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Many semiarid streams are threatened both by human demands and climatic effects (global warming), but little is known about their algal biodiversity and their conservation value, especially in Mediterranean areas. The aim of the present study was to evaluate annual changes in the structure and species richness of diatom communities in runs and pools of an oligohaline, semiarid stream in south-eastern Spain, and their relationship with nutrients and hydrology. The pool substrate was covered most of the time by a yellow-brown coat, mainly composed of diatoms and a few cyanophytes (Leptolyngbya). In the runs, cyanophytes were dominant (Rivularia) and diatoms occurred as epiphytes. The diversity of these diatom assemblages was high, particularly in the pools. Some species were present throughout the year, whereas others showed a bimodal growth pattern. Maximum biomasses were reached in summer in the pools and in autumn in the runs, when levels of phosphorus increased as a result of flooding. Species richness and diversity were correlated with hydrology and sulfate concentrations, whereas biomass (as chlorophyll a and ash-free dry weight) was associated with variations in temperature, conductivity and ammonium. These semiarid streams are refuges of biodiversity, representing habitats reliant on cycles of drought and floods, which should be considered in proposals for their conservation., Ministerio de Educación. Gobierno de España., Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

Mammalian digit tip regeneration is linked to the presence of nail tissue, but a nail-explicit model is missing. Here, we report that nail-less double-ventral digits of dLARM1/2 mutants that lack limb-specific Lmx1b enhancers fail to regenerate. To separate the nail's effect from the lack of dorsoventral (DV) polarity, we also interrogate double-dorsal double-nail digits and show that they regenerate. Thus, DV polarity is not a prerequisite for regeneration, and the nail requirement is supported. Transcriptomic comparison between wild-type and non-regenerative dLARM1/2 mutant blastemas reveals differential upregulation of vascularization and connective tissue functional signatures in wild type versus upregulation of inflammation in the mutant. These results, together with the finding of Lmx1b expression in the postnatal dorsal dermis underneath the nail and uniformly in the regenerative blastema, open the possibility of additional Lmx1b roles in digit tip regeneration, in addition to the indirect effect of mediating the formation of the nail.

The developing limb is one of the main model systems to study biological pattern formation and evolutionary adaptation. Of particular importance is the establishment of dorso-ventral patterning (DV) as it gives rise to crucial DV asymmetries in the skeletal elements, nerves and vessels that enable the appendages to serve crucial functions such as stable locomotion on land and detailed manipulation.

n our role as medical educators and researchers, we support in the strongest manner possible the personal autonomy and self-determination of our students, patients, and colleagues. A fundamental value of the medical profession is for the patient to have personal autonomy in their healthcare decisions, including how they would like to be identified. It is, and will continue to be, an important priority to be taught and encouraged throughout healthcare education,1 and it begins in the anatomy laboratory with the proper and respectful treatment of body donors.2 Learning this value continues with respecting and accepting this same autonomy in fellow students, staff, faculty, and patients.

Complex signalling between the apical ectodermal ridge (AER - a thickening of the distal epithelium) and the mesoderm controls limb patterning along the proximo-distal axis (humerus to digits). However, the essential in vivo requirement for AER-Fgf signalling makes it difficult to understand the exact roles that it fulfils. To overcome this barrier, we developed an amenable ex vivo chick wing tissue explant system that faithfully replicates in vivo parameters. Using inhibition experiments and RNA-sequencing, we identify a transient role for Fgfs in triggering the distal patterning phase. Fgfs are then dispensable for the maintenance of an intrinsic mesodermal transcriptome, which controls proliferation/differentiation timing and the duration of patterning. We also uncover additional roles for Fgf signalling in maintaining AER-related gene expression and in suppressing myogenesis. We describe a simple logic for limb patterning duration, which is potentially applicable to other systems, including the main body axis.

The dorso-ventral (DV) organization of limb morphology is crucial for its function but little is known about how it is implemented during morphogenesis. Lmx1b is the key regulator of DV patterning as it is both necessary and sufficient to specify dorsal fates, but its regulation remains poorly known. Recently, we reported that Lmx1b is subject to a complex and autoregulatory mode of transcriptional regulation mediated by two limb-specific enhancers termed LARM1 and LARM2. Interestingly, these enhancers display anterior-posterior (AP) spatial modularity with LARM1 controlling Lmx1b expression in the anterior and LARM2 in the posterior half of the limb. To investigate the regulatory logic of the Lmx1b locus in higher detail, we have dissected the LARM1 enhancer and explored the functional contribution of two Lmx1b associated lncRNAs. The results confirm the presence of a functional repressor and underscore the AP positional specificity of the LARM sequences. Conjointly, the data suggests that Lmx1b transcription uses different regulatory elements in different positions probably reflecting the integration of the local transcriptional environment. Finally, since the double-ventral limbs of dLARM1/2 mutants fail to support locomotion, we hypothesized that the elaboration of the DV axis, possible driven by changes in the regulatory genome, had to accompany the fin transition towards appendages capable of lifting the body weight. In this direction, we have identified orthologs of the LARM sequences from chondrichthyans. All these sequences show activity in the pectoral fin in zebrafish reporter assays, revealing the deep roots of Lmx1b regulation.

[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 regeneration of the digit tip is an exceptional model of multi-tissue regeneration in mammals, with remarkable potential to advance regenerative medicine. Digit tip regeneration positively correlates with the presence of the nail organ, most likely because of the environment of the nail stem cells, but the underlying mechanisms are still unclear. The fact that the nail is a dorsal structure links regeneration with dorso-ventral (DV) polarity. Here, to evaluate the nail function more directly, and to disentangle it from DV positional information, we tested the regenerative ability of double-ventral digits and double-dorsal digits resulting from the limb-specific loss of Lmx1b and of En1 expression, respectively. Our results indicate that DV polarity is not required for digit tip regeneration but that an excessive inflammatory response prevents regeneration in the absence of Lmx1b. The possible causes of the exacerbated inflammation in the absence of Lmx1b are discussed.

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.

Mammalian limb regeneration is restricted to the distal third of terminal phalanges. Amputation of distal digit tips results in blastema formation and restoration of the missing part. This regenerative response has been linked to the presence of the nail, but digits without nails have not been evaluated. Here we interrogate the regenerative ability of the double-ventral digits of dLARM1/2 mutants, which lack dorsal nails due to the limb-specific absence of Lmx1b. We report that these nail-less digits fail to regenerate, supporting the nail requirement. Because the absence of dorso-ventral (DV) polarity could also impair regeneration, as reported in amphibians, we tested the double-dorsal double-nail digits of Del(27) mutants, which lack DV polarity due to the limb-restricted loss of En1. Since Del(27) digits did regenerate, we concluded that DV polarity is not required for digit tip regeneration. We documented continued dorsal expression of Lmx1b postnatally in the wild-type (WT) nail dermis. Moreover, there is uniform Lmx1b expression in WT regeneration-competent blastemas. Transcriptomic comparison between WT and dLARM1/2 mutant blastemas revealed differential up-regulation of gene signatures functionally associated with vascularization and connective tissue remodeling in WT mice. In contrast, the dLARM1/2 non-regenerative blastemas that lack Lmx1b expression show differential up-regulation of genes associated with inflammation and inflammatory processes. Conjointly, our data implicate a direct role for Lmx1b in digit tip regeneration by maintaining the nail dermis and promoting a competent blastema.

Regeneration in mammals is limited to the digit tips. The amputation of the distal tip of the terminal phalanx triggers the formation of an undifferentiated pool of progenitor cells called a blastema that will regenerate the multiple tissues required to restore the missing part. Digit tip regeneration is linked to the presence of the dorsal nail organ, as only amputations that conserve the nail matrix regenerate. However, the regenerative potential of digits without nails has not yet been tested. To evaluate nailless digits, we used dLARM1/2 mutants that develop digits with no nails due to the limb-specific loss of the dorsal limb determinant Lmx1b. We report that dLARM1/2 mutant digits are unable to regenerate, although they form a blastema. To test whether the lack of regeneration was due to the absence of dorso-ventral (DV) polarity, a concept suggested in amphibia, we evaluated Del(27) mutants that also lack DV polarity with nails on both dorsal and ventral aspects of distal phalanges due to ventral overexpression of Lmx1b. Unlike dLARM1/2 mutants, Del(27) mutant digits regenerated indicating that DV polarity is dispensable for regeneration. Interestingly, Lmx1b expression (which continues in the nail dermis postnatally) is detected uniformly in the blastema of WT, but not dLARM1/2 mutant mice. In addition, comparison of dLARM1/2 vs WT blastemal transcriptomes revealed that the mutants had defective pro regenerative processes such as vasculogenesis and extracellular matrix remodeling, while inflammatory pathways were exacerbated. Gene expressions typical of the nail dermis were also lost in dLARM1/2 digit tips, suggesting regulation by Lmx1b. Collectively, our data suggest a direct role for Lmx1b in digit tip regeneration maintaining the nail dermis and promoting a competent blastema.

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

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.

[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.

[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] 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.

[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.

Lmx1b is the key regulator of dorso-ventral (DV) pattern in the limb, as it is both necessary and sufficient to specify dorsal fates. Accordingly, targeted disruption of Lmx1b results in double ventral limbs. In humans, mutations in the LMX1B gene are responsible for the autosomal dominant Nail-Patella Syndrome (OMIM #161200), characterized by incomplete limb dorsalization. Here, we report on two enhancers located upstream of the gene, LARM1 and LARM2, that regulate Lmx1b specifically in the limb. These sequences contain several canonical Lmx1b binding sites that mediate a positive loop of Lmx1b autoregulation. Deletion of these enhancers together results in mice with double ventral limbs, like these of Lmx1b-nul mice. Remarkably, deletion of each individual enhancer produced a double ventral phenotype clearly restricted to the posterior half of the limb in LARM1 mutants and to the anterior in LARM2 mutants, indicating differential activity of these enhancers along the anterior-posterior axis. These results reflect an unexpected complex, modular and autoregulatory mode of Lmx1b transcriptional regulation during limb development. We also report on several NPS patient families with normal LMX1B coding sequence but variations in the LARM region that exhibit limb restricted phenotypes, stressing the role of regulatory modules in disease pathogenesis.

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.

During the last few decades, the genomes from hundreds of different living organisms have been fully sequenced. Decoding this vast amount of genetic information promises to unlock the molecular secrets of life in our planet as well as the molecular basis of human disease. However, this is far from trivial since in many species the protein-coding sequences (i.e. genes) only account for a small fraction of their genome, with the remaining non-coding sequences potentially playing regulatory functions. Moreover, within multicellular organisms, the genome is uniquely used in each cell type by introducing epigenetic modifications and/or adopting particular 3D conformations that can affect gene activity and expression without changing the underlying DNA sequences. Despite this complexity, recent advances in various -omics and genome editing technologies have dramatically improved our current understanding of genome function. Therefore, we are now in a unique position to sequence, analyze and modify genomes, and, thus, to improve not only our quality of life but also that of our planet.

[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.

[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.

[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.

[ES] El proyecto trata el desarrollo de un nuevo modelo habitacional entendido como un organismo con vida propia que apuesta por la autosuficiencia y proporciona al habitante el poder de la acción individual. Se trata de una comunidad transitoria localizada en el periurbano este del barrio de Benimaclet, en Valencia, cuyo eje motor es la recuperación de la huerta, ahondando en sus valores productivo, paisajístico y natural. Las viviendas, reciclables y sostenibles, acogerán a personas en riesgo de exclusión social por lo que la comunidad será un punto de confluencia de diferentes culturas e ideologías. Además, se dará cabida a usos auxiliares vinculados a la acción social, educación y relaciones interpersonales., [EN] The project deals with the development of a new housing model understood as a living organism base on the self-sufficiency and the power of individual action. It is a transitory community located in the eastern peri-urban neighborhood of Benimaclet, in Valencia, whose driving force is the recovery of the vegetable garden, delving into its productive, landscape and natural values. The homes, recyclable and sustainable, will welcome people at risk of social exclusion so the community will be a point of confluence of different cultures and ideologies. In addition, the community will develop auxiliary uses about social action, education and interpersonal relationships.

[ES] El proyecto trata el desarrollo de un nuevo modelo habitacional entendido como un organismo con vida propia que apuesta por la autosuficiencia y proporciona al habitante el poder de la acción individual. Se trata de una comunidad transitoria localizada en el periurbano este del barrio de Benimaclet, en Valencia, cuyo eje motor es la recuperación de la huerta, ahondando en sus valores productivo, paisajístico y natural. Las viviendas, reciclables y sostenibles, acogerán a personas en riesgo de exclusión social por lo que la comunidad será un punto de confluencia de diferentes culturas e ideologías. Además, se dará cabida a usos auxiliares vinculados a la acción social, educación y relaciones interpersonales., [EN] The project deals with the development of a new housing model understood as a living organism base on the self-sufficiency and the power of individual action. It is a transitory community located in the eastern peri-urban neighborhood of Benimaclet, in Valencia, whose driving force is the recovery of the vegetable garden, delving into its productive, landscape and natural values. The homes, recyclable and sustainable, will welcome people at risk of social exclusion so the community will be a point of confluence of different cultures and ideologies. In addition, the community will develop auxiliary uses about social action, education and interpersonal relationships.

[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.

[ES] El proyecto trata el desarrollo de un nuevo modelo habitacional entendido como un organismo con vida propia que apuesta por la autosuficiencia y proporciona al habitante el poder de la acción individual. Se trata de una comunidad transitoria localizada en el periurbano este del barrio de Benimaclet, en Valencia, cuyo eje motor es la recuperación de la huerta, ahondando en sus valores productivo, paisajístico y natural. Las viviendas, reciclables y sostenibles, acogerán a personas en riesgo de exclusión social por lo que la comunidad será un punto de confluencia de diferentes culturas e ideologías. Además, se dará cabida a usos auxiliares vinculados a la acción social, educación y relaciones interpersonales., [EN] The project deals with the development of a new housing model understood as a living organism base on the self-sufficiency and the power of individual action. It is a transitory community located in the eastern peri-urban neighborhood of Benimaclet, in Valencia, whose driving force is the recovery of the vegetable garden, delving into its productive, landscape and natural values. The homes, recyclable and sustainable, will welcome people at risk of social exclusion so the community will be a point of confluence of different cultures and ideologies. In addition, the community will develop auxiliary uses about social action, education and interpersonal relationships.

[ES] El proyecto trata el desarrollo de un nuevo modelo habitacional entendido como un organismo con vida propia que apuesta por la autosuficiencia y proporciona al habitante el poder de la acción individual. Se trata de una comunidad transitoria localizada en el periurbano este del barrio de Benimaclet, en Valencia, cuyo eje motor es la recuperación de la huerta, ahondando en sus valores productivo, paisajístico y natural. Las viviendas, reciclables y sostenibles, acogerán a personas en riesgo de exclusión social por lo que la comunidad será un punto de confluencia de diferentes culturas e ideologías. Además, se dará cabida a usos auxiliares vinculados a la acción social, educación y relaciones interpersonales., [EN] The project deals with the development of a new housing model understood as a living organism base on the self-sufficiency and the power of individual action. It is a transitory community located in the eastern peri-urban neighborhood of Benimaclet, in Valencia, whose driving force is the recovery of the vegetable garden, delving into its productive, landscape and natural values. The homes, recyclable and sustainable, will welcome people at risk of social exclusion so the community will be a point of confluence of different cultures and ideologies. In addition, the community will develop auxiliary uses about social action, education and interpersonal relationships.

In recent years, the development of CRISPR technologies has provided an excellent tool for genomic editing. We have taken advantage of this technology to set up a CRISPR service at our Institute that based on the electroporation of mouse zygotes (day 0.5) generates desired mouse genomic modifications in a highly efficient, rapid and unexpensive manner. We have applied this methodology to the generation of several mouse models of human congenital hand malformations, especially focussing on the Split Hand Foot Malformation (SHFM), a rare and highly variable malformation characterized by the loss or deformity of the central digit rays. We have previously shown the involvement of Sp6 and Sp8 transcription factors in the generation of SHFM phenotypes in mouse and the interaction of these two factors with Dlx family members. Dlx genes are the genes involved in human SHFM types 1 (Dlx5/Dlx6) and 5 (Dlx1/Dlx2). With the aim of fully investigating the implication of the Dlx-Sp interactions in the pathogenesis of SHFM, we have generated a couple of mouse models: the Dlx5/Dlx6 double deletion and the Sp6- V5 tagged knock-in. Additionally, and based on studies in Drosophila indicating that Sp factors control appendage development through the Notch pathway, we have also generated a Jag2 KO mutant. We are currently using the generated animal models to advance our understanding of the mechanisms subjacent to the SHFM.

Lmx1b is a LIM homeodomain transcription factor that plays essential roles in a wide range of developmental processes including the development of the kidney, the eye, the dopaminergic and serotonergic neurons and the limb. In the limb, Lmx1b expression is restricted to the dorsal mesenchyme and is responsible for limb dorsalization. Mice lacking functional Lmx1b die at birth because of multisystemic malformations. Homozygous Lmx1b mutants develop a double-ventral limb phenotype with loss of dorsal structures such as the patella, whereas the ventral ones such as the sesamoid bones are duplicated and dorsal tendons and muscles are transformed into mirror images of ventral ones. At superficial level, hair and nails are absent while typical ventral features such as pads are also duplicated. Herein, we report on two conserved Lmx1bassociated cis-regulatory modules (LARM1 and LARM2) located upstream of Lmx1b that are bound by Lmx1b and mediate an autoregulatory loop required to provide adequate levels of Lmx1b expression necessary for limb dorsalization. Combined removal of both enhancer lead to a limb restricted phenotype identical to that of the Lmx1b Knockout mice with no other Lmx1b-related systemic defects, indicating that they are limb specific enhancers. Unexpectedly, the CRISPR/Cas9 deletion of each individual enhancer produced a double ventral phenotype clearly restricted to the posterior or anterior half of the autopod in LARM1 and LARM2 respectively, unveiling spatial modularity in the transcriptional control of Lmx1b in the limb. In humans, LMX1B haploinsufficiency causes Nail-patella syndrome (NPS; MIM 161200), characterized by nail dysplasia, absent/hypoplastic patellae, chronic kidney disease, and glaucoma. Of most interest, we report on two NPS patient families with normal LMX1B coding sequence and limb-restricted phenotype, but with loss-of-function variations in the LARM1/2 region, stressing the role of regulatory modules in disease pathogenesis.

Epimorphic regeneration is a type of multi-tissue regeneration defined by the formation of a blastema. In contrast to amphibians, which can regenerate their entire limbs, mammals can only regenerate the distal tip of their digits, hence investigating the mechanisms involved is of maximum interest for regenerative medicine. Interestingly, in mice and humans this regeneration associates with the nail organ, particularly with the Wnt/ß-catenin active nail matrix. Nails are ectodermal appendages of the dorsal tip of the digits. Their development reflects the dorso-ventral polarity of the limb, established in the early limb bud ectoderm by the interaction of three central molecules. En1, expressed in the ventral ectoderm, restricts Wnt7a to the dorsal ectoderm. Wnt7a induces Lmx1b, the dorsal determinant, in the subjacent mesoderm. Lmx1b-null mice display bi-ventral distal limbs and die perinatally due to multisystemic defects. Recent studies have identified two Lmx1b limb-specific enhancers named LARM1 and LARM2. CRISPR/Cas9-mediated deletion of these two enhancers (LARM1/2-/-) yielded mice with a limb-restricted Lmx1b-null phenotype, but no other systemic defects. LARM1/2 null mutants show absence of nails in their digit tips, providing an opportunity to directly test the involvement of the nail in digit tip regeneration. As expected, LARM1/2 mutants fail to regenerate their digit tips, as histological and ¿CT analyses demonstrate. Importantly, disregarding the lack of regeneration, a blastema does form at the tip of the LARM1/2 stump. Our preliminary results point to a reduction of proliferation in the LARM1/2 blastema compared to wild-type, and to an absence of Wnt/ß-catenin active epidermis in mutants, as an explanation of the regenerative failure. Our results confirm that bi-ventral digits do not regenerate their digit tips and set the LARM1/2 mutant as a useful model to investigate the mechanisms of regenerative blastema, with the aim of enhancing regeneration.