Development of a high-throughput screening platform to identify new therapeutic agents for Medulloblastoma Group 3.

Pediatric brain tumors (PBTs) represent about 25 % of all pediatric cancers and are the most common solid tumors in children and adolescents. Medulloblastoma (MB) is the most frequently occurring malignant PBT, accounting for almost 10 % of all pediatric cancer deaths. MB Group 3 (MB G3) accounts for 25-30 % of all MB cases and has the worst outcome, particularly when associated with MYC amplification. However, no targeted treatments for this group have been developed so far. Here we describe a unique high throughput screening (HTS) platform specifically designed to identify new therapies for MB G3. The platform incorporates optimized and validated 2D and 3D efficacy and toxicity models, that account for tumor heterogenicity, limited efficacy and unacceptable toxicity from the very early stage of drug discovery. The platform has been validated by conducting a pilot HTS campaign with a 1280 lead-like compound library. Results showed 8 active compounds, targeting MB reported targets and several are currently approved or in clinical trials for pediatric patients with PBTs, including MB. Moreover, hits were combined to avoid tumor resistance, identifying 3 synergistic pairs, one of which is currently under clinical study for recurrent MB and other PBTs.

Tracking Openness and Topic Evolution of COVID-19 Publications January 2020-March 2021: Comprehensive Bibliometric and Topic Modeling Analysis.

BACKGROUND: The COVID-19 outbreak highlighted the importance of rapid access to research. OBJECTIVE: The aim of this study was to investigate research communication related to COVID-19, the level of openness of papers, and the main topics of research into this disease. METHODS: Open access (OA) uptake (typologies, license use) and the topic evolution of publications were analyzed from the start of the pandemic (January 1, 2020) until the end of a year of widespread lockdown (March 1, 2021). RESULTS: The sample included 95,605 publications; 94.1% were published in an OA form, 44% of which were published as Bronze OA. Among these OA publications, 42% do not have a license, which can limit the number of citations and thus the impact. Using a topic modeling approach, we found that articles in Hybrid and Green OA publications are more focused on patients and their effects, whereas the strategy to combat the pandemic adopted by different countries was the main topic of articles selecting publication via the Gold OA route. CONCLUSIONS: Although OA scientific production has increased, some weaknesses in OA practice, such as lack of licensing or under-researched topics, still hold back its effective use for further research.

Survey of Candidatus Liberibacter Solanacearum and Its Associated Vectors in Potato Crop in Spain.

'Candidatus Liberibacter solanacearum' (CaLsol), the etiological agent of potato zebra chip (ZC), is transmitted to potato plants by the psyllid Bactericera cockerelli (Sulc, 1909) in North and Central America and New Zealand. The risk of the dispersion of ZC in Spain depends on the presence of an efficient vector. This work studies the presence and abundance of ZC symptoms and CaLsol in potato plants, as well as the presence and abundance of psyllid species associated with potato crops in the main producing areas in Spain. Eighty-eight plots were surveyed punctually to detect ZC symptoms and psyllid species in the main potato-producing areas. Furthermore, fourteen potato plots were surveyed by different sampling methods during the cropping season to detect psyllid species from 2016 to 2018. Very few symptomatic and CaLsol-positive plants were detected in Mainland Spain, and any positive plant was detected in the Canary Islands. Most of the adult psyllids captured were identified as Bactericera nigricornis (Foerster, 1848), and some of them as Bactericera trigonica, but no B. cockerelli was detected. B. nigricornis was found widely distributed in the northern half of the Iberian Peninsula; however, this psyllid does not seem sufficient to pose a threat to potato production, due to the scarce number of specimens and because the frequency of B. nigricornis specimens that were CaLsol+ was very low.

FAIR4Health: Findable, Accessible, Interoperable and Reusable data to foster Health Research.

Due to the nature of health data, its sharing and reuse for research are limited by ethical, legal and technical barriers. The FAIR4Health project facilitated and promoted the application of FAIR principles in health research data, derived from the publicly funded health research initiatives to make them Findable, Accessible, Interoperable, and Reusable (FAIR). To confirm the feasibility of the FAIR4Health solution, we performed two pathfinder case studies to carry out federated machine learning algorithms on FAIRified datasets from five health research organizations. The case studies demonstrated the potential impact of the developed FAIR4Health solution on health outcomes and social care research. Finally, we promoted the FAIRified data to share and reuse in the European Union Health Research community, defining an effective EU-wide strategy for the use of FAIR principles in health research and preparing the ground for a roadmap for health research institutions. This scientific report presents a general overview of the FAIR4Health solution: from the FAIRification workflow design to translate raw data/metadata to FAIR data/metadata in the health research domain to the FAIR4Health demonstrators' performance.

Analysis of shared research data in Spanish scientific papers about COVID-19: A first approach.

During the coronavirus pandemic, changes in the way science is done and shared occurred, which motivates meta-research to help understand science communication in crises and improve its effectiveness. The objective is to study how many Spanish scientific papers on COVID-19 published during 2020 share their research data. Qualitative and descriptive study applying nine attributes: (a) availability, (b) accessibility, (c) format, (d) licensing, (e) linkage, (f) funding, (g) editorial policy, (h) content, and (i) statistics. We analyzed 1,340 papers, 1,173 (87.5%) did not have research data. A total of 12.5% share their research data of which 2.1% share their data in repositories, 5% share their data through a simple request, 0.2% do not have permission to share their data, and 5.2% share their data as supplementary material. There is a small percentage that shares their research data; however, it demonstrates the researchers' poor knowledge on how to properly share their research data and their lack of knowledge on what is research data.

Community-driven governance of FAIRness assessment: an open issue, an open discussion.

Although FAIR Research Data Principles are targeted at and implemented by different communities, research disciplines, and research stakeholders (data stewards, curators, etc.), there is no conclusive way to determine the level of FAIRness intended or required to make research artefacts (including, but not limited to, research data) Findable, Accessible, Interoperable, and Reusable. The FAIR Principles cover all types of digital objects, metadata, and infrastructures. However, they focus their narrative on data features that support their reusability. FAIR defines principles, not standards, and therefore they do not propose a mechanism to achieve the behaviours they describe in an attempt to be technology/implementation neutral. Various FAIR assessment metrics and tools have been designed to measure FAIRness. Unfortunately, the same digital objects assessed by different tools often exhibit widely different outcomes because of these independent interpretations of FAIR. This results in confusion among the publishers, the funders, and the users of digital research objects. Moreover, in the absence of a standard and transparent definition of what constitutes FAIR behaviours, there is a temptation to define existing approaches as being FAIR-compliant rather than having FAIR define the expected behaviours. This whitepaper identifies three high-level stakeholder categories -FAIR decision and policymakers, FAIR custodians, and FAIR practitioners - and provides examples outlining specific stakeholders' (hypothetical but anticipated) needs. It also examines possible models for governance based on the existing peer efforts, standardisation bodies, and other ways to acknowledge specifications and potential benefits. This whitepaper can serve as a starting point to foster an open discussion around FAIRness governance and the mechanism(s) that could be used to implement it, to be trusted, broadly representative, appropriately scoped, and sustainable. We invite engagement in this conversation in an open Google Group fair-assessment-governance@googlegroups.com.

Assessment of Multilocus Sequence Analysis (MLSA) for Identification of Candidatus Liberibacter Solanacearum from Different Host Plants in Spain.

Liberibacter is a bacterial group causing different diseases and disorders in plants. Among liberibacters, Candidatus Liberibacter solanaceraum (CLso) produces disorders in several species mainly within Apiaceae and Solanaceae families. CLso isolates are usually grouped in defined haplotypes according to single nucleotide polymorphisms in genes associated with ribosomal elements. In order to characterize more precisely isolates of CLso identified in potato in Spain, a Multilocus Sequence Analysis (MLSA) was applied. This methodology was validated by a complete analysis of ten housekeeping genes that showed an absence of positive selection and a nearly neutral mechanism for their evolution. Most of the analysis performed with single housekeeping genes, as well as MLSA, grouped together isolates of CLso detected in potato crops in Spain within the haplotype E, undistinguishable from those infecting carrots, parsnips or celery. Moreover, the information from these housekeeping genes was used to estimate the evolutionary divergence among the different CLso by using the concatenated sequences of the genes assayed. Data obtained on the divergence among CLso haplotypes support the hypothesis of evolutionary events connected with different hosts, in different geographic areas, and possibly associated with different vectors. Our results demonstrate the absence in Spain of CLso isolates molecularly classified as haplotypes A and B, traditionally considered causal agents of zebra chip in potato, as well as the uncertain possibility of the present haplotype to produce major disease outbreaks in potato that may depend on many factors that should be further evaluated in future works.

Open Access of COVID-19-related publications in the first quarter of 2020: a preliminary study based in PubMed.

Background: The COVID-19 outbreak has made funders, researchers and publishers agree to have research publications, as well as other research outputs, such as data, become openly available. In this extraordinary research context of the SARS CoV-2 pandemic, publishers are announcing that their coronavirus-related articles will be made immediately accessible in appropriate open repositories, like PubMed Central, agreeing upon funders' and researchers' instigation. Methods: This work uses Unpaywall, OpenRefine and PubMed to analyse the level of openness of articles about COVID-19, published during the first quarter of 2020. It also analyses Open Access (OA) articles published about previous coronavirus (SARS CoV-1 and MERS CoV) as a means of comparison. Results: A total of 5,611 COVID-19-related articles were analysed from PubMed. This is a much higher amount for a period of 4 months compared to those found for SARS CoV-1 and MERS during the first year of their first outbreaks (335 and 116 articles, respectively).  Regarding the levels of openness, 88.8% of the SARS CoV-2 papers are freely available; similar rates were found for the other coronaviruses. Deeper analysis showed that (i) 67.4% of articles belong to an undefined Bronze category; (ii) 76.4% of all OA papers don't carry any license, followed by 10.4% which display restricted licensing. These patterns were found to be repeated in the three most frequent publishers: Elsevier, Springer and Wiley. Conclusions: Our results suggest that, although scientific production is much higher than during previous epidemics and is open, there is a caveat to this opening, characterized by the absence of fundamental elements and values ​​on which Open Science is based, such as licensing.

Fostering global data sharing: highlighting the recommendations of the Research Data Alliance COVID-19 working group.

The systemic challenges of the COVID-19 pandemic require cross-disciplinary collaboration in a global and timely fashion. Such collaboration needs open research practices and the sharing of research outputs, such as data and code, thereby facilitating research and research reproducibility and timely collaboration beyond borders. The Research Data Alliance COVID-19 Working Group recently published a set of recommendations and guidelines on data sharing and related best practices for COVID-19 research. These guidelines include recommendations for clinicians, researchers, policy- and decision-makers, funders, publishers, public health experts, disaster preparedness and response experts, infrastructure providers from the perspective of different domains (Clinical Medicine, Omics, Epidemiology, Social Sciences, Community Participation, Indigenous Peoples, Research Software, Legal and Ethical Considerations), and other potential users. These guidelines include recommendations for researchers, policymakers, funders, publishers and infrastructure providers from the perspective of different domains (Clinical Medicine, Omics, Epidemiology, Social Sciences, Community Participation, Indigenous Peoples, Research Software, Legal and Ethical Considerations). Several overarching themes have emerged from this document such as the need to balance the creation of data adherent to FAIR principles (findable, accessible, interoperable and reusable), with the need for quick data release; the use of trustworthy research data repositories; the use of well-annotated data with meaningful metadata; and practices of documenting methods and software. The resulting document marks an unprecedented cross-disciplinary, cross-sectoral, and cross-jurisdictional effort authored by over 160 experts from around the globe. This letter summarises key points of the Recommendations and Guidelines, highlights the relevant findings, shines a spotlight on the process, and suggests how these developments can be leveraged by the wider scientific community.

The testis-specific Cα2 subunit of PKA is kinetically indistinguishable from the common Cα1 subunit of PKA.

BACKGROUND: The two variants of the α-form of the catalytic (C) subunit of protein kinase A (PKA), designated Cα1 and Cα2, are encoded by the PRKACA gene. Whereas Cα1 is ubiquitous, Cα2 expression is restricted to the sperm cell. Cα1 and Cα2 are encoded with different N-terminal domains. In Cα1 but not Cα2 the N-terminal end introduces three sites for posttranslational modifications which include myristylation at Gly1, Asp-specific deamidation at Asn2 and autophosphorylation at Ser10. Previous reports have implicated specific biological features correlating with these modifications on Cα1. Since Cα2 is not modified in the same way as Cα1 we tested if they have distinct biochemical activities that may be reflected in different biological properties. RESULTS: We show that Cα2 interacts with the two major forms of the regulatory subunit (R) of PKA, RI and RII, to form cAMP-sensitive PKAI and PKAII holoenzymes both in vitro and in vivo as is also the case with Cα1. Moreover, using Surface Plasmon Resonance (SPR), we show that the interaction patterns of the physiological inhibitors RI, RII and PKI were comparable for Cα2 and Cα1. This is also the case for their potency to inhibit catalytic activities of Cα2 and Cα1. CONCLUSION: We conclude that the regulatory complexes formed with either Cα1 or Cα2, respectively, are indistinguishable.

Diagnóstico genético preimplantacional en un caso de una paciente portadora de una inmunodeficiencia combinada severa. Revisión crítica de este procedimiento en ciclos de FIV-ICSI / Preimplantation genetic diagnosis in a patient carrier of severe combined immunodeficiency. Critical review of this procedure in IFV-ICSI cycles

Objetivo: Realizar un análisis crítico, a partir de un caso clínico del diagnóstico genético preimplantacional (DGP).Pacientes y métodos: Se describe a una paciente con una enfermedad de origen genético (inmunodeficiencia combinada severa) en la que el DGP fue de vital importancia para su tratamiento.Conclusiones: El DGP es un arma terapéutica de gran relevancia en la reproducción asistida pero sus indicaciones, salvo en enfermedades monogénicas y las ligadas al sexo están todavía en debate (AU)

Objective: To perform a critical analysis from a clinical case of preimplantation genetic diagnosis (PGD).Patients and methods: A patient is described who has a disease of genetic origin (severe combined immunodeficiency) in which PGD was of vital importance for her treatment.Conclusions: PGD is a very important tool in assisted reproduction but its indications, except in monogenic diseases and those linked to sex, are still under debate (AU)

Synthesis, pharmacology and molecular modeling of N-substituted 2-phenyl-indoles and benzimidazoles as potent GABA(A) agonists.

Among the known non-benzodiazepine hypnotic drugs, Zolpidem (1a), Indiplon (2a) and Zaleplon (2b) have shown high affinity and selectivity for the alpha(1) subunit of the GABA-A receptor. Our group has performed pharmacophoric and ADMET-prediction studies to evaluate a virtual library of new molecules based on privileged structures. Among these, we have synthesized a library of N-substituted indoles and a library of N-substituted benzimidazoles. Afterwards, in vitro screening and in vivo spontaneous motor activity in mice has revealed molecules with good in vitro affinities for the alpha(1) receptor and potent in vivo induction of sedation.

Design, synthesis and biological activity of acyl substituted 3-amino-5-methyl-1,4,5,7-tetrahydropyrazolo[3,4-b]pyridin-6-ones as potential hypnotic drugs.

Among the known non-benzodiazepinic hypnotic drugs acting on the alpha1 subunit of the GABA-A receptor, Zolpidem, Zaleplon and Indiplon have showed high affinity and selectivity. Following a design methodology including pharmacophoric requirements and ADME-predicted properties, we have synthesized a library of 3-amino-4,5-dihydro-1H-pyrazolo[3,4-b]pyridin-6(7H)-ones and their N1-alkyl derivatives as new scaffolds for designing non-benzodiazepine BZ receptor ligands.

A spectrophotometric assay for the determination of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase activity.

We report an assay for the determination of the activity of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, the enzyme which catalyzes the fourth reaction step of the 2-C-methyl-D-erythritol 4-phosphate pathway for the synthesis of isoprenoids, which is based on the spectrophotometrical determination of adenosine 5'-diphosphate using pyruvate kinase and L-lactate dehydrogenase as auxiliary enzymes. This method can be adapted to microtiter plates, can be automated, and because of its simplicity and speed can be useful for the functional characterization of the enzyme and for the screening of inhibitors with potential antibiotic or antimalarial action.

Modulation of the steroidogenic activity of luteinizing hormone by insulin and insulin-like growth factor-I through interaction with the cAMP-dependent protein kinase signaling pathway in the trout ovary.

In the salmonid ovary, luteinizing hormone (LH) is the major gonadotropic hormone stimulating the production of steroids during the periovulatory period and its effects are mediated by the cAMP-dependent protein kinase (PKA) signaling pathway. We have previously shown that the in vitro steroidogenic activity of LH in the salmonid ovary is inhibited by insulin-like growth factor I (IGF-I) which, like insulin, has specific receptors in both theca and granulosa layers. In the present study, we have investigated the modulatory effects of insulin on salmon LH (sLH)-stimulated steroid production in preovulatory theca layers of brown trout (Salmo trutta) and the effects of both insulin and IGF-I on the sLH-stimulated cAMP/PKA signaling pathway. Our results show that insulin, like IGF-I, blocked the stimulatory effects of sLH, dibutyryl cAMP and IBMX on testosterone (T) production but not those of sLH on cAMP production. Furthermore, insulin and IGF-I blocked the activation of PKA induced by sLH and these effects were correlated with changes in the total protein content of the catalytic (C) and type II regulatory (RII) subunits of PKA. Interestingly, insulin and IGF-I had different effects on total PKA subunit content since insulin potentiated the sLH-stimulated increase in RII subunit content whereas IGF-I blocked the sLH-stimulated increase in total C subunit content. The effects of insulin and IGF-I in trout theca layers appeared to be mediated by the mitogen-activated protein kinase (MAPK) signaling pathway because inhibition of extracellular signal-regulated kinase 1/2(ERK1/2) activity completely blocked the inhibitory effects of insulin and IGF-I on the sLH-stimulated production of T and because insulin and IGF-I increased the total protein content of ERK1/2 in trout theca layers. Therefore, our results suggest that insulin and IGF-I, probably through the MAPK pathway, block the action of sLH in trout theca layers by modulating the cAMP/PKA signaling pathway.

Effects of follicle stimulating hormone on estradiol-17 beta production and P-450 aromatase (CYP19) activity and mRNA expression in brown trout vitellogenic ovarian follicles in vitro.

In order to determine whether follicle stimulating hormone (FSH) regulates P-450 aromatase (P-450 arom) in salmonid fish, we investigated the in vitro effects of FSH on estradiol (E(2)) production and P-450 arom activity and expression in brown trout (Salmo trutta) vitellogenic ovarian follicles. Brown trout ovarian follicles were incubated in the presence of coho salmon FSH and the production of E(2) into the medium was measured by RIA, the activity of P-450 arom by the tritiated water release assay and the expression of P-450 arom by Northern blotting using a homologous cDNA probe obtained by RT-PCR. Results from this study indicate that the dose- and time-dependent stimulatory effects of FSH on E(2) production are dependent on new RNA and protein synthesis. The basal and FSH-stimulated E(2) production was completely blocked by fadrozole, a specific aromatase inhibitor. FSH was capable of stimulating P-450 arom activity but this stimulation was only detectable with short incubation times (30 min) since longer incubation times with FSH resulted in the inhibition of P-450 arom activity. In addition, FSH increased the steady-state P-450 arom mRNA levels. In conclusion, our results indicate, for the first time in teleost fish, that FSH stimulates the expression of P-450 arom, as well as its activity, albeit after a short-term treatment with FSH, and that FSH plays a fundamental role in the regulation of the production of E(2) in the salmonid ovary.

Activation of the cAMP-dependent protein kinase signaling pathway by luteinizing hormone in trout theca layers.

In the fish ovary, LH is the main factor regulating the production of steroids during the periovulatory period and its effects are believed to be mediated, at least partially, through the cAMP-dependent protein kinase (PKA) signaling pathway. However, there is no direct evidence for the presence of PKA in the fish ovary nor on the regulation of its activity by fish LH. Here, we show the identification of regulatory (R) and catalytic (C) subunits of PKA in trout theca cells by immunoblotting. DEAE-cellulose chromatography of theca cell extracts indicated the presence of PKA type I and II and showed that trout theca cells display PKA-specific phosphotransferase and cAMP-binding activities. Salmon LH (sLH) stimulated PKA activity and increased the levels of immunoreactive RIIalpha, RIIbeta and C subunits in trout theca layers. These observations, coupled with the sLH-dependent decrease in the half-life of the C subunit, as shown by pulse-chase experiments, strongly suggest that sLH activates PKA in trout theca cells. Furthermore, our results suggest that ovarian PKA activity and its regulation by LH has been well conserved from fish to humans.