Substrate-selective repair and restart of replication forks by DNA translocases

Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases.

dKDM5/LID regulates H3K4me3 dynamics at the transcription-start site (TSS) of actively transcribed developmental genes

H3K4me3 is a histone modification that accumulates at the transcription-start site (TSS) of active genes and is known to be important for transcription activation. The way in which H3K4me3 is regulated at TSS and the actual molecular basis of its contribution to transcription remain largely unanswered. To address these questions, we have analyzed the contribution of dKDM5/LID, the main H3K4me3 demethylase in Drosophila, to the regulation of the pattern of H3K4me3. ChIP-seq results show that, at developmental genes, dKDM5/LID localizes at TSS and regulates H3K4me3. dKDM5/LID target genes are highly transcribed and enriched in active RNApol II and H3K36me3, suggesting a positive contribution to transcription. Expression-profiling show that, though weakly, dKDM5/LID target genes are significantly downregulated upon dKDM5/LID depletion. Furthermore, dKDM5/LID depletion results in decreased RNApol II occupancy, particularly by the promoter-proximal Pol lloser5 form. Our results also show that ASH2, an evolutionarily conserved factor that locates at TSS and is required for H3K4me3, binds and positively regulates dKDM5/LID target genes. However, dKDM5/LID and ASH2 do not bind simultaneously and recognize different chromatin states, enriched in H3K4me3 and not, respectively. These results indicate that, at developmental genes, dKDM5/LID and ASH2 coordinately regulate H3K4me3 at TSS and that this dynamic regulation contributes to transcription.

Important role of ventromedial hypothalamic carnitine palmitoyltransferase-1a in the control of food intake

Carnitine palmitoyltransferase-1 (CPT-1) liver isoform or CPT-1a is implicated in CNS control of food intake. However, the exact brain nucleus site(s) in mediating this action of CPT-1a has not been identified. In this report, we assess the role of CPT-1a in hypothalamic ventromedial nucleus (VMN). We stereotaxically injected an adenoviral vector containing CPT-1a coding sequence into the VMN of rats to induce overexpression and activation of CPT-1a. The VMN-selective activation of CPT-1a induced orexigenic effect, suggesting CPT-1a in the VMN is involved in the central control of feeding. Intracerebroventricular administration of etomoxir, a CPT-1 inhibitor, decreases food intake. Importantly, in the animals with VMN-overexpression of a CPT-1a mutant that antagonizes the CPT-1 inhibition by etomoxir, the anorectic response to etomoxir was attenuated. This suggests that VMN is involved in mediating the anorectic effect of central inhibition of CPT-1a. In contrast, Arc overexpression of the mutant did not alter etomoxir-induced inhibition of food intake, suggesting that Arc CPT-1a does not play significant roles in this anorectic action. Furthermore, in the VMN, CPT-1a appears to act downstream of hypothalamic malonyl-CoA action of feeding. Finally, we show that in the VMN, CPT-1 activity altered in concert with fasting and refeeding states, supporting a physiological role of CPT-1a in mediating the control of feeding. Taking together, CPT-1a in the hypothalamic VMN appears to play an important role in the central control of food intake. VMN-selective modulation of CPT-1a activity may therefore be a promising strategy in controlling food intake and maintaining normal body weight.

LRCW.NET: A web site with a virtual laboratory for the study of coarse and cooking wares in the Late Antique Mediterranean

This contribution presents the LRCW.net a website with a virtual laboratory intranet devoted to the study of coarse and cooking wares in the late Antique Mediterranean. It is designed as a public website with a virtual laboratory intranet. There, all institutions and researchers interested in the subject can work together towards a specific purpose such as the creation of an on-line ‘encyclopedia’ for these categories of ceramics. The LRCW.net website and the associated virtual laboratory are just a small part of a wider initiative that aims to create an on-line Encyclopedia for Ancient Ceramics in the Mediterranean.

1,2,3,4-Tetrahydrobenzo[h][1,6]naphthyridines as a new family of potent peripheral-to-midgorge-site inhibitors of acetylcholinesterase: synthesis, pharmacological evaluation and mechanistic studies

A series of 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridines differently substituted at positions 1, 5, and 9 have been designed from the pyrano[3,2-c]quinoline derivative 1, a weak inhibitor of acetylcholinesterase (AChE) with predicted ability to bind to the AChE peripheral anionic site (PAS), at the entrance of the catalytic gorge. Fourteen novel benzonaphthyridines have been synthesized through synthetic sequences involving as the key step a multicomponent Povarov reaction between an aldehyde, an aniline and an enamine or an enamide as the activated alkene. The novel compounds have been tested against Electrophorus electricus AChE (EeAChE), human recombinant AChE (hAChE), and human serum butyrylcholinesterase (hBChE), and their brain penetration has been assessed using the PAMPA-BBB assay. Also, the mechanism of AChE inhibition of the most potent compounds has been thoroughly studied by kinetic studies, a propidium displacement assay, and molecular modelling. We have found that a seemingly small structural change such as a double O → NH bioisosteric replacement from the hit 1 to 16a results in a dramatic increase of EeAChE and hAChE inhibitory activities (>217- and >154-fold, respectively), and in a notable increase in hBChE inhibitory activity (> 11-fold), as well. An optimized binding at the PAS besides additional interactions with AChE midgorge residues seem to account for the high hAChE inhibitory potency of 16a (IC50 = 65 nM), which emerges as an interesting anti-Alzheimer lead compound with potent dual AChE and BChE inhibitory activities.

1,2,3,4-Tetrahydrobenzo[h][1,6]naphthyridines as a new family of potent peripheral-to-midgorge-site inhibitors of acetylcholinesterase: synthesis, pharmacological evaluation and mechanistic studies

A series of 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridines differently substituted at positions 1, 5, and 9 have been designed from the pyrano[3,2-c]quinoline derivative 1, a weak inhibitor of acetylcholinesterase (AChE) with predicted ability to bind to the AChE peripheral anionic site (PAS), at the entrance of the catalytic gorge. Fourteen novel benzonaphthyridines have been synthesized through synthetic sequences involving as the key step a multicomponent Povarov reaction between an aldehyde, an aniline and an enamine or an enamide as the activated alkene. The novel compounds have been tested against Electrophorus electricus AChE (EeAChE), human recombinant AChE (hAChE), and human serum butyrylcholinesterase (hBChE), and their brain penetration has been assessed using the PAMPA-BBB assay. Also, the mechanism of AChE inhibition of the most potent compounds has been thoroughly studied by kinetic studies, a propidium displacement assay, and molecular modelling. We have found that a seemingly small structural change such as a double O → NH bioisosteric replacement from the hit 1 to 16a results in a dramatic increase of EeAChE and hAChE inhibitory activities (>217- and >154-fold, respectively), and in a notable increase in hBChE inhibitory activity (> 11-fold), as well. An optimized binding at the PAS besides additional interactions with AChE midgorge residues seem to account for the high hAChE inhibitory potency of 16a (IC50 = 65 nM), which emerges as an interesting anti-Alzheimer lead compound with potent dual AChE and BChE inhibitory activities.

Effect of selective recording on estimates of heritability

En este trabajo se analiza el efecto de la selección de datos sobre las estimaciones de heredabilidad. Se estimó el valor de heredabilidad del tamaño de camada en una población porcina en la que los datos correspondientes a las cerdas más viejas eran una muestra seleccionada. Las estimaciones se obtuvieron usando distintos conjuntos de datos derivados de toda la información disponible. Esos conjunto de datos se compararon evaluando su capacidad predictiva. Se vio que las estimaciones de heredabilidad obtenidas utilizando todos los datos disponibles correspondían a valores infraestimados. También se simuló un carácter materno y se generó un conjunto de datos seleccionados eliminando aquellos correspondientes a las hembras sin padres conocidos. Distintos modelos, habitualmente empleados cuando no existe selección de registros, se consideraron para estimar el valor de heredabilidad. Los resultados mostraron que ninguno de esos modelos ofrecía estimaciones insesgadas. Sólo los modelos que tenían en cuenta el efecto de la selección sobre la media residual y la media y varianza genéticas ofrecían estimaciones poco sesgadas. Sin embargo, para poder aplicarlos se debe conocer la selección realizada. El problema de la selección de datos es difícil de abordar cuando se desconoce cual es el proceso de selección que se ha realizado en una población.

Clustering of grape yield maps to delineate site-specific management zones

Zonal management in vineyards requires the prior delineation of stable yield zones within the parcel. Among the different methodologies used for zone delineation, cluster analysis of yield data from several years is one of the possibilities cited in scientific literature. However, there exist reasonable doubts concerning the cluster algorithm to be used and the number of zones that have to be delineated within a field. In this paper two different cluster algorithms have been compared (k-means and fuzzy c-means) using the grape yield data corresponding to three successive years (2002, 2003 and 2004), for a ‘Pinot Noir’ vineyard parcel. Final choice of the most recommendable algorithm has been linked to obtaining a stable pattern of spatial yield distribution and to allowing for the delineation of compact and average sized areas. The general recommendation is to use reclassified maps of two clusters or yield classes (low yield zone and high yield zone) and, consequently, the site-specific vineyard management should be based on the prior delineation of just two different zones or sub-parcels. The two tested algorithms are good options for this purpose. However, the fuzzy c-means algorithm allows for a better zoning of the parcel, forming more compact areas and with more equilibrated zonal differences over time.

Dose and time-dependent selective neurotoxicity induced by mephedrone in mice.

Mephedrone is a drug of abuse marketed as 'bath salts'. There are discrepancies concerning its long-term effects. We have investigated the neurotoxicity of mephedrone in mice following different exposition schedules. Schedule 1: four doses of 50 mg/kg. Schedule 2: four doses of 25 mg/kg. Schedule 3: three daily doses of 25 mg/kg, for two consecutive days. All schedules induced, in some animals, an aggressive behavior and hyperthermia as well as a decrease in weight gain. Mephedrone (schedule 1) induced dopaminergic and serotoninergic neurotoxicity that persisted 7 days after exposition. At a lower dose (schedule 2) only a transient dopaminergic injury was found. In the weekend consumption pattern (schedule 3), mephedrone induced dopamine and serotonin transporter loss that was accompanied by a decrease in tyrosine hydroxylase and tryptophan hydroxylase 2 expression one week after exposition. Also, mephedrone induced a depressive-like behavior, as well as a reduction in striatal D2 density, suggesting higher susceptibility to addictive drugs. In cultured cortical neurons, mephedrone induced a concentration-dependent cytotoxic effect. Using repeated doses for 2 days in an elevated ambient temperature we evidenced a loss of frontal cortex dopaminergic and hippocampal serotoninergic neuronal markers that suggest injuries at nerve endings.

Dose and time-dependent selective neurotoxicity induced by mephedrone in mice.

Mephedrone is a drug of abuse marketed as 'bath salts'. There are discrepancies concerning its long-term effects. We have investigated the neurotoxicity of mephedrone in mice following different exposition schedules. Schedule 1: four doses of 50 mg/kg. Schedule 2: four doses of 25 mg/kg. Schedule 3: three daily doses of 25 mg/kg, for two consecutive days. All schedules induced, in some animals, an aggressive behavior and hyperthermia as well as a decrease in weight gain. Mephedrone (schedule 1) induced dopaminergic and serotoninergic neurotoxicity that persisted 7 days after exposition. At a lower dose (schedule 2) only a transient dopaminergic injury was found. In the weekend consumption pattern (schedule 3), mephedrone induced dopamine and serotonin transporter loss that was accompanied by a decrease in tyrosine hydroxylase and tryptophan hydroxylase 2 expression one week after exposition. Also, mephedrone induced a depressive-like behavior, as well as a reduction in striatal D2 density, suggesting higher susceptibility to addictive drugs. In cultured cortical neurons, mephedrone induced a concentration-dependent cytotoxic effect. Using repeated doses for 2 days in an elevated ambient temperature we evidenced a loss of frontal cortex dopaminergic and hippocampal serotoninergic neuronal markers that suggest injuries at nerve endings.

Spatiotemporal variability in radial growth of trees in the Central Pyrenees: climatic and site influences.

To understand how tree growth has responded to recent climate warming, an understanding of the tree-climate-site complex is necessary. To achieve this, radial growth variability among 204 trees established before 1850 was studied in relation to both climatic and site factors. Seventeen forest stands were sampled in the Spanish Central Pyrenees. Three species were studied: Pinus uncinata, Abies alba, and Pinus sylvestris. For each tree, a ring-width residual chronology was built. All trees cross-dated well, indicating a common influence of the regional climate. For the 1952-1993 period, the radial growth of all species, especially P. uncinata, was positively correlated with warm Novembers during the year before ring formation and warm Mays of the year the annual ring formed. Differences in species-stand elevation modulated the growth-climate associations. Radial growth in P. uncinata at high elevation sites was reduced when May temperatures were colder and May precipitation more abundant. In the 20th century, two contrasting periods in radial growth were observed: one (1900-1949) with low frequency of narrow and wide rings, low mean annual sensitivity, and low common growth variation; and another (1950-1994) with the reverse characteristics. The increased variability in radial growth since the 1950s was observed for all species and sites, which suggests a climatic cause. The low shared variance among tree chronologies during the first half of the 20th century may result from a"relaxation" of the elevation gradient, allowing local site conditions to dominate macroclimatic influence. These temporal trends may be related to the recently reported increase of climatic variability and warmer conditions. This study emphasizes the need to carefully assess the relationships between radial growth and site conditions along ecological gradients to improve dendroclimatic reconstructions.

Drosophilid collection in the Font Groga site, Barcelona (Spain)

During October 23rd and 24th and November 2012 we collected a sample of drosophilids at Font Groga (Barcelona). This site is located on the foothills of the Tibidabo mountain, which is located on the northwest edge of Barcelona and at approximately 400m above sea level. The vegetation is typical for the area, and it is mainly composed of a sparse pine forest (Pinus pinea) with some oaks (Quercus ilex) and Mediterranean brushwood. Flies were netted over 12 baits containing fermenting bananas. A large proportion of D. simulans males was found. The invasive species D. suzukii (Calabria et al. 2010; Cini et al. 2012) was detected in a non-negligible quantity. Taking into account the number of males and females, the estimated Ne for D. suzukii in the Font Groga sample was 33.70. A similar value was obtained for D. subobscura (34.97). Finally, in the study of species diversity the values obtained for H" (Shannon diversity index) and J (Shannon uniformity index) were 0.678 and 0.421, respectively. These estimates are very similar to those obtained in September 2009 in Montpellier by Calabria (2012), who reported H" = 0.679 and J = 0.422, but differ from those reported by the same author in a Font Groga sample of October 2007 (H" = 0.904 and J = 0.505).

Buccal dental microwear and stable isotopes of El Collado: A mesolithic site from Spain,

This study examines the correlation between buccal dental microwear and stable isotopes. The buccal surface of post-canine teeth casts from El Collado, the largest Mesolithic site in Spain, were examined under Scanning Electron Microscope; photomicrographs were taken from the middle third of the buccal surface with magnification 100X. Only six individuals passed the criteria for buccal dental microwear analysis. The photomicrographs were treated by adobe Photoshop 8.01 to cover an area 0.56 mm² of middle third of buccal surface, the output photomicrographs were digitized using Sigmascan Pro 5 by SPSS. Then the correlation between buccal microwear pattern and stable isotopes of the same individuals, of the previous study of Guixe et al., 2006, was examined using a Pearson test. Statistical analysis revealed that there is no significant correlation between stable isotopes and buccal dental microwear of the people of the Mesolithic site of El Collado. The historical and archaeological documentation suggest that the Mesolithic people tended to consume marine food. Fish-drying techniques were used during the Mesolithic period which allowed the introduction of dust and sand to the fish. These abrasive particles affected the buccal dental microwear pattern, so that no correlation between the isotopes and microwear may be expected. This also suggests that the buccal dental microwear pattern exceeds dietary reconstruction to reconstruct food processing techniques.