adjuvant-
engineered DC in vitro (p=0.001 and p=0.0039; respectively). In contrast, CD8+ T cells from PLWH on ART
for less than a decade (ST-ARTp) were less responsive to DC (p=0.0024) and unable to increase cytotoxic function
(p=0.0156). This was associated with lower frequencies of central memory CD8+ T cells, increased co- expression
of PD1 and TIGIT (p=0.0362) and reduced mitochondrial respiration and glycolytic induction after TCR activation
(p=0.002). In contrast, enrichment on TIM3+ PD1- cells (p=0.001) and preserved glycolytic induction (p=0.0005)
was observed in CD8+ T cells from LT-ARTp. Finally, combined treatment of anti-PD1, anti-TIGIT antibodies and
metformin restored cytotoxic properties of dysfunctional CD8+ T cells from ST-ARTp (p=0.0156).

Conclusions: We identified new immunometabolic parameters potentially useful to personalize DC-based HIV-1
vaccines and improve specific CD8+ T cell response in different PLWH populations.

e00005
Dmrt5 beyond the cortex: early role in the sexual
differentiation of the mouse limbic system.

Rafael Casado-Navarro1, Rodrigo Torrillas-de la Cal1, Javier Macho Rendón2, Juan Ramón Martínez-Morales2,
Esther Serrano-Saiz1..

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00005

Copyright: © 2022 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: Casado-Navarro R., Torrillas-de la Cal R., Macho Rendón J., Martínez-Morales JR. and
Serrano-Saiz E., Dmrt5 beyond the cortex: early role in the sexual
differentiation of the mouse limbic system. IBJ Plus 2022 1(s5) e00005. DOI: 10.24217/2531-0151.22v1s5.00005.

Edited: Madrid, España.

Abstract

The mammalian limbic system is sexually dimorphic and underlies sex-specific innate behaviors such as mating,
maternal care, and aggression. However, the genetic programs involved in the sexual differentiation of the limbic
system are poorly understood. Dmrt transcription factors have emerged as conserved regulators of sex-specific
traits across animal kingdom. They have been studied mostly in invertebrates, where they play an important role
in nervous system sexual differentiation. In mammals, Dmrt genes have been predominantly studied in the gonads,
however, Dmrt5, controls cortex development in humans and mice. In fact, mutations in DMRTA2 (Dmrt5)
lead to a recessive human condition characterized by microcephaly and lissencephaly. Surprisingly, there are no
studies beyond the cortex nor comparing males and females. In this work we use Dmrt5 null mutant mice, RNAseq
and histological techniques to study Dmrt5 function in the developing mammalian nervous system beyond
the cortex and comparing males and females. It is plausible that genetic factors involved in brain sexual differentiation,
like Dmrt, may represent also etiologic causes for mental disorders, explaining the observed male-female
sexual bias.
In this study we showed that Dmrt5 is broadly expressed in the mouse nervous system, including the vomeronasal
organ, olfactory tubercle, or ventral tegmental area (all key nodes in the control of innate behaviors).
Furthermore, we showed that Dmrt5 is necessary for the correct specification of the main olfactory epithelium,
the olfactory bulb, and several hypothalamic nuclei. Additionally, we found a novel function for Dmrt5 as a
suppressor of sex differences in gene expression in the posterior hypothalamic area and midbrain. In this region,
Dmrt5 could be specifying and maintaining a neuronal subpopulation or a group of neurons only in males. Taken
together, Dmrt5 comes out as a crucial transcription factor for the accurate differentiation of limbic regions, integrating
sex information. By studying the genetic factors involved in sexual differentiation of the nervous system,
we might find potential factors to either afford protection or generate vulnerability in one sex versus the other
for sex-biased mental disorders.

e00006
A unique F-actin and junctional organization that maintains
the corneal endothelial barrier.

Cerro-Tello,G1; Benito-Martínez,S1; Cacho-Navas,C1; Barroso, S1; de Rivas-Hidalgo, G1; Millán, J1*.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00006

Copyright: © 2022 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: Cerro-Tello G., Benito-Martínez S., Cacho-Navas C., Barroso S., De Rivas-Hidalgo G. and Millán J., A unique F-actin
and junctional organization that maintains the corneal endothelial barrier. IBJ Plus 2022 1(s5) e00006. DOI: 10.24217/2531-
0151.22v1s5.00006.

Edited: Madrid, España.

Abstract

Introduction: The corneal endothelium is responsible for the correct hydration of the corneal stroma and for an
adequate transport through this tissue. Corneal endothelial layer has a particular organization to maintain its
curved shape and function. The corneal endothelium has low proliferative capacity and form a monolayer that
is subjected to an even mechanical tension coming from the positive pressure of the aqueous humor. Preserving
their barrier function under suboptimal conditions, such as corneal pathologies, age and transplantation, is
essential for maintaining corneal transparency.

Material and methods: We have investigated the structure and the proteins in charge of maintaining corneal
endothelial barrier function by confocal microscopy and time lapse spinning disk microscopy in murine corneas
ex vivo, and hepatic organoids.

Results: We have characterized a novel filamentous actin network that organizes into radial structures arising
from the center of the cell under the nucleus towards cell-cell junctions. This structure is also observed in spherical
epithelial organoids whose cells are also exposed to positive luminal pressure.
Due to the simplicity of the model, it can be easily implemented in any clinic which leads to increasing ADR and
preventing CRC, but requires validation in large multicenter trials.

e00007
In vivo nanotracer for the detection of brain thrombi in an
AD mouse model.

Carlos Cerón1, Marta Casquero-Veiga1, Irene Fernandez-Barahona2,3, Irene Fernandez-Nueda1, Valentín Fuster1,4,
María Ángeles Moro1, Fernando Herranz2,3, Marta Cortes-Canteli*1,5.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00007

Copyright: © 2022 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: Cerón C., Casquero-Veiga M., Fernandez-Barahona I., Fernandez-Nueda I., Fuster V., Moro MA., Herranz F. and Cortes-Canteli
M., In vivo nanotracer for the detection of brain thrombi in an AD mouse model. IBJ Plus 2022 1(s5) e00007. DOI: 10.24217/2531-
0151.22v1s5.00007.

Edited: Madrid, España.

Abstract

Background: Alzheimer’s disease (AD) is the most common cause of dementia. It is a multifactorial degenerative disease
pathologically characterized by intracellular neurofibrillary tangles and extracellular deposition of amyloid. An
early hemostatic dysregulation is also present and contributes to an increment in clot formation, leading to hypoperfusion,
blood brain barrier disruption and neuronal loss. The detection of this prothrombotic state is of the upmost
importance in diagnostic approaches to identify AD patients who would benefit from anticoagulation.

Method: The aim of this project is to use an in vivo nanotracer for the detection of brain thrombi in an AD mouse
model by fast pre-targeted positron emission tomography (PET) imaging.
For that purpose, AD animals and their wild-type littermates were intravenously injected with the antiplatelet antibody
against CD41 conjugated with transcyclooctene (TCO-antiCD41). Twenty-four hours later, [68Ga]core-doped
iron oxide nanoparticles (NP) functionalized with tetrazine (TZ) were intravenously administered. TCO and TZ produce
a rapid in vivo reaction by means of bioorthogonal chemistry, allowing to non-invasively evaluate platelets’ levels
by PET. Two hours after [68Ga]NP-TZ injection, a static PET study of each mouse was acquired with a scanner for
small animals (nanoScan® PET/CT, Mediso, USA). Finally, biodistribution assays of different organs after the PET study
were performed. All PET images were analyzed by regions of interest and voxel-wise analyses.
Conclusions: Our results provide a neuroimaging strategy to diagnose the prothrombotic state towards the personalization
of anticoagulation treatment in AD patients.

e00008
Regulation of BMP8A expression during
hepatic fibrogenesis process.

Stephania C Isaza1, Patricia Marañón1, Esther Rey1, Carmelo García-Monzón1, Águeda González Rodríguez1,2#.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00008

Copyright: © 2022 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: Isaza S.C., Marañón P., Rey E., García-Monzón C. and González Rodríguez A., Regulation of BMP8A expression during
hepatic fibrogenesis process. IBJ Plus 2022 1(s5) e00008. DOI: 10.24217/2531-0151.22v1s5.00008.

Edited: Madrid, España.

Abstract

Introduction: Hepatocellular injury is the main triggering event of wound healing response that leads to liver fibrosis.
Hepatic stellate cell (HSC) activation is crucial in the progression of fibrogenic process since they represent
the major source of extracellular matrix components and contribute to the inflammatory response by secreting
proinflammatory cytokines. Bone morphogenetic proteins (BMPs) are soluble growth factors which exert pleiotropic
effects in various tissues regulating different physiological processes of cellular homeostasis. Regarding
BMP8A, its implication in liver damage has been poorly investigated. Therefore, the aim of this study was to
determine BMP8A expression in different fibrosis-mediated liver damage scenarios.

Materials and methods: Histological study of livers was performed and hepatic BMP8A expression levels were
determined by RT-qPCR in different experimental models of hepatic fibrosis: carbon tetrachloride injected mice,
as a classic hepatic fibrosis model, mice subjected to bile duct ligation, as a model of cholestatic damage-derived
hepatic fibrosis, and high fat diet fed mice, reproducing the progression of non-alcoholic fatty liver disease
(NAFLD) which curses with variable states of concomitant fibrosis in advanced stages. Likewise, the same analysis
was conducted in livers from 11 patients with biopsy proven NAFLD-derived fibrosis and 25 NAFLD patients without
fibrosis. To reproduce the experimental conditions of the murine models, BMP8A levels were determined
in hepatocytes stimulated with conditioned media derived from TGFbeta-stimulated hepatic stellate cells (LX2).

Results: Firstly, murine models were validated through a histological examination of the liver and a molecular
analysis of fibrotic specific markers. Next, hepatic BMP8A mRNA expression was significant increased in all
studied models of hepatic fibrosis comparing with the respective controls. In fact, there is a positive correlation
between hepatic BMP8A levels and fibrosis stage as well as with markers of fibrosis. Accordingly, the clinical
study revealed an elevated BMP8A expression in fibrotic livers, in comparison with those without any fibrotic
sign. Furthermore, in vitro experiments also showed an increased BMP8A expression in Huh7 cells treated with
conditioned media derived from TGFbeta-activated LX2 cells.

Conclusions: This study reveals for the first time an increased BMP8A hepatic expression in the context of hepatic
fibrosis. Its detection in serum might be useful as a non-invasive tool for the diagnosis/prognosis of patients
affected by this liver disease. Moreover, these results suggest that BMP8A is involved in hepatic fibrosis progression,
being possibly relevant in its therapeutic manage.

e00009
The Release of Exosomes from Astrocytes in Response to Fatty Acid
Alters the Metabolism of Proopiomelanocortin (POMC) Neurons.

Collado-Pérez R1,2, Jiménez-Hernaiz M1,2, García-Piqueras J1, Guerra-Cantera S1,2,3, Argente J1,2,3,4,
Fernández-Marcos PJ5, Frago LM1,2,3, Chowen JA1,3,4.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00009

Copyright: © 2022 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: Collado-Pérez R., Jiménez-Hernaiz M., García-Piqueras J., Guerra-Cantera S., Argente J., Fernández-Marcos PJ., Frago LM. and Chowen JA.,
The Release of Exosomes from Astrocytes in Response to Fatty Acid Alters the Metabolism of Proopiomelanocortin (POMC) Neurons. IBJ Plus 2022
1(s5) e00009. DOI: 10.24217/2531-0151.22v1s5.00009.

Edited: Madrid, España.

Abstract

Exosomes are extracellular micro-vesicles with a complex content that include a vast heterogeneity of molecules
such as growth factors, cytokines and RNAs, including micro-RNAs (mi-RNAs) that affect target cells. Neurons are
influenced by neighboring astrocytes through the release of exosomes amongst other signals. We postulated that
the metabolic status is communicated by hypothalamic astrocytes via exosomes to neighboring POMC neurons
to modify their functions in the promotion of energy expenditure and satiety. With this aim, cultures of primary
hypothalamic astrocytes were treated with both palmitic or oleic acid (PA or OA; 0.5 mM) for 24 hours. Exosomes
were isolated and purified from the media culture and used to treat (1.25 or 2.50 μg/mL) a POMC neuronal cell line
for 24 hours. Exosomes released in response to PA (E-PA) or (E-OA) did not change the expression of the markers
of inflammation [interleukin (IL)-6] and Endoplasmic Reticle stress (CHOP) comparing to exosomes released in response
to vehicle (E-V) or with no exosomes (control). Next-Generation miRNA sequencing analysis was performed
to reveal modifications in miRNAs contained in exosomes. The content of miR-199a-3p and miR-145-5p was higher
in E-PA compared to E-V. POMC neurons transfected with a mimetic of miR-199a-3p (1.5 pmol) increased insulin-like
growth factor 1 receptor (IGF1r) protein levels (p < 0.05), as well as POMC expression (ns). Moreover, levels of
mTOR and p70S6k, reported targets of miR-199a-3p, were decreased (p < 0.05, both). Mimetic overexpression
of miR- 145-5p reduced POMC expression (p < 0.001) and protein levels of insulin receptor substrate 1 (IRS1; p <
0.001), which is a known target of this miRNA. To determine modifications in cellular metabolism in the POMC neurons
in response to exosomal astrocytes, Seahorse Cell Mito Stress test was performed. The mitochondrial spare
respiratory capacity of neurons was increased (p < 0.0001) in response to both doses (1.25 or 2.50 μg/mL) of E-PA
and E-OA, with the maximal respiration (p < 0.0001) also increasing with E-PA or E-OA (both doses) compared to
E-V or control. Our results suggest that astrocytes may communicate directly with neurons via exosomes, and that
the exosomes content is modulated by the source cell in response to the nutritional environment. The messages
contained in exosomes from astrocytes can directly modulate the levels of cellular receptors and factors involved
in cell proliferation, protection, metabolism, and nutrient sensing. Specific miRNAs participate in these processes
as well as on the neuropeptide expression in targeted neurons. In addition, cellular respiration is affected in POMC
neurons treated with exosomes from astrocytes in response to fatty acid in a way that suggests that cells are preparing
for a possible respiratory stress by increasing their spare respiratory capacity and maximal respiration.

e00010
Prioritization and validation of asthma and respiratory
allergy biomarkers using in silico analysis: Systems Biology.

Lucía Cremades-Jimeno1*, María Ángeles De Pedro1, María López-Ramos1, Joaquín Sastre2, 3, Pablo Mínguez4,5, Ignacio
Mahillo-Fernandez6, Selene Baos1, Blanca Cárdaba1, 3.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00010

Copyright: © 2022 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: Cremades-Jimeno L., De Pedro MA., López-Ramos M., Sastre J., Mínguez P., Mahillo-Fernandez I., Baos S. and Cárdaba B., Prioritization and
validation of asthma and respiratory allergy biomarkers using in silico analysis: Systems Biology. IBJ Plus 2022 1(s5) e00010. DOI: 10.24217/2531-
0151.22v1s5.00010.

Edited: Madrid, España.

Abstract

Introduction. Asthma and respiratory allergy are chronic inflammatory diseases, with high prevalence and a wide
clinical spectrum. Due to their heterogeneity, it is difficult to diagnose some patients and predict their response to
treatments. Moreover, although allergic mechanisms have been implicated in most asthma diagnostics, there are still a
10-33% of patients with nonallergic asthma, less studied and understood. Thus, there is a need to define new biomarkers
capable of classify patients correctly. At this respect, we defined a group of 94 potential biomarkers with the
ability to differentiate clinical phenotypes and disease severity. Here, the objective was to theoretically prioritize those
biomarkers using systems biology, based on their association with the studied diseases.

Methods. Anaxomics’ TPMS technology (Therapeutic Performance Mapping System) was used to create one mathematical
model, according to molecular motifs, per disease: respiratory allergy (RA), allergic asthma (AA) and nonallergic
asthma (NA). The relationship of each candidate with the diseases was analyzed by artificial neural networks (ANNs)
scores, according to their specificity. A validation of the theoretical results was performed through a study of their sensitivity
and specificity, through ROC curve analysis, using gene expression data obtained from peripheral samples from
healthy control subjects, RA patients, and asthmatic patients (AA and NA). Finally, a triggering analysis was performed,
and possible pathways connecting triggering and specific proteins were created using Cytoscape program (Pathlinkers).

Results. First, two molecular motifs were defined for RA, shared with AA; three motifs were specific for AA; and two
for NA. According to these molecular motifs, 21 from the 94 candidate biomarkers showed the highest specificity for at
least one of the diseases studied: 7 for RA, 12 for AA and 2 for NA. Regarding the experimental validation, ROC curves
analysis highlighted 13 genes with the potential to discriminate between phenotypes and severity according to the
AUC (Area Under the Curve) obtained, confirming the correlation between theoretical and experimental specificity
results in some of the genes analyzed. Finally, this study also revealed the ability of AKT1, STAT1 and MAPK13 to trigger
the three conditions, along with TLR4 in asthma; and possible pathways connecting the 4 triggering proteins with the
21 specific proteins were built.

Conclusion. We theoretically prioritize 21 possible new molecular biomarkers according to their specificity with different
respiratory diseases, and validated some of them. Also, 4 proteins were defined as triggers of the diseases, giving
potential targets to future therapies.

e00011
Neutralizing serum amyloid A1 as a therapeutic strategy for
traumatic brain injury.

Céline Decouty Pérez1,2, Víctor Farré Alins1,2, Alejandra Palomino Antolín1,2, Paloma Narros Fernández1,2, María José
Calzada3, Ana Belén López Rodríguez1,2 and Javier Egea1,2.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00011

Copyright: © 2022 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: Decouty Pérez C., Farré Alins V., Palomino Antolín A., Narros Fernández P., Calzada MJ., López Rodríguez AB. and Egea J., Neutralizing serum
amyloid A1 as a therapeutic strategy for traumatic brain injury. IBJ Plus 2022 1(s5) e00011. DOI: 10.24217/2531-0151.22v1s5.00011

Edited: Madrid, España.

Abstract

Traumatic brain injury (TBI) is the most common cause of disability for millions of people worldwide. It is not only
a health problem, but also an economic problem, as there is no effective treatment yet and the sequelae last for
the rest of the patient’s life. After a TBI, acute phase proteins (APP) like serum amyloid A1 (SAA1) are released
into the serum, promoting neuroinflammation among other things. It has been recently demonstrated that SAA1
binds to TLR4 and activates the production of proinflammatory cytokines, which in turn increases the production
of SAA1.
TAK242 administration, an antagonist of TLR4, shows protective effects in mice models after a TBI. Therefore, we
hypothesize that using a molecule to neutralize SAA1 in serum will also reduce the neurological severity score
(NSS) after inducing the closed head injury (CHI). The results showed that the intraperitoneal administration of
this molecule can reduce the neurological damage in animals after TBI. Regarding the effects of this molecule at
the inflammatory level, we have seen that the administration of this inhibitory compound of the SAA1 pathway
produces a decrease in the gene expression of proinflammatory cytokines (IL-1β, IL6 and TNFα). This study
demonstrates that SAA1 blockade can reduce neurological damage and the inflammatory genetic profile after
trauma in an animal model.

e00012
Inflammasome Activation: A Keystone of Proinflammatory
Response in Obstructive Sleep Apnea.

Elena Díaz-García1,2, Sara García-Tovar2, Enrique Alfaro2, Ana Jaureguizar1,2, Raquel Casitas1,2, Begoña Sánchez-
Sánchez1,2, Ester Zamarrón1,2, Juan Fernández-Lahera1,2, Eduardo López-Collazo1,3, Carolina Cubillos-Zapata1,2, and
Francisco García-Río1,2,4.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00012

Copyright: © 2022 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: Díaz-García E., García-Tovar S., Alfaro E., Jaureguizar A., Casitas R., Sánchez-
Sánchez B., Zamarrón E., Fernández-Lahera J., López-Collazo E., Cubillos-Zapata C. and
García-Río F., Inflammasome Activation: A Keystone of Proinflammatory Response in Obstructive Sleep Apnea. IBJ Plus 2022 1(s5) e00012. DOI:
10.24217/2531-0151.22v1s5.00012.

Edited: Madrid, España.

Abstract

Introduction: As the mechanism that links obstructive sleep apnea (OSA) with the regulation of inflammatory response
is not well known, it is important to understand the inflammasome activation, mainly of NLRP3 (nucleotide-binding
oligomerization domain-like receptor 3).To assess the NLRP3 activity in patients with severe OSA and to identify its role
in the systemic inflammatory response of patients with OSA.

Materials and Methods: We analyzed the NLRP3 activity as well as key components of the inflammasome cascade,
such as adaptor molecule apoptosis-associated speck-like protein, caspase-1, Gasdermin D, IL-1b, IL-18, and tissue
factor, in monocytes and plasma from patients with severe OSA and control subjects without sleep apnea. We explored
the association of the different key markers with inflammatory comorbidities.

Results: Monocytes from patients with severe OSA presented higher NLRP3 activity than those from control subjects,
which directly correlated with the apnea–hypopnea index and hypoxemic indices. NLRP3 overactivity triggered inflammatory
cytokines (Il-1b and IL-18) via caspase-1 and increased Gasdermin D, allowing for tissue factor to be released.
In vitro models confirmed that monocytes increase NLRP3 signaling under intermittent hypoxia in a hypoxia- inducible
factor-1a–dependent manner, and/or in combination with plasma from patients with OSA. Plasma concentrations of
tissue factor were higher in patients with OSA with systemic inflammatory comorbidities than in those without them.

Conclusions: In patients with severe OSA, NLRP3 activation might be a linking mechanism between intermittent
hypoxia and other OSA-induced immediate changes with the development of systemic inflammatory response.

e00013
An inhibitor of the interaction between the transcription
factor NRF2 and the E3 ligase adapter β-TrCP suppresses
lipopolysaccharide- mediated inflammation.

Raquel Fernández-Ginés 1, Ana I. Rojo 1, José Antonio Encinar2, and Antonio Cuadrado 1.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00013

Copyright: © 2022 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-Ginés R., Rojo AI., Antonio Encinar J. and Cuadrado A. An inhibitor of the interaction between the transcription
factor NRF2 and the E3 ligase adapter β-TrCP suppresses lipopolysaccharide- mediated inflammation. IBJ Plus 2022 1(s5) e00013.
DOI: 10.24217/2531-0151.22v1s5.00013.

Edited: Madrid, España.

Abstract

Inflammation plays a crucial role in the pathology of most chronic diseases, such as neurodegenerative and
metabolic disorders. Transcription factor NRF2 has been proposed recently as a promising target to generate a
beneficial therapeutic effect in most chronic diseases characterized by low-grade oxidative stress and inflammation.
Most of the compounds identified as NRF2 activators are electrophiles that inhibit its main repressor:
KEAP1. However, electrophiles display many off-target effects and elicit a supra-physiological NRF2 activation. As
an alternative, we identified a small molecule that disrupts the interaction between NRF2 and its other repressor
E3 ubiquitin ligase β- TrCP. In vitro and cell culture experiments demonstrated that our hit small molecule is a
β-TrCP/NRF2 interaction inhibitor. This compound is specific for NRF2 and not for the other substrates described
for β-TrCP, such as β-Catenin. Moreover, it attenuates the production of pro-inflammatory markers in cultured
macrophages submitted to the endotoxin lipopolysaccharide (LPS). In vivo pharmacodynamics studies demonstrated
selective exposure and NRF2 activation in liver. In mice submitted to LPS-induced acute liver inflammation,
the compound greatly attenuated Kupffer cells activation and the NFkB-mediated inflammatory response. These
findings report an innovative mechanism to activate NRF2 and it could be used as an alternative to conventional
anti-inflammatory therapies.

e00014
SARS-CoV-2 Membrane protein-specific antibodies from
critically ill COVID-19-infected individuals are potent
stimulators of NK cell activation.

Daniel Fernández-Soto1, Paula Bueno2, Urtzi Garaigorta2, Pablo Gastaminza2, José L. Bueno3, Rafael F. Duarte3,
Ricardo Jara4, Mar Valés-Gómez1, Hugh T. Reyburn1

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00014

Copyright: © 2022 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-Soto D., Bueno P., Garaigorta U., Gastaminza P., Bueno JL., Duarte RF., Jara R., Valés-Gómez M. and Reyburn HT., SARS-CoV-2
Membrane protein-specific antibodies from critically ill COVID-19-infected individuals are potent stimulators of NK cell activation. IBJ Plus 2022 1(s5)
e00014. DOI: 10.24217/2531-0151.22v1s5.00014.

Edited: Madrid, España.

Abstract

Introduction. The M glycoprotein is the most abundant structural protein of the SARS-CoV-2 particle and is one
of the key components for virion assembly and morphogenesis. It is a major source of peptide antigens driving
T-cell responses and previous studies have shown that most patients make antibodies against its N-terminal domain.
Nevertheless, no studies of the function of M-specific antibodies have been reported.

Materials and Methods. We expressed the first 23 amino-acids of the M protein as a GST-fusion protein in E.coli.
Sera from 37 COVID-19 patients (20 of them critically ill, admitted to ICU; 17 of them with mild disease) were
assayed in a standard ELISA assay to detect reactive antibodies. The neutralization capacity of M-specific antibodies
was assessed in neutralization experiments against SARS-CoV-2 virus using polyclonal antibodies purified by
affinity chromatography from patient sera, while the ability of the antibodies in patients’ sera to activate Natural
Killer cells was measured in an ELISA-based assay in which antigen-bound antibodies were incubated with PBMC
and NK cell activation was detected by flow cytometry.

Results. We could detect M-specific antibodies in most of the patients, and they could discriminate between
infected and non-infected individuals with around 90% efficacy. No significant difference in the quantity of the
antibody response was found between mild and critical patients. No polyclonal M-specific antibodies showed
neutralization activity in the in vitro system used. Nevertheless, when PBMC from healthy donors were incubated
with patients’ M- specific antibodies bound to the GST-M fusion protein in an ELISA plate, NK cells were significantly
activated, as measured by LAMP1+ degranulation and cytokine (MIP1β) production.

Conclusion. We show that most SARS-CoV-2 infected individuals produce specific antibodies against a surfaceexposed
epitope at the N-terminus of M glycoprotein. Although these antibodies do not appear to neutralize the
virus efficiently, they are able to mediate Fc/FcγR interactions to drive the activation of NK cells.

e00015
Characterization of the mitochondrial GlutamyltRNAGln
amidotransferase (GatCAB) as a new model for
mitochondrial translation disorders.

Fitch SJ.1,2, Bosch-Pastor I.1, Antolinez A. 1, Gutiérrez-García L.1, Marty J.1, Garesse R.1,2, Fernández-Moreno MA.1,2

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00015

Copyright: © 2022 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: Fitch SJ., Bosch-Pastor I., Antolinez A., Gutiérrez-García L., Marty J., Garesse R. and Fernández-Moreno MA., Characterization of the
mitochondrial Glutamyl-tRNAGln
amidotransferase (GatCAB) as a new model for
mitochondrial translation disorders. IBJ Plus 2022 1(s5) e00015. DOI: 10.24217/2531-0151.22v1s5.00015.

Edited: Madrid, España.

Abstract

Mitochondrial OXPHOS diseases are provoked by dysfunction of the OXPHOS system, showing heterogeneity from a
genetic, biochemical and clinical perspective. This complexity is partly due to the involvement of proteins encoded in
two genomes located in different cellular compartments, nuclear and mitochondrial DNA (mtDNA). mtDNA encodes
part of the mtRNAs translation machinery, including 22 tRNAs. All these are charged by their corresponding aminoacyl-
tRNA synthetases (ARS2), except mt-Gln-tRNAGln whose synthesis is carried out through an indirect pathway.
The mt-tRNAGln is charged with glutamic acid (Glu) by a non-discriminating mitochondrial glutamyl-tRNA synthetase
(EARS2), then the Glu is converted to Gln by a Glutamyl-tRNAGln amidotransferase, using free glutamine as an amide
donor, yielding Gln- tRNAGln. This mitochondrial amidotransferase activity lies in the GatCAB complex, which is formed
by three subunits: GatA (QSRL1), GatB (GATB), and GatC (GATC). Patients with mutations in these genes presented with
severe cardiomyopathy and lactic acidosis, which underscores the importance of the GatCAB complex as an essential
component in the translational machinery of mitochondrial protein synthesis.
We have generated Knockout (KO) lines for all subunits using the genomic editing system CRISPR/Cas9 in HEK293T
cells. We measured the levels of the GatCAB subunits and mitochondrial proteins by western blot. We studied the
oxygen consumption levels using a Clark electrode and detected the GatCAB complex by Blue Native-western blotting.
In all cases, KO cells show reduced levels of mitochondrial proteins, decreased oxygen consumption and an absence
of the GatCAB complex compared to wildtype cells. In the QRSL1 and GATC KO cells the levels of all subunits diminish,
but in GATB KO cells the levels of subunits A and C remain stable. Possibly, QRSL1 and GATC could form a stable but
nonfunctional dimer, to which GATB would join.
GATB has two potential ATG codons at the start of its sequence separated by 12 nucleotides. From over 90 clones
analyzed, we only obtained one KO, by generating an upstream ORF, and two cell lines with very low expression of
GATB. The latter present a duplicated region at the beginning of GATB which changes the reading frame from the first
ATG; suggesting the downstream ATG as an alternative translation start codon for those ribosome small subunits that
do not assemble on the first one, yielding a GATB protein missing 4 amino acids.
Characterization of the molecular mechanisms that lead to the synthesis of mt-Gln-tRNAGln and its role in the physiology
of the cell could allow us to comprehend the pathological manifestation of the defects in these genes and propose
possible therapeutic avenues.

e00016
Use of anti-GD2 (Dinutuximab) as a target for CAR-T cells
immunotherapy in neuroblastoma.

García-García Lorena1, Pastora Keren1, González-Murillo África1*, Ramírez M2.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00016

Copyright: © 2022 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-García L., Pastora K, González-Murillo A. and Ramírez M., Use of anti-GD2 (Dinutuximab) as a target for CAR-T cells
immunotherapy in neuroblastoma. IBJ Plus 2022 1(s5) e00016. DOI: 10.24217/2531-0151.22v1s5.00016.

Edited: Madrid, España.

Abstract

Treating solid tumours with chimeric antigen receptors-modified T cells (CAR-T cells) has shown limited efficacy
due to the lack of cancer-type specific antigens. Dinutuximab is a monoclonal antibody that recognizes a sphingolipid,
disialoganglioside GD2, which has limited expression in normal tissues but is overexpressed in paediatric
tumours, mainly neuroblastoma (NB) and diffuse intrinsic pontine glioma (DIPG) and other histone H3 K27M
(H3K27M) mutated diffuse midline gliomas. Dinutuximab, an anti-GD2 antibody, is currently standard of care
in the treatment of NB. We are contemplating the possibility of using Dinutuximab as target element for anti-
NB-CAR-T cells. Our group have explored the feasibility of producing CAR-T cells from peripheral blood (PB),
post-apheresis CD45RA+ fraction and cord blood (CB) using similar protocols of T cell transduction and expansion
ability as a way to favour the persistence of CAR-T cells in the host. Afterward, we performed a deeply characterization
of phenotype and functionality of anti-FITC-CAR-T cells derived from different sources, and the cytotoxic
effect against anti-GD2-FITC- NB labelled cell line.
T cells purified from PB, CB and 45RA fraction after apheresis were cultured in vitro with cytokines IL-7+IL-15+IL-
21 to maintain early memory phenotypes as Naïve T cells (TN). A second generation lentiviral expression vector
(LV) was used to generate second generation CARs (FITC-L-BBz CAR). After 48h of activation in culture with anti-
CD3/CD28 and cytokines, T cells were transduced with viral supernatants. Next, we performed a characterization
of phenotype of anti-FITC-CAR-T cells by flow cytometry studying how the culture conditions enhanced a TN phenotype
expressing CD45RA+ and CCR7+. Cytotoxic function of CAR-T cells was analysed in cocultures with LAN-1
NB cell line labelled with Dinutuximab conjugated with FITC.
Preliminary results showed that culture condition with IL-7/15/21 maintained more primitive phenotypes in
45RA (71% TN CD4+ and 66% TN CD8+) and CB (60% of TN CD4+ and 77% of TN CD8+) derived T cells than PB T
cells (only a 20% of TN CD4+ and TN CD8+). PB and CB-derived CAR-T cells showed higher cytolitic function than
45RA fraction, resulting in a 30% of dead cells in both cases in a 24h-culture assay. Both, PB and CB derived CAR-T
cells showed the best CAR-T activation capacity with 19,6% of cells from PB and 33,9% of cells from CB expressing
early activation markers as CD25 and CD134. In vivo experiments are currently on-going to test the efficacy
of CAR-T cells in combination with GD2 treatment.
Our strategy may complement the current use of Dinutuximab in the treatment of NB through its combination
with a targeted CAR-T cell approach.

e00017
At-home monitorization using artificial noses and
multimodal sensors: a noninvasive approach to human
routine certification.

*Carlos Garcia-Saura, Irene Rodriguez-Lujan, Eduardo Serrano, Francisco B. Rodríguez, Pablo Varona.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00017

Copyright: © 2022 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: Garcia-Saura C., Rodriguez-Lujan I., Serrano E., Rodríguez F.B. and Varona P., At-home monitorization using artificial noses
and multimodal sensors: a noninvasive approach to human routine certification. IBJ Plus 2022 1(s5) e00017. DOI: 10.24217/2531-
0151.22v1s5.00017.

Edited: Madrid, España.

Abstract

Latest advances in e-health highlight the importance of gathering accurate data from patients, to allow for a better
diagnosis, and in turn provide more effective treatments. The remote communication with patients however makes
it difficult to gather accurate information, as it is often biased or limited to what the patient can communicate to the
doctor. We have developed a noninvasive integrated system that can monitor human routines more effectively in a
noninvasive manner using multimodal sensors.
The human body releases multiple substances into the air through its natural biological functions. Breathing, sweat,
digestion, etc., all release chemical components (I.e., CO2, amines, methane) which we readily measure with
specialized olfactory sensors, effectively “artificial noses”. Our device registers the variability in these air parameters
associated with cognitive activity and complements this data with other multimodal sensors (presence sensors,
luminosity, loudness, etc.). In the integrated system proposed the data is then transmitted in real-time into a server
where it can be securely stored, even for years, and quickly accessed and analyzed.
We have tested the use of the proposed noninvasive monitoring technology in university classrooms and in a primary
school. Our results show that all routines in these environments are reflected in the sensor signals and that
the artificial nose can be used to certify the corresponding cognitive activities. The noninvasive approach allows to
obtain valuable data over long periods, and in the context of e-health this could allow a much better understanding
of patient routines and the changes associated with health conditions. Also, the real time nature of this approach
allows to implement “early warning” or notification strategies to quickly react upon changes in routines that signal a
health problem.
Artificial noses and associated information systems to store and analyze the data online and offline can be used to
noninvasively monitor human activity and, in particular, cognitive activity. We argue that this technology can be
directly applied to monitor the elderly, people at early stages of neurogenerative diseases, any progress in cognitive
disease development, and to ensure that patients correctly follow treatment.

e00018
Regulation of β-amyloid clearance in APP/PS1 astrocytes by
AMPK activation and MTORC1 inhibition.

Marta García-Juan1*, Lara Ordóñez-Gutiérrez 1, 2, Francisco Wandosell1, 2.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00018

Copyright: © 2022 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-Juan M., Ordóñez-Gutiérrez L. and Wandosell F., Regulation of β-amyloid clearance in APP/PS1 astrocytes by AMPK
activation and MTORC1 inhibition. IBJ Plus 2022 1(s5) e00018. DOI: 10.24217/2531-0151.22v1s5.00018

Edited: Madrid, España.

Abstract

Alzheimer disease (AD) is the most common neurodegenerative dementia. It is characterized by the progressive
accumulation of amyloid beta (Aβ) aggregates and hyperphosphorylated tau. This aberrant accumulation has been
associated with autophagy dysfunction, occurring in early stages of the disease. AMPK and AKT-MTORC1 signaling
pathways are central nodes in the balance between anabolism and catabolism. It is generally accepted that MTORC1
activation leads to the inhibition of autophagy, whereas AMPK activation is supposed to enhance this process. Some
regulatory cross-talk between both pathways has been reported. Accordingly, autophagy inducers such as MTORC1
inhibitors may have beneficial effects in the clearance/prevention of protein aggregates in the brain, as we previously
reported in the APP/PS1 AD mouse model. Until recently, most of the mechanisms that mediate protein clearance
have been studied in neurons and the contribution of glial cells remains to be elucidated. The aim of this work is to
determine the contribution of astrocytes to Aβ clearance through autophagy.
In an attempt to analyze the effect of autophagic flux modulation, we performed primary cultures of astrocytes form
APP/PS1 mice and their wild type littermates. We treated them with different described drugs that act as autophagy
inductors through the modulation of MTORC1 and AMPK pathways and evaluated the activation of these pathways
and their effect in the autophagic flux by western blot. We found a slight increase of autophagy with rapamycin,
a well-known MTORC1 inhibitor. Surprisingly, AMPK activation with metformin did not enhance autophagy, which
could be due, at least in part, to an insufficient inhibition of MTORC1. Finally, we measured the direct effect of this
modulation on autophagy-dependent amyloidosis through ELISA.
These results suggest that AMPK activation, in contrast to mTORC1 inhibition, is not sufficient to enhance autophagy
in primary astrocytes, as we previously described in neurons. Thus, the protective mechanisms of autophagy against
neurodegeneration must be further examined, with the final aim of describing mechanisms that allow an effective
treatment for this neurodegenerative disorder.

e00019
Brain Tumor Modelling Using the CRISPR-Cas9 Base
Editing Technology.

María del Mar Gardeazabal Bataller1, Massimo Squatrito1.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00019

Copyright: © 2022 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: Gardeazabal Bataller MM. and Squatrito M., Brain Tumor Modelling Using the CRISPR-Cas9 Base Editing Technology. IBJ Plus
2022 1(s5) e00019. DOI: 10.24217/2531-0151.22v1s5.00019

Edited: Madrid, España.

Abstract

Introduction: In common solid tumors, a plethora of genetic alterations can act as disease-driver mutation.
Approximately, 95% of these variants are single-base substitutions, being cytidine-to-thymidine (C-to-T) conversion
the most common among them. Cytosine base editors (CBEs) enable efficient C-to-T substitutions at
targeted loci without double-stranded breaks, overcoming one of the main limitations in classical CRISPR-Cas9
technology.
A major application of base editing technology is the study or treatment of disease-associated point mutations.
In this project, we will focus on the most common point mutations in different genes across paediatric and adult
brain tumors: e.g. CTNNB1 and TP53, frequently altered in the medulloblastoma, and PIK3CA and TP53, commonly
associated with adult gliomas.

Material and methods: To properly recapitulate some of the genetic alterations identified in brain tumors, we
have combined the RCAS-TVA model and the CBE system for somatic genome editing. We have generated an
animal model expressing TVA receptor under the control of specific promoters (GFAP or Nestin) together with
an inducible CBE in order to exploit the base editing technology in the neural stem cells compartment in vivo. By
intracranial injection of these mice with sgRNAs for specific genetic alterations, we will be able to recapitulate
these point mutations in vivo.

Results: We have successfully generated a novel mouse model for generation of brain tumors driven by point
mutations in several genes that are known to drive tumorigenesis in humans. Characterization of these tumors
allowed the detection by Sanger sequencing of C-to-T conversion in all samples, as well as the activation of
downstream signaling derived from each mutations. We observed a complete loss of TP53 expression upon
Tp53Q97*, which translates into an early stop codon in Tp53 sequence. On the other hand, we also validated the
overexpression of TP53 as a consequence of Tp53R270C mutations with no major changes in downstream effectors,
as it has been previously described in the literature.

Conclusion: By integrating the RCAS-TVA system together with BE we have developed a more precise and flexible
tool to better recapitulate in a more efficient way many point mutations identified in different brain tumor types.
Using the RCAS-TVA-BE model we were able to model common point mutations in brain cancer and confirm their
contribution in tumor formation.

e00020
Adenine nucleotides transport across the inner mitochondrial
membrane in cancer cells: role of ANTs and SCaMCs.

Luis González-Moreno1, Inés Juaristi1, Jorgina Satrústegui1 and Araceli del Arco1,2.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00020

Copyright: © 2022 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: González-Moreno L., Juaristi I., Satrústegui J. and Del Arco A., Adenine nucleotides transport across the inner mitochondrial
membrane in cancer cells: role of ANTs and SCaMCs. IBJ Plus 2022 1(s5) e00020. DOI: 10.24217/2531-0151.22v1s5.00020.

Edited: Madrid, España.

Abstract

Introduction: The regulation of adenine nucleotide levels in the mitochondria and the cytosol is essential for cell
metabolism. To sustain mitochondrial respiration, ATP generated by ATP synthase must be exported to the cytosol,
whereas mitochondria must be refilled with ADP and Pi. Adenine Nucleotide Translocases (ANTs), which catalyze the
electrogenic ATP4-/ADP3- exchange, have been proposed to work together with ATP synthase and the mitochondrial
phosphate carrier to support mitochondrial oxidative phosphorylation by forming the so-called ATP synthasome.
Nevertheless, disruption of the main ANT isoform or pharmacological inhibition of ANTs in cancer cells does not
decrease oxidative phosphorylation. Furthermore, the import of cytosolic ATP to the mitochondria when oxidative
phosphorylation is inhibited (required, for example, for the maintenance of the ΔΨm) is neither mediated by ANTs
in cancer cells. Here, we study the possibility that calcium-regulated mitochondrial ATP-Mg2+/Pi or ADP/Pi carriers,
(also called SCaMCs), might participate in the mitochondrial transport of adenine nucleotides in cancer cells.

Material and methods: To study the function of SCaMC-1, the main isoform in cancer cells, we have generated
SCaMC-1 KO HeLa cells using CRISPR-Cas9 genome editing. These cells have been characterized in terms of OCR
(with Seahorse XF24), mitochondrial membrane potential (with TMRM) and mitochondrial ATP levels (with mito-
GoATeam genetic probe) in basal conditions and in the presence of ETC inhibitors.

Results and conclusions: We show that SCaMC-1, the isoform that is abundant in tumor cells, mediates ATP import
to the mitochondria after histamine stimulation of HeLa cells, probably to buffer calcium entry to the mitochondria.
However, SCaMC-1 does not participate in the transport of cytosolic ATP for the maintenance of ΔΨm after OXPHOS
inhibition, neither decreases mitochondrial respiration. Future studies based on the deletion of the other SCaMCs
isoforms (SCaMC-2, SCaMC-3, SCaMC-3 like) together with simultaneous deletion of ANTs and SCaMCs will be
required to shed light on the control of adenine nucleotides transport between the mitochondria and the cytosol in
cancer cells.

e00021
Monitoring One-carbon metabolism by mass spectrometry
for early diagnosis of cirrhosis and HCC

Laura Guerrero1, Alberto Paradela1, Fernando J. Corrales1.2

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00021

Copyright: © 2022 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: Guerrero L., Paradela A. and Corrales FJ., Monitoring One-carbon metabolism by mass spectrometry
for early diagnosis of cirrhosis and HCC. IBJ Plus 2022 1(s5) e00021. DOI: 10.24217/2531-0151.22v1s5.00021.

Edited: Madrid, España.

Abstract

Introduction: Liver cancer represents one of the most frequent causes of death by cancer, ranked as the sixth most
prevalent and the second in lethality. Hepatocellular carcinoma (HCC) is the predominant type of liver cancer and presents
an increasing incidence during the last years. Late diagnosis is one of the reasons explaining the low survival rate
of HCC patients (5 years survival after diagnosis below 20%). Remarkably, 80% of HCC cases develop in cirrhotic tissue
(1). Main risk factors for HCC are well known and include hepatitis B and C viral infections or abusive alcohol consumption.
However, the underlying molecular mechanisms remain unknown and its research will lead to the characterization
of new biomolecular markers for the early diagnosis, prognosis and therapy of HCC.
Metabolic remodeling is a common feature among several hepatic disorders, from steatosis to HCC (2). Tumoral hepatocytes
modify their metabolism to satisfy cancer’s proliferative requirements. One-carbon metabolism (OCM) plays
a fundamental role maintaining the differentiation and quiescent state of hepatocytes, and is recognized as the link
between intermediate metabolism and epigenetic regulation. Owing to these reasons, it might be a potential source of
biomarkers for early diagnosis and prognosis of HCC. Accordingly, it has been demonstrated that some OCM enzymes
are differentially expressed in murine HCC models (3).

Materials and methods: We have developed a robust targeted mass spectrometry-based method, using SRM mode
(Selected Reaction Monitoring), for the systematic quantification of 13 enzymes that participate in OCM. For this
purpose, purified synthetic heavy standard peptides, as well as OCM recombinant proteins have been used. Sixty-four
human liver samples, including 28 control samples, 21 tumoral samples and15 cirrhotic samples have been used.

Results and conclusions: We have demonstrated that there is a profound remodeling of the OCM cycle in HCC versus
control samples, while cirrhotic samples tend to show intermediate expression levels between both physiological
situations. Machine learning- based analysis of our results suggests that monitoring a panel of functionally related proteins
might be useful for future clinical developments and improve the management of HCC patients. However, further
experiments with larger cohorts are required to confirm these findings.
References
1) Ryerson, A. B., et al (2016). Annual Report to the Nation on the Status of Cancer, 1975‐2012, featuring the
increasing incidence of liver cancer. Cancer, 122(9), 1312-1337.
2) Vander Heiden, et al (2009). Understanding the Warburg effect: the metabolic requirements of cell proliferation.
Science, 324(5930), 1029-1033
3) Mora, M. I., Corrales, F. J., et al (2017). Prioritizing popular proteins in liver cancer: remodelling one-carbon
metabolism. Journal of proteome research, 16(12), 4506-4514.

e00022
Volumetric parameters calculated with 2-[18F]FDG PET/CT
and their correlation with biochemical analysis in patients
with diffuse large B- cell lymphoma

Stefanía Guzmán Ortiz1, Jorge Mucientes Rasilla2, Juan Antoni Varga Núñez3, Ana Royuela4, María Belén Navarro
Matilla5, Mercedes Mitjavila Casanovas2

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00022

Copyright: © 2022 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: Guzmán Ortiz S., Mucientes Rasilla J., Varga Núñez JA., Royuela A., Navarro Matilla MB. and Mitjavila Casanovas M., Volumetric parameters
calculated with 2-[18F]FDG PET/CT and their correlation with biochemical analysis in patients with diffuse large B- cell lymphoma. IBJ Plus 2022 1(s5)
e00022. DOI: 10.24217/2531-0151.22v1s5.00022.

Edited: Madrid, España.

Abstract

Introduction/Aim: Diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS) is a heterogeneous and
aggressive group with a high mortality rate, thus, to improve risk stratification in terms of survival, several studies
have been performed with 18F-Fluorodeoxyglucose positron emission tomography/computed tomography (2-[18F]
FDG PET/CT) to assess the total tumor burden, through volumetric parameters such as tumor metabolic volume
(MTV) and total lesion glycolysis (TLG). The aim is to evaluate the relationship of MTV and TLG with progression-free
survival (PFS) and overall survival (OS), and their correlation with prognostic factors Beta-2-microglobulin (B2M) and
lactate dehydrogenase (LDH).

Methodology: Retrospective study of 44 patients with LBDCG undergoing baseline 2-[18F]FDG PET/CT between
January 2012 and December 2018. The calculation method for VMT and TLG was the SUV2.5 threshold. For the evaluation
of VMT and TLG with PFS and OS, Harrell’s C-index was used, after performing a Cox proportional hazards regression
model. Pearson’s correlation coefficient was used to correlate VMT and TLG with B2M and LDH.

Results: In relation to OS and PFS the VMT2.5 (p 0.006; p <0.001) showed statistically significant differences, while
TLG2.5 (p 0.078; p <0.001) was statistically significant only in PFS. When comparing VMT2.5 and TLG2.5, VMT2.5 obtained
a higher Harrell’s C statistical concordance index for both OS (p 0.025) and PFS (p 0.008) showing great ability to
discriminate between patients in whom the event does or does not occur. In the Pearson correlation analysis, VMT2.5
showed a good correlation with LDH (0.676) and a poor correlation with B2M (0.348). The TLG2.5 presented lower
correlation with LDH (0.629) and poor correlation with B2M (0.274).
Table 1. Univariate analysis to evaluate the discriminatory capacity of each variable for both OS and PFS.

Conclusion: The volumetric parameter MTV2.5 calculated with 2-[18F]FDG PET/CT can be a good prognostic indicator
to predict PFS and OS in patients with DLBCL-NOS. In addition, it presents a good correlation with LDH.

e00023
Gasdermin B over-expression modulates HER2-targeted
therapy resistance through LC3B/Rab7 interaction

Gámez-Chiachio, Manuel1,2; Ramos-Nebot, Carmen1,2; Molina-Crespo, Ángela1,2; Pérez-Torres, Bárbara1; Martínez,
Lidia1,2; Sarrió, David1,2; Moreno- Bueno, Gema1,2,3.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00023

Copyright: © 2022 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: Gámez-Chiachio M., Ramos-Nebot C., Molina-Crespo A., Pérez-Torres B., Martínez L., Sarrió D. and Moreno-Bueno G.,
Gasdermin B over-expression modulates HER2-targeted therapy resistance through LC3B/Rab7 interaction. IBJ Plus 2022 1(s5)
e00023. DOI: 10.24217/2531-0151.22v1s5.00023.

Edited: Madrid, España.

Abstract

Tumor resistance to therapy is one of the most important challenges in current oncology. A clear example of this
major issue is the HER2-positive tumors characterized by ERBB2 (HER2/neu) overexpression. HER2+ overexpression/
amplification occurs in several tumors, especially in breast and gastric carcinomas that show an aggressive phenotype
traditionally associated to poor prognosis. In fact, despite the clinical outcome of these tumors has gradually
improved, due to the development of different antiHER2 therapies, many patients will not respond to these treatments
or develop acquired resistance. Several potential resistance mechanisms to these targeted therapies have
been described. Among them, our laboratory established, for the first time, that the amplification/overexpression of
Gasdermin B (GSDMB), which occurs in more than 60% HER2+ breast cancer patients, is a marker of poor prognosis.
Moreover, our data in HER2+ tumors revealed that GSDMB overexpression correlated to distant metastasis, poor
clinical outcome, and reduced response to anti-HER2 therapies. In this context, we have attempted to elucidate the
molecular mechanism whereby GSDMB collaborates in the resistance to anti-HER2 therapies.
To decipher the functional relevance of GSDMB in promoting resistance to HER2-targeted therapies we performed
several molecular approaches (immunoblot, flow cytometry, immunoprecipitation, etc.) in different breast and gastric
carcinoma cell models. Finally, we validated the efficacy of the identified targeted treatment using two complementary
in vivo preclinical models (mice and zebrafish).
Our results confirmed that similarly to HER2 breast tumors, GSDMB is also over-expressing in more than 50% in
HER2 gastric tumors. Furthermore, this over-expression renders HER2 breast and gastric cancer cells more resistant
to anti- HER2 agents by promoting protective autophagy. Consistent with this, we proved that the combination of
lapatinib with the autophagy inhibitor chloroquine increases the therapeutic response specifically in GSDMB-positive
tumors in vitro and in vivo. Mechanistically, we also confirmed that GSDMB forms a complex with LC3B and Rab7.
Finally, we validated these results in clinical samples of HER2 breast and gastric cancers, where GSDMB/LC3B/Rab7
co- expression associates significantly with relapse. In conclusion, our data decipher the molecular mechanism that
underlies the relation between Gasdermin B and pro-survival autophagy with the final goal of finding a therapy that
would be effective in overcoming resistance to anti-HER2 standard therapies in HER2/GSDMB+ tumors.

e00024
Activation of the mTORC1 and mitochondrial signaling under
diabetic and hypertensive cardiomyopathy

Hang T1, Corrales S1, Azkargorta M2, Lumpuy-Castillo J1, Elortza F2, Martínez-Chantar M2, and Lorenzo O1.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00024

Copyright: © 2022 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: Hang T., Corrales S., Azkargorta M., Lumpuy-Castillo J., Elortza F., Martínez-Chantar M., and Lorenzo O., Activation of
the mTORC1 and mitochondrial signaling under diabetic and hypertensive cardiomyopathy. IBJ Plus 2022 1(s5) e00024. DOI:
10.24217/2531-0151.22v1s5.00024.

Edited: Madrid, España.

Abstract

Introduction: Type 2 Diabetes Mellitus (T2DM) and hypertension (HTN) are correlated with cardiovascular disease
(CVD). The presence of HTN in T2DM will lead to risk elevation in CVD. Inflammation, hypertrophy, apoptosis and fibrosis
can be found in these patients. However, the mechanisms involved haven’t been fully depicted.

Methodology: Cardiac biopsies from interventricular septum were isolated from patients with T2DM and/or HTN.
Differential protein expression was evaluated by proteomics (hybrid trapped ion mobility spectrometry) and PEAKS
software. Ingenuity Pathway Analysis (IPA, Qiagen) was used to predict the implication of molecular pathways. Cultured
cardiomyocytes were used to reveal the alteration of relevant pathways under hyperglycemic, hyperlipidemic and
pro-hypertensive milieu mimicked by high glucose, high fatty-acid (HF) and/or angiotensin-II.

Results: By proteomics, there is a huge difference between T2DM/HTN group and HTN in number of altered factors
and its fold-change when compared with control. HTN induced a decrease of cardiac factors related to carbohydrate
metabolism, mitochondrial homeostasis and respiration, while factors decreased in T2DM/HTN most related to metabolism
and mitochondrial dysfunction. Also, T2DM/HTN increased fibrosis and apoptosis related factors. Interestingly,
a potential regulation of mTOR complexes was suggested by bioinformatics. Thus, we confirmed the phosphorylation
on Thr421/Ser424 of p70S6, a mTORC1 downstream mediator, under HF in cardiomyocytes. Also, we observed an
enhanced phosphorylation on Thr172 of AMPKα, and a decreased expression of PGC1α and ACADm, in parallel to a
lessening of ATP production. These alterations were abolished by rapamycin, an inhibitor of mTORC1 and metformin.
Also, the silencing of a subunit of mTORC2, Rictor, induced a decrease of p-p70s6 under HF.

Conclusion: T2DM/ HTN can lead to dramatic protein changes in heart and induce a synergy cooperation when combined.
The mitochondrial alteration may be major responsible of cardiac dysfunction. In particular, mTORC1 can be
activated under hyperlipidemia to further lead to β-oxidation reduction and ATP synthesis. Therefore, the regulation of
the mTOR-mitochondria signaling could be essential for prognosis of heart failure in T2DM and HTN patients.

e00025
Generation and characterization of the adult neuron-specific
Aralar/AGC1 knock-out mice.

Eduardo Herrada1,2,3, Araceli del Arco2,3,4, Jorgina Satrustegui1,2,3, Beatriz Pardo1,2,3..

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00025

Copyright: © 2022 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: Herrada E., Del Arco A., Satrustegui J. and Pardo B., Generation and characterization of the adult neuron-specific Aralar/
AGC1 knock-out mice. IBJ Plus 2022 1(s5) e00025. DOI: 10.24217/2531-0151.22v1s5.00025.

Edited: Madrid, España.

Abstract

Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier expressed in neurons, is the regulatory
component of the NADH malate-aspartate shuttle. AGC1 deficiency is a neuropediatric rare disease (OMIM
#612949, also named early infantile epileptic encephalopathy 39) characterized by hypomyelination, hypotonia,
developmental arrest and epilepsy. In mice, the global aralar knockout shows hyperreactivity, growth retardation,
motor discoordination, seizures and hypomyelination; thus, recapitulating the pathology of the human disease.
Aralar brain expression is mainly restricted to neurons, while glial expression of Aralar has been demonstrated to
be scarce. However, Aralar deficiency manifests alteration in fundamental glial functions such as glutamine and
myelination synthesis by astrocytes and oligodendrocytes respectively. In order to dissect the role of neuronal
Aralar in the disease mechanism of AGC1 deficiency, we have ablated the expression of Aralar in mature (from
PND30 onward) excitatory cortical and hippocampal neurons using the CaMKIIα-driven Cre recombinase expression
in Aralarlox/lox mice.
Here, we present the characterization of the mature neuron-specific aralar knock out mice. We demonstrated that
the excision of exon 3 aralar starts from PND30 and continues to occur during mouse adulthood. Consequently,
Aralar protein levels gradually decay across the 2nd to 6th month up to a 70- 75% decrease in the cortex and a 50%
decrease in the hippocampus. The drop in Aralar levels drive an alteration in cortical metabolites causing a 25% and
50% decrease in aspartate and serine levels, the metabolites most affected in the brain of the global aralar-KO mice.
This partial decreases in ARALAR and metabolites caused by cortical CaMKIIα-driven aralar ablation are not accompanied
by growth or behavioral alterations in a battery of motor, memory and cognitive tests. The results suggest
that the absence of Aralar in CaMKIIα expressing neurons (cortical and hippocampal mature excitatory neurons),
does not mimic the alterations caused by global deficiency of Aralar/AGC1. However, since the levels of cortical
ARALAR in this model resemble those of healthy aralar+/- mice, further experiments are being carried out to explore
the origin (or location) of the ARALAR protein remnants and the possible metabolic compensation by other neurons
and/or cell types in these mice. Without ruling out either that the ARALAR-MAS pathway could play a more critical
role during neurodevelopment than adulthood in brain physiology and metabolism.

e00026
Selective autophagy plays a protective role against acute and
age- related retinal degeneration.

Juan Ignacio Jiménez-Loygorri1, Ignacio Ramírez-Pardo1, Beatriz Villarejo-Zori1, Álvaro Viedma-Poyatos1, Elena Sierra-
Filardi1, Sandra Alonso-Gil1, Raquel Gómez-Sintes1 and Patricia Boya1*.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00026

Copyright: © 2022 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-Loygorri JI., Ramírez-Pardo I., Villarejo-Zori B., Viedma-Poyatos A., Sierra-Filardi E., Alonso-Gil S., Gómez-Sintes
R. and Boya P., Selective autophagy plays a protective role against acute and age- related retinal degeneration. IBJ Plus 2022 1(s5)
e00026. DOI: 10.24217/2531-0151.22v1s5.00026.

Edited: Madrid, España.

Abstract

Objective. Age-related macular degeneration (AMD) is the leading cause of blindness in elderly people in the developed
world, and the number of people affected is expected to almost double by 2040. The retina presents one of the
highest metabolic demands that is partially or fully fulfilled by mitochondria in the neuroretina and retinal pigment
epithelium (RPE), respectively. Together with its post-mitotic status, this context requires a tightly-regulated housekeeping
system that includes selective mitochondrial autophagy. We want to assess the effects of selective autophagy
deficit or induction in the retina given an AMD-like paradigm.
Materials and methods. Eyes from Ambra1+/+, Ambra1+/gt and mito-QC mice were analysed using flatmount or cryosection
immunostaining. Sodium iodate (SI) was used as a model of AMD-like damage and Urolithin A (UA) as a mitophagy
inducer. ARPE-19 human cells were used as an in vitro model and analysed by immunostaining, flow cytometry and
RT-qPCR. Bioinformatic analysis of public human datasets was also performed.
Results. Ambra1+/gt autophagy-deficient mice present alterations in the RPE, similar to those observed in human AMD
patients, such as abnormal morphology or lipofuscin accumulation, which appear in an age-dependent manner. Furthermore,
Ambra1+/gt mice are more sensitive to acute SI-induced retinal degeneration than their Ambra+/+ littermates.
UA induced mitophagy in vivo and prevented degeneration both in the neuroretina and RPE. This amelioration was
also associated with decreased lipid peroxidation, gliosis and increased photoreceptor survival. In vitro, inhibition of
mitophagy, or general macroautophagy, abolished this rescue.
Conclusions. Selective autophagy plays a protective role in the retina and can be exploited to preserve vision in physiological
or pathological conditions.

e00027
Transcription factor NRF2 participates in cell cycle
progression at the level of G1/S and mitotic checkpoints.

Diego Lastra1,2,3,4,*, Maribel Escoll 1,2,3,4 and Antonio Cuadrado1,2,3,4

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00027

Copyright: © 2022 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: Lastra D., Escoll M. and Cuadrado A., Transcription factor NRF2 participates in cell cycle progression at the level of G1/S and
mitotic checkpoints. IBJ Plus 2022 1(s5) e00027. DOI: 10.24217/2531-0151.22v1s5.00027.

Edited: Madrid, España.

Abstract

Transcription factor NRF2 is considered a master regulator of cell homeostasis, coordinating multiple cytoprotective
responses that confer growth advantage to cells. Although its implication in redox homeostasis, detoxification or metabolic
reprogramming has been widely studied, its participation in cell cycle division and the mechanisms governing
this process has not been explored in detail.
In this study, we used several standard methods of synchronization of proliferating cells in G1, before S entry and during
mitosis. Together with flow cytometry analysis, we monitored the participation of NRF2 along the cell cycle by
knockdown of its gene expression. Our results showed that NRF2 levels are maximal at S phase entry, and minimal
during mitosis. Besides, NRF2 absence caused both G1 and M arrest. A targeted transcriptomics analysis of cell cycle
regulators showed that NRF2 depletion leads to changes in key cell cycle regulators, including CDK2, TFDP1, CDK6,
CDKN1A (p21), CDKN1B (p27), CCNG1 and RAD51. This study gives a new dimension to NRF2 effects, showing its
implication in cell cycle progression.

e00028
Regulation of the DNA damage response by E2F4 phosphorylation in
its T249/T251 conserved motif and Alzheimer’s disease.

A.M. Llabrés-Mas1, A. Garrido-García,1,2 J.M. Frade1

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00028

Copyright: © 2022 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: Llabrés-Mas A.M., Garrido-García A. and Frade J.M., Regulation of the DNA damage response by E2F4 phosphorylation in its
T249/T251 conserved motif and Alzheimer’s disease. IBJ Plus 2022 1(s5) e00028. DOI: 10.24217/2531-0151.22v1s5.00028.

Edited: Madrid, España.

Abstract

Alzheimer’s disease (AD) has a multifactorial etiology that includes DNA damage in neurons. The transcription
factor E2F4, which can potentially regulate DNA repair, has two conserved threonines (T249/T251 in the mouse)
that can be phosphorylated by p38MAPK (p38). The expression in vivo of the T249A/T251A E2F4 mutant form
(E2F4DN) has been shown to be a multifactorial therapeutic agent against AD.
In this work, we have analyzed the effects of E2F4 phosphorylation in T249/T251 on the DNA repair response
(DDR). To this aim, we used N2a mouse neuroblastoma cells treated with 10 μM camptothecin (CPT), a treatment
known to induce Cited2 expression and subsequent cell death.
In this paradigm, the repression of Cited2 by E2F4 is abolished upon CPT treatment, thus allowing its E2F1-dependent
expression followed by cell death. While E2F4 can be detected in both the nucleus and cytoplasm of
N2a cells, phosphoT249-E2F4-specific immunoreactivity is specifically observed in the nucleus 4 h after treatment
with CPT. Therefore, the known activation of p38 in response to CPT could lead to T249 phosphorylation
of E2F4, thus suppressing its inhibition on E2F1 activity and allowing Cited2 expression. This hypothesis, is being
tested in CPT- treated N2a cells co-transduced with adenoviral vectors expressing E2F1 together with either
wild-type E2F4 or E2F4DN, in either the presence or absence of p38 inhibitors.
In summary, our work provides support for a novel mechanism used by E2F4 to regulate the response to DNA
damage in pathological situations, which could participate in the therapeutic capacity of E2F4DN against AD.

e00029
Alteration of PGC1-α in the mitochondrial homeostasis of
cardiomyocytes under hyperglycemia. Role of the GLP-1R activation.

Jairo Lumpuy-Castillo1, Tianyu Hang1, Óscar Lorenzo-González1,2

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00029

Copyright: © 2022 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: Lumpuy-Castillo J., Hang T. and Lorenzo-González O., Alteration of PGC1-α in the mitochondrial homeostasis of cardiomyocytes under
hyperglycemia. Role of the GLP-1R activation. IBJ Plus 2022 1(s5) e00029. DOI: 10.24217/2531-0151.22v1s5.00029.

Edited: Madrid, España.

Abstract

Introduction: Cardiovascular disease is the major cause of morbidity and mortality in diabetic patients, and mitochondrial
homeostasis can be a key organelle in cardiomyocytes. Peroxisome proliferator-activated receptor- gamma
coactivator-alpha (PGC-1α), a master transcription factor of mitochondrial function, may be regulated under hyperglycemic
stimulus and anti-diabetic drugs, such as Glucagon-like protein-1 receptor agonists (GLP-1RA).

Methodology: The mitochondrial protein expression of PGC-1α and Cytochrome-C was evaluated under 25 mM glucose
(HG) and/or GLP-1RA stimulation in H9c2 cardiomyocytes after 18-48 hours. Western blotting was performed
from cytosolic and enriched mitochondrial fractions. Also, PGC-1α related genes (Cpt1a, Sdhb, Mfn1 and Nrf1) were
quantified by qPCR assay.

Results: Mitochondrial PGC-1α and Cytochrome-C, but not TFAM and PPARd, were increased only after 24h of HG.
However, at this time, the levels of Cpt1a, Sdhb, Mfn1 and Nrf1 were differentially regulated. Interestingly, co- incubation
with GLP-1 and GLP-1RA attenuated expression of both PGC-1 and Cytochrome-C at the mitochondrial location.

Conclusions: Hyperglycemia may damage the myocardium by increase of cellular oxidation, but it also could enhance
early-responses of cardioprotection by enhancing PGC-1α and Cytochrome-C at the mitochondria. Interestingly,
activation of GLP-1RA may attenuate this effect, suggesting a new mechanism of action for GLP-1RA drugs.

e00030
Bone morphogenetic protein 2 is a new molecular target
linked to nonalcoholic fatty liver disease with potential value
as non-invasive screening tool.

Patricia Marañón1, Carlos Ernesto Fernández-García1*, Stephania C Isaza1*, Esther Rey1*, Rocío Gallego-Durán2,3, Rocío
Montero-Vallejo2,3, Javier Rodríguez de Cía1, Javier Ampuero2,3, Manuel Romero-Gómez2,3, Carmelo García-Monzón1,3,
Águeda González-Rodríguez1,4#

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00030

Copyright: © 2022 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: Marañón P., Fernández-García CE., Isaza S., Rey E., Gallego-Durán R., Montero-Vallejo R., Rodríguez de Cía J., Ampuero J., Romero-Gómez
M., García-Monzón C. and
González-Rodríguez A., Bone morphogenetic protein 2 is a new molecular target linked to nonalcoholic fatty liver disease with potential value as
non-invasive screening tool. IBJ Plus 2022 1(s5) e00030. DOI: 10.24217/2531-0151.22v1s5.00030.

Edited: Madrid, España.

Abstract

Background: Nonalcoholic fatty liver disease (NAFLD) is the commonest cause of chronic liver disease worldwide,
being nonalcoholic steatohepatitis (NASH) its most clinically relevant form. Given the risks associated with taking
a liver biopsy, the design of accurate non-invasive methods to identify NASH patients is of upmost importance.
BMP2 plays a key role in metabolic homeostasis; however, little is known about its involvement in NAFLD onset
and progression. This study aimed to elucidate the impact of BMP2 in NAFLD pathophysiology.

Methods: Hepatic and circulating levels of BMP2 were quantified in serum and liver specimens from 115 biopsyproven
NAFLD patients and 75 subjects with histologically normal liver (NL). In addition, BMP2 content and
release was determined in cultured human hepatocytes upon palmitic acid (PA) overload.

Results: We found that BMP2 expression was abnormally increased in livers from NAFLD patients than in subjects
with NL and this was reflected in higher serum BMP2 levels. Notably, we observed that PA upregulated BMP2 expression
and secretion by human hepatocytes. An algorithm based on serum BMP2 levels and clinically relevant
variables to NAFLD showed an AUROC of 0.886 (95%CI, 0.83–0.94) to discriminate NASH. We used this algorithm
to develop SAN (Screening Algorithm for NASH): a SAN < 0.2 implied a low risk and a SAN ≥ 0.6 indicated high risk
of NASH diagnosis.

Conclusion: This proof-of-concept study shows BMP2 as a new molecular target linked to NAFLD and introduces
SAN as a simple and efficient algorithm to screen individuals at risk for NASH.

e00031
Astrocytes of Nucleus Accumbens control the impairments
derived from chronic exposure of THC.

Cristina Martín-Monteagudo1,2, Julio Esparza1, Marta Navarrete1.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00031

Copyright: © 2022 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: Martín-Monteagudo C., Esparza J. and Navarrete M., Astrocytes of Nucleus Accumbens control the impairments derived
from chronic exposure of THC. IBJ Plus 2022 1(s5) e00031. DOI: 10.24217/2531-0151.22v1s5.00031.

Edited: Madrid, España.

Abstract

Background: Nonalcoholic fatty liver disease (NAFLD) is the commonest cause of chronic liver disease worldwide,
being nonalcoholic steatohepatitis (NASH) its most clinically relevant form. Given the risks associated with taking
a liver biopsy, the design of accurate non-invasive methods to identify NASH patients is of upmost importance.
BMP2 plays a key role in metabolic homeostasis; however, little is known about its involvement in NAFLD onset
and progression. This study aimed to elucidate the impact of BMP2 in NAFLD pathophysiology.

Methods: Hepatic and circulating levels of BMP2 were quantified in serum and liver specimens from 115 biopsyproven
NAFLD patients and 75 subjects with histologically normal liver (NL). In addition, BMP2 content and
release was determined in cultured human hepatocytes upon palmitic acid (PA) overload.

Results: We found that BMP2 expression was abnormally increased in livers from NAFLD patients than in subjects
with NL and this was reflected in higher serum BMP2 levels. Notably, we observed that PA upregulated BMP2 expression
and secretion by human hepatocytes. An algorithm based on serum BMP2 levels and clinically relevant
variables to NAFLD showed an AUROC of 0.886 (95%CI, 0.83–0.94) to discriminate NASH. We used this algorithm
to develop SAN (Screening Algorithm for NASH): a SAN < 0.2 implied a low risk and a SAN ≥ 0.6 indicated high risk
of NASH diagnosis.

Conclusion: This proof-of-concept study shows BMP2 as a new molecular target linked to NAFLD and introduces
SAN as a simple and efficient algorithm to screen individuals at risk for NASH.

e00031
Astrocytes of Nucleus Accumbens control the impairments
derived from chronic exposure of THC.

Cristina Martín-Monteagudo1,2, Julio Esparza1, Marta Navarrete1.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00031

Copyright: © 2022 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: Martín-Monteagudo C., Esparza J. and Navarrete M., Astrocytes of Nucleus Accumbens control the impairments derived
from chronic exposure of THC. IBJ Plus 2022 1(s5) e00031. DOI: 10.24217/2531-0151.22v1s5.00031.

Edited: Madrid, España.

Abstract

Drug use is a growing problem in actual society. Usually, the first experience with drugs takes place during adolescence,
being cannabis the most used illicit drug. Although cannabis could be considered a harmless drug, we are beginning
to appreciate its consequences. Chronic exposure to addictive drugs has shown to imbalance glutamate homeostasis
in Nucleus Accumbens (NAc), altering plasticity mechanisms such as long-term depression. Therefore, it is
crucial to elucidate the mechanisms underlying these alterations and how to reverse them. It is known the activation
of cannabinoid receptors in astrocytes modulate synaptic plasticity and could be involved in glutamate homeostasis.
However, the functional role of astrocytes in alterations derived from chronic drug exposure is not fully understood.
In this study, we analyzed how astrocytes contribute to alterations produced by tetrahydrocannabinol (THC).
Using fiber photometry in vivo we analyzed astrocytic activity (Ca2+ and glutamate dynamics) in NAc after 1mg/kg
THC chronic administration in wildtype and p38αMAPK-/- (Astrop38α) mice4 and we performed electrophysiology
experiments to analyze synaptic plasticity. Moreover, we performed behavioral tests to assess whether THC had
reinforcing properties or affected learning and memory. Furthermore, using a chemogenetic approaches (DREADDs)
we activated NAc astrocytes to analyze their behavioral implications.
We observed: 1)THC increases astrocytic calcium activity; 2)THC induces glutamate release in NAc in wildtype,
but not Astrop38α; 3)NAc astrocytes are involved in learning; 4)Removal of p38αMAPK in NAc astrocytes restores
THC-related impairments.
Altogether, our results reveal astrocytes as critical elements for the maintenance of glutamate signaling, with a significant
role in drug-use-related alterations.

e00032
Looking for novel interactors of IL-13 receptor α2 (IL13Rα2)
and tyrosine-protein phosphatase non-receptor type 1 (PTPN1/
PTP1B) by using proximity-dependent biotinylation (BioID).

Ángela Martín-Regalado1, Rubén Á. Bartolomé1, Javier Robles1,2, Marta Jaén1, Vivian de los Ríos1 and J. Ignacio Casal1*.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00032

Copyright: © 2022 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: Martín-Regalado A., Bartolomé RA., Robles J., Jaén M., De los Ríos V. and Casal JI., Looking for novel interactors of IL-
13 receptor α2 (IL13Rα2) and tyrosine-protein phosphatase non-receptor type 1 (PTPN1/PTP1B) by using proximity-dependent
biotinylation (BioID). IBJ Plus 2022 1(s5) e00032. DOI: 10.24217/2531-0151.22v1s5.00032.

Edited: Madrid, España.

Abstract

Introduction: IL13Rα2 is a high affinity receptor for IL-13 that is re-expressed in late neoplastic stages. The IL- 13/IL-
13Rα2 axis triggers invasion in colorectal cancer (CRC) and other cancers in a process mediated, among others, by the
phosphatase PTPN1 (aka PTP1B), involved in the dephosphorylation of the autoinhibitory Src Tyr416, and further Src
activation. PTPN1 takes part in IL13Rα2 internalization and associates with poor outcome in glioblastoma and CRC patients.
Inhibition of IL13Rα2 pathway decreases invasion of multiple cancer cell lines (i.e., SKOV-3, PC-3 and KM12SM).
To better define the oncogenic properties of IL13Rα2 and PTPN1, we aim to identify novel interactors by using proximity-
dependent biotinylation coupled to mass spectrometry.
Materials and methods: PTPN1 and IL13Rα2 were cloned into pcDNA.5-pDEST-BirA-Flag-Ct by using LR clonase
enzyme, obtaining the desired plasmids containing fusion genes PTPN1-BirA and IL13Rα2-BirA. Flp-In™ T-REx™ cells
were co-transfected with pOG44 and pcDNA.5 vectors and selected with hygromycin. Then doxycycline and biotin
were added to induce the expression of the fusion gene and proximity labelling. Biotinylated proteins were purified by
affinity using streptavidin-sepharose, trypsin-digested and analysed in a Q-Exactive mass spectrometer. Raw data were
analysed with MaxQuant, followed by Perseus for statistical analysis and SAINT for probabilistic scoring of protein-protein
interaction data, which were subjected to gene ontology (GO) analysis.

Results: For IL13Rα2, 735 proteins were identified and 94 were scored as significant interactors by SAINT. GO enrichment
analysis showed association with cadherin binding and response to reticulum stress, among others. 1009
proteins were identified and 217 were scored as significant PTPN1 interactors. GO enrichment analysis showed important
association with mitotic cell cycle regulation, such as CDK1 (G2/M transition) or PCTN (centrosome component).
Among the 19 IL13Rα2 and PTPN1 common interactors stand out those related to SNARE complex (YKT6, SNAP29 and
VAMP3), which is involved in regulation of cancer invasion, drug resistance and kinase phosphorylation. Another interesting
finding was the presence of VRK2, a kinase involved in MAPK pathway.

Conclusions: This study reveals novel individual interactors of IL13Rα2 and PTPN1, but also identifies common interactors
of both proteins such as members of the SNARE complex and VRK2, opening new strategies for the inhibition of
IL13Rα2 and PTPN1 in cancer metastasis.

e00033
Association of mutant spectra of SARS-CoV-2 with COVID-19 disease severity.

Brenda Martínez-González1,2, María Eugenia Soria1,3, Lucía Vázquez-Sirvent1, Cristina Ferrer-Orta4, Pablo Mínguez5,6,7, Lorena de la
Fuente5,6,7, Carlos Llorens8, Beatriz Soriano8, Ricardo Ramos9, Marta Cortón5,6, Carlos García-Crespo3, Isabel Gallego3,10, Ana Isabel
de Ávila3, Jordi Gómez10,11, Llanos Salar-Vidal1, Jaime Esteban1, Ricardo Fernandez-Roblas1, Ignacio Gadea1, Carmen Ayuso6, Javier
Ruíz-Hornillos12,13,14, Nuria Verdaguer4, Esteban Domingo3,10, Celia Perales1,2,10,*..

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00033

Copyright: © 2022 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: Martínez-González B., Soria ME., Vázquez-Sirvent L., Ferrer-Orta C., Mínguez P., De la Fuente L., Llorens C., Soriano B., Ramos R., Cortón M., García-
Crespo C., Gallego I., De Ávila AI., Gómez J., Salar-Vidal LL., Esteban J., Fernandez-Roblas R., Gadea I., Ayuso C., Ruíz-Hornillos J., Verdaguer N., Domingo E.. and
Perales C., Association of mutant spectra of SARS-CoV-2 with COVID-19 disease severity. IBJ Plus 2022 1(s5) e00033. DOI: 10.24217/2531-0151.22v1s5.00033.

Edited: Madrid, España.

Abstract

Introduction: Replication of RNA virus, as SARS-CoV-2, leads to composition of sequences which are at different frequencies
in the infected host. Minority variants present in mutant spectra can have an epidemiological importance generating
more infectious variants, resistance to vaccines and drugs and affecting to the resolution of the disease. How different are
the mutant spectra of virus isolated from patients with different disease outcome has not yet been studied.

Material and Methods: To approach this question, 30 patients infected with SARS-CoV-2 during the first pandemic wave in
Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain were classified as mild, moderate and exitus according to the
disease severity of COVID-19. RNA presents in the nasopharyngeal swabs of these patients was extracted. Four amplicons
of nsp12 (polymerase) and two amplicons of spike (S) coding regions were amplified. PCR products were sequenced by
ultra-deep sequencing (UDS) using MiSeq platform (Illumina). Sequences were analyzed with two pipelines and differences
between the mutant spectra of each virus were determined. Possible structural and functional alterations of mutations
detected have been studied.

Results: Most of the substitutions were found at frequencies between 0.5% to 30% in the mutant spectra. The number of
mutations was significantly higher in patients with mild symptoms. The analysis of the mutant spectra resulted in a higher
number of transitions and non-synonymous substitutions for in all COVID-19 categories. A major complexity of mutant
spectra of virus isolated from mild patients were reflected in the study of several diversity indices, with significant statistical
differences between COVID-19 categories. Structural analysis of nsp12 and spike substitutions, detected in the mutant spectra,
showed possible alterations in the structure or function of the proteins.

Conclusion: The study of mutant spectra shows a positive association between the number of point mutations and the
complexity of mutant spectra with a mild disease outcome. Possible models for this association are discussed. Structural and
functional alterations are under study right now.

e00034
Multiplex Immunofluorescence reveals specific subsets of
immune cell populations expressing CD137/TNFRSF9 as
predictors of unfavorable outcomes in Hodgkin Lymphoma.

José L. Solórzano 1,†, Victoria Menéndez 2,†, Sara Fernández 2, Carlos Montalbán 3, and Juan F. García 1,4.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00034

Copyright: © 2022 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: Solórzano JL., Menéndez V., Fernández S., Montalbán C., and García JF., Multiplex Immunofluorescence reveals specific
subsets of immune cell populations expressing CD137/TNFRSF9 as predictors of unfavorable outcomes in Hodgkin Lymphoma. IBJ
Plus 2022 1(s5) e00034. DOI: 10.24217/2531-0151.22v1s5.00034.

Edited: Madrid, España.

Abstract

The tumor microenvironment (TME) is a crucial determinant of tumor growth, progression, and resistance to
chemotherapy in cancer, with classical Hodgkin’s lymphoma (cHL) being one of the most representative examples.
The major component of the tumor mass in cHL is a rich and vast immune microenvironment, comprising a diverse
repertoire of cell populations, many of which remain unknown. The specific contribution of the different functional
cell subsets in the TME, such as T lymphocytes, monocytic populations, or dendritic cells, is also undiscovered.
Currently, around 30% of advanced-disease patients or some atypical cases of primary refractory tumors do not respond
to treatment or relapse shortly afterwards. Response predicting features of TME have been recently identified,
which suggest a rationale for alternative therapeutic strategies. All existing evidence indicates that HRS cells actively
coordinate and remodel their complex TME and that its diverse components influence the therapeutic response in
cHL patients.
The sensitivity of current technology enables analysis of specific functional phenotypes in the TME as diagnostic and
predictive biomarkers. One example is the quantitative dissection of the TME with in situ multispectral imaging using
formalin-fixed, paraffin-embedded (FFPE) tissues, with which we could analyze simultaneously CD68, CD137, CD30,
CD3, PDL1, and PD1 markers to identify specific cell subsets.
Remarkably, some cell populations expressing CD137 were associated with unfavorable responses in cHL patients,
such as the activated monocytic cells (CD68+ CD137+), the tumor-reactive T cells (CD3+ CD137+), and the tumor
CD30+ CD137+ PDL1+ cell subset. By contrast, the CD3+ CD137- T-cell population was associated with better prognosis,
suggesting that CD137 could be a new prognostic marker in cHL for poor outcomes. Moreover, some cell subsets
that could not be studied until now due to the lack of appropriate technology were found to be more abundant in
the first stages of the disease, whereas others were less abundant than in stage IV. For instance, we could study
exhausted T cells (CD3+ PD-L1+ PD-1+) and other related cells that were largely unexplored in cHL. Finally, these
findings were validated with independent series of patient samples, including immunohistochemical studies with
specific antibodies and gene-expression analysis.

e00035
Targeted endothelial knockout of caveolin-1 in established
atherosclerosis does not inhibit plaque progression.

Rocío Muñiz-Anquela1, Ibon Redondo1,2, Leticia González-Cintado1, Marta Pulgarín-Alfaro2, Fidel Romero2, Miguel
Angel del Pozo2, Jacob Fog Bentzon1.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00035

Copyright: © 2022 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: Muñiz-Anquela R., Redondo I., González-Cintado L., Pulgarín-Alfaro M., Romero F., Del Pozo MA. and Fog Bentzon J.,
Targeted endothelial knockout of caveolin-1 in established atherosclerosis does not inhibit plaque progression. IBJ Plus 2022 1(s5)
e00035. DOI: 10.24217/2531-0151.22v1s5.00035.

Edited: Madrid, España.

Abstract

Caveolae are small invaginations of the plasma membrane with functions in mechano-sensing and -adaption,
membrane transport and transcytosis. Caveolin-1 (Cav1) is required for caveolae to form, and previous research
has shown that global Cav1 knockout substantially reduces early atherosclerosis in mice – an effect suggested to be
mediated by reduced endothelial LDL transcytosis. In the present study, we tested whether targeting caveolin 1 in
endothelium of already established atherosclerosis in mice can be a potential therapy to limit plaque progression.
To allow the manipulation of endothelial Cav1 in atherosclerotic plaques, we bred mice that harbored a tamoxifeninducible
Cre recombinase transgene expressed under the control of the endothelium-specific cadherin 5 (Cdh5)
promoter and were homozygous for either the wildtype (control, n= 32 males and n= 36 females) or a floxed Cav1
(Cav1ECKO, n=31 males and n= 31 females) allele. Atherosclerosis was induced by injecting a single dose of adeno-associated
virus vector encoding a gain of function mutant proprotein convertase subtilisin/kexin type 9 (PCSK9)
followed by feeding high cholesterol diet for 20 weeks. At 8 and 4 weeks prior to study end, 5 doses of tamoxifen (20
mg/ml) were administered to control and Cav1ECKO mice. Recombination efficiency was assessed by immunofluorescence
for Cav1. Plasma lipids were measured by standard techniques. Atherosclerotic plaque size, structure (necrotic
core, lipid and collagen content), content of macrophages (CD68+) and smooth muscle cells (ACTA2+), and the
expression level of fibronectin were studied by histological and immune fluorescence techniques in sections of the
aortic root.
Caveolin 1 protein expression in plaque endothelium was reduced by 90% in Cav1ECKO compared with control mice
by immunofluorescence microscopy (%area fraction of cav-1 signal in endothelium: 67.49 ± 16.87 Cav1WT vs 6.89 ±
8.66 Cav1ECKO in females and 59.85 ± 16.41 Cav1WT vs 5.47 ± 3.85 Cav1ECKO in males). In Cav1ECKO mice, plasma total
and LDL cholesterol were slightly increased (15%) at endpoint (10.67 ± 2.82 for Cav1ECKO vs 9.6 ± 3.52 for Cav1WT in
females and 14.31 ± 4.61 for Cav1ECKO and 11.48 ± 4.42 for Cav1WT mmol/L). No significant differences in plaque
size, necrotic cores, lipid deposits, collagen composition, or content of macrophages and smooth muscle cells were
observed. Fibronectin was reduced in Cav1ECKO males but not in females.
Efficient targeting of Cav-1 in the endothelium of murine plaques cannot stop the progression of already established
atherosclerotic lesions.

e00036
Hypoxia classifier for transcriptome datasets.

Laura Puente-Santamariaa,b,c,*, Lucia Sanchez-Gonzaleza, Ricardo Ramos-Ruizb and Luis del Pesoa,b,d,e,f,*.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00036

Copyright: © 2022 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: Puente-Santamaria L., Sanchez-Gonzaleza L., Ramos-Ruiz R. and Del Peso L., Hypoxia classifier for transcriptome datasets. IBJ Plus 2022 1(s5)
e00036. DOI: 10.24217/2531-0151.22v1s5.00036.

Edited: Madrid, España.

Abstract

developed under a narrow range of conditions and cell types and are often restricted to a set of gene identities.
Focusing on the transcriptional response to hypoxia, we aimed to generate widely applicable classifiers sourced from the
results of a meta-analysis of 69 differential expression datasets which included 425 individual RNA-seq experiments from
33 different human cell types exposed to different degrees of hypoxia (0.1-5\%O\textsubscript{2}) for 2-48h.
The resulting decision trees include both gene identities and quantitative boundaries, allowing for easy classification
of individual samples without control or normoxic reference. Each tree is composed by 3-5 genes mostly drawn from a
small set of just 8 genes (EGLN1, MIR210HG, NDRG1, ANKRD37, TCAF2, PFKFB3, BHLHE40, and MAFF). In spite of
their simplicity, these classifiers achieve over 95\% accuracy in cross validation and over 80\% accuracy when applied
to additional challenging datasets. Our results indicate that the classifiers are able to identify hypoxic tumor samples
from bulk RNAseq and hypoxic regions within tumor from spatially resolved transcriptomics datasets. Moreover, application
of the classifiers to histological sections from normal tissues suggest the presence of a hypoxic gene expression
pattern in the kidney cortex not observed in other normoxic organs. Finally, tree classifiers described herein outperform
traditional hypoxic gene signatures when compared against a wide range of datasets.
This work describes a set of hypoxic gene signatures, structured as simple decision tress, that identify hypoxic samples
and regions with high accuracy and can be applied to a broad variety of gene expression datasets and formats.

e00037
Building insights into CDC37-RAF1 interaction: an opportunity
for design new strategies to treat KRAS driven tumors.

Laura de-la-Puente-Ovejero 1, Gonzalo Aizpurua1, Carmen G Lechuga1, Pablo Mesa2, Guillermo Montoya2, Sara
García-Alonso1*, Mariano Barbacid1*.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00037

Copyright: © 2022 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: De-la-Puente-Ovejero L., Aizpurua G., Lechuga C.G., Mesa P., Montoya G., García-Alonso S. and Barbacid M., Building insights into CDC37-RAF1
interaction: an opportunity for design new strategies to treat KRAS driven tumors. IBJ Plus 2022 1(s5) e00037. DOI: 10.24217/2531-0151.22v1s5.00037.

Edited: Madrid, España.

Abstract

Introduction: genetic interrogation of the KRAS signaling pathway in genetically engineered mouse models harboring
a KRASG12V mutation has shown that RAF1 ablation promotes tumor regression, and is therefore highlighted as
a promising therapeutic target. Recently, our group has been able to purify the full-length form of RAF1 in its native
state and resolve part of its structure by Cryo-Electronic Microscopy. RAF1 is found as a component of a protein
complex (RHC complex), including the chaperone HSP90 and its cochaperone CDC37, being this interaction crucial
for RAF1 stability. In this work, we interrogate the role of CDC37-RAF1 interaction in the stability of RAF1 in order to
identify effective approaches to degrade this kinase.

Materials and Methods: by site-directed mutagenesis we generated a plethora of RAF1 mutants, altering key residues
located in its CDC37-binding region. Then, the relative levels of the RAF1 mutant proteins in the isolated RHC
complexes were assessed by mass spectrometry (MS) and compared to those formed by the wild type protein.
To further evaluate the relevance of CDC37-RAF1 disruption, we designed small peptides covering the region of
interaction. The binding affinity of these peptides was assessed by incubating the cochaperone with a nitrocellulosebound
dodecapeptide array (PepScan) displaying the corresponding RAF1 sequences. Then, to assess the phenotypic
effect of the selected peptides, we functionalized them for in vivo delivery, using a TAT cell penetrating motif. After
treating cells with RAF1 specific and non-specific peptides, culture proliferation was measured.

Results: according to MS results, some of the mutants resulted in RAF1 natural degradation, and same results were
observed when the key residues involved in the interaction were mutated in CDC37 instead of RAF1. On the other
hand, the treatment with CDC37-RAF1 interfering peptides selectively inhibited growth in lung cancer cell lines.
These observations suggest that these RAF1-derived peptides may prevent RHC complex assembly, hindering RAF1
activity and thereby affecting cell proliferation.

Conclusions: altogether, our results highlight the importance of chaperon association for RAF1 stability, raising the
possibility that the interface between RAF1 and CDC37 may represent a vulnerable region, which could be targeted
to induce the degradation of RAF1. Our preliminary data point out to the use of peptidomimetics as a potential
pharmacological approach capable of reproducing the therapeutic results obtained in experimental mouse models
of lung cancer upon ablation of RAF1 expression, representing a potential strategy to treat KRAS driven tumors.

e00039
Automatic synapse parameter exploration for the interaction
of living neurons and models in hybrid circuits and hybrots.

Manuel Reyes-Sanchez 1, Rodrigo Amaducci1, Irene Elices1,2, Pablo Sánchez-Martín 1, Alicia Garrido-Peña 1, Francisco
B. Rodriguez 1, Pablo Varona 1.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00039

Copyright: © 2022 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: Reyes-Sanchez M., Amaducci R., Elices I., Sánchez-Martín P., Garrido-Peña A., Rodriguez FB. and Varona P., Automatic synapse parameter
exploration for the interaction of living neurons and models in hybrid circuits and hybrots. IBJ Plus 2022 1(s5) e00039. DOI: 10.24217/2531-
0151.22v1s5.00039.

Edited: Madrid, España.

Abstract

Hybrid circuits that connect living and model neurons allow studying neural dynamics to assess the role of specific
cells and synapses in emergent phenomena of neural computation (Reyes-Sanchez et al. 2020). In this work, we
deal with the automatic adaptation and mapping of parameters in hybrid circuits and, in particular, those that yield
dynamical invariants. Such invariants take the form of robust relationships between the intervals that build robust
sequences arising from the cell interactions and have been recently unveiled in well-known CPGs (Elices et al. 2019).
In our methodology, we input biological time series with a characteristic temporal structure of spiking-bursting dynamics
to different model neurons with monodirectional and bidirectional synapses implemented with dynamic clamp.

To illustrate the protocol, we searched for dynamical invariants established between a living pyloric CPG cell and
a Komendantov-Kononenko model neuron connected through a graded synapse model. The biological recordings
were preprocessed to automatically adapt the corresponding time and amplitude scales to those of the synapse and
neuron models employed. Our automatic experimental protocol then mapped the neuron and synapse parameters
that yielded a predefined dynamical invariant. By using parallel computing, this approach readily achieved a full
characterization of the parameter space that resulted in the predefined target dynamics. To search for dynamical
invariants in real-time bidirectional connections, we also developed a genetic search that found valid set of parameters
to reproduce the target dynamical invariant in a few iterations.

We illustrated this methodology in the study of the coordination generated by the dynamical invariants to balance
flexibility and robustness in neural rhythms. Our results demonstrate that maps showing the presence of dynamical
invariants can be built in a few minutes for monodirectional hybrid circuits and that the genetic algorithm can readily
find dynamical invariants in bidirectional connections between living and model neurons. The proposed strategy
can be generalized for any hybrid circuit and can also be used in the design of hybrots, i.e., robots whose locomotion
is controlled by living neural circuits with feedback from the sensor robots.

e00040
The secretome of highly metastatic cells as a source of biomarkers
and metastatic effectors in colorectal cancer patients.

Javier Robles1,2, Laura Pintado2, Issam Boukich2, Beatriz Escudero2, Juan Ignacio Imbaud1 and José Ignacio Casal2.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00040

Copyright: © 2022 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: Robles J., Pintado L., Boukich I., Escudero B., Imbaud JA. and Casal JA., The secretome of highly metastatic cells as a
source of biomarkers and metastatic effectors in colorectal cancer patients. IBJ Plus 2022 1(s5) e00040. DOI: 10.24217/2531-
0151.22v1s5.00040.

Edited: Madrid, España.

Abstract

Introduction: Colorectal cancer (CRC) is the second leading cause of cancer death in developed countries, mainly
as a consequence of metastatic spread. Secreted proteins are essential to communicate the cancer cells with
the tumor- microenvironment, favoring tumor progression and metastasis. The conditioned medium or cellular
secretome, including exosomes, has demonstrated to be a rich source of metastatic effectors and biomarkers of
metastatization in different tumors. Our goal was to identify proteins altered in the secreted fraction of highly
metastatic cells in order to determine altered biological pathways and potential biomarkers of prognosis.

Material and methods: A quantitative label-free proteomic analysis was carried out on the secretome of highly
and poorly metastatic CRC cell lines from different genetic background. Proteomic results were validated using
unbiased transcriptomic analyses. Hazard ratios and long-rank tests for the differentially-secreted proteins were
determined in four different external datasets in order to select the proteins with clinical relevance for further
analysis. Gene ontology was performed to determine the biological processes altered in highly metastatic cells.

Results: 221 differentially-secreted proteins were found to be significantly associated to metastasis. Some corresponding
genes were able to predict the overall and progression free survival in stage II and stage III CRC patients.
In addition, they showed higher expression in the stem cell-like and CMS4 subtypes of CRC, associated to worse
prognosis. These genes were also associated to deficient mismatch repair, CpG-island methylator positive status
and BRAF mutation. It is also remarkable that a significant number of altered proteins were involved in cholesterol
metabolism, suggesting an enhancement of the LDL uptake and metabolism in highly metastatic cells.
Extracellular matrix constituents involved in cell adhesion and migration were also overrepresented in the highly
metastatic fraction.

Conclusion: Secretome of highly metastatic cells is enriched in proteins involved in essential pathways for metastasis
such us adhesion, migration and lipid metabolism. In addition, several proteins with a robust and significant
prognosis value were detected howing the value of the fraction as a source of biomarkers.

e00041
Does MAPT have anything new to say? Discovery of novel nonaggregative
Tau isoforms that are decreased in Alzheimer’s disease.

Daniel Ruiz-Gabarre1, 2, 3 ‡, Vega García-Escudero1, 2, 3 ‡, Ricardo Gargini3, 4, Mar Pérez1,3, Esther García3, Raquel Cuadros3, Ivó H
Hernández3, Jorge R Cabrera5, Ramón García-Escudero6, 7, 8, José J Lucas3, 9, Félix Hernández3, 9, Jesús Ávila3, 9 *.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00041

Copyright: © 2022 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: Ruiz-Gabarre D., García-Escudero V., Gargini R., Pérez M., García E., Cuadros R., Hernández IH., Cabrera JR., García-Escudero R., Lucas JJ.,
Hernández F. and Ávila J., Does MAPT have anything new to say? Discovery of novel nonaggregative Tau isoforms that are decreased in Alzheimer’s
disease. IBJ Plus 2022 1(s5) e00041. DOI: 10.24217/2531-0151.22v1s5.00041.

Edited: Madrid, España.

Abstract

Introduction: Tauopathies, including Alzheimer’s disease (AD), are a group of neurodegenerative disorders characterised
by Tau hyperphosphorylation. Post-translational modifications of Tau such as phosphorylation and truncation
have been demonstrated to be an essential step in the molecular pathogenesis of tauopathies. Alternative splicing is
the process by which a single gene can produce multiple transcripts and, potentially, as many different proteins. Tau
isoforms are generated by alternative splicing from a single gene, MAPT. In this work, we demonstrate the existence
of a new, human- specific truncated form of Tau generated by intron 12 retention.
Materials and methods: Intron-12-retaining MAPT transcripts were detected using qPCR with SH-SY5Y neuroblastoma
human cells and confirmed in human brain samples. Results were validated on a database of human RNA-seq
samples (363 samples from three brain regions). Functional assays were carried out after cloning the isoform on the
eukaryotic and prokariotic expression vectors pSG5 and pRK172. Functional analysis evaluated sarkosyl solubility,
heparin-induced self-aggregation, microtubule stabilisation and binding affinity and epitope phosphorylation; all of
them evaluated by Western blot. Heparin-induced aggregation and microtubule stabilisation were also analysed by
electron microscopy. Protein levels on human samples were analysed by Western blot on Alzheimer’s brain classified
according to Braak stages I (n = 3), II (n = 6), III (n = 4), IV (n = 1), V (n = 10) and VI (n = 8), and non-demented control
subjects (n = 10).
Results and conclusions: In this work, we demonstrated the existence of a previously undescribed, human-specific
truncated tau isoform generated by intron 12 retention. RNA transcripts retaining intron 12 were detected in SHSY5Y
cells and, to a greater extent, in human brains, which were then further confirmed on a larger RNAseq public
database. Functional analysis demonstrated that, while this new Tau isoform exhibits similar post-transcriptional
modifications by phosphorylation and affinity for microtubule binding, it is less prone to aggregate than other Tau
isoforms. Importantly, diminished protein levels of this new Tau isoform are found in Alzheimer’s patients’ brains
with respect to non-demented control subjects, suggesting that the lack of this truncated isoform may play an
important role in the pathology. Our results open up new research avenues focused on the exploration of MAPT
alternative splicing and the striking characteristics of novel isoforms, that may help develop future therapies for
Alzheimer’s disease and other tauopathies.

e00042
Unravelling neuron-astrocyte communication in the dorsal
raphe nucleus.

Andrea Sánchez-Ruiz1, Candela González-Arias1, Gertrudis Perea1.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00042

Copyright: © 2022 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: Sánchez-Ruiz A., González-Arias C. and Perea G., Unravelling neuron-astrocyte communication in the dorsal raphe nucleus.
IBJ Plus 2022 1(s5) e00042. DOI: 10.24217/2531-0151.22v1s5.00042.

Edited: Madrid, España.

Abstract

Introduction: Serotonin is a neuromodulator widely spread throughout the central nervous system and involved in
a vast variety of behaviors, such as cognitive functions and emotional states. On this basis, serotonergic dysfunction
has been related to several psychiatric disorders such as Major Depressive Disorder, schizophrenia, or autism.
Despite serotonin’s widespread distribution, the location of serotonergic neurons is restricted to the midbrain raphe
nuclei. In this context, astrocytes are known to participate in synaptic transmission modulating neural circuits; however,
their role in the serotonergic system remains largely unknown. My study aims to elucidate the neuron-astrocyte
signaling at serotonergic nuclei, looking at the dorsal raphe nucleus (DRN).
Materials and methods: For the purpose of this project, we performed intracranial injections of viral constructs in
adult mice to express the Ca2+ indicator GCaMP6 specifically in DRN astrocytes, Ca2+ imaging experiments to record
astrocytic activity, electrophysiological recordings of neuronal activity using patch-clamp and immunohistochemistry
techniques.
Results and conclusions: Preliminary data show that astrocytes from DRN respond to serotonin with an increase in
the amplitude and frequency of intracellular Ca2+ events, which is partly mediated by serotonin type 2 receptors. As
a consequence of triggering Ca2+ signaling, we found that serotonin induces gliotransmission, shown by an increased
frequency of Slow Inward Currents (SICs) and modulation of excitatory synaptic transmission in dorsal raphe
neurons. In conclusion, these data suggest that astrocytes might play a role in synaptic transmission and neuronal
excitability in the DRN and, therefore, in the serotonergic-mediated actions of DRN.

e00043
Resolvin D2 prevents cardiovascular damage in angiotensin
II-induced hypertension.

Lucía Serrano Díaz del Campo1, Ana B. García-Redondo1,2,3, Mercedes Salaices1,2,3, Ana M. Briones1,2,3.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00043

Copyright: © 2022 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: Serrano Díaz del Campo L., García-Redondo AB., Salaices M. and Briones AM., Resolvin D2 prevents cardiovascular damage in angiotensin
II-induced hypertension. IBJ Plus 2022 1(s5) e00043. DOI: 10.24217/2531-0151.22v1s5.00043

Edited: Madrid, España.

Abstract

Introduction: Vascular functional and structural alterations induced by hypertension are greatly influenced by
low- grade chronic inflammation. Resolution of inflammation is orchestrated by specialized lipid pro-resolving
mediators (SPMs), which derive from n3 fatty acids (PUFAs). Previous evidence suggest that SPM prevent vascular
damage in several pathological situations including atherosclerosis or vascular restenosis. Among SPMs,
resolvins (Rv) seem to have beneficial effects in some cardiovascular pathologies, but little is known about their
effect on cardiovascular damage in hypertension.

Objective: The aim of this study was to evaluate the effects of resolvin D2 (RvD2) in blood pressure and cardiovascular
damage associated with hypertension.
Material and methods: aorta, mesenteric resistance arteries (MRA), heart and peritoneal macrophages were
taken from C57BL/6J mice, infused or not with angiotensin II (AngII; 1,44mg/kg/day; for 14 days) in presence or
absence of RvD2 (100ng/mice, every second day, started before AngII infusion). Blood pressure was measured by
tail-cuff plethysmography. Cardiac and vascular function and structure were studied with wire and pressure myographs,
confocal microscopy, histological staining, and echocardiography. Circulating leucocyte were analyzed by
flow cytometry and macrophages behavior by electrophysiology. Gene expression was analyzed with RT-PCR.

Results: Aorta or heart from AngII-infused mice showed altered expression of enzymes and receptors involved in
SPMs biosynthesis and signaling. We also observed a downregulation of SPMs in heart tissues from these mice
including 17R-RvD1 and RvE3. Treatment with RvD2 partially prevented the increase in blood pressure and in the
content of circulating immune cells induced by AngII. RvD2 treatment also improved cardiac hypertrophy, fibrosis
and dysfunction. Moreover, RvD2 treatment reduced vascular hypercontractility and endothelial dysfunction
induced by AngII likely because of enhanced NO and PGI2 availability. RvD2 normalized AngII-induced vascular
remodeling by decreasing media thickness and number of vascular smooth muscle cell, while it did not affect
vascular stiffness. Finally, RvD2 reduced aortic and cardiac leukocyte infiltration and shifted macrophage phenotype
towards a pro-resolving phenotype.

Conclusion: Our data shows that RvD2 treatment limits the cardiovascular alterations induced by hypertension.
These findings highlight that activating resolution mechanisms by treatment with RvD2 may represent a novel
therapeutic strategy for the treatment of cardiovascular alterations associated to hypertension.

e00044
Resolvin E1 attenuates endothelial senescence induced
by doxorubicin through the modulation of NLRP3
inflammasome activation.

Licia Shamoon 1,2,3, Jenaro A. Espitia-Corredor 1,2,4, Pilar Dongil 1,3, Marta Menéndez-Ribes 1,3, Alejandra Romero 1,3,
Inés Valencia 1,3, Guillermo Díaz- Araya 4,5, Carlos F. Sánchez-Ferrer 1,3, Concepción Peiró 1,3.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00044

Copyright: © 2022 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: Shamoon L., Espitia-Corredor JA., Dongil P., Menéndez-Ribes M., Romero A., Valencia I., Díaz-Araya G., Sánchez-Ferrer CF. and Peiró C.,
Resolvin E1 attenuates endothelial senescence induced by doxorubicin through the modulation of NLRP3 inflammasome activation. IBJ Plus 2022
1(s5) e00044. DOI: 10.24217/2531-0151.22v1s5.00044.

Edited: Madrid, España.

Abstract

Introduction: Vascular aging is associated with endothelial cell senescence, favoring low-grade inflammation,
endothelial dysfunction, and cardiovascular diseases. Cell senescence arises from a wide variety of endogenous
and exogenous stressors including some anticancer agents such as doxorubicin. Recently, doxorubicin
was linked to the innate immunity component NLRP3 inflammasome which is implicated in many vascular
inflammatory disorders. There is a need for therapeutic tools to help cancer patients who have been exposed
to cardiovascular toxic chemotherapy averting premature vascular complications. We investigated whether resolvin
E1 (RvE1), an endogenous lipid mediator of the inflammation resolution phase, could prevent doxorubicin-
induced senescence in cultured human umbilical veins endothelial cells (HUVEC) with focus on a potential
involvement of the NLRP3 inflammasome.

Materials and Methods: Cell senescence was quantified by senescence-associated-β-galactosidase (SA-β-gal)
staining. The expression of senescence markers (γH2AX, p21, p53) and inflammatory markers (pP65, NLRP3) was
determined via Western blot. NLRP3 inflammasome activation was determined by visualizing the formation of
ASC specks by indirect immunofluorescence.

Results: Doxorubicin (25 nmol/L) augmented the number of SA-β-gal positive HUVEC and the levels of γH2AX,
p21 and p53 which were all reduced by RvE1 (10 nmol/L). In doxorubicin-treated cells, RvE1 further reduced
the expression of pP65 and NLRP3 proteins and the formation of ASC specks as did the inflammasome assembly
inhibitor MCC950 (1μmol/L). Additionally, both MCC950 and interleukin-1 receptor inhibitor anakinra diminished
SA-β-gal positive staining induced by doxorubicin.

Conclusion: RvE1 offers a novel therapeutic approach against doxorubicin-induced cardiovascular toxicity and
subsequent age-related vascular disorders by counteracting endothelial senescence through the modulation of
NLRP3- inflammasome activation.

e00046
Infections in children with Juvenile Idiopathic Arthritis, more frequent
than in healthy children? Prospective multi-center observational study.

Clara Udaondo Gascón1*, Esmeralda Núñez Cuadros14, Sara Murias Loza1, Agustín Remesal Camba1, Rosa Alcobendas Rueda1,
Concepción Guerrero López2, Sara Guillén Martín3, Marta Escuredo Bergua4, Esther Aleo Lujan5, Daniel Enrique Alonso Martín6,
Alfredo Tagarro García7, Eloísa De Santiago García-Caro8, Marisol Camacho Lovillo9, Fátima Díaz Fernández10, Dolores Arenas
Cabrera11, María José Lirola Cruz12, Pilar Camacho Conde13, Cristina Calvo Rey1.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00046

Copyright: © 2022 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: Udaondo Gascón C., Núñez Cuadros E., Murias Loza S., Remesal Camba A., Alcobendas Rueda R.,
Guerrero López C., Guillén Martín S., Escuredo Bergua M., Aleo Lujan E., Alonso Martín DE., Tagarro García A., De Santiago García-Caro E., Camacho Lovillo
M., Díaz Fernández F., Arenas Cabrera D., Lirola Cruz MJ., Camacho Conde P. and Calvo Rey C., Infections in children with Juvenile Idiopathic Arthritis, more
frequenthan in healthy children? Prospective multi-center observational study. IBJ Plus 2022 1(s5) e00046. DOI: 10.24217/2531-0151.22v1s5.00046.

Edited: Madrid, España.

Abstract

Background
Children with JIA may have a higher risk of infection. Our objectives are to describe and compare infection rates in JIA
patients vs non-JIA counterparts.
Methods A prospective, multicenter observational study was performed in Spain from 01/2017 to 06/2019. JIA
patients from 7 participating hospitals and children without JIA (siblings of JIA patients, and non-JIA children from
primary health centers) were followed up with quarterly questionnaires recording infection episodes. Tuberculosis,
Herpes Zoster, and infections requiring hospital admission were considered severe infections. Rate of infection (episodes/
patient/year) was compared using a Generalized Estimating Equations Model.

Results 371 children (181 JIA and 190 non-JIA) were included. Median age was 8.8years (IQR 5.5 – 11.3); 75% of JIA patients
received immunosuppressive treatment (24% methotrexate, 22% biologic, 26% both). 667 infections were recorded,
15(2.2%) considered severe. Infection rate was 1.31 (95%CI 1.1 – 1.5) in JIA and 1.12 (95%CI 0.9 – 1.3) in non-JIA
participants (p0.19). Age <4 years increased 2.5 times infection rate (2.51 vs. 0.98, p<0.001) in both groups. The most
frequent infection sites were upper respiratory (62.6% vs. 74.5%) and gastrointestinal (18.8%vs 11.4%). There were
no differences in severe infections (2.5%vs 2%, p0.65) among groups. In JIA children, younger age and higher disease
activity (JADAS71) were associated with a higher infection rate.

Conclusions Despite being a potential risk group, we found no differences in infection rate, severity, type and antibiotic
treatment between patients with and without JIA. Most infections were mild. Age below 4 years increased infection
risk in both groups. Higher disease activity was associated with a higher infection rate in JIA patients.

e00047
THE SONIC HEDGEHOG AGONIST SAG ATTENUATES MITOCHONDRIAL
DYSFUNCTION AND DECREASES THE NEUROTOXOCITY INDUCED BY
FRATAXIN-DEFICIENT ASTROCYTES.

Andrés Vicente-Acosta1,2,3,4, Alfredo Giménez-Cassina1,2, Javier Díaz-Nido1,2,3, Frida Loria1,2,5 *.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00047

Copyright: © 2022 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: Vicente-Acosta A., Giménez-Cassina A., Díaz-Nido J. and Loria F., The sonic hedgehog agonist sag attenuates mitochondrial Dysfunction and
decreases the neurotoxocity induced by Frataxin-deficient astrocytes. IBJ Plus 2022 1(s5) e00047. DOI: 10.24217/2531-0151.22v1s5.00047.

Edited: Madrid, España.

Abstract

Friedreich’s ataxia (FRDA) is predominantly a neurodegenerative disease caused by the deficiency of a protein called frataxin
(FXN). Although the main pathological alterations are observed in neurons, it is becoming clear that other non-neuronal
cells such as astrocytes may be actively involved in the neurodegenerative process associated with the disease.
Depending on the stimuli they respond to, astrocytes acquire different activation states in a process called astrogliosis.

Neuroinflammatory stimuli induce the formation of A1 reactive astrocytes, which upregulate proinflammatory
genes, being harmful for neurons. A1 astrocytes have been detected in post-mortem tissue of patients with different
neurodegenerative disorders, being hypothesized that they might have deleterious effects on neurons, exacerbating
the neurodegenerative process. Recent studies have demonstrated positive effects of Sonic Hedgehog (SHH) agonists
in astrocyte viability and proliferation, astrocyte-mediated neuroprotection, and also positive effects in mitochondrial
activity and dynamics. As mitochondrial changes are important components in the etiology of neurodegenerative
disorders, the influence of SHH agonists in mitochondrial physiology could be of therapeutic relevance. In this work,
we have thoroughly characterized astrocyte reactivity phenotype and mitochondrial status of FXN-deficient human
astrocytes, evaluating as well the effect of SHH agonists on astrocyte reactivity, viability, and function.
We used an in vitro model based on a short hairpin RNA packaged in a lentiviral vector, which allowed us to decrease
FXN levels in human cortical astrocytes, to similar levels as those observed in FRDA patients, and found that FXN-deficient
cells had less cell viability and higher expression of several A1 reactive astrocyte markers, than control cells.
Both phenomena were prevented by a chronic treatment with the smoothened agonist (SAG), a SHH signaling agonist.
Moreover, FXN-deficient astrocytes showed defects in mitochondrial function and dynamics, which were partially rescued
by SAG. Regarding the possible neuroprotective effects of SHH agonists, previous results showed that FXN-deficient astrocytes
are able to induce neurodegeneration, and we have observed that the chronic treatment with SAG attenuated the
neurotoxicity triggered by the treatment of mouse cortical neurons with conditioned medium of FXN-deficient astrocytes.

Overall, our results suggest that the treatment of FXN-deficient astrocytes with a SHH agonist like SAG, could be used
as a possible target to reduce FRDA-associated neurodegeneration.

e00048
Mitophagy boosting protects cells against MNU toxicity.

Beatriz Villarejo Zori1, Juan Zapata Muñoz1, Elena Sierra Filardi1, Ignacio Ramírez Pardo2 and Patricia Boya1*.

Published June 2022

DOI: 10.24217/2531-0151.22v1s5.00047

Copyright: © 2022 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: Vicente-Acosta A., Giménez-Cassina A., Díaz-Nido J. and Loria F., The sonic hedgehog agonist sag attenuates mitochondrial Dysfunction and
decreases the neurotoxocity induced by Frataxin-deficient astrocytes. IBJ Plus 2022 1(s5) e00047. DOI: 10.24217/2531-0151.22v1s5.00047.

Edited: Madrid, España.

Abstract

Objective: A pharmacological model to study the human disease Retinitis Pigmentosa is N-methyl-N-nitrosourea
(MNU) injection which results in retinal degeneration. Our objective is to study the autophagy and mitophagy
status of the cells in this disease model.
Materials and methods: The mitophagy reporter (MitoQC) and autophagy reporter mice (mCherry-GFP-LC3)
have been used to determine the mitophagic and autophagic flux in the retina after MNU injection. Retinal pigment
epithelium cell line (ARPE-19) MitoQC has been used to elucidate the cellular and molecular mechanisms
after MNU treatment.

Results: Neurodegeneration events appear one day after intraperitoneal MNU injection: reduction of photoreceptors
thickness layer and increase of TUNEL and GFAP staining. Mitophagosomes and autophagosomes are less
frequent in the retina of mice treated with MNU, but they are bigger and they tend to accumulate in the external
limiting membrane. ARPE-19 cells are also vulnerable to MNU in a dose-dependent manner producing DNA damage,
cytoplasm vacuolization, organelle alterations and cell death. Mitophagy levels depend on the MNU dose:
an increase of the mitophagic flux is observed at low doses, whereas it seems to be blocked at high doses. We
demonstrate this mitophagy is PINK-Parkin dependent and boosting this pathway makes cells more resistant to
MNU. Finally, we achieve to protect MNU-treated retinae explants with the mitophagy inductor, DFP.

Conclusion: MNU treatment induces activation of PINK-Parkin dependent mitophagy. We propose that mitophagy
could act as a defense mechanism in this disease model.