Search

[Background]: The development of the amniote limb has been an important model system to study patterning mechanisms and morphogenesis. For proper growth and patterning, it requires the interaction between the distal sub-apical mesenchyme and the apical ectodermal ridge (AER) that involve the separate implementation of coordinated and tissue-specific genetic programs., [Results]: Here, we produce and analyze the transcriptomes of both distal limb mesenchymal progenitors and the overlying ectodermal cells, following time-coursed dissections that cover from limb bud initiation to fully patterned limbs. The comparison of transcriptomes within each layer as well as between layers over time, allowed the identification of specific transcriptional signatures for each of the developmental stages. Special attention was given to the identification of genes whose transcription dynamics suggest a previously unnoticed role in the context of limb development and also to signaling pathways enriched between layers., [Conclusion]: We interpret the transcriptomic data in light of the known development pattern and we conclude that a major transcriptional transition occurs in distal limb buds between E9.5 and E10.5, coincident with the switch from an early phase continuation of the signature of trunk progenitors, related to the initial proximo distal specification, to a late intrinsic phase of development.

The dorso-ventral (DV) limb asymmetry is crucial for its function; however, little is known about how it is accomplished during morphogenesis. Lmx1b is the key regulator of DV patterning during limb development as it is both necessary and sufficient to specify dorsal fates, but its regulation remains poorly known. We recently demonstrated a complex autoregulatory mode of transcriptional Lmx1b regulation mediated by two limb-specific enhancers, LARM1 and LARM2. The deletion of both enhancers (dLARM1/2) reproduces the Lmx1b-null phenotype in the limb, while the removal of each enhancer produced a double ventral phenotype restricted to the posterior (dLARM1) or anterior (dLARM2) half of the limb. To investigate the regulatory logic of the Lmx1b locus in detail, we dissected the LARM1 element into its enhancer and silencer sequences and explored the functional contribution of the two Lmx1b-associated lncRNAs. The results confirm the presence of a functional repressor contained in LARM1 and underscore the positional specificity of the LARM sequences. The data suggests that Lmx1b transcription uses different regulatory elements in different positions probably reflecting the integration of the local transcriptional environment. Indeed, published ChIP-seq data showed the Gli3 and Hox13 are significantly enriched in the LARM sequences exposing their interaction with major limb regulators. Finally, considering that the double-ventral limbs of dLARM1/2 mutants fail to support locomotion, even to lift the body weight, we hypothesized that the elaboration of the DV asymmetry by modification of Lmx1b regulation, or its functional targets, had to accompany the fin to limb transition. We have identified orthologs of the LARMs in fishes and all of them show activity in the pectoral fin in zebrafish reporter essays, revealing the deep roots of Lmx1b regulation.

[Background]: The phalanges are the final skeletal elements to form in the vertebrate limb and their identity is regulated by signaling at the phalanx forming region (PFR) located at the tip of the developing digit ray. Here, we seek to explore the relationship between PFR activity and phalanx morphogenesis, which define the most distal limb skeletal elements, and signals associated with termination of limb outgrowth. [Results]: As Grem1 is extinguished in the distal chick limb mesoderm, the chondrogenesis marker Aggrecan is up-regulated in the metatarsals and phalanges. Fate mapping confirms that subridge mesoderm cells contribute to the metatarsal and phalanges when subridge Grem1 is down-regulated. Grem1 overexpression specifically blocks chick phalanx development by inhibiting PFR activity. PFR activity and digit development are also disrupted following overexpression of a Gli3 repressor, which results in Grem1 expression in the distal limb and downregulation of Bmpr1b. [Conclusions]: Based on expression and fate mapping studies, we propose that downregulation of Grem1 in the distal limb marks the transition from metatarsal to phalanx development. This suggests that downregulation of Grem1 in the distal limb mesoderm is necessary for phalanx development. Grem1 downregulation allows for full PFR activity and phalanx progenitor cell commitment to digit fate.

The aim of the present study was to assess the presence of perfluoroalkyl acids (PFAAs), namely perfluoroalkane sulfonates and perfluoroalkyl carboxylic acids, in Spanish river basins in order to: identify potential spatiotemporal variations; evaluate the effectiveness of the measures implemented for the reduction/elimination of these pollutants; verify the fulfillment of the Environmental Quality Standards (EQSs) in the European Union. PFOS and PFOA were determined in 116 water samples from four sites in the Duero basin, the largest in the Iberian Peninsula, collected seasonally from 2013 to 2020. In addition, 30 fish sample composites from the sample banks of Duero, Tagus, Ebro, Eastern Cantabrian and Catalonian basins were analyzed for 15 PFAAs. Median PFOS and PFOA concentrations were 0.72 and 0.42 ng/L, ranging from values below the limit of quantification (LOQ) to 81 and 22 ng/L, respectively. During the studied period, 51% of water samples were above the EQS of 0.65 ng/L for PFOS. In the case of fish, the PFOS range was

Targeting early-stage lung cancer is vital to improve survival. However, the mechanisms and components of the early tumor suppressor response in lung cancer are not well understood. In this report, we study the role of Toll-like receptor 2 (TLR2), a regulator of oncogene-induced senescence, which is a key tumor suppressor response in premalignancy. Using human lung cancer samples and genetically engineered mouse models, we show that TLR2 is active early in lung tumorigenesis, where it correlates with improved survival and clinical regression. Mechanistically, TLR2 impairs early lung cancer progression via activation of cell intrinsic cell cycle arrest pathways and the proinflammatory senescence-associated secretory phenotype (SASP). The SASP regulates non-cell autonomous anti-tumor responses, such as immune surveillance of premalignant cells, and we observe impaired myeloid cell recruitment to lung tumors after Tlr2 loss. Last, we show that administration of a TLR2 agonist reduces lung tumor growth, highlighting TLR2 as a possible therapeutic target.

[ES] El clordiazepóxido es un fármaco que pertenece al grupo de las benzodiazepinas y se utiliza para tratar problemas asociados a la ansiedad. En relación a la biotransformación de fármacos, esta se asocia a una mayor facilidad para ser eliminados del organismo y generalmente los convierte en menos tóxicos. Sin embargo, para algunos fármacos existen evidencias de la generación de metabolitos reactivos durante este proceso de biotransformación, llegando en algunos casos a ser los metabolitos más tóxicos que el propio fármaco original. En el presente trabajo se ha extraído el clordiazepóxido de comprimidos del ansiolítico comercial Huberplex. Se han estudiado sus propiedades fotofísicas, fotoquímicas y fotobiológicas, con el fin de obtener información acerca de una posible fotosensibilización debida a este fármaco. Además, se ha realizado una caracterización fotofísica y fotoquímica de uno de sus metabolitos principales, el demoxepam. En este contexto, se observaron diferencias en los espectros de absorción del fármaco y del metabolito. El fármaco absorbe hasta longitudes de onda mayores en el UVA. Por otra parte, la fluorescencia de los dos compuestos resultó ser prácticamente nula. Respecto a la fotoreactividad en disolución (en PBS, cloroformo y acetonitrilo) se observó la degradación del compuesto, dando lugar a fotoproducto(s) cuya estructura química está por determinar. La evolución de las irradiaciones se monitorizó por espectroscopia de absorción, fluorescencia y RMN-1H. Respecto a los ensayos en medio celular, se realizó un ensayo de viabilidad celular de captación del rojo neutro (NRU) para evaluar la posible fototoxicidad del fármaco. Así, el factor de fotoirritación calculado indicó el alto potencial fototóxico de este fármaco. Teniendo en cuenta estos resultados, se procedió a investigar si CDZ y DEM pueden dar lugar a fotoalergia, usando como sistemas modelo albúmina sérica humana y CDZ o DEM. En primer lugar, se estudió la posible formación de complejo, [EN] Chlordiazepoxide is a drug that belongs to benzodiazepines family. It is used to treat problems associated with anxiety. The biotransformation of drugs is associated with a better clearance from the body and generally it makes them less toxic. However, for some drugs there is evidence of the generation of reactive metabolites during the biotransformation process; thus, in some cases, the metabolites are more toxic than the parent drug. In the present work, chlordiazepoxide has been extracted from commercially available tablets of anxiolytic Huberplex. Its photophysical, photochemical and photobiological properties have been studied, in order to obtain information about a possible photosensitization due to this drug. In addition, a photophysical and photochemical characterization of one of its main metabolites, demoxepam, has been carried out. In this context, differences were observed in the absorption spectra of the drug and the metabolite. The absorption spectrum of the drug reaches longer wavelengths in the UVA region. On the other hand, the fluorescence of the two compounds is negligible. Concerning the photoreactivity in solution (in PBS, chloroform and acetonitrile), degradation of the compound was observed, giving rise to photoproduct(s) whose chemical structure remains still undetermined. The evolution of the irradiations was monitored by absorption spectroscopy, fluorescence and 1H-NMR. Regarding the assays in cellular medium, the neutral red uptake cell viability test (NRU) was carried out to evaluate the possible phototoxicity of the drug. Thus, the calculated photoirritation factor indicated a high phototoxic potential for this drug. Taking into account these results, we proceeded to investigate whether CDZ and DEM can lead to photoallergy, using CDZ or DEM and human serum albumin as model protein. First, the possible formation of intermolecular complexes between the protein and the substrates was studied using UV-Vis absorption spectroscopy and flu, [CA] El clordiazepòxid és un fàrmac que pertany al grup de les benzodiazepines i s’utilitza per a tractar problemes associats a l’ansietat. En relació amb la biotransformació de fàrmacs, aquesta s’associa a una major facilitat per a ser eliminats de l'organisme i, en general, els converteix en menys tòxics. No obstant això, per a alguns fàrmacs existeixen evidències de la generació de metabòlits reactius durant el procés de biotransformació, arribant en algunes ocasions a ser els metabòlits més tòxics i reactius que el mateix fàrmac original. En el present treball s’ha extret el clordiazepòxid de comprimits de l’ansiolític comercial Huberplex. S’han estudiat les seues propietats fotofísiques, fotoquímiques i fotobiològiques, amb la finalitat d’obtenir informació sobre una possible fotosensibilització deguda a aquest fàrmac. A més a més, s’ha realitzat una caracterització fotofísica i fotoquímica d’un dels seus metabòlits principals, el demoxepam. En aquest context, s’observaren diferències en els espectres d’absorció del fàrmac i del metabòlit. El fàrmac absorbeix fins a longituds d’ona majors en l’UVA. Per una altra banda, la fluorescència d’ambdós compostos va resultar ser pràcticament nul·la. Pel que respecta a la fotoreactivitat en dissolucions (en PBS, cloroform i acetonitril) s’observà la degradació del compost, donant lloc a fotoproducte(s) l’estructura química del(s) qual(s) es troba per determinar. L’evolució de les irradiacions es monitoren per espectroscòpia d’absorció, fluorescència i 1H-RMN. Fent referència als assajos en medi cel·lular, es va realitzar un assaig de viabilitat cel·lular de captació del roig neutre (NRU) per tal d’avaluar la possible fototoxicitat del fàrmac. Així, el factor de fotoirritació calculat va indicar l’elevat potencial fototòxic d’aquest fàrmac. Tenint en compte aquests resultats, es prosseguí a investigar si el CDZ i el DEM podien donar lloc a fotoal·lèrgia, utilitzant com a sistemes model albúmina sèrica humana i CDZ o DEM.

[ES] La sulfasalazina (SSZ) es un fármaco empleado para el tratamiento de la artritis reumatoide, la colitis ulcerosa y la enfermedad de Crohn. Entre otras reacciones adversas asociadas a este fármaco se han descrito los trastornos de fotosensibilidad (fototoxicidad y fotoalergia). Por tanto, es relevante investigar la fotoquímica y la fotofísica de la SSZ en sistemas supramoleculares tales como micelas de ácidos grasos, la albúmina sérica humana y la alfa glicoproteína ácida. Estos estudios se llevarán a cabo combinando técnicas fotofísicas (fluorescencia y fotólisis de destello láser) con técnicas propias de un laboratorio de química orgánica para analizar, separar, y caracterizar los fotoproductos que se puedan generar tras irradiación del fármaco en estos medios., [EN] Sulfasalazine (SSZ) is a drug used for the treatment of rheumatoid arthritis, ulcerative colitis and Crohn's disease. Among other adverse reactions associated with this drug, photosensitivity disorders (phototoxicity and photoallergy) have been described. It is therefore relevant to investigate the photochemistry and photophysics of SSZ in supramolecular systems such as fatty acid micelles, human serum albumin and acid alpha-glycoprotein. These studies will be carried out by combining photophysical techniques (fluorescence and laser flash photolysis) with organic chemistry laboratory techniques to analyse, separate, and characterise the photoproducts that may be generated after irradiation of the drug in these media.

EXECUTIVE SUMMARY In the last two decades, we have witnessed major technological advances that have announced the advent of the genomic era. Great international efforts have provided us with the genomic information not only of many different species, but even now from different individuals within the same species. The development of the -omics techniques, genomic, transcriptomic, epigenomic, proteomic, metabolomic, etc., also provide us with multiple layers of information to analyse. And finally, a major breakthrough has been made when the possibility to alter virtually any genome of any species at will has been made possible with the identification and popularisation of genomic editing tools. Thus, currently we are in the verge of being able, for the first time, to really understand genomic information and manipulate it for basic research or specific applications. In this chapter, we will revise the current state-of-the-art of the available methods to analyse and modify the genome. Moreover, we will identify the major challenges that this field is facing and should be tackled in the next years. Among them, we will highlight the need to implement and improve tools to extract and read the information from -omic data as well as to modify the genome. Ultimately, we analyse CSIC strengths and weakness for research in this area. CSIC is in a unique position to contribute to it, thanks to its broad expertise in many different sciences, from physics to biology, from informatics to chemistry, and in a wide variety of experimental models. Such multidisciplinary setup will benefit the development of these tools. Also, we recognize the existence of specific institutes and laboratories within CSIC that are experts and world leaders in specific topics required for understanding and further improve these technologies.

LMX1B haploinsufficiency causes Nail-patella syndrome (NPS; MIM 161200), characterized by nail dysplasia, absent/hypoplastic patellae, chronic kidney disease, and glaucoma. Accordingly in mice, Lmx1b has been shown to play crucial roles in the development of the limb, kidney and eye. Although one functional allele of Lmx1b appears adequate for development, Lmx1b null mice display ventral-ventral distal limbs with abnormal kidney, eye and cerebellar development, more disruptive, but fully concordant with NPS. In Lmx1b functional knockouts (KOs), Lmx1b transcription in the limb is decreased nearly 6-fold, indicating autoregulation. Herein, we report on two conserved Lmx1b-associated cis-regulatory modules (LARM1 and LARM2) that are bound by Lmx1b, amplify Lmx1b expression with unique spatial modularity in the limb, and are necessary for Lmx1b-mediated limb dorsalization. These enhancers, being conserved across vertebrates (including coelacanth, but not other fish species), and required for normal locomotion, provide a unique opportunity to study the role of dorsalization in the fin to limb transition. We also report on two NPS patient families with normal LMX1B coding sequence, but with loss-of-function variations in the LARM1/2 region, stressing the role of regulatory modules in disease pathogenesis.

[EN] BACKGROUND: Quinolones are the second most frequent cause of hypersensitivity reactions (HSRs) to antibiotics. A marked increase in the number of patients with HSRs to quinolones has been detected. OBJECTIVE: To describe the clinical characteristics of patients with HSRs to quinolones and present methods for their diagnosis. METHODS: Patients attending the allergy unit due to reactions suggestive of HSRs to quinolones were prospectively evaluated between 2005 and 2018. Diagnosis was achieved using clinical history, skin tests (STs), basophil activation tests (BATs), and drug provocation tests (DPTs) if ST and BAT results were negative. RESULTS: We included 128 subjects confirmed as having HSRs to quinolones and 42 found to be tolerant. Anaphylaxis was the most frequent entity in immediate HSRs and was most commonly induced by moxifloxacin. Patients were evaluated a median of 150 days (interquartile range, 60-365 days) after the reaction. Of patients who underwent ST and BAT, 40.7% and 70%, respectively, were positive. DPT with a quinolone was performed in 48 cases, giving results depending on the culprit drug: when moxifloxacin was involved, 62.5% of patients gave a positive DPT result to ciprofloxacin, whereas none reacted to levofloxacin. The risk of HSR was 96 times higher in subjects who reported moxifloxacin-induced anaphylaxis and 18 times higher in those reporting immediate reactions compared with clinical entities induced by quinolones other than moxi-floxacin and nonimmediate reactions. CONCLUSIONS: The diagnosis of HSR to quinolones is complex. The use of clinical history is essential as a first step. BAT shows higher sensitivity than STs. DPTs can be useful for finding safe alternative quinolones.

[EN] Lapatinib (LAP) is an anticancer drug generally used to treat breast and lung cancer. It exhibits hypersensitivity reactions in addition to dermatological adverse effects and photosensitivity. Moreover, LAP binds to serum proteins and is readily biotransformed in humans, giving rise to several metabolites, such as N- and O-dealkylated products (N-LAP and O-LAP, respectively). In this context, the aim of the present work is to obtain key information on drug@protein complexation, the first step involved in a number of hypersensitivity reactions, by a combination of fluorescence, femtosecond transient absorption spectroscopy and molecular dynamics (MD) simulations. Following this approach, the behavior of LAP and its metabolites has been investigated in the presence of serum proteins, such as albumins and alpha(1)-acid glycoproteins (SAs and AGs, respectively) from human and bovine origin. Fluorescence results pointed to a higher affinity of LAP and its metabolites to human proteins; the highest one was found for LAP@HSA. This is associated to the coplanar orientation adopted by the furan and quinazoline rings of LAP, which favors emission from long-lived (up to the ns time-scale) locally-excited (LE) states, disfavoring population of intramolecular charge transfer (ICT) states. Moreover, the highly constrained environment provided by subdomain IB of HSA resulted in a frozen conformation of the ligand, contributing to fluorescence enhancement. Computational studies were clearly in line with the experimental observations, providing valuable insight into the nature of the binding sites and the conformational arrangement of the ligands inside the protein cavities. Besides, a good correlation was found between the calculated binding energies for each ligand@protein complex and the relative affinities observed in competition experiments.

This is the peer reviewed version of the following article: I. Vayá, I. Andreu, E. Lence, C. González-Bello, M. Consuelo Cuquerella, M. Navarrete-Miguel, D. Roca-Sanjuán, M. A. Miranda, Chem. Eur. J. 2020, 26, 15922, which has been published in final form at https://doi.org/10.1002/chem.202001336. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving., [EN] Lapatinib (LAP) is an anticancer drug, which is metabolized to theN- and O-dealkylated products (N-LAP andO-LAP, respectively). In view of the photosensitizing potential of related drugs, a complete experimental and theoretical study has been performed on LAP,N-LAP andO-LAP, both in solution and upon complexation with human serum albumin (HSA). In organic solvents, coplanar locally excited (LE) emissive states are generated; they rapidly evolve towards twisted intramolecular charge-transfer (ICT) states. By contrast, within HSA only LE states are detected. Accordingly, femtosecond transient absorption reveals a very fast switching (ca. 2 ps) from LE (lambda(max)=550 nm) to ICT states (lambda(max)=480 nm) in solution, whereas within HSA the LE species become stabilized and live much longer (up to the ns scale). Interestingly, molecular dynamics simulation studies confirm that the coplanar orientation is preferred for LAP (or to a lesser extentN-LAP) within HSA, explaining the experimental results.

[EN] Transient absorption spectroscopy in combination with in silico methods has been employed to study the interactions between human serum albumin (HSA) and the anti-psychotic agent chlorpromazine (CPZ) as well as its two demethylated metabolites (MCPZ and DCPZ). Thus, solutions containing CPZ, MCPZ or DCPZ and HSA (molar ligand:protein ratios between 1:0 and 1:3) were submitted to laser flash photolysis and the Delta A(max) value at lambda = 470 nm, corresponding to the triplet excited state, was monitored. In all cases, the protein-bound ligand exhibited higher Delta Amax values measured after the laser pulse and were also considerably longer-lived than the non-complexed forms. This is in agreement with an enhanced hydrophilicity of the metabolites, due to the replacement of methyl groups with H that led to a lower extent of protein binding. For the three compounds, laser flash photolysis displacement experiments using warfarin or ibuprofen indicated Sudlow site I as the main binding site. Docking and molecular dynamics simulation studies revealed that the binding mode of the two demethylated ligands with HSA would be remarkable different from CPZ, specially for DCPZ, which appears to come from the different ability of their terminal ammonium groups to stablish hydrogen bonding interactions with the negatively charged residues within the protein pocket (Glu153, Glu292) as well as to allocate the methyl groups in an apolar environment. DCPZ would be rotated 180 degrees in relation to CPZ locating the aromatic ring away from the Sudlow site I of HSA. (C) 2019 Elsevier B.V. All rights reserved.

[EN] The human genome is constantly attacked by endogenous and exogenous agents (ultraviolet light, xenobiotics, reactive oxygen species), which can induce chemical transformations leading to DNA lesions. To combat DNA damage, cells have developed several repair mechanisms; however, if the repair is defective, DNA lesions lead to permanent mutations. Single-cell gel electrophoresis (COMET assay) is a sensitive and well-established technique for quantifying DNA damage in individual cells. Nevertheless, this tool lacks relationship with mutagenesis. Therefore, to identify errors that give rise to mutations it would be convenient to test an alternative known procedure, such as next generation sequencing (NGS). Thus, the present work aims to evaluate the photomutagenicity of neuroleptic drug chlorpromazine (CPZ), and its N-demethylated metabolites using COMET assay and to test NGS as an alternative method to assess photomutagenesis. In this context, upon exposure to UVA radiation, COMET assay reveals CPZ-photosensitized DNA damage partially repaired by cells. Conversely with this result, metabolites demethylchlorpromazine (DMCPZ) and didemethylchlorpromazine (DDMCPZ) promote extensive DNA-photodamage, hardly repaired under the same conditions. Parallel assessment of mutagenesis by NGS is consistent with these results with minor discrepancies for DDMCPZ. To our knowledge, this is the first example demonstrating the utility of NGS for evaluating drug-induced photomutagenicity.