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Despite the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway being frequently altered in T-ALL/LBL, no specific therapy has been approved for T-ALL/LBL patients with constitutive signalling by JAK/STAT, so there is an urgent need to identify pathway members that may be potential therapeutic targets. In the present study, we searched for JAK/STAT pathway members potentially modulated through aberrant methylation and identified SOCS3 hypermethylation as a recurrent event in T-ALL/LBL. Additionally, we explored the implications of SOCS3 deregulation in T-ALL/LBL and demonstrated that SOCS3 counteracts the constitutive activation of the JAK/STAT pathway through different molecular mechanisms. Therefore, SOCS3 emerges as a potential therapeutic target in T-ALL/LBL, Comunidad de Madrid, Grant/Award Number: B2017/BMD-3778; LINFOMAS-CM; Fundación Científica Asociación Española Contra el Cáncer, Grant/Award Number: PROYE18054PIRI; Fundación Ramón Areces, Grant/Award Number: CIVP19S7917; Instituto de Investigación Sanitaria Fundación Jiménez Díaz; Ministerio de Ciencia, Innovación y Universidades, Grant/ Award Number: RTI2018- 093330-B-I00 and MCIU/FEDER; Ministerio de Economía y Competitividad, Grant/Award Number: SAF2015-70561-R and MINECO/FEDER

The emergent human coronavirus SARS-CoV-2 and its high infectivity rate has highlighted the strong need for new virucidal treatments. In this sense, the use of photodynamic therapy (PDT) with white light, to take advantage of the sunlight, is a potent strategy for decreasing the virulence and pathogenicity of the virus. Here, we report the virucidal effect of PDT based on Hypericum extract (HE) in combination with white light, which exhibits an inhibitory activity of the human coronavirus HCoV-229E on hepatocarcinoma Huh-7 cells. Moreover, despite continuous exposure to white light, HE has long durability, being able to maintain the prevention of viral infection. Given its potent in vitro virucidal capacity, we propose HE in combination with white light as a promising candidate to fight against SARS-CoV-2 as a virucidal compound., This research was funded by Fundación Universidad Autónoma de Madrid, grant number PI21/00315 and by Instituto de Salud Carlos iii, grant number PI21/00953.

A striking feature of the nervous system pertains to the appearance of different neural cell subtypes at different axial levels. Studies in the Drosophila central nervous system reveal that one mechanism underlying such segmental differences pertains to the segment-specific removal of cells by programmed cell death (PCD). One group of genes involved in segment-specific PCD is the Hox homeotic genes. However, while segment-specific PCD is highly precise, Hox gene expression is evident in gradients, raising the issue of how the Hox gene function is precisely gated to trigger PCD in specific segments at the outer limits of Hox expression. The Drosophila Va neurons are initially generated in all nerve cord segments but removed by PCD in posterior segments. Va PCD is triggered by the posteriorly expressed Hox gene Abdominal-B (Abd-B). However, Va PCD is highly reproducible despite exceedingly weak Abd-B expression in the anterior frontiers of its expression. Here, we found that the transcriptional cofactor Dachshund supports Abd-B-mediated PCD in its anterior domain. In vivo bimolecular fluorescence complementation analysis lends support to the idea that the Dachshund/Abd-B interplay may involve physical interactions. These findings provide an example of how combinatorial codes of transcription factors ensure precision in Hox-mediated PCD in specific segments at the outer limits of Hox expression, Ministerio de Ciencia y Educación, Grant/Award Number: PID2019-110952GB-I00

Regeneration, the ability to replace lost body parts, is a widespread phenomenon in the animal kingdom often connected to asexual reproduction or fission, since the only difference between the two appears to be the stimulus that triggers them. Both developmental processes have largely been characterized; however, the molecular toolkit and genetic mechanisms underlying these events remain poorly unexplored. Annelids, in particular the oligochaete Pristina leidyi, provide a good model system to investigate these processes as they show diverse ways to regenerate, and can reproduce asexually through fission under laboratory conditions. Here, we used a comparative transcriptomics approach based on RNA‐sequencing and differential gene expression analyses to understand the molecular mechanisms involved in anterior regeneration and asexual reproduction. We found 291 genes upregulated during anterior regeneration, including several regeneration‐related genes previously reported in other annelids such as frizzled, paics, and vdra. On the other hand, during asexual reproduction, 130 genes were found upregulated, and unexpectedly, many of them were related to germline development during sexual reproduction. We also found important differences between anterior regeneration and asexual reproduc tion, with the latter showing a gene expression profile more similar to that of control individuals. Nevertheless, we identified 35 genes that were upregulated in both conditions, many of them related to cell pluripotency, stem cells, and cell proliferation. Overall, our results shed light on the molecular mechanisms that control anterior regeneration and asexual reproduction in annelids and reveal similarities with other animals, suggesting that the genetic machinery controlling these processes is conserved across metazoans., Irene del Olmo was supported by the program “Ayudas para el Fomento de la Investigación en Estudios de Máster‐UAM 2020 en la Facultad de Ciencias” financed by the Universidad Autónoma de Madrid (3931‐ 326F‐726DP6A38‐4F77). Aida Verdes was funded by the European Union's Horizon 2020 research and innovation program through a Marie Skłodowska‐Curie individual fellowship (grant 841576). This study was funded through the Madrid Government (Comunidad de Madrid‐Spain) under the Multiannual Agreement with Universidad Autónoma de Madrid in the line of action encouraging youth research doctors, in the context of the V PRICIT (Regional Program of Research and Technological Innovation) (SI1/PJI/2019‐00532), and the EMBO Long Term Fellowship (ALTF‐217‐2018) to Patricia Álvarez‐Campos.

Propionic acidemia (PA) disorder shows major involvement of the heart, among other alterations. A significant number of PA patients develop cardiac complications, and available evidence suggests that this cardiac dysfunction is driven mainly by the accumulation of toxic metabolites. To contribute to the elucidation of the mechanistic basis underlying this dysfunction, we have successfully generated cardiomyocytes through the differentiation of induced pluripotent stem cells (iPSCs) from a PCCB patient and its isogenic control. In this human cellular model, we aimed to examine microRNAs (miRNAs) profiles and analyze several cellular pathways to determine miRNAs activity patterns associated with PA cardiac phenotypes. We have identified a series of upregulated cardiac-enriched miRNAs and alterations in some of their regulated signaling pathways, including an increase in the expression of cardiac damage markers and cardiac channels, an increase in oxidative stress, a decrease in mitochondrial respiration and autophagy; and lipid accumulation. Our findings indicate that miRNA activity patterns from PA iPSC-derived cardiomyocytes are biologically informative and advance the understanding of the molecular mechanisms of this rare disease, providing a basis for identifying new therapeutic targets for intervention strategies.

© 2020 by the authors., Photodynamic Therapy (PDT) with methyl-aminolevulinate (MAL-PDT) is being used for the treatment of Basal Cell Carcinoma (BCC), although resistant cells may appear. Normal differentiated cells depend primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate energy, but cancer cells switch this metabolism to aerobic glycolysis (Warburg effect), influencing the response to therapies. We have analyzed the expression of metabolic markers (β-F1-ATPase/GAPDH (glyceraldehyde-3-phosphate dehydrogenase) ratio, pyruvate kinase M2 (PKM2), oxygen consume ratio, and lactate extracellular production) in the resistance to PDT of mouse BCC cell lines (named ASZ and CSZ, heterozygous for ptch1). We have also evaluated the ability of metformin (Metf), an antidiabetic type II compound that acts through inhibition of the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway to sensitize resistant cells to PDT. The results obtained indicated that resistant cells showed an aerobic glycolysis metabolism. The treatment with Metf induced arrest in the G0/G1 phase and a reduction in the lactate extracellular production in all cell lines. The addition of Metf to MAL-PDT improved the cytotoxic effect on parental and resistant cells, which was not dependent on the PS protoporphyrin IX (PpIX) production. After Metf + MAL-PDT treatment, activation of pAMPK was detected, suppressing the mTOR pathway in most of the cells. Enhanced PDT-response with Metf was also observed in ASZ tumors. In conclusion, Metf increased the response to MAL-PDT in murine BCC cells resistant to PDT with aerobic glycolysis., This research was funded by Spanish grants from Instituto de Salud Carlos III MINECO and Feder Funds (FIS PI15/00974 and PI18/00708) and Ministerio de Ciencia, Innovación y Universidades (SAF2016-75916-R).

[Background] Viruses are key players regulating microbial ecosystems. Exploration of viral assemblages is now possible thanks to the development of metagenomics, the most powerful tool available for studying viral ecology and discovering new viruses. Unfortunately, several sources of bias lead to the misrepresentation of certain viruses within metagenomics workflows, hindering the shift from merely descriptive studies towards quantitative comparisons of communities. Therefore, benchmark studies on virus enrichment and random amplification protocols are required to better understand the sources of bias. [Results] We assessed the bias introduced by viral enrichment on mock assemblages composed of seven DNA viruses, and the bias from random amplification methods on human saliva DNA viromes, using qPCR and deep sequencing, respectively. While iodixanol cushions and 0.45 μm filtration preserved the original composition of nuclease-protected viral genomes, low-force centrifugation and 0.22 μm filtration removed large viruses. Comparison of unamplified and randomly amplified saliva viromes revealed that multiple displacement amplification (MDA) induced stochastic bias from picograms of DNA template. However, the type of bias shifted to systematic using 1 ng, with only a marginal influence by amplification time. Systematic bias consisted of over-amplification of small circular genomes, and under-amplification of those with extreme GC content, a negative bias that was shared with the PCR-based sequence-independent, single-primer amplification (SISPA) method. MDA based on random priming provided by a DNA primase activity slightly outperformed those based on random hexamers and SISPA, which may reflect differences in ability to handle sequences with extreme GC content. SISPA viromes showed uneven coverage profiles, with high coverage peaks in regions with low linguistic sequence complexity. Despite misrepresentation of certain viruses after random amplification, ordination plots based on dissimilarities among contig profiles showed perfect overlapping of related amplified and unamplified saliva viromes and strong separation from unrelated saliva viromes. This result suggests that random amplification bias has a minor impact on beta diversity studies. [Conclusions] Benchmark analyses of mock and natural communities of viruses improve understanding and mitigate bias in metagenomics surveys. Bias induced by random amplification methods has only a minor impact on beta diversity studies of human saliva viromes., Institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMSO are acknowledged.This work was supported by the Spanish Ministry of Economy and Competitiveness through grant SAF2012-38421 and a “Formación de Personal Investigador” Ph.D. studentship to MP-M

Root-knot nematodes (RKNs) are obligate endoparasites that feed on their host plants to complete its life cycle, representing a major threat to agriculture and economy worldwide. The development of new management strategies becomes essential as effective chemical nematicides are progressively being restricted. Hence, we analysed grape pomace-derived biochars, pyrolysed at 350 °C (BC350) and 700 °C (BC700), focusing on their potential for RKN control. The thermal treatment of grape pomace caused an increase in the concentration of carbon and plant macro- and micronutrients, which were largely present in a water-soluble form. Synchrotron radiation-based Fourier transform infrared microspectroscopy data showed a general loss of carboxylic functional groups during pyrolysis, partially contributing to the alkalinisation of both biochars, mostly in BC700. Scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy analysis revealed a highly porous structure filled with different crystals composed of elements such as K, Ca, Mg, P, Si or Al, which could be a suitable environment for the growth of microorganisms. Biochar-derived aqueous extracts showed phytotoxicity to tomato seedlings at high concentrations, and disappeared upon dilution, but no toxic effect was observed on the nematode’s infective stage. However, the infective and reproductive traits of a Meloidogyne javanica population in tomato were significantly reduced (i.e. egg masses and eggs per plant) in washed-biochar-treated soil in pots (0.75%; BC350W). Therefore, the large amount of grape waste generated after wine production can be transformed into a valuable product such as biochar, effective for RKNs control, thus reducing the waste management problem and contributing to a circular economy, This work was supported by the Spanish Government (PID2019-105924RB-I00 MCIN/AEI/10.13039/501100011033; RED2018-102407-T) to CE, and (AGL201453771-R; AGL2017-87591-R) to LEH, and by the Castilla-La Mancha Govern ment (SBPLY/17/180501/000287 and SBPLY/21/180501/000033) to CE. AM-G was recipient of a PhD grant from Fundación Tatiana Pérez de Guzmán el Bueno. EU FEDER funds complemented all the grants

Photodynamic Therapy (PDT) with methyl-aminolevulinate acid (MAL-PDT) is being used for the treatment of Basal cell carcinoma (BCC), but recurrences have been reported. In this work, we have evaluated resistance mechanisms to MAL-PDT developed by three BCC cell lines (ASZ, BSZ and CSZ), derived from mice on a ptch+/− background and with or without p53 expression, subjected to 10 cycles of PDT (10thG). The resistant populations showed mesenchymal-like structure and diminished proliferative capacity and size compared to the parental (P) cells. The resistance was dependent on the production of the endogenous photosensitiser protoporphyrin IX in the CSZ cell line and on its cellular localisation in ASZ and BSZ cells. Moreover, resistant cells expressing the p53 gene presented lower proliferation rate and increased expression levels of N-cadherin and Gsk3β (a component of the Wnt/β-catenin pathway) than P cells. In contrast, 10thG cells lacking the p53 gene showed lower levels of expression of Gsk3β in the cytoplasm and of E-cadherin and β-catenin in the membrane. In addition, resistant cells presented higher tumorigenic ability in immunosuppressed mice. Altogether, these results shed light on resistance mechanisms of BCC to PDT and may help to improve the use of this therapeutic approach.

Syllis Savigny in Lamarck, 1818, the type genus of Syllidae, is the largest and most diverse within the family. This genus presents many taxonomic, biogeographic, and phylogenetic problems due mainly to the lack of molecular data and morphological synapomorphies for many of the species, but also due to poor or inadequate descriptions. In order to improve the knowledge of the genus, we have undertaken a morphological revision of Australian species, based on type material and material collected over years by the Australian Museum. This is the third paper dealing with this family. Eighteen species are herein described, of which five are new and twelve are redescriptions of already known species, with four new combinations: Syllis imajimai, n. sp.; S. narranuk, n. sp.; S. noolinga, n. sp.; S. similisunzima, n. sp.; S. thylacine, n. sp.; S. busseltonensis (Hartmann-Schröder, 1982) n. comb.; S. patriciae (Hartmann-Schröder, 1981) n. comb.; S. pharobroomensis (Hartmann-Schröder, 1979) n. comb., S. pharynxcircunfusata (Hartmann-Schröder, 1979) n. comb, S. armillaris (O. F. Müller, 1771); S. augeneri Haswell, 1920; S. benelihauae (Campoy & Alquézar, 1982); S. hyalina Grube, 1863; S. cf. nigrescens Grube, 1878; S. profunda Cognetti, 1955; S. truncata Haswell, 1920; and S. variegata Grube, 1860.

Aim: The latitudinal biodiversity gradient is considered a first-order biogeographical pattern for most taxonomic groups. Latitudinal variation in plant diversity is not always consistent, and this could be related to the particular characteristics of different forest types. In this study, we compare latitudinal changes in floristic diversity (alpha diversity), composition (beta diversity) and dominance across different tropical forest types: floodplain, terra firme and submontane forests. Location: Western Amazonia (Ecuador, Peru and Bolivia). Taxon: Woody plants. Methods: We inventoried 1978 species and 31,203 individuals of vascular plants with a diameter at breast height ≥ 2.5 cm in 118 0.1-ha plots over an 1800 km latitudinal gradient in three different forest types. The relationships between alpha diversity, latitude and forest type were analysed using generalised linear mixed models. Semi-parametric permutational multivariate analysis of variance was used to investigate the effects of latitude and forest type on beta diversity. Dominant species abundances were correlated with non-metric multidimensional scaling ordination axes to reflect their contributions in shaping changes in beta diversity. Results: Alpha diversity increased towards equatorial latitudes in terra firme and submontane forests but remained relatively constant in floodplains. Beta diversity of all forest types changed with latitude, although less clearly in floodplains. Also, in floodplain forests, there were fewer dominant species contributing to beta diversity and more species homogeneous along the gradient. Main Conclusions: Latitudinal diversity patterns are manifested in alpha and beta diversity since latitude summarizes climatic and edaphic changes. However, we found different responses of each forest type. In floodplain forests, inundation regime is a stronger predictor than latitude, limiting floristic diversity and composition. Changes in dominant species abundance over gradients explained species composition, but floodplain forests harboured more homogeneous dominant species than well drained forests. It is key to study environmental trends and habitat characteristics of each forest type to understand their species diversity and dominance patterns

The 10boron neutron-capture therapy (BNCT) is an emerging antitumoral method that shows increasing biomedical interest. BNCT is based on the selective accumulation of the 10boron isotope within the tumor, which is then irradiated with low-energy thermal neutrons, generating nuclear fission that produces 7lithium, 4helium, and γ rays. Simple catechol-borate esters have been rather overlooked as precursors of melanin biosynthesis, and therefore, a proof-of-concept approach for using dopamine-borate (DABO) as a suitable boron-containing candidate for potential BNCT is presented here. DABO can spontaneously oxidize and autopolymerize in vitro, giving a soluble, eumelanin-like brown-black poly-DABO product. Melanotic melanoma cell cultures treated with 1 mM DABO for 24 and 48 h were viable and showed no signs of damage or cell death. The stability and possible trans-esterification of DABO is shortly discussed. Chemical calculations and quantitative structure-activity relationships (QSAR) analysis of DABO and the BNCT agent BPA indicated that they should be cell permeant and accumulate within lysosomes and melanosomes. Molecular modeling allows visualization of both the DABO precursor and the structure of a borate derivative of the proposed catechol-porphycene model for eumelanin, showing interesting features from molecular orbital calculations. The main difference between DABO and other agents, such as BPA, is that it is not a boronic acid nor a boron cluster. This simple catechol-borate ester (protected from oxidation and blackening) could be administrated to living cells and organisms, in which biosynthesis of boron-melanin in melanoma melanocytes can lead to improved BNCT, These authors received no specific funding for this study

The knowledge and control of invasive species are essential in the management of marine ecosystems. The Mediterranean Sea is one of the marine areas with the highest number of invasive species detected, most of them invertebrates. On a smaller scale, colonization by non-native species in coastal lagoons depends directly on their connectivity with the adjacent sea and on the changes in their environmental conditions induced mainly by human interventions. In this study, the cirratulid species Timarete caribous Grube, 1859, native from the Western and Central Atlantic Ocean, was detected for the first time in a Western Mediterranean coastal lagoon, the Mar Menor. It has been found in dense aggregates on both artificial and natural rocky substrates, mainly in the most important communication channel with the Mediterranean Sea. Following the evolution of this NIS will be important for understanding the dynamic and spread of the species and its possible effects on the indigenous biological communities

Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories, This work was supported by grants BFU2014-55478-R from Ministerio de Economía y Competitividad, and from Fundación Ramón Areces to MY-M; grants PI13/00050 from the “Fondo de Investigación Sanitaria” (FISISCIII), 2014SGR804 from Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la“Generalitat de Catalunya” and REDinREN (16/0009 Feder Funds) to FEB; BFU2014-54181-P from Ministerio de Economía y Competitividad to JLC, and by SAF2016-77096-R from Ministerio de Economía y Competitividad to CC. H.S. was supported by a FPI-UAM and GEIVEX Mobility fellowship; AGV is sponsored by a Grant (482/U/2014) from Fundació La Marató TV3; FEB is sponsored by the “Researchers Stabilization Program” from the Spanish “Sistema Nacional de Salud” (SNS-ISCIII) and “Direcció d’Estratègia i Coordinació”, Catalan Health Dept. (CES07/015) and MY-M by Ramón y Cajal contract RYC-2012-11025

Mismanaged plastic litter submitted to environmental conditions may breakdown into smaller fragments, eventually reaching nano-scale particles (nanoplastics, NPLs). In this study, pristine beads of four different types of polymers, three oil-based (polypropylene, PP; polystyrene, PS; and low-density polyethylene, LDPE) and one bio-based (polylactic acid, PLA) were mechanically broken down to obtain more environmentally realistic NPLs and its toxicity to two freshwater secondary consumers was assessed. Thus, effects on the cnidarian Hydra viridissima (mortality, morphology, regeneration ability, and feeding behavior) and the fish Danio rerio (mortality, morphological alterations, and swimming behavior) were tested at NPLs concentrations in the 0.001 to 100 mg/L range. Mortality and several morphological alterations were observed on hydras exposed to 10 and 100 mg/L PP and 100 mg/L LDPE, whilst regeneration capacity was overall accelerated. The locomotory activity of D. rerio larvae was affected by NPLs (decreased swimming time, distance or turning frequency) at environmentally realistic concentrations (as low as 0.001 mg/L). Overall, petroleum- and bio-based NPLs elicited pernicious effects on tested model organisms, especially PP, LDPE and PLA. Data allowed the estimation of NPLs effective concentrations and showed that biopolymers may also induce relevant toxic effects, The authors acknowledge the financial support provided by the Spanish Government (Ministerio de Ciencia e Innovación): PID2020-113769RBC21/22, PLEC2021-007693 and the Thematic Network of Micro- and Nanoplastics in the Environment (RED2018-102345-T, EnviroPlaNet Network) and by the Portuguese Government: Project NanoPlanet (2022.02340.PTDC) and financial support to CESAM (UIDP/ 50017/ 2020+UIDB/50017/2020+LA/P/0094/2020), through FCT/MCTES. MTB is the recipient of a FPU (FPU17/01789) pre-doctoral contract by the Spanish Ministerio de Ciencia, Innovación y Universidades

Glycerol fermentation for 1,3-propanediol (1,3-PDO) production was studied in an anaerobic up-flow reactor with biomass attached to silicone support. A mixed microbial culture was activated to perform the biofilm formation and attachment to a silicone hose prior the reactor operation. The reactor was operated over 362 days divided into two phases (P). In P1 and P2, the reactor was continuously fed with pure and crude glycerol, respectively. The operation consisted of increasing the glycerol loading rates (gly-LR). The achieved highest 1,3-PDO average yields were 0.43 mol mol-gly−1 and 0.62 mol mol-gly−1 when applied gly-LR was 18 and 46 g L−1 d−1 in P1 and P2, respectively. A maximum 1,3-PDO productivity of 14.7 g L−1 d−1 was obtained in P2. The higher yields of 1,3-PDO when the feed changed from pure to crude glycerol indicated a change in the microbial community. These results show that the studied system can be very promising and cost-effective for converting crude glycerol into value-added products on large scale even at high loads, This study was funded by Spanish Ministry of Science and Innovation (Grant no: PID2019-104812GBI00X), UAM-Santander Inter-university Cooperation Projects with Latin America (CEAL-AL/2017-14), and CAPES-PrInt (fnance code 001, Process no 88887.467533/2019-00)

Accurate assessment of predation risk is critical for prey survival during predator–prey interactions. Prey can assess predation risk by the presence of cues dropped by predators themselves, but they can also gather information about risk level through cues released by other prey, avoiding the hazard of being in close proximity to predators. In this study, we examine the ability of anuran larvae (Pelobates cultripes) to detect predation risk indirectly when they are in contact with conspecifics that have been recently exposed to chemical stimuli from natural predators (larvae of aquatic beetles). In a first experiment, we confirmed that larvae exposed to predator cues exhibited innate defensive behavior, indicating that they perceived the risk of predation and, thus, could potentially act as risk indicators for naïve conspecifics. In a second experiment, we observed that unexposed larvae paired with a startled conspecific adjusted their antipredator behavior, presumably by mirroring conspecifics’ behavior and/or using chemical cues from their partners as a risk information source. This cognitive ability of tadpoles to assess predation risk through conspecific cues might play an important role in their interaction with predators, facilitating the early detection of potential threats to elicit appropriate antipredator responses and increase the chances of survival, Financial support was provided by the Spanish Ministry of Science and Innovation (MICINN), under Grant CGL2015-68670-R

Tau is a well-known microtubule-associated protein related to its cytoplasmic localization in a neuronal cell. However, tau has been located at the cell nucleus where it could be a nucleic acid-associated protein by its preferential binding to DNA sequences present in the nucleolus and pericentromeric heterochromatin. This less well-known localization of tau could not be trivial, since during aging, an increase in the amount of nuclear tau takes place and it may be related to the described role of tau in the activation of transposons and further aging acceleration, Work in the laboratory of JA is funded by grants from the Spanish Ministry of Economy and Competitiveness (PGC-2018-09177-B-100). Work in the laboratory of FH is funded by grants from the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía, Industria y Competitividad, Gobierno de España, PID2020-113204GB-I00) and was co-financed from the Comunidad de Madrid through Structural Funds of the European Union [S2017/BMD-3700 (NEUROMETAB-CM)]

The emergent human coronavirus SARS-CoV-2 and its resistance to current drugs makes the need for new potent treatments for COVID-19 patients strongly necessary. Dextran sulfate (DS) polysaccharides have long demonstrated antiviral activity against different enveloped viruses in vitro. However, their poor bioavailability has led to their abandonment as antiviral candidates. Here, we report for the first time the broad-spectrum antiviral activity of a DS-based extrapolymeric substance produced by the lactic acid bacterium Leuconostoc mesenteroides B512F. Time of addition assays with SARS-CoV-2 pseudoviruses in in vitro models confirm the inhibitory activity of DSs in the early stages of viral infection (viral entry). In addition, this exopolysaccharide substance also reports broad-spectrum antiviral activity against several enveloped viruses such as SARS-CoV-2, HCoV229E, HSV-1, in in vitro models and in human lung tissue. The toxicity and antiviral capacity of DS from L. mesenteroides was tested in vivo in mouse models which are susceptible to SARS-CoV-2 infection. The described DS, administered by inhalation, a new route of administration for these types of polymers, shows strong inhibition of SARS-CoV-2 infection in vivo, significantly reducing animal mortality and morbidity at non-toxic doses. Therefore, we suggest that it may be considered as a potential candidate for antiviral therapy against SARS-CoV-2, Financial support for the study was provided by the REACT-EU 2021 grant from Comunidad de Madrid to the Project COVTRAVI19-CM, Plataformas y modelos preclínicos para el abordaje multidisciplinar en COVID-19 y en respuesta a futuras pandemias

The hollow protein capsids from a number of different viruses are being considered for multiple biomedical or nanotechnological applications. In order to improve the applied potential of a given viral capsid as a nanocarrier or nanocontainer, specific conditions must be found for achieving its faithful and efficient assembly in vitro. The small size, adequate physical properties and specialized biological functions of the capsids of parvoviruses such as the minute virus of mice (MVM) make them excellent choices as nanocarriers and nanocontainers. In this study we analyzed the effects of protein concentration, macromolecular crowding, temperature, pH, ionic strength, or a combination of some of those variables on the fidelity and efficiency of self-assembly of the MVM capsid in vitro. The results revealed that the in vitro reassembly of the MVM capsid is an efficient and faithful process. Under some conditions, up to ~40% of the starting virus capsids were reassembled in vitro as free, non aggregated, correctly assembled particles. These results open up the possibility of encapsidating different compounds in VP2-only capsids of MVM during its reassembly in vitro, and encourage the use of virus-like particles of MVM as nanocontainers, This research was funded by grants from MICINN/FEDER EU (Spain, RTI2018-096635-B-100 and PID2021-126973OB-I00) to M.G.M. and by an institutional grant from Fundación Ramón Areces

In eutherian mammals, hundreds of programmed DNA double-strand breaks (DSBs) are generated at the onset of meiosis. The DNA damage response is then triggered. Although the dynamics of this response is well studied in eutherian mammals, recent findings have revealed different patterns of DNA damage signaling and repair in marsupial mammals. To better characterize these differences, here we analyzed synapsis and the chromosomal distribution of meiotic DSBs markers in three different marsupial species (Thylamys elegans, Dromiciops gliorides, and Macropus eugenii) that represent South American and Australian Orders. Our results revealed inter-specific differences in the chromosomal distribution of DNA damage and repair proteins, which were associated with differing synapsis patterns. In the American species T. elegans and D. gliroides, chromosomal ends were conspicuously polarized in a bouquet configuration and synapsis progressed exclusively from the telomeres towards interstitial regions. This was accompanied by sparse H2AX phosphorylation, mainly accumulating at chromosomal ends. Accordingly, RAD51 and RPA were mainly localized at chromosomal ends throughout prophase I in both American marsupials, likely resulting in reduced recombination rates at interstitial positions. In sharp contrast, synapsis initiated at both interstitial and distal chromosomal regions in the Australian representative M. eugenii, the bouquet polarization was incomplete and ephemeral, γH2AX had a broad nuclear distribution, and RAD51 and RPA foci displayed an even chromosomal distribution. Given the basal evolutionary position of T. elegans, it is likely that the meiotic features reported in this species represent an ancestral pattern in marsupials and that a shift in the meiotic program occurred after the split of D. gliroides and the Australian marsupial clade. Our results open intriguing questions about the regulation and homeostasis of meiotic DSBs in marsupials. The low recombination rates observed at the interstitial chromosomal regions in American marsupials can result in the formation of large linkage groups, thus having an impact in the evolution of their genomes, This work was supported by grants CGL 2014-53106-P to JP (Ministerio de Ecomonía y Competitividad, Spain), BIOUAM02- 2020 to JP and RG (Departamento de Biología, Universidad Autónoma de Madrid), PID 2020-112557 GB-I00 to AR-H. (Ministerio de Ciencia e Innovación, Spain) and from the Australian Research Council to MBR, GS, and PDW. (DP21103512 and DP220101429). PW is also supported by the NHMRC (APP1182667 and APP2021172). LM-G was supported by a FPU predoctoral fellowship from the Ministry of Science, Innovation and University (FPU18/03867)

Background: Alzheimer’s disease (AD) and type 2 diabetes mellitus (T2DM) share metabolic alterations such as abnormal insulin and lipid metabolism and have some common genetic factors such as APOE genotype. Taking this into account, we hypothesized that we could identify common genetic factors involved in the development of diabetes and cardiovascular diseases. Methodology: We first genotyped 48 single nucleotide polymorphisms (SNPs) previously associated with AD in a cohort composed of 330 patients with cognitive impairment (CI) to assess their association with plasma lipids. Second, we conducted pleiotropy-informed conjunctional false discovery rate (FDR) analysis designed to identify shared variants between AD and plasma lipid levels. Finally, we used the SNPs to be found associated with lipid parameters and AD to search for associations with lipoprotein parameters in 281 patients with cardiometabolic risk. Results: Five SNPs were significantly associated with lower levels of cholesterol transported in remnant lipoprotein particles (RLPc) in subjects with CI; among these SNPs was the rs73572039 variant in PVRL2. Stratified QQ-plots were conducted on GWAS designed for AD and triglycerides (TG). The cross-trait analysis resulted in a total of 22 independent genomic loci associated with both AD and TG levels with a conjFDR < 0.05. Among these loci, two pleiotropic variants were located in PVRL2 (rs12978931 and rs11667640). The three SNPs in PVRL2 were significantly associated with RLPc, TG, and number of circulating VLDL and HDL particles in subjects with cardiometabolic risk. Conclusions: We have identified three variants in PVRL2 that predispose individuals to AD that also influence the lipid profile that confers cardiovascular risk in T2DM subjects. PVRL2 is a potential new modulating factor of atherogenic dyslipidemia, This work was supported by a research grant from the Spanish Ministerio de Ciencia, Innovación y Universidades (PI19/00832), Fondo Europeo de Desarrollo Regional (FEDER), and CIBERDEM (CIBER de Diabetes y Enfermedades Metabólicas Asociadas), which are initiatives of Instituto de Salud Carlos III (ISCIII); by Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (2017SGR0444); and by the Cerca Programme, Generalitat de Catalunya

A limited number of effective therapies are currently available to treat human coronavirus SARS-CoV-2 and other human coronaviruses, which are responsible for nearly a third of global cases of the common cold. The possibility of new emerging coronaviruses demands powerful new antiviral strategies. Lactoferrin is a well-known protein that possesses anti-inflammatory and immunomodulatory activities, and it has previously shown antiviral activity against several viruses, including SARS-CoV-2. To increase this antiviral activity, here we present bovine liposomal lactoferrin. Liposomal encapsulation of the compound was proven to increase permeability, bioavailability, and time release. In the present work, we compare the antiviral activity of free and liposomal bovine lactoferrin against HCoV229E and SARS-CoV-2 in vitro and in human primary bronchial epithelial cells, and we demonstrated that the liposomal form exerts a more potent antiviral activity than its free form at non-cytotoxic doses.

The species Coniopteris laciniata and Sphenopteris wonnacottii are heterophyllous ferns recovered from two Spanish Barremian localities, Las Hoyas and El Montsec respectively. The similarities between these species, observed in a study of a total of 66 hand specimens from both localities, indicate that they are conspecific. Following the rule of priority in botanical nomenclature, and because there are doubts about the assignment of this species to a higher taxonomic rank, the name Coniopteris is maintained. Characters of this fern and additional characters of other species of Coniopteris would support a diversification of polypod ferns during the Early Cretaceous. Metric architecture analyses suggest that the heterophylly of the plant was probably caused by the submersion of the apical part of fronds in water during their development, which would be consistent with the taphonomy of the remains in both localities, We would like to thank El Montsec housed at the Muséum national d’Histoire naturelle (Paris, France), which was visited with support of a grant from the SYNTHESYS Project (http://www.synthesys.info/), financed by the European Community Research Infrastructure Action under the FP7 Integrating Activities Program. Candela Blanco-Moreno is supported by a Margarita Salas Postdoc CA1/RSUE/2021-00703 scholarship funded by the Spanish Ministry of Universities and Universidad Autónoma de Madrid. This work is part of the project PID2019-105546GB-I00 of the Spanish Ministry of Science, Innovation and Universities

Leishmania infantum is one of the causative agents of visceral leishmaniases, the most severe form of leishmaniasis. An improved assembly for the L. infantum genome was published five years ago, yet delineation of its transcriptome remained to be accomplished. In this work, the transcriptome annotation was attained by a combination of both short and long RNA-seq reads. The good agreement between the results derived from both methodologies confirmed that transcript assembly based on Illumina RNA-seq and further delimitation according to the positions of spliced leader (SAS) and poly-A (PAS) addition sites is an adequate strategy to annotate the transcriptomes of Leishmania, a procedure previously used for transcriptome annotation in other Leishmania species and related trypanosomatids. These analyses also confirmed that the Leishmania transcripts boundaries are relatively slippery, showing extensive heterogeneity at the 5′- and 3′-ends. However, the use of RNA-seq reads derived from the PacBio technology (referred to as Iso-Seq) allowed the authors to uncover some complex transcription patterns occurring at particular loci that would be unnoticed by the use of short RNA-seq reads alone. Thus, Iso-Seq analysis provided evidence that transcript processing at particular loci would be more dynamic than expected. Another noticeable finding was the observation of a case of allelic heterozygosity based on the existence of chimeric Iso-Seq reads that might be generated by an event of intrachromosomal recombination. In addition, we are providing the L. infantum gene models, including both UTRs and CDS regions, that would be helpful for undertaking whole-genome expression studies. Moreover, we have built the foundations of a communal database for the active curation of both gene/transcript models and functional annotations for genes and proteins, This research was supported by the Spanish Ministerio de Ciencia, Innovación (MICINN), Agencia Estatal deInvestigación(AEI), grant number PID2020-117916RB-I00, and Instituto de Salud Carlos III, grant CB21/13/00018 (CIBERINFEC). An institutional grant from Fundacion RamonAreces is also acknowledged

The hippocampus hosts the continuous addition of new neurons throughout life—a phenomenon named adult hippocampal neurogenesis (AHN). Here we revisit the occurrence of AHN in more than 110 mammalian species, including humans, and discuss the further validation of these data by single-cell RNAseq and other alternative techniques. In this regard, our recent studies have addressed the long-standing controversy in the field, namely whether cells positive for AHN markers are present in the adult human dentate gyrus (DG). Here we review how we developed a tightly controlled methodology, based on the use of high-quality brain samples (characterized by short postmortem delays and ≤24 h of fixation in freshly prepared 4% paraformaldehyde), to address human AHN. We review that the detection of AHN markers in samples fixed for 24 h required mild antigen retrieval and chemical elimination of autofluorescence. However, these steps were not necessary for samples subjected to shorter fixation periods. Moreover, the detection of labile epitopes (such as Nestin) in the human hippocampus required the use of mild detergents. The application of this strictly controlled methodology allowed reconstruction of the entire AHN process, thus revealing the presence of neural stem cells, proliferative progenitors, neuroblasts, and immature neurons at distinct stages of differentiation in the human DG. The data reviewed here demonstrate that methodology is of utmost importance when studying AHN by means of distinct techniques across the phylogenetic scale. In this regard, we summarize the major findings made by our group that emphasize that overlooking fundamental technical principles might have consequences for any given research field, Association for Frontotemporal Degeneration; Banco de Santander; Center for Networked Biomedical Research on Neurodegenerative Diseases; Consejo Nacional de Ciencia y Tecnología (CONACYT), Grant/Award Number: 385084; European Research Council, Grant/Award Number: ERC-CoG2020-101001916; Fundacion Ram on Areces; Secretaria de Educacion, Ciencia Tecnología e Innovacion (SECTEI) of the Regional Government of Ciudad de México (CDMX), Grant/Award Number: SECTEI/159/2021; Spanish Ministry of Economy and Competitiveness, Grant/Award Numbers: PID2020-113007RB-I00, RYC-2015-171899, SAF-2017-82185-R; The Alzheimer's Association, Grant/Award Numbers: 2015-NIRG-340709, AARG-17-528125, AARG-17-528125-RAPID

Among the extracellular vesicles, apoptotic bodies (ABs) are only formed during the apoptosis and perform a relevant role in the pathogenesis of different diseases. Recently, it has been demonstrated that ABs from human renal proximal tubular HK-2 cells, either induced by cisplatin or by UV light, can lead to further apoptotic death in naïve HK-2 cells. Thus, the aim of this work was to carry out a non-targeted metabolomic approach to study if the apoptotic stimulus (cisplatin or UV light) affects in a different way the metabolites involved in the propagation of apoptosis. Both ABs and their extracellular fluid were analyzed using a reverse-phase liquid chromatography-mass spectrometry setup. Principal components analysis showed a tight clustering of each experimental group and partial least square discriminant analysis was used to assess the metabolic differences existing between these groups. Considering the variable importance in the projection values, molecular features were selected and some of them could be identified either unequivocally or tentatively. The resulting pathways indicated that there are significant, stimulus-specific differences in metabolites abundancies that may propagate apoptosis to healthy proximal tubular cells; thus, we hypothesize that the share in apoptosis of these metabolites might vary depending on the apoptotic stimulus, This research was funded by the Spanish Ministry of Science and Innovation (project PID2019-104913GB-I00, Agencia Estatal de Investigación, Referencia del Proyecto/AEI/10.13039/ 501100011033) and the Community of Madrid, I+D Biomedicine Activities Program 2022 (General Direction of Research and Technological Innovation (project P2022/BMD7223 CIFRA_CORCM)

Thethermochemical treatments of biowaste yield alternative renewable carbon-rich materials, while achieving waste valorization. These technologies allow to reduce amount of biowaste and enhance its life span avoiding the landfill disposal. The agronomic application as a soil amendment strategy using fresh hydrochar (obtained via the hydrothermal treatment of garden and park waste (GPW) at 180 °C for 1 h), post-treated hydrochar (washed, aged, and thermally treated), and biochar (obtained via the pyrolysis of GPW at 900 °C for 90 min) has been studied and compared in order to stablish the best approach for its valorization. We evaluated the effects of mixing fresh hydrochar (1–5% on dry weight) with different peat-based growth substrates on the seed germination index as well as the fresh and dry weights of Arabidopsis thaliana, Chenopodium quinoa, and Solanum lycopersicum (tomato). We also performed a germination assay with marginal agricultural soil mixed with fresh and post-treated chars as well as biochar using the same doses. All carbonaceous materials complied with the European legal framework being categorized as a class A amendment and present a favorable chemical composition for their agronomic use, carbon sources with a low heavy-metal content, and a high mineral and organic matter content. Application of post-treated hydrochar to the agricultural marginal soil improved the germination index of tomato seeds (by 10–20%) at low dosages (< 3% on dry weight) when washed and thermally treated hydrochar was used. However, fresh hydrochar negatively affected seed germination and plant growth when applied to marginal soil and peat-based growth media, particularly sandy substrates. Washing improved the germination index (by approximately 18%), reduced 90% of the total volatile fatty acid content, and effectively removed furans, amines, amides, pyridines, pyrazines, benzoic compounds, and organic acids that can affect seed germination and plant growth. Because the use of hydrochar in soils for agricultural purposes requires post-treatment to alleviate germination and plant growth inhibition, washing is the most suitable option considering the energy and technological requirements, Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. The authors would like to thank MICINN (PID2019-108445RB-I00, PDC2021-120755-I00, TED2021- 130287B-I00) and Madrid Regional Government (S2018/EMT-4344 and PEJD-2019-PRE/AMB-14231) for funding this work

12 Pág., In this work, we used palladium-doped polystyrene NPLs (PS-NPLs with a primary size of 286 ± 4 nm) with an irregular surface morphology which allowed for particle tracking and evaluation of their toxicity on two primary producers (cyanobacterium, Anabaena sp. PCC7120 and green algae, Chlamydomonas reinhardtii) and one primary consumer (crustacean, Daphnia magna). the concentration range for Anabaena and C. reinhardtii was from 0.01 to 1000 mg/L and for D. magna, the range was from 7.5 to 120 mg/L.EC50 s ranged from 49 mg NPLs/L for D. magna (48hEC50 s) to 248 mg NPLs/L (72hEC50 s for C. reinhardtii). PS-NPLs induced dose-dependent reactive oxygen species overproduction, membrane damage and metabolic alterations. To shed light on the environmental fate of PS-NPLs, the short-term distribution of PS-NPLs under static (using lake water and sediments) and stirring (using river water and sediments) conditions was studied at laboratory scale. The results showed that most NPLs remained in the water column over the course of 48 h. The maximum percentage of settled particles (∼ 30 %) was found under stirring conditions in comparison with the ∼ 10 % observed under static ones. Natural organic matter increased the stability of the NPLs under colloidal state while organisms favored their settlement. This study expands the current knowledge of the biological effects and fate of NPLs in freshwater environments., The authors acknowledge the financial support provided by the Spanish Government (Ministerio de Ciencia e Innovación, MICIN): PID2020-113769RB-C21/22, PLEC2021-007693 (Funded by MCIN/AEI/10.13039/501100011033 and by the European Union “NextGenerationEU”/PRTR”), TED2021-131609B-C32/33 grants and the Thematic Network of Micro- and Nanoplastics in the Environment (RED2018-102345-T, EnviroPlaNet Network). MTB is the recipient of a FPU (FPU17/01789) pre-doctoral contract by the Spanish Ministerio de Universidades. D.M.M. was funded through the Swiss National Science Foundation (SNF), Grant nos. PZ00P2_168105 and PCEFP2_186856. The authors gratefully acknowledge the support of IESMAT during physicochemical characterization of the nanoplastics.

The enormous societal impact of the ongoing COVID-19 pandemic has been particularly harsh for some social groups, such as the elderly. Recently, it has been suggested that senescent cells could play a central role in pathogenesis by exacerbating the pro-inflammatory immune response against SARS-CoV-2. Therefore, the selective clearance of senescent cells by senolytic drugs may be useful as a therapy to ameliorate the symptoms of COVID-19 in some cases. Using the established COVID-19 murine model K18-hACE2, we demonstrated that a combination of the senolytics dasatinib and quercetin (D/Q) significantly reduced SARS-CoV-2-related mortality, delayed its onset, and reduced the number of other clinical symptoms. The increase in senescent markers that we detected in the lungs in response to SARS-CoV-2 may be related to the post-COVID-19 sequelae described to date. These results place senescent cells as central targets for the treatment of COVID-19, and make D/Q a new and promising therapeutic tool., This work was supported by grants from the Instituto de Salud Carlos III (ISCIII), COV20-00755 (C.V.K.) and COV20-00792 (M.B., M.A.R.), Apadrina la Ciencia (C.S., C.O., G.G.A.), the AECC Scientific Foundation, PRDMA18011PAST, SIRTBIO- LABAE18008FERN (P.J.F.M., A.P., A.S.R.), the RETOS projects Programme of Ministerio de Ciencia e Innovación (MICINN), SAF2017-85766-R and PID2020-114077RB-I00 (P.J.F.M., A.P., A.S.R.), Ramon y Cajal Award from MICINN, RYC-2017-22335 (P.J.F.M., A.P., A.S.R.), Proyecto REACT from REACT/European Union/FEDER, COVTRAVI-19-CM U (J.M.S., M.F., N.G.), grant from Consejo Superior de Investigaciones Científicas (CSIC), CSIC-COV19-014 (A.I.d.Á), PID2020-113888RB-I00 from MICINN (A.I.d.Á), and the European Commission–Next Generation EU (regulation EU 2020/2094), through the CSIC's Global Health Platform (PTI+ Global Health) (A.I.d.Á). The RT–qPCR reactions were provided by the Genomics and NGS Core Facility (GENGS) at the Centro de Biología Molecular Severo Ochoa (CBMSO, CSIC-UAM) Madrid, Spain, which obtains general funding from both institutions. The GENGS facility is part of the PTI+ Global Health (CSIC) (http://www.cbm.uam.es/genomica).

The current distribution of most species results from ecological niche, past distribution, and migrations during glacial–interglacial periods and in situ evolution. Here, we disentangle the colonization history of Saxifraga longifolia Lapeyr., a limestone plant abundant in the Pyrenees and rare in other Iberian mountains and the African Atlas. Our working hypothesis is that the current distribution results from the shrinkage of a more extensive distribution in previous cold periods. We sampled 160 individuals of 32 populations across the whole distribution range and sequenced four DNA regions (rpl32‐trnL, rps16‐trnQ, trnS‐trnG, and ITS). Ecological conditions were modeled to identify factors promoting high genetic diversity and long‐term persistence areas for S. longifolia. In addition, we inferred phylogenetic relationships, phylogeographic divergence, genetic diversity, and migration routes. Seven plastid haplotypes were found, of which six occur in the Pyrenees and one in the High Atlas (Morocco). Discrete phylogeographic analysis (DPA) estimated migration routes predominantly from the Pyrenees to the other areas. Colonization events to those areas appear to have taken place recently given that the rest of the Iberian mountains do not harbor exclusive haplotypes. Iberian–Northern African distribution was inferred to be the result of long‐distance dispersal because the split between Iberian and High Atlas haplotypes is estimated to have taken place in the last 4 million years ago when the Strait of Gibraltar was already open. Migrations from the Pyrenees to the south may have been favored by a corridor of predominant limestone rocks along Eastern Iberia, followed by successful overcoming the Strait of Gibraltar to reach northern Africa., This research was partially financed by the “Ministerio de Economía, Industria y Competitividad (Spain),” project VULBIMON (CGL2017‐90040‐R). FP‐G. was supported by the Youth Employment Initiative (European Social Fund and Community of Madrid; reference PEJ‐2018‐AI/ AMB‐10790).

Antigen cross-presentation is a vital mechanism of dendritic cells and other antigen presenting cells to orchestrate the priming of cytotoxic responses towards killing of infected or cancer cells. In this process, exogenous antigens are internalized by dendritic cells, processed, loaded onto MHC class I molecules and presented to CD8+ T cells to activate them. Sec22b is an ER-Golgi Intermediate Compartment resident SNARE protein that, in partnership with sintaxin4, coordinates the recruitment of the transporter associated with antigen processing protein and the peptide loading complex to phagosomes, where antigenic peptides that have been proteolyzed in the cytosol are loaded in MHC class I molecules and transported to the cell membrane. The silencing of Sec22b in dendritic cells primary cultures and conditionally in dendritic cells of C57BL/6 mice, critically impairs antigen cross-presentation, but neither affects other antigen presentation routes nor cytokine production and secretion. Mice with Sec22b conditionally silenced in dendritic cells (Sec22b−/− ) show deficient priming of CD8+ T lymphocytes, fail to control tumor growth, and are resistant to anti-checkpoint immunotherapy. In this work, we show that Sec22b−/− mice elicit a deficient specific CD8+ T cell response when challenged with sublethal doses of Trypanosoma cruzi trypomastigotes that is associated with increased blood parasitemia and diminished survival, This research was funded by grants from “Ministerio de Ciencia e Innovación” (SAF 2016–75988-R. PID-2019-104760RB-100), “Comunidad de Madrid (S2017/BMD-3671. INFLAMUNE-CM) to MF “Ministerio de Ciencia, Innovación y Universidades Agencia Estatal de Investigación and Fondo Europeo de Desarrollo Regional (PGC 2018–096132-B-I00 and PID 2021–123389OB-I00 (MICINN/FEDER) to NG CBMSO institutional grants from “Fundación Ramón Areces” and “Banco de Santander” are also acknowledged. AA was suported by the Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación of Argentina through the grants: PICT 2018–4664, PICT 2017–1367, PICT 2020-SERIEA-01643. AA was also supported by Fundación Carolina and the Ministerio de Educación de la República Argentina

Glucose transporter 1 deficiency syndrome (GLUT1DS) is a neurometabolic disorder caused by haploinsufficiency of the GLUT1 glucose transporter (encoded by SLC2A1) leading to defective glucose transport across the blood–brain barrier. This work describes the genetic analysis of 56 patients with clinical or biochemical GLUT1DS hallmarks. 55.4% of these patients had a pathogenic variant of SLC2A1, and 23.2% had a variant in one of 13 different genes. No pathogenic variant was identified for the remaining patients. Expression analysis of SLC2A1 indicated a reduction in SLC2A1 mRNA in patients with pathogenic variants of this gene, as well as in one patient with a pathogenic variant in SLC9A6, and in three for whom no candidate variant was identified. Thus, the clinical and biochemical hallmarks generally associated with GLUT1DS may be caused by defects in genes other than SLC2A1, Carlos III Institute (ISCIII), European Regional Development Funds (PI19/01155); CIBERER (ERTRLE0I1); Consejería de Educacion, Juventud y Deporte, Comunidad de Madrid (B2017/BMD3721); Fundacion Isabel Gemio, the Fundacion La Caixa (LCF/PR/ PR16/11110018)

Trapania is the second largest genus belonging to the family Goniodorididae, of which most of the species are reported from Indo-Pacific waters. To date, there are nine species of Trapania distributed along the temperate coasts of the East Atlantic Ocean and Mediterranean Sea: Trapania fusca, Trapania graeffei, Trapania hispalensis, Trapania lineata, Trapania maculata, Trapania orteai, Trapania pallida, Trapania sanctipetrensis and Trapania tartanella. However, the validity of some of these species has been problematic due to uncertain taxonomic characteristics used for the differentiation of the species. The genus Trapania has a very uniformly external morphology and very similar internal anatomy. As a consequence, the features most commonly used to differentiate species have been the colour pattern of the body and the morphology of the radula. In the present study, we perform a morphological and molecular revision of the East Atlantic-Mediterranean species of the genus Trapania. Morphological analyses include dissections and scanning electron microscope photographs of radulae, labial cuticles and penises. Molecular work includes phylogenetic, species delimitation and haplotype network analyses. Our results bring doubt on the taxonomic characteristics used so far, suggesting that the richness of the North Eastern Atlantic-Mediterranean species has been overestimated. Trapania hispalensis, T. lineata and T. pallida are shown to belong to the same taxa, with Trapania lineata as senior synonym

25 páginas, 6 figuras, 2 tablas, Characterization of the genetic landscape of Alzheimer's disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/'proxy' AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele., This work was funded by a grant (EADB) from the EU Joint Programme – Neurodegenerative Disease Research. INSERM UMR1167 is also funded by the INSERM, Institut Pasteur de Lille, Lille Métropole Communauté Urbaine and French government’s LABEX DISTALZ program (development of innovative strategies for a transdisciplinary approach to AD). Full consortium acknowledgements and funding are in the Supplementary Not

The genome of viruses classified as picornaviruses consists of a single monocistronic positive strand RNA. The coding capacity of these RNA viruses is rather limited, and thus, they rely on the cellular machinery for their viral replication cycle. Upon the entry of the virus into susceptible cells, the viral RNA initially competes with cellular mRNAs for access to the protein synthesis machinery. Not surprisingly, picornaviruses have evolved specialized strategies that successfully allow the expression of viral gene products, which we outline in this review. The main feature of all picornavirus genomes is the presence of a heavily structured RNA element on the 5´UTR, referred to as an internal ribosome entry site (IRES) element, which directs viral protein synthesis as well and, consequently, triggers the subsequent steps required for viral replication. Here, we will summarize recent studies showing that picornavirus IRES elements consist of a modular structure, providing sites of interaction for ribosome subunits, eIFs, and a selective group of RNA-binding proteins, This work was supported by grants PID2020-115096RB-I00 (MICIN), B2017/BMD-3770 (cofinanced by Autonomous Community of Madrid and FEDER funds), and an Institutional grant from Fundación Ramón Areces

The process of species formation is characterized by the accumulation of multiple reproductive barriers. The evolution of hybrid male sterility, or Haldane's rule, typically characterizes later stages of species formation, when reproductive isolation is strongest. Yet, understanding how quickly reproductive barriers evolve and their consequences for maintaining genetic boundaries between emerging species remains a challenging task because it requires studying taxa that hybridize in nature. Here, we address these questions using the meadow grasshopper Pseudochorthippus parallelus, where populations that show multiple reproductive barriers, including hybrid male sterility, hybridize in two natural hybrid zones. Using mitochondrial data, we infer that such populations diverged some 100,000 years ago, at the beginning of the last glacial cycle in Europe. Nuclear data show that contractions at multiple glacial refugia, and post-glacial expansions have facilitated genetic differentiation between lineages that today interact in hybrid zones. We find extensive introgression throughout the sampled species range, irrespective of the current strength of reproductive isolation. Populations exhibiting hybrid male sterility in two hybrid zones show repeatable patterns of genomic differentiation, consistent with shared genomic constraints affecting ancestral divergence or with the role of those regions in reproductive isolation. Together, our results suggest that reproductive barriers that characterize late stages of species formation can evolve relatively quickly, particularly when associated with strong demographic changes. Moreover, we show that such barriers persist in the face of extensive gene flow, allowing future studies to identify associated genomic regions.

Human activities involving noise emission can affect wild animals. European mink was exposed to road noise and human voice playbacks to analyze how sound intensity level and duration of both noises altered the time that individuals were active and if their fecal cortisol metabolite (FCM) levels varied. A Hierarchical Analysis Cluster was performed to establish 2 mink groups with respect to both noise source type: short duration/low intensity (SL) and long duration/high intensity (LH). We performed general linear mixed models to evaluate the variation in locomotor activity duration (s) and FCM (nanogram per gram) levels, respectively. The results showed both road noise and human voices decreased locomotor activity duration in SL more sharply compared with LH, and human voices were the triggers that induced the most pronounced response to both exposure conditions. FCM (ng/g) levels increased in SL compared with LH during road noise while the opposite happened during human voices. Differences based on sex and age of individuals were observed. In conclusion, noise characteristics given by the sound type determined the variations in locomotor activity duration while noise exposure level determined the variations in FCM (ng/g) levels. Attention should be paid to noisy activities (e.g., recreational activities for visitors in protected natural areas) and loud groups of people to conserve wildlife, especially noise sensitive species.

Taxa restricted to mountains may be vulnerable to global warming, unless localscale topographic variation and conservation actions can protect them against expected changes to the climate. We tested how climate change will affect the 19 mountain-restricted Erebia species of the Iberian Peninsula, of which 7 are endemic. To examine the scope for local topographic variation to protect against warming, we applied species distribution models (HadGEM2 and MPI) at two spatial scales (10 10 and 1 1 km) for two representative concentration pathways (RCP4.5 and RCP8.5) in 2050 and 2070. We also superimposed current and future ranges on the protected area (PA) network to identify priority areas for adapting Erebia conservation to climate change. 4. In 10 10 km HadGEM2 models, climatically suitable areas for all species decreased in 2050 and 2070 (average 95.7%). Modelled decreases at 1 1 km were marginally less drastic (95.3%), and 14 out of 19 species were still expected to lose their entire climatically favourable range by 2070. The PA network is well located to conserve the species that are expected to retain some climatically suitable areas in 2070. However, we identify 25 separate 10 10 km squares where new PAs would help to adapt the network to expected range shifts or contractions by Erebia. Based on our results, adapting the conservation of range-restricted mountain taxa to projected climate change will require the implementation of complementary in situ and ex situ measures alongside urgent climate change mitigation., This study was supported by the project BIOUAM07-2019 founded by Biology Department from Universidad Autonoma de Madrid, the project IBERARTRO (SBPLY/17/180501/000492) funded by European Regional Development Fund (ERDF) through the Consejería de Educacion, Ciencia y Cultura, Junta de Comunidades de Castilla-La Mancha, the project RTI2018-096739-B-C21 funded by MCIU/AEI/ FEDER, UE, and the NexTdive project (PID2021-124187NB-I00) funded by the Spanish Ministry of Science and Innovation.

In the infection cycle, viruses release their genome in the host cell during uncoating. Here we use a variety of physicochemical procedures to induce and monitor the in vitro uncoating of ssDNA from individual Minute Virus of Mice (MVM) particles. Our experiments revealed two pathways of genome release: i) filamentous ssDNA appearing around intact virus particles when using gradual mechanical fatigue and heating at moderate temperature (50 °C). ii) thick structures of condensed ssDNA appearing when the virus particle is disrupted by mechanical nanoindentations, denaturing agent guanidinium chloride and high temperature (70 °C). We propose that in the case of filamentous ssDNA, when the capsid integrity is conserved, the genome is externalized through one channel of the capsid pores. However, the disruption of virus particles revealed a native structure of condensed genome. The mechanical analysis of intact particles after DNA strands ejection confirm the stabilization role of ssDNA in MVM

This is a pre-copyedited, author-produced PDF of an article accepted for publication in Molecular biology and evolution following peer review. The version of record Molecular biology and evolution 40.2 (2023): msac270 is available online at: https://academic.oup.com/search-results?page=1&q=10.1093%2Fmolbev%2Fmsac270&fl_SiteID=191&SearchSourceType=1&allJournals=1, Asymmetries are essential for proper organization and function of organ systems. Genetic studies in bilaterians have shown signaling through the Nodal/Smad2 pathway plays a key, conserved role in the establishment of body asymmetries. Although the main molecular players in the network for the establishment of left-right asymmetry (LRA) have been deeply described in deuterostomes, little is known about the regulation of Nodal signaling in spiralians. Here, we identified orthologs of the egf-cfc gene, a master regulator of the Nodal pathway in vertebrates, in several invertebrate species, which includes the first evidence of its presence in non-deuterostomes. Our functional experiments indicate that despite being present, egf-cfc does not play a role in the establishment of LRA in gastropods. However, experiments in zebrafish suggest that a single amino acid mutation in the egf-cfc gene in at least the common ancestor of chordates was the necessary step to induce a gain of function in LRA regulation. This study shows that the egf-cfc gene likely appeared in the ancestors of deuterostomes and "protostomes", before being adopted as a mechanism to regulate the Nodal pathway and the establishment of LRA in some lineages of deuterostomes

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 03-02-2023, La enfermedad de Alzheimer (EA) es un trastorno neurodegenerativo progresivo con una etiología compleja, que requiere de un abordaje multifactorial para un tratamiento eficaz. Se ha descrito que el factor de transcripción E2F4, que regula la quiescencia celular y la homeostasis tisular, también controla genes afectados en la EA. Además, E2F4 está regulado positivamente en los cerebros de pacientes con alzhéimer y del modelo murino de alzhéimer 5xFAD. E2F4 contiene dos residuos de Thr conservados evolutivamente que, cuando se fosforilan, modulan su actividad, constituyendo así una diana potencial sobre la que intervenir. En este estudio, se generó una cepa knock-in de ratón cuyas neuronas expresan una variante dominante negativa de E2F4 de ratón que carece de estos residuos de Thr (E2F4DN). Estos ratones, junto con sus controles knock-in para EGFP, fueron cruzados con ratones 5xFAD para analizar los efectos de la expresión de E2F4DN en dicho modelo murino de alzhéimer. Los resultados obtenidos en este trabajo demuestran que la expresión neuronal de E2F4DN en ratones 5xFAD atenúa la respuesta inmune. Este efecto correlaciona con una microgliosis y astrogliosis reducidas, y con la modulación de la proteostasis del péptido β amiloide (βA). Además, se ha observado que la expresión de E2F4DN en las neuronas conduce a una comunicación entre estas células y la microglía mediada por un factor soluble. Una terapia génica basada en E2F4DN podría ser un buen enfoque multifactorial contra la EA. Es por ello por lo que se estudió la expresión de una forma dominante negativa de E2F4 humano (hE2F4DN) en las neuronas de ratones homocigotos 5xFAD (h5xFAD), gracias a la administración sistémica de un vector AAV.PHP.B-hSyn1.hE2F4DN. De igual modo que en los ratones transgénicos 5xFAD/E2F4DN, la expresión de hE2F4DN tras la administración intravenosa del vector en los ratones h5xFAD, condujo a la atenuación de la astrocitosis reactiva y de la microgliosis, y redujo la acumulación de βA en el hipocampo. Después de décadas de trabajo infructuoso, no hay terapias efectivas disponibles para la EA, probablemente debido a su compleja etiología que requiere de un abordaje terapéutico multifactorial. Los resultados del presente estudio indican que la terapia génica basada en E2F4DN es un enfoque terapéutico prometedor contra la EA, Alzheimer's disease (AD) is a progressive neurodegenerative disorder with a complex etiology, which requires a multifactorial approach for an efficient treatment. Transcription factor E2F4, which regulates cellular quiescence and tissue homeostasis, has been shown to control gene networks affected in AD. Furthermore, E2F4 is upregulated in the brains of Alzheimer's patients and in the murine model of Alzheimer's disease 5xFAD. E2F4 contains two evolutionarily conserved Thr-motifs that, when phosphorylated, modulates its activity, thus constituting a potential target for intervention. In this study, a knock-in mouse strain was generated, with neuronal expression of a mouse E2F4 variant lacking these Thr residues (E2F4DN). These mice, together with their EGFP knock-in controls, were bred with 5xFAD mice to analyze the effects of the expression of E2F4DN in such a murine model of Alzheimer's disease. Results in this work show that neuronal expression of E2F4DN in 5xFAD mice mitigates the immune response. Reduced microgliosis and astrogliosis are correlated with this effect, as well as modulation of amyloid-β peptide (Aβ) proteostasis. In addition, the expression of E2F4DN in neurons has been observed to lead to soluble factor-mediated communication between these cells and microglia. A gene therapy based on E2F4DN could be a good multifactorial approach against AD. Therefore, the expression of a dominant negative form of human E2F4 (hE2F4DN) in neurons of homozygous 5xFAD (h5xFAD) mice, due to the systemic administration of an AAV.PHP.B-hSyn1.hE2F4DN vector, has been under study. As in the 5xFAD/E2F4DN transgenic mice, the expression of hE2F4DN followed by the intravenous administration of the vector in h5xFAD mice led to the attenuation of reactive astrocytosis and microgliosis, and reduced βA accumulation in the hippocampus. After decades of unsuccessful work, no effective therapies are available for AD, probably due to its complex etiology requiring a multifactorial therapeutic approach. These results show that E2F4DNbased gene therapy is a promising therapeutic approach against AD, La realización de esta tesis doctoral ha sido posible gracias a la concesión de la ayuda para la formación de doctores en empresas "Doctorados Industriales” (referencia de la ayuda DI-17-09661) del Ministerio de Economía, Industria y Competitividad del Subprograma Estatal de Formación, convocatoria 2017, del Programa Estatal de Promoción del Talento y su Empleabilidad, en el marco del Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016. El contrato predoctoral fue cofinanciado por esta ayuda y por la empresa Tetraneuron, S. L.

Taxonomic studies of the phylum Kinorhyncha follow a standardised structure, which is extremely useful in many aspects, such as making it easier to read and compare the different species descriptions. Nevertheless, the morphological measurements methods, essential for formal description of species, may differ according to the authors. In the present contribution, we propose a standardised method of taking and representing the measurements, with the aim of obtaining comparable and repeatable morphometric results. Additionally, we propose an online repository to make the measurements accessible to all researchers in the same format, facilitating future comparisons and studies. Finally, a glossary that compiles and defines all the measurements that may be included in Kinorhyncha descriptions is presented. Taxonomic studies of the phylum Kinorhyncha follow a standardised structure, which is extremely useful in many aspects, such as making it easier to read and compare the different species descriptions. Nevertheless, the morphological measurements methods, essential for formal description of species, may differ according to the authors. In the present contribution, we propose a standardised method of taking and representing the measurements, with the aim of obtaining comparable and repeatable morphometric results. Additionally, we propose an online repository to make the measurements accessible to all researchers in the same format, facilitating future comparisons and studies. Finally, a glossary that compiles and defines all the measurements that may be included in Kinorhyncha descriptions is presented.

15 pags., 6 figs., The RNA binding protein TDP-43 forms cytoplasmic inclusions via its C-terminal prion-like domain in several neurodegenerative diseases. Aberrant TDP-43 aggregation arises upon phase de-mixing and transitions from liquid to solid states, following still unknown structural conversions which are primed by oxidative stress and chaperone inhibition. Despite the well-established protective roles for molecular chaperones against protein aggregation pathologies, knowledge on the determinants of chaperone recognition in disease-related prions is scarce. Here we show that chaperones and co-chaperones primarily recognize the structured elements in TDP-43´s prion-like domain. Significantly, while HSP70 and HSP90 chaperones promote TDP-43 phase separation, co-chaperones from the three classes of the large human HSP40 family (namely DNAJA2, DNAJB1, DNAJB4 and DNAJC7) show strikingly different effects on TDP-43 de-mixing. Dismantling of the second helical element in TDP-43 prion-like domain by methionine sulfoxidation impacts phase separation and amyloid formation, abrogates chaperone recognition and alters phosphorylation by casein kinase-1δ. Our results show that metamorphism in the post-translationally modified TDP-43 prion-like domain encodes determinants that command mechanisms with major relevance in disease., This work was supported by grants PID2019-109276RA-I00/AEI/ 10.13039/501100011033 (to J.O.), PID2019-103845RB-C21-AEI/10.13039/ 501100011033 (to M.G.) and SAF2016-76678-C2-2-R (MINECO/AEI/ FEDER UE to D.V.L.) from the Spanish AEI-Ministry of Science and Innovation and Enhanced New Staff Start-up Research Grant (The University of Hong Kong) to R.H. J.O. was a recipient of a Leonardo Grant from the Spanish BBVA Foundation (BBM_TRA_0203) and is a Ramón y Cajal Fellow of the Spanish AEI-Ministry of Science and Innovation (RYC2018-026042-I funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”). NMR experiments were performed in the “Manuel Rico” NMR Laboratory (LMR) of the Spanish National Research Council (CSIC), a node of the Spanish Large-Scale National Facility (ICTS R-LRB).

Parvoviruses are promising anticancer and gene therapy agents, but a deep knowledge of the entry process is crucial to exploit their therapeutic potential. We addressed this issue while attempting to retarget the oncolytic parvovirus minute virus of mice (MVMp) to the tumor vasculature. Residues at three functional domains of the icosahedral capsid were substituted by rational design with peptides competing with the vascular endothelial growth factor. Most substitutions impaired virus maturation, though some yielded infectious chimeric virions, and substitutions in a dimple at the twofold axis that allocates sialic acid (SIA) receptors altered viral tropism. One dimple-modified chimeric virion was efficiently attached as MVMp to α2-linked SIA moieties, but the infection was impaired by the binding to some inhibitory α2-3,-6,-8 SIA pseudoreceptors, which hampers intracellular virus traffic to the endosome in a cell type-dependent manner. Infectious from nonproductive traffic could be mechanistically discriminated by an endosomal drastic capsid structural transition comprising the cleavage of some VP2-Nt sequences and its associated VP1-Nt exposure. Correspondingly, neuraminidase removal of inhibitory SIA moieties enhanced the infection quantitatively, correlating to the restored virus traffic to the endosome and the extent of VP2-Nt cleavage/VP1-Nt exposure. This study illustrates (i) structural constraints to retarget parvoviruses with evolutionary adopted narrow grooves allocating small SIA receptors, (ii) the possibility to enhance parvovirus oncolysis by relaxing the glycan network on the cancer cell surface, and (iii) the major role played by the attachment to cell type-specific SIAs in the intracellular virus traffic to the endosome, which may determine parvovirus tropism and host range.

Nitro-fatty acids (NO2-FAs) are unsaturated fatty acid nitration products that exhibit anti-inflammatory actions in experimental mouse models of autoimmune and allergic diseases. These electrophilic molecules interfere with intracellular signaling pathways by reversible post-translational modification of nucleophilic amino-acid residues. Several regulatory proteins have been identified as targets of NO2-FAs, modifying their activity and promoting gene expression changes that result in anti-inflammatory effects. Herein, we report the effects of nitro-oleic acid (NO2-OA) on pro-inflammatory T cell functions, showing that 9- and 10-NOA, but not their oleic acid precursor, decrease T cell proliferation, expression of activation markers CD25 and CD71 on the plasma membrane, and IL-2, IL-4, and IFN-γ cytokine gene expressions. Moreover, we have found that NO2-OA inhibits the transcriptional activity of nuclear factor of activated T cells (NFAT) and that this inhibition takes place through the regulation of the phosphatase activity of calcineurin (CaN), hindering NFAT dephosphorylation, and nuclear translocation in activated T cells. Finally, using mass spectrometry-based approaches, we have found that NO2-OA nitroalkylates CaNA on four Cys (Cys129, 228, 266, and 372), of which only nitroalkylation on Cys372 was of importance for the regulation of CaN phosphatase activity in cells, disturbing functional CaNA/CaNB heterodimer formation. These results provide evidence for an additional mechanism by which NO2-FAs exert their anti-inflammatory actions, pointing to their potential as therapeutic bioactive lipids for the modulation of harmful T cell-mediated immune responses., This work was supported by grant RTI2018-100815-B-I00 Ministerio de Ciencia e Innovación (MICIU)/Fondo Europeo de Desarrollo Regional (FEDER) and the accompanying 2021 CSIC Exceptional Grant to M.A.Í. and J.M.S. Additional funding was obtained from grants PID2021-122348NB-I00 (MICIU) and “La Caixa” Banking Foundation (HR22-00253) to E.C. and J.V., R01GM125944-05 and DK112854-04 to F.J.S., and from Universidad de la República Comisión Intersectorial de Investigación Científica Grupos_2018, Espacio Interdisciplinario_2020 to R.R. The CBMSO receives an institutional grant from the Fundación Ramón Areces.

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 16-01-2023, El cáncer de pulmón es la principal causa de mortalidad relacionada con el cáncer, diagnosticándose en estadios avanzados en el 80% de los casos. El manejo del cáncer de pulmón de células no pequeñas (CPCNP) avanzado ha mejorado notablemente gracias a la identificación de alteraciones moleculares y al desarrollo de fármacos dirigidos frente a las mismas. Las mutaciones más relevantes en el CPCNP ocurren en los genes EGFR, ALK y ROS1, dianas de terapias dirigidas basadas en inhibidores de tirosina kinasa (TKIs). En concreto, el 3-7% de los pacientes con CPCNP presentan translocaciones en ALK, originando un subtipo molecular observado especialmente en pacientes jóvenes, no fumadores y con histología de adenocarcinoma. El correcto diagnóstico de estos pacientes es crucial dada la mejora significativa en supervivencia y calidad de vida gracias al amplio abanico de TKIs dirigidos frente ALK (ALK-TKIs). Sin embargo, alrededor del 70% de los pacientes progresan tras 1 o 2 años de tratamiento debido a mutaciones de resistencia. Estas mutaciones, no obstante, no confieren resistencia a todos los TKIs disponibles. En este sentido, el estudio del perfil mutacional del tumor a la progresión, mediante técnicas de alta sensibilidad, puede suponer una estrategia eficiente para establecer una secuencia adecuada y personalizada de ALK-TKIs. Sin embargo, la biopsia tumoral en los pacientes con CPCNP no siempre es factible. La biopsia líquida, entendida como sangre y otros fluidos corporales, permite la detección tanto de las fusiones en ALK como de mecanismos de resistencia a ALK-TKIs de forma mínimamente invasiva. Sin embargo, la detección de fusiones en biopsia líquida sigue siendo un desafío dada su complejidad genómica. La Tesis doctoral se presenta como un compendio de publicaciones que tienen como objetivo perfeccionar los métodos diagnósticos y de seguimiento de los pacientes con CPCNP, especialmente aquellos con fusión en ALK. El primer trabajo demostró que la secuenciación masiva (NGS) del ADN circulante libre (cfDNA) basada en identificadores moleculares únicos (UMIs), con sensibilidad similar a técnicas como la PCR digital (dPCR), permite conocer el perfil mutacional del tumor con una alta concordancia con la dPCR. En el segundo trabajo, se demostró que el cfDNA procedente de fluidos corporales representa una fuente de información importante para identificar biomarcadores, en cáncer de pulmón, con mayor sensibilidad que el plasma. En el tercer artículo se analizaron mediante NGS 26 muestras de plasma y dos de líquido cefalorraquídeo (LCR), procedentes de 24 pacientes con CPCNP ALK-positivos, obtenidas en el momento de la progresión a un ALK-TKI. Mediante el desarrollo de un algoritmo bioinformático se identificaron mutaciones en el locus de ALK en el 35% de los pacientes, así como potenciales mutaciones de resistencia en otros genes. Estos resultados, junto con los del caso clínico descrito en el cuarto artículo, demostraron la validez de la NGS de muestras de biopsia líquida para la detección de mutaciones de resistencia en el locus de ALK y la utilidad de este abordaje para la toma de decisiones terapéuticas en los pacientes ALK-positivos. Por último, nuestra estrategia para el quinto trabajo fue el estudio de transcritos de fusión de ALK (ARN mensajero, mRNA) contenidos en vesículas extracelulares (EVs), donde se encuentran protegidos de las RNAasas del torrente sanguíneo. Así, la fusión de EML4-ALK, tanto en su forma proteica como en mRNA, se identificó en EVs aisladas de líneas celulares y, posteriormente, en 8 de las 16 muestras clínicas de plasma derivadas de pacientes con CPCNP ALK-positivos analizadas, especialmente en aquellas con mayor expresión de la tetraspanina CD81. El aislamiento de EVs supone una estrategia alternativa al tejido, no invasiva, automatizable y aplicable a la práctica clínica para la detección de fusiones como la translocación de ALK

PLK1 is a serine/threonine kinase with crucial roles during mitosis. However, its involvement during mammalian male meiosis remains largely unexplored. By inhibiting the kinase activity of PLK1 using BI 2536 on organotypic cultures of seminiferous tubules, we found that the disassembly of SYCP3 and HORMAD1 from the lateral elements of the synaptonemal complex during diakinesis is impeded. We also found that the normal recruitment of SYCP3 and HORMAD1 to the inner centromere in prometaphase I spermatocytes did not occur. Additionally, we analyzed the participation of PLK1 in the assembly of the inner centromere by studying its implication in the Bub1-H2AT120ph-dependent recruitment of shugoshin SGO2, and the Haspin-H3T3ph-dependent recruitment of Aurora B/C and Borealin. Our results indicated that both pathways are regulated by PLK1. Altogether, our results demonstrate that PLK1 is a master regulator of the late prophase I/metaphase I transition in mouse spermatocytes.

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de Lectura: 12-01-2023

Cell differentiation involves profound changes in global gene expression that often have to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibility data, we find that p27 is coincidentally necessary to repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2, and ASCL1. Our data reveal both a direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of Sox2 leads to reduced levels of its downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.