Unidades Centrales Científico-Técnicas (UCCTs)

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12105/25179

Las Unidades Centrales Científico-técnicas (UCCTs) de la SGSAFI es un área integrada por nueve Unidades, Veterinaria, Microcirugía Experimental-Micro manipulación de gametos (MEMIG), Histología, Microscopia Electrónica, Microscopía Óptica Avanzada, Citometría de Flujo, Proteómica, Genómica, y Bioinformática. Las actividades técnicas que desarrollan van orientadas a dar el soporte a los investigadores de todos los centros del ISCIII que soliciten la asistencia científico-técnica y prestación de servicios de alta calidad. Los miembros de las UCCTs desarrollan una triple actividad. Además de ofrecer servicios técnicos de alta calidad efectúan una labor investigadora mediante proyectos propios o en colaboración con otros grupos, y se implican en la formación de personal técnico o científico, participando en actividades inherentes a cada Unidad, en las encuadradas dentro del programa de formación del ISCIII y en los programas formativos de IMIENS- Instituto Mixto de Investigación UNED.

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Elucidating the functional role of the novel BdP50 protein and extracellular vesicles in the human erythrocyte infection by Babesia divergens
(Public Library of Science (PLOS), 2025-08-13) Gonzalez, Luis Miguel; Revuelta, Belén; Gil, Aitor; Terrón-Orellana, Maria Carmen; Sachse, Martin; Sotillo, Javier; Luque, Daniel; Rahman, S M Raihan; Bastos, Reginaldo G.; Suarez, Carlos Esteban; Montero-Clemente, Estrella; Instituto de Salud Carlos III; Comunidad de Madrid (España)
Babesia divergens is a blood-borne parasite that invades, replicates within and destroys red blood cells (RBCs) during its asexual life cycle, causing babesiosis in humans and cattle. This study focuses on BdP50, a putative B. divergens glycosylphosphatidylinositol-anchored protein involved in the parasite life cycle. BdP50 is found on the surface of B. divergens invasive parasites (merozoites) as well as on extracellular vesicles (Bd-derived EVs). These EVs are secreted by parasites cultured in fresh human RBCs and, in addition to BdP50, are enriched in human and parasite proteins, including proteins related to the parasite invasion process. BdP50 binds to RBCs and could mediate interactions of free merozoites and Bd-derived EVs with the host cell. Anti-BdP50 antibodies support this by blocking the BdP50 protein and inhibiting up to 88% of merozoite entry into naïve RBCs. This reinforces the role of BdP50 in parasite-host cell interactions and invasion. However, the inhibitory effect of BdP50 antibodies begins to gradually decrease slightly several hours after invasion, leading to a progressive increase in B. divergens infected RBCs over time. Consistent with these findings, our in vitro de novo infection assays showed that Bd-derived EVs, in addition to promoting parasite propagation, display proteins such as BdP50 that mimic the merozoite surface to likely attenuate the blocking effect of antibodies, thereby ensuring the parasite survival during subsequent rounds of invasion and growth. Given the role of BdP50 and Bd-derived EVs in the B. divergens life cycle, this study could have future implications for developing new approaches to interfere with parasite invasion proteins and Bd-derived EVs functions.
Dasatinib interferes with HIV-1 proviral integration and the inflammatory potential of monocyte-derived macrophages from people with HIV
(Elsevier, 2024-11) Rodriguez Mora, Sara; Sánchez-Menéndez, Clara; Bautista-Carrascosa, Guiomar; Mateos, Elena; Moreno-Serna, Lucia; Megias Vazquez, Diego; Cantón, Juan; García-Gutiérrez, Valentín; Murciano-Antón, María Aranzazu; Cervero, Miguel; Spivak, Adam; Planelles, Vicente; Coiras, Mayte; National Institutes of Health (Estados Unidos); Ministerio de Ciencia e Innovación (España); Agencia Estatal de Investigación (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Centro de Investigación Biomédica en Red - CIBERINFEC (Enfermedades Infecciosas); Fundación para la Investigación Biomédica del Hospital Universitario Ramón y Cajal
HIV-1 infection is efficiently controlled by the antiretroviral treatment (ART) but viral persistence in long-lived reservoirs formed by CD4 + T cells and macrophages impedes viral eradication and creates a chronic inflammatory environment. Dasatinib is a tyrosine kinase inhibitor clinically used against chronic myeloid leukemia (CML) that has also showed an anti-inflammatory potential. We previously reported that dasatinib is very efficient at interfering with HIV-1 infection of CD4 + T cells by preserving the antiviral activity of SAMHD1, an innate immune factor that blocks T-cell activation and proliferation and that is inactivated by phosphorylation at T592 (pSAMHD1). We observed that short-term treatment in vitro with dasatinib significantly reduced pSAMHD1 in monocyte-derived macrophages (MDMs) isolated from people with HIV (PWH) and healthy donors, interfering with HIV-1 infection. This inhibition was based on low levels of 2-LTR circles and proviral integration, while viral reverse transcription was not affected. MDMs isolated from people with CML on long-term treatment with dasatinib also showed low levels of pSAMHD1 and were resistant to HIV-1 infection. In addition, dasatinib decreased the inflammatory potential of MDMs by reducing the release of M1-related cytokines like TNFα, IL-1β, IL-6, CXCL8, and CXCL9, but preserving the antiviral activity through normal levels of IL-12 and IFNγ. Due to the production of M2-related anti-inflammatory cytokines like IL-1RA and IL-10 was also impaired, dasatinib appeared to interfere with MDMs differentiation. The use of dasatinib along with ART could be used against HIV-1 reservoir in CD4 and macrophages and to alleviate the chronic inflammation characteristic of PWH.
Evolution of invasive pneumococcal disease by serotype 3 in adults: a Spanish three-decade retrospective study
(Elsevier, 2024-06) Calvo-Silveria, Sara; González-Díaz, Aida; Grau, Inmaculada; Marimón, José María; Cercenado, Emilia; Quesada, M Dolores; Casabella, Antonio; Larrosa, Nieves; Yuste, Jose Enrique; Berbel, Dàmaris; Alonso, Marta; Tubau, Fe; Belman, Sophie; Cadenas-Jiménez, Irene; Martin-Galiano, Antonio Javier; Domínguez, María Ángeles; Martí, Sara; Liñares, Josefina; Pallarés, Román; Càmara, Jordi; Ardanuy, Carmen; Instituto de Salud Carlos III; Centro de Investigación Biomédica en Red - CIBERES (Enfermedades Respiratorias); Centro de Investigación Biomédica en Red - CIBERINFEC (Enfermedades Infecciosas)
Background: Invasive pneumococcal disease due to serotype 3 (S3-IPD) is associated with high mortality rates and long-term adverse effects. The introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into the Spanish paediatric immunisation programme has not led to a decrease in the adult S3-IPD. We aimed to analyse the incidence, clinical characteristics and genomics of S3-IPD in adults in Spain. Methods: Adult IPD episodes hospitalized in a Southern Barcelona hospital were prospectively collected (1994-2020). For genomic comparison, S3-IPD isolates from six Spanish hospitals (2008-2020) and historical isolates (1989-1993) were analysed by WGS (Illumina and/or MinION). Findings: From 1994 to 2020, 270 S3-IPD episodes were detected. When comparing pre-PCV (1994-2001) and late-PCV13 (2016-2020) periods, only modest changes in S3-IPD were observed (from 1.58 to 1.28 episodes per 100,000 inhabitants year). In this period, the incidence of the two main lineages shifted from 0.38 to 0.67 (CC180-GPSC12) and from 1.18 to 0.55 (CC260-GPSC83). The overall 30-day mortality remained high (24.1%), though a decrease was observed between the pre-PCV (32.4%; 95.0% CI, 22.0-45.0) and the late-PCV13 period (16.7%; 95.0% CI, 7.5-32.0) (p = 0.06). At the same time, comorbidities increased from 77.3% (95.0% CI, 65.0-86.0) to 85.7% (95.0% CI, 71.0-94.0) (p = 0.69). There were no differences in clinical characteristics or 30-day mortality between the two S3 lineages. Although both lineages were genetically homogeneous, the CC180-GPSC12 lineage presented a higher SNP density, a more open pan-genome, and a major presence of prophages and mobile genetic elements carrying resistance genes. Interpretation: Adult S3-IPD remained stable in our area over the study period despite PCV13 introduction in children. However, a clonal shift was observed. The decrease in mortality rates and the increase in comorbidities suggest a change in clinical management and overall population characteristics. The low genetic variability and absence of clinical differences between lineages highlight the role of the S3 capsule in the disease severity.
First isolation of the Sindbis virus in mosquitoes from southwestern Spain reveals a new recent introduction from Africa
(Elsevier, 2025) Gutiérrez-López, Rafael; Ruiz-López, María José; Ledesma, Juan; Ledesma, Juan; Magallanes, Sergio; Nieto, Cristina; Ruiz, Santiago; Sánchez-Peña, Carolina; Ameyugo, Ulises; Camacho, Juan; Varona Fernandez, Sarai; Cuesta de la Plaza, Isabel; Jado, Isabel; Sánchez-Seco, María Paz; Figuerola, Jordi; Vazquez, Ana; Agencia Estatal de Investigación (España); Ministerio de Ciencia e Innovación (España); Unión Europea. Comisión Europea. NextGenerationEU; Regional Government of Andalusia (España); Instituto de Salud Carlos III
Sindbis virus (SINV), is an of the family . This zoonotic arbovirus is transmitted by mosquitoes, primarily from the genus, with bird species acting as amplifying vertebrate hosts. Occasionally it can also affect humans that are accidental hosts. SINV genotype I (SINV-I) has been isolated in mosquitoes and birds in South Africa and Northern Europe, producing fever outbreaks. In the last decades, there were several detections of SINV in Europe. In 2022, during the West Nile virus (WNV) mosquito surveillance program in Andalucía (Spain) implemented by the regional health administration, we detected the presence of both SINV and WNV in a pool, representing the first detection of SINV in Spain. After this finding, we screened 1149 mosquito pools to determine the status of SINV circulation in western Andalucía. We identified for the first time the presence of SINV in five different mosquito species. presented the highest infection rate by SINV. In addition, SINV was geographically widespread and distributed in four out of the five Andalucía's provinces studied, with Cadiz presenting the highest infection rate. All SINV genomes from Southwestern Spain characterised in this study belonged to SINV-I, previously detected in Europe and Africa. These isolated SINV-I strains presented low molecular variation among them and in the phylogenomic analyses they formed a monophyletic group that clustered with strains from Algeria and Kenya. These results suggest that, around 2017, a single new SINV introduction into the European continent occurred, probably from Northern (Algeria) or Central Africa.
Internalization and toxicity of polystyrene nanoplastics on inmortalized human neural stem cells
(Elsevier, 2024-05) González-Caballero, MCarmen; De Alba-González, Mercedes; Torres-Ruiz, Mónica; Iglesias-Hernandez, Patricia; Zapata, Verónica; Terrón-Orellana, Maria Carmen; Sachse, Martin; Morales, Mónica; Martin-Folgar, Raquel; Liste-Noya, Isabel; Cañas Portilla, Ana Isabel; Instituto de Salud Carlos III; Agencia Estatal de Investigación (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF)
Global plastic production has increased exponentially in recent decades, and a significant part of it persists in the environment, where it degrades into microplastics and nanoplastics (MPs and NPs). These can enter in humans by ingestion, inhalation, and dermal routes, and there is scientific evidence that they are able to reach the systemic circulation and penetrate and accumulate in various tissues and organs. Neurodevelopmental toxicity of NPs is one of the most worrying effects, as they can cross the blood-brain barrier. In the following study, we analyzed, by transmission electron microscopy, the in vitro uptake of 30-nm polystyrene nanoplastics (PS-NPs) into human neural stem cells (NSCs), their accumulation and subcellular localization within the cell. Furthermore, we studied the effects of different concentrations of PS-NPs on cell death, proliferation, and cell differentiation using immunocytochemistry and quantitative real time PCR for specific markers. This study demonstrated that PS-NPs were able to enter the cell, probably by endocytosis, accumulate, and aggregated in human NSCs, without being detected in the nucleus, causing cell death by apoptosis and decreased cell proliferation. This study provides new insights into the interaction and effects of PS-NPs in human NSC and supports the scientific evidence for the involvement of nanoplastic in neurodevelopmental disorders.
CRISPR targeting of FOXL2 c.402C>G mutation reduces malignant phenotype in granulosa tumor cells and identifies anti-tumoral compounds
(Wiley, 2025-01-08) Amarilla-Quintana, Sandra; Navarro, Paloma; Hernández Martínez, Iván; Ramos, Alejandra; Montero-Calle, Ana Maria; Cabezas-Sainz, Pablo; Barrero, Maria; Megias Vazquez, Diego; Vilaplana-Marti, Borja; Epifano, Carolina; Gomez-Dominguez, Deborah; Monzon-Fernandez, Sara; Cuesta de la Plaza, Isabel; Sánchez, Laura; Barderas Manchado, Rodrigo; Garcia-Donas, Jesus; Martin, Alberto; Perez de Castro, Ignacio
Forkhead box L2 (FOXL2) encodes a transcription factor essential for sex determination, and ovary development and maintenance. Mutations in this gene are implicated in syndromes involving premature ovarian failure and granulosa cell tumors (GCTs). This rare cancer accounts for less than 5% of diagnosed ovarian cancers and is causally associated with the FOXL2 c.402C>G, p.C134W mutation in 97% of the adult cases (AGCTs). In this study, we employed CRISPR technology to specifically eliminate the FOXL2 c.402C>G mutation in granulosa tumor cells. Our results show that this Cas9-mediated strategy selectively targets the mutation without affecting the wild-type allele. Granulosa cells lacking FOXL2 c.402C>G exhibit a reduced malignant phenotype, with significant changes in cell proliferation and invasion. Furthermore, these modified cells are more susceptible to dasatinib and ketoconazole. Transcriptomic and proteomic analyses reveal that CRISPR-modified granulosa tumor cells shift their expression profiles towards a wild-type-like phenotype. Additionally, this altered expression signature has led to the identification of new compounds with antiproliferative and pro-apoptotic effects on granulosa tumor cells. Our findings demonstrate the potential of CRISPR technology for the specific targeting and elimination of a mutation causing GCTs, highlighting its therapeutic promise for treating this rare ovarian cancer.
Monkeypox outbreak in Madrid (Spain): Clinical and virological aspects.
(Elsevier, 2022-07-10) Orviz, Eva; Negredo, Anabel; Ayerdi, Oskar; Vazquez, Ana; Muñoz-Gómez, Ana; Monzon-Fernandez, Sara; Clavo, Petunia; Zaballos, Ángel; Vera, Mar; Sánchez, Patricia; Cabello, Noemi; Jimenez Sancho, Maria Pilar; Pérez-García, Jorge A; Varona Fernandez, Sarai; Del Romero, Jorge; Cuesta de la Plaza, Isabel; Delgado-Iribarren, Alberto; Torres, Montse; Sagastagoitia, Iñigo; Palacios, Gustavo; Estrada, Vicente; Sánchez-Seco, María Paz; Grupo Viruela del Simio Madrid CNM/ISCIII/HCSC/Sandoval; Centro de Investigación Biomédica en Red - CIBERINFEC (Enfermedades Infecciosas)
Background: Monkeypox is the most prevalent Orthopoxvirus zoonosis infection since the eradication of smallpox. The current multi-country outbreak involves five WHO regions affecting mainly Europe. Accurate clinical and virological aspects of the disease outside endemic areas are needed. Methods: We performed an observational study of cases diagnosed in Madrid (Spain) (May/June 2022). Confirmation from vesicular lesions swabs, Orthopoxvirus real-time PCR, sequencing, phylogenetic analysis, and direct detection by Electron microscopy was performed. In addition, a structured epidemiological questionnaire was completed systematically to gather sociodemographic, clinical, and behavioral data from all confirmed cases. Findings: We extracted data from 48 patients, all cisgender men. The median age was 35 years (IQR 29 – 44), and 87.5% were MSM. The most prevalent symptoms were the presence of vesicular-umbilicated and pseudo-pustular skin lesions (93.8%), asthenia (66.6%), and fever (52.1%). In addition, the location of the lesions in the genital or perianal area was related to the role in sexual intercourse (p<0.001). Sequencing analysis indicated the virus circulating in Spain belongs to the western African clade. Like the other European cases in the outbreak, the Spanish isolates are a direct descendant of viruses previously detected in Nigeria, the UK, Singapore, and Israel in 2017–2018. Conclusions: Monkeypox is an emerging infectious disease in Europe where community transmission is reported, mainly in MSM. The first symptom was skin lesions instead of classical fever and rash. The disease follows a self-limited course, and there have been no cases with a serious presentation or severe complications.
Multiresistance to Nonazole Fungicides in Aspergillus fumigatus TR/L98H Azole-Resistant Isolates
(American Society for Microbiology (ASM), 2021-08-17) Gonzalez-Jimenez, Irene; Garcia-Rubio, Rocio; Monzon-Fernandez, Sara; Lucio, Jose; Cuesta de la Plaza, Isabel; Mellado, Emilia; Instituto de Salud Carlos III; Plan Nacional de I+D+i (España); Ministerio de Economía, Industria y Competitividad (España); RETICS-Investigación en Patología Infecciosa (REIPI-ISCIII) (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF)
Drug resistance is a worldwide problem affecting all pathogens. The human fungal pathogen Aspergillus fumigatus coexists in the environment with other fungi targeted by crop protection compounds, being unintentionally exposed to the selective pressure of multiple antifungal classes and leading to the selection of resistant strains. A. fumigatus azole-resistant isolates are emerging in both clinical and environmental settings. Since their approval, azole drugs have dominated clinical treatment for aspergillosis infections and the agriculture fungicide market. However, other antifungal classes are used for crop protection, including benzimidazoles (methyl benzimidazole carbamates [MBCs]), strobilurins (quinolone oxidation inhibitors [QoIs]), and succinate dehydrogenase inhibitors (SDHIs). Mutations responsible for resistance to these fungicides have been widely researched in plant pathogens, but resistance has not been explored in A. fumigatus. In this work, the genetic basis underlying resistance to MBCs, QoIs, and SDHIs was studied in azole-susceptible and -resistant A. fumigatus strains. E198A/Q and F200Y mutations in β-tubulin conferred resistance to MBCs, G143A and F129L substitutions in cytochrome conferred resistance to QoIs, and H270R/Y mutations in SdhB conferred resistance to SDHIs. Characterization of susceptibility to azoles showed a correlation between strains resistant to these fungicides and the ones with tandem-repeat (TR)-based azole resistance mechanisms. Whole-genome sequencing analysis showed a genetic relationship among fungicide multiresistant strains, which grouped into subclusters that included only strains carrying the TR-based azole resistance mechanisms, indicating a common ancestor/evolution pattern and confirming the environmental origin of this type of azole-resistant A. fumigatus.
Loss of Lipooligosaccharide Synthesis in Produces Changes in Outer Membrane Vesicle Protein Content
(Multidisciplinary Digital Publishing Institute (MDPI), 2024-08-27) Cano-Castaño, Beatriz; Corral-Lugo, Andres; Gato, Eva; Terrón-Orellana, Maria Carmen; Martin-Galiano, Antonio Javier; Sotillo, Javier; Perez Gomez, Astrid; McConnell, Michael J; Instituto de Salud Carlos III; Ministerio de Economía, Industria y Competitividad (España)
Outer membrane vesicles (OMVs) are nanostructures derived from the outer membrane of Gram-negative bacteria. We previously demonstrated that vaccination with endotoxin-free OMVs isolated from an strain lacking lipooligosaccharide (LOS) biosynthesis, due to a mutation in , provides full protection in a murine sepsis model. The present study characterizes the protein content of highly-purified OMVs isolated from LOS-replete and LOS-deficient strains. Four purification methods were evaluated to obtain highly purified OMV preparations: ultracentrifugation, size exclusion chromatography (SEC), ultracentrifugation followed by SEC, and Optiprep™. OMVs from each method were characterized using nanoparticle tracking analysis and electron microscopy. OMVs from LOS-deficient and LOS-replete strains purified using the Optiprep™ method were subjected to LC-MS/MS analysis to determine protein content. Significant differences in protein composition between OMVs from LOS-deficient and LOS-replete strains were found. Computational analyses using Bepipred 3.0 and SEMA 2.0 indicated that the lack of LOS led to the overexpression of immunogenic proteins found in LOS-containing OMVs and the presence of immune-stimulating proteins absent in LOS-replete OMVs. These findings have important implications for developing OMV-based vaccines against , using both LOS-containing and LOS-free OMVs preparations.
Molecular and Epidemiological Characterization of Toxigenic and Nontoxigenic Corynebacterium diphtheriae, Corynebacterium belfantii, Corynebacterium rouxii, and Corynebacterium ulcerans Isolates Identified in Spain from 2014 to 2019
(American Society for Microbiology (ASM), 2021-02-18) Hoefer, Andreas; Pampaka, Despina; Herrera-León, Silvia; Peiró, Sonia; Varona Fernandez, Sarai; Lopez-Perea, Noemi; Masa-Calles, Josefa; Herrera-Leon, Laura
This study examines the microbiological and epidemiological characteristics of toxigenic and nontoxigenic isolates submitted to the national reference laboratory in Spain, between 2014 and 2019, in order to describe the current situation and improve our knowledge regarding these emerging pathogens. Epidemiological information was extracted from the Spanish Surveillance System. Microbiological and molecular characterization was carried out using phenotypic methods, multilocus sequence typing (MLST), whole-genome sequencing (WGS), and core genome MLST (cgMLST). Thirty-nine isolates were analyzed. Twenty-one isolates were identified as (6 toxigenic), 14 as , 4 as (3 toxigenic), and 1 as One isolate was identified as nontoxigenic gene bearing (NTTB). Ages of patients ranged from 1 to 89 years, with 10% (3/30) of nontoxigenic and 22% (2/9) of toxigenic isolates collected from children less than 15 years. Twenty-five of the patients were males (17/30 in nontoxigenic; 8/9 in toxigenic). MLST identified 28 sequence types (STs), of which 7 were described for the first time in Spain. WGS analysis showed that 10 isolates, including 3 toxigenic isolates, harbored a variety of antibiotic resistance genes in addition to the high prevalence of penicillin resistance phenotypically demonstrated. Phylogenetic analysis revealed one cluster of isolates from family members. Risk information was available for toxigenic isolates (9/39); 3 patients reported recent travels to countries of endemicity and 3 had contact with cats/dogs. One unvaccinated child with respiratory diphtheria had a fatal outcome. Including nontoxigenic infections in disease surveillance and using WGS could further improve current surveillance.
CRISPR targeting of FOXL2 c.402C>G mutation reduces malignant phenotype in granulosa tumor cells and identifies anti-tumoral compounds
(2024-07-03) Amarilla-Quintana, Sandra; Navarro, Paloma; Ramos, Alejandra; Montero-Calle, Ana Maria; Cabezas-Sainz, Pablo; Barrero, Maria; Megias Vazquez, Diego; Vilaplana-Marti, Borja; Epifano, Carolina; Gomez-Dominguez, Deborah; Hernández Martínez, Iván; Monzon-Fernandez, Sara; Cuesta de la Plaza, Isabel; Sánchez, Laura; Barderas Manchado, Rodrigo; Garcia-Donas, Jesus; Martin, Alberto; Perez de Castro, Ignacio; Instituto de Salud Carlos III; GETTHI; Asociación Española Contra el Cáncer
FOXL2 is a transcription factor essential for sex determination and ovary development and maintenance. Mutations in this gene are implicated in syndromes involving premature ovarian failure and granulosa cell tumors (GCTs). This rare cancer accounts for less than 5% of diagnosed ovarian cancers and is causally associated with the FOXL2 c.402C>G, p.C134W mutation in 97% of the adult cases (AGCTs). In this study, we employed CRISPR technology to specifically eliminate the FOXL2 c.402C>G mutation in granulosa tumor cells. Our results show that this Cas9-mediated strategy selectively targets the mutation without affecting the wild-type allele. Granulosa cells lacking the FOXL2 c.402C>G exhibit a reduced malignant phenotype, with significant changes in cell proliferation, invasion, and cell death. Furthermore, these modified cells are more susceptible to Dasatinib and Ketoconazole. Transcriptomic and proteomic analyses reveal that CRISPR-modified granulosa tumor cells shift their expression profiles towards a wild-type like phenotype. Additionally, this altered expression signature has led to the identification of new compounds with antiproliferative and pro-apoptotic effects on granulosa tumor cells. Our findings demonstrate the potential of CRISPR technology for the specific targeting and elimination of a mutation causing granulosa cell tumors, highlighting its therapeutic promise for treating this rare ovarian cancer.
Deep Intraclonal Analysis for the Development of Vaccines against Drug-Resistant Lineages
(Multidisciplinary Digital Publishing Institute (MDPI), 2024-09-11) Tajuelo, Ana; Gato, Eva; Oteo-Iglesias, Jesus; Perez-Vazquez, Maria; McConnell, Michael J; Martin-Galiano, Antonio Javier; Perez Gomez, Astrid; McConnell, Michael J; Instituto de Salud Carlos III; Ministerio de Ciencia e Innovación (España); Unión Europea. Comisión Europea. NextGenerationEU; National University of Distance Education (España)
Despite its medical relevance, there is no commercial vaccine that protects the population at risk from multidrug-resistant (MDR) infections. The availability of massive omic data and novel algorithms may improve antigen selection to develop effective prophylactic strategies. Up to 133 exposed proteins in the core proteomes, between 516 and 8666 genome samples, of the six most relevant MDR clonal groups (CGs) carried conserved B-cell epitopes, suggesting minimized future evasion if utilized for vaccination. Antigens showed a range of epitopicity, functional constraints, and potential side effects. Eleven antigens, including three sugar porins, were represented in all MDR-CGs, constitutively expressed, and showed limited reactivity with gut microbiota. Some of these antigens had important interactomic interactions and may elicit adhesion-neutralizing antibodies. Synergistic bivalent to pentavalent combinations that address expression conditions, interactome location, virulence activities, and clone-specific proteins may overcome the limiting protection of univalent vaccines. The combination of five central antigens accounted for 41% of all non-redundant interacting partners of the antigen dataset. Specific antigen mixtures represented in a few or just one MDR-CG further reduced the chance of microbiota interference. Rational antigen selection schemes facilitate the design of high-coverage and "magic bullet" multivalent vaccines against recalcitrant lineages.
Metabolic and mitochondria alterations induced by SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10
(Wiley, 2024-07) López-Ayllón, Blanca D; Marin, Silvia; Fariñas Fernández, Marco; García-García, Tránsito; Fernández-Rodríguez, Raúl; de Lucas-Rius, Ana; Redondo, Natalia; Mendoza-García, Laura; Foguet, Carles; Grigas, Juozas; Calvet, Alba; Villalba, José Manuel; Rodríguez, María Josefa; Megías, Diego; Mandracchia, Biagio; Luque, Daniel; Lozano, Juan José; Calvo, Cristina; Merino Herrán, Unai; Thomson, Timothy M; Garrido, Juan J; Cascante, Marta; Montoya, Maria; López-Ayllón, Blanca D; Marin, Silvia; Fariñas Fernández, Marco; García-García, Tránsito; Fernández-Rodríguez, Raúl; de Lucas-Rius, Ana; Redondo, Natalia; Mendoza-García, Laura; Foguet, Carles; Grigas, Juozas; Calvet, Alba; Villalba, José Manuel; Megías, Diego; Mandracchia, Biagio; Luque, Daniel; Lozano, Juan José; Calvo, Cristina; Merino Herrán, Unai; Thomson, Timothy M; Garrido, Juan J; Cascante, Marta; Montoya, María; Unión Europea. Comisión Europea. NextGenerationEU; Consejo Superior de Investigaciones Científicas (España); Regional Government of Andalusia (España); Ministerio de Ciencia (España); Agència de Gestió d´Ajuts Universitaris i de Recerca (AGAUR); Institució Catalana de Recerca i Estudis Avançats; Instituto de Salud Carlos III; Agencia Estatal de Investigación (España)
Antiviral signaling, immune response and cell metabolism are dysregulated by SARS-CoV-2, the causative agent of COVID-19. Here, we show that SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes with critical roles in mitochondrial function and morphology. On the other hand, all four ORFs altered mitochondrial dynamics and function, but only ORF3a and ORF9c induced a marked alteration in mitochondrial cristae structure. Genome-Scale Metabolic Models identified both metabolic flux reprogramming features both shared across all accessory proteins and specific for each accessory protein. Notably, a downregulated amino acid metabolism was observed in ORF9b, ORF9c and ORF10, while an upregulated lipid metabolism was distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention.
Human cerebral organoids: cellular composition and subcellular morphological features
(Frontiers Media, 2024) Mateos-Martínez, Patricia; Coronel Lopez, Raquel; Sachse, Martin; González-Sastre, Rosa; Maeso Cuesta, Laura; Rodríguez, María Josefa; Terrón-Orellana, Maria Carmen; López-Alonso, Victoria; Liste-Noya, Isabel; Ministerio de Ciencia e Innovación (España); Agencia Estatal de Investigación (España); Instituto de Salud Carlos III; Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF)
Introduction: Human cerebral organoids (hCOs) derived from pluripotent stem cells are very promising for the study of neurodevelopment and the investigation of the healthy or diseased brain. To help establish hCOs as a powerful research model, it is essential to perform the morphological characterization of their cellular components in depth. Methods: In this study, we analyzed the cell types consisting of hCOs after culturing for 45 days using immunofluorescence and reverse transcriptase qualitative polymerase chain reaction (RT-qPCR) assays. We also analyzed their subcellular morphological characteristics by transmission electron microscopy (TEM). Results: Our results show the development of proliferative zones to be remarkably similar to those found in human brain development with cells having a polarized structure surrounding a central cavity with tight junctions and cilia. In addition, we describe the presence of immature and mature migrating neurons, astrocytes, oligodendrocyte precursor cells, and microglia-like cells. Discussion: The ultrastructural characterization presented in this study provides valuable information on the structural development and morphology of the hCO, and this information is of general interest for future research on the mechanisms that alter the cell structure or function of hCOs.
The proteomic signature of circulating extracellular vesicles following intracerebral hemorrhage: Novel insights into mechanisms underlying recovery
(Elsevier, 2024-10-15) Casado-Fernández, Laura; Laso-García, Fernando; Piniella, Dolores; Gómez-de Frutos, Mari Carmen; Otero-Ortega, Laura; Bravo, Susana-Belén; Fuentes-Gimeno, Blanca; Docando, Felix; Alonso-López, Elisa; Ruiz-Ares, Gerardo; Rodríguez-Pardo, Jorge; Rigual, Ricardo; de Celis-Ruiz, Elena; Hervás, Carlos; Díez-Tejedor, Exuperio; Gutiérrez-Fernández, María; Alonso de Leciñana, Maria; Instituto de Salud Carlos III; Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); RETICS-Enfermedades Vasculares Cerebrales (ICTUS) (INVICTUS-ISCIII) (España); Unión Europea. Comisión Europea. NextGenerationEU; Ministerio de Universidades (España); Plan de Recuperación, Transformación y Resiliencia (España); Autonomous University of Madrid (España); Ministerio de Sanidad (España)
Circulating extracellular vesicles (EVs) can participate in innate repair processes triggered after intracerebral hemorrhage (ICH). We aimed to describe changes in the proteomic profile of circulating EVs between the acute and subacute phases of ICH and to compare the findings depending on outcomes, as an approach to unraveling such repair mechanisms. This was a prospective observational study including patients with non-traumatic supratentorial ICH. Exclusion criteria were previous disability, signs of herniation on baseline computed tomography, or limited life expectancy. EVs were isolated from blood samples at 24 h and 7 days after symptom onset. After 6-months' follow-up, patients were dichotomized into poor and good outcomes, defining good as an improvement of >10 points or > 50 % on the National Institutes of Health Stroke Scale and a modified Rankin Scale of 0-2. The protein cargo was analyzed by quantitative mass spectrometry and compared according to outcomes. Forty-four patients completed follow-up, 16 (35.5 %) having good outcomes. We identified 1321 proteins in EVs, 37 with differential abundance. In patients with good outcomes, proteins related to stress response (DERA, VNN2, TOMM34) and angiogenesis (RHG01) had increased abundance at 7 days. EVs from patients with poor outcomes showed higher levels of acute-phase reactants (CRP, SAA2) at 7 days compared with 24 h. In conclusion, the protein content of circulating EVs in patients with ICH changes over time, the changes varying depending on the clinical outcome, with greater abundance of proteins potentially involved in the repair processes of patients with good outcomes.
Renal fibroblasts are involved in fibrogenic changes in kidney fibrosis associated with dysfunctional telomeres
(Nature Publishing Group, 2024-10-01) Saraswati, Sarita; Martínez, Paula; Serrano, Rosa; Mejías, Diego; Graña-Castro, Osvaldo; Álvarez Díaz, Ruth; Blasco, Maria A; Agencia Estatal de Investigación (España); Ministerio de Ciencia e Innovación (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Comunidad de Madrid (España); Unión Europea. Comisión Europea. European Research Council (ERC); Botín Foundation; World Cancer Research Fund International
Tubulointerstitial fibrosis associated with chronic kidney disease (CKD) represents a global health care problem. We previously reported that short and dysfunctional telomeres lead to interstitial renal fibrosis; however, the cell-of-origin of kidney fibrosis associated with telomere dysfunction is currently unknown. We induced telomere dysfunction by deleting the Trf1 gene encoding a telomere-binding factor specifically in renal fibroblasts in both short-term and long-term life-long experiments in mice to identify the role of fibroblasts in renal fibrosis. Short-term Trf1 deletion in renal fibroblasts was not sufficient to trigger kidney fibrosis but was sufficient to induce inflammatory responses, ECM deposition, cell cycle arrest, fibrogenesis, and vascular rarefaction. However, long-term persistent deletion of Trf1 in fibroblasts resulted in kidney fibrosis accompanied by an elevated urinary albumin-to-creatinine ratio (uACR) and a decrease in mouse survival. These cellular responses lead to the macrophage-to-myofibroblast transition (MMT), endothelial-to-mesenchymal transition (EndMT), and partial epithelial-to-mesenchymal transition (EMT), ultimately causing kidney fibrosis at the humane endpoint (HEP) when the deletion of Trf1 in fibroblasts is maintained throughout the lifespan of mice. Our findings contribute to a better understanding of the role of dysfunctional telomeres in the onset of the profibrotic alterations that lead to kidney fibrosis.
Conservation of HLA Spike Protein Epitopes Supports T Cell Cross-Protection in SARS-CoV-2 Vaccinated Individuals against the Potentially Zoonotic Coronavirus Khosta-2
(Multidisciplinary Digital Publishing Institute (MDPI), 2024-05-31) Martin-Galiano, Antonio Javier; Lopez, Daniel; Ministerio de Ciencia e Innovación (España)
Heterologous vaccines, which induce immunity against several related pathogens, can be a very useful and rapid way to deal with new pandemics. In this study, the potential impact of licensed COVID-19 vaccines on cytotoxic and helper cell immune responses against Khosta-2, a novel sarbecovirus that productively infects human cells, was analyzed for the 567 and 41 most common HLA class I and II alleles, respectively. Computational predictions indicated that most of these 608 alleles, covering more than 90% of the human population, contain sufficient fully conserved T-cell epitopes between the Khosta-2 and SARS-CoV-2 spike-in proteins. Ninety percent of these fully conserved peptides for class I and 93% for class II HLA molecules were verified as epitopes recognized by CD8+ or CD4+ T lymphocytes, respectively. These results show a very high correlation between bioinformatic prediction and experimental assays, which strongly validates this study. This immunoinformatics analysis allowed a broader assessment of the alleles that recognize these peptides, a global approach at the population level that is not possible with experimental assays. In summary, these findings suggest that both cytotoxic and helper cell immune protection elicited by currently licensed COVID-19 vaccines should be effective against Khosta-2 virus infection. Finally, by being rapidly adaptable to future coronavirus pandemics, this study has potential public health implications.
Monkeypox virus genomic accordion strategies
(Nature Publishing Group, 2024-04-18) Monzon-Fernandez, Sara; Varona Fernandez, Sarai; Negredo, Anabel; Vidal-Freire, Santiago; Patiño-Galindo, Juan Angel; Ferressini-Gerpe, Natalia; Zaballos, Ángel; Orviz, Eva; Ayerdi, Oskar; Muñoz-Gómez, Ana; Delgado-Iribarren, Alberto; Estrada, Vicente; Garcia-Amil, Cristina; Molero-Sanz, Francisca; Sánchez-Mora, Patricia; Torres, Montserrat; Vazquez, Ana; Galán, Juan Carlos; Torres, Ignacio; Causse Del Río, Manuel; Merino-Diaz, Laura; López, Marcos; Galar, Alicia; Cardeñoso, Laura; Gutiérrez, Almudena; Loras, Cristina; Escribano, Isabel; Alvarez-Argüelles, Marta E; Del Río, Leticia; Simón, María; Meléndez, María Angeles; Camacho, Juan; Herrero, Laura; Jimenez Sancho, Maria Pilar; Navarro-Rico, María Luisa; Jado, Isabel; Giannetti, Elaina; Kuhn, Jens H; Sanchez-Lockhart, Mariano; Di Paola, Nicholas; Kugelman, Jeffrey R; Guerra, Susana; García-Sastre, Adolfo; Cuesta de la Plaza, Isabel; Sánchez-Seco, María Paz; Palacios, Gustavo; Instituto de Salud Carlos III; Centro de Investigación Biomédica en Red - CIBERINFEC (Enfermedades Infecciosas); Icahn School of Medicine at Mount Sinai; NIH - National Institute of Allergy and Infectious Diseases (NIAID) (Estados Unidos)
The 2023 monkeypox (mpox) epidemic was caused by a subclade IIb descendant of a monkeypox virus (MPXV) lineage traced back to Nigeria in 1971. Person-to-person transmission appears higher than for clade I or subclade IIa MPXV, possibly caused by genomic changes in subclade IIb MPXV. Key genomic changes could occur in the genome's low-complexity regions (LCRs), which are challenging to sequence and are often dismissed as uninformative. Here, using a combination of highly sensitive techniques, we determine a high-quality MPXV genome sequence of a representative of the current epidemic with LCRs resolved at unprecedented accuracy. This reveals significant variation in short tandem repeats within LCRs. We demonstrate that LCR entropy in the MPXV genome is significantly higher than that of single-nucleotide polymorphisms (SNPs) and that LCRs are not randomly distributed. In silico analyses indicate that expression, translation, stability, or function of MPXV orthologous poxvirus genes (OPGs), including OPG153, OPG204, and OPG208, could be affected in a manner consistent with the established "genomic accordion" evolutionary strategies of orthopoxviruses. We posit that genomic studies focusing on phenotypic MPXV differences should consider LCR variability.
RAD51 restricts DNA over-replication from re-activated origins
(EMBO Press, 2024-03) Muñoz, Sergio; Blanco-Romero, Elena; Gonzalez-Acosta, Daniel; Rodriguez-Acebes, Sara; Megías, Diego; Lopes, Massimo; Mendez, Juan; Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Amigos CNIO; Agencia Estatal de Investigación (España); Ministerio de Ciencia e Innovación (España); European Molecular Biology Organization; Swiss National Science Foundation
Eukaryotic cells rely on several mechanisms to ensure that the genome is duplicated precisely once in each cell division cycle, preventing DNA over-replication and genomic instability. Most of these mechanisms limit the activity of origin licensing proteins to prevent the reactivation of origins that have already been used. Here, we have investigated whether additional controls restrict the extension of re-replicated DNA in the event of origin re-activation. In a genetic screening in cells forced to re-activate origins, we found that re-replication is limited by RAD51 and enhanced by FBH1, a RAD51 antagonist. In the presence of chromatin-bound RAD51, forks stemming from re-fired origins are slowed down, leading to frequent events of fork reversal. Eventual re-initiation of DNA synthesis mediated by PRIMPOL creates ssDNA gaps that facilitate the partial elimination of re-duplicated DNA by MRE11 exonuclease. In the absence of RAD51, these controls are abrogated and re-replication forks progress much longer than in normal conditions. Our study uncovers a safeguard mechanism to protect genome stability in the event of origin reactivation.
Re-Emergence of a West Nile Virus (WNV) Variant in South Spain with Rapid Spread Capacity
(Multidisciplinary Digital Publishing Institute (MDPI), 2023-12-01) Ruiz-López, María José; Aguilera-Sepúlveda, Pilar; Cebrián-Camisón, Sonia; Figuerola, Jordi; Magallanes, Sergio; Varona Fernandez, Sarai; Cuesta de la Plaza, Isabel; Cano-Gómez, Cristina; Sánchez-Mora, Patricia; Camacho, Juan; Sánchez-Peña, Carolina; Marchena, Francisco José; Ameyugo, Ulises; Ruíz, Santiago; Sánchez-Seco, María Paz; Agüero, Montserrat; Jiménez-Clavero, Miguel Ángel; Fernández-Pinero, Jovita; Vazquez, Ana; Regional Government of Andalusia (España); Agencia Estatal de Investigación (España); Instituto de Salud Carlos III; Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (España); Unión Europea. Comisión Europea. NextGenerationEU
West Nile Virus (WNV) is a mosquito vector-borne zoonosis with an increasing incidence in Europe that has become a public health concern. In Spain, although local circulation has been known for decades, until 2020, when a large outbreak occurred, West Nile Virus cases were scarce and mostly occurred in southern Spain. Since then, there have been new cases every year and the pathogen has spread to new regions. Thus, monitoring of circulating variants and lineages plays a fundamental role in understanding WNV evolution, spread and dynamics. In this study, we sequenced WNV consensus genomes from mosquito pools captured in 2022 as part of a newly implemented surveillance program in southern Spain and compared it to other European, African and Spanish sequences. Characterization of WNV genomes in mosquitoes captured in 2022 reveals the co-circulation of two WNV lineage 1 variants, the one that caused the outbreak in 2020 and another variant that is closely related to variants reported in Spain in 2012, France in 2015, Italy in 2021-2022 and Senegal in 2012-2018. The geographic distribution of these variants indicates that WNV L1 dynamics in southern Europe include an alternating dominance of variants in some territories.

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