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A combined study of rock-magnetism and electronic-paramagnetic resonance (EPR) was performed in core samples from an oil well in the Vaca Muerta Formation (SW Argentina). The aim of this work was to characterize the effects of hydrocarbon-related diagenesis on the magnetic signature of oil shales. Similar research has been previously conducted in near-surface sediments affected by oil and gas microseepage, so as to establish a relationship between micromagnetic anomalies and the underlying reservoirs. The EPR technique was employed to measure minute concentrations of organic matter free radicals (OMFRs), Fe3+ and Mn2+. These results, and the concentrations of extractable organic matter (EOM), were compared with mass-specific magnetic susceptibility (χ) and natural remanent magnetization (NRM). The reactive OMFRs, resulting from the thermal degradation of a kerogen that yields a slightly-biodegraded crude oil, seem to have acted on the primary Fe oxides and sulfides through two diagenetic stages. The first stage might have partially dissolved these minerals. Consequently, a number of depth levels show a decrease of both χ and NRM, against an increase of the OMFRs. On the other hand, for a core-interval between 2663 and 2695 m (P18/P24), a second diagenetic stage could have produced partial replacement of framboidal pyrite by authigenic pyrrhotite, as recognized by scanning electron microscopy (SEM), electron X-ray energy dispersion (EDX) experiments and the analysis of the thermomagnetic and IRM curves. Thus, within P18/P24, magnetic parameters increase in direct proportion with the amount of OMFRs. The lithologies encompassed by P18/P24 show small levels of Mn2+, a proxy directly related to calcareous cementation. Moreover, Gamma Ray, resistivity and neutron porosity well logs reveal distinctive features for P18/P24, as well as an increase of hydrocarbon content, contrasting with the rest of the section analyzed. The hydrocarbon-induced magnetic anomalies in these oil shales seem to be conditioned mostly by their petrophysical properties. Fil: Costanzo Álvarez, Vincenzo. Universidad Simon Bolivar; Venezuela. Escuela Superior Politécnica del Litoral. Facultad de Ingeniería en Ciencias de la Tierra; Ecuador Fil: Rapalini, Augusto Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Geociencias Básicas, Aplicadas y Ambientales de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Geociencias Básicas, Aplicadas y Ambientales de Buenos Aires; Argentina Fil: Aldana, Milagrosa. Universidad Simon Bolivar; Venezuela Fil: Díaz, Marisel. Instituto Venezolano de Investigaciones Científicas. Centro de Física; Venezuela Fil: Kietzmann, Diego Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Geociencias Básicas, Aplicadas y Ambientales de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Geociencias Básicas, Aplicadas y Ambientales de Buenos Aires; Argentina Fil: Iglesia Llanos, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Geociencias Básicas, Aplicadas y Ambientales de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Geociencias Básicas, Aplicadas y Ambientales de Buenos Aires; Argentina Fil: Cabrera, Ana. Universidad Simon Bolivar; Venezuela Fil: Luppo, Tomas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Geociencias Básicas, Aplicadas y Ambientales de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Geociencias Básicas, Aplicadas y Ambientales de Buenos Aires; Argentina Fil: Vallejo, María D.. Chevrón Argentina; Argentina Fil: Walther, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Geociencias Básicas, Aplicadas y Ambientales de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Geociencias Básicas, Aplicadas y Ambientales de Buenos Aires; Argentina

Nephrogenic diabetes insipidus (NDI) is a rare tubulopathy characterized by urinary concentration defect due to renal resistance to vasopressin. Loss-of-function mutations of vasopressin V2 receptor (V2R) gene (AVPR2) is the most common cause of the disease. We have identified five novel mutations L86P, R113Q, C192S, M272R, and W323_I324insR from NDI-affected patients. Functional characterization of these mutants revealed that R113Q and C192S were normally localized at the basolateral membrane of polarized Madin-Darby Canine Kidney (MDCK) cells and presented proper glycosylation maturation. On the other side, L86P, M272R, and W323_I324insR mutants were retained in endoplasmic reticulum and exhibited immature glycosylation and considerably reduced stability. All five mutants were resistant to administration of vasopressin analogues as evaluated by defective response in cAMP release. In order to rescue the function of the mutated V2R, we tested VX-809, sildenafil citrate, ibuprofen and tolvaptan in MDCK cells. Among these, tolvaptan was effective in rescuing the function of M272R mutation, by both allowing proper glycosylation maturation, membrane sorting and response to dDAVP. These results show an important proof of concept for the use of tolvaptan in patients affected by M272R mutation of V2R causing NDI.

Summary: It has been long assumed that normally leading strand synthesis must proceed coordinated with the lagging strand to prevent strand uncoupling and the pathological accumulation of single-stranded DNA (ssDNA) in the cell, a dogma recently challenged by in vitro studies in prokaryotes. Here, we report that human DNA polymerases can function independently at each strand in vivo and that the resulting strand uncoupling is supported physiologically by a cellular tolerance to ssDNA. Active forks rapidly accumulate ssDNA at the lagging strand when POLA1 is inhibited without triggering a stress response, despite ssDNA formation being considered a hallmark of replication stress. Acute POLA1 inhibition causes a lethal RPA exhaustion, but cells can duplicate their DNA with limited POLA1 activity and exacerbated strand uncoupling as long as RPA molecules suffice to protect the elevated ssDNA. Although robust, this uncoupled mode of DNA replication is also an in-built weakness that can be targeted for cancer treatment. : Using specific POLA1 inhibitors, Ercilla et al. show that DNA synthesis works independently at leading and lagging strands and can be “uncoupled” in vivo. The authors show that cells can naturally tolerate ssDNA formation during DNA replication as long as it is protected by the surplus of RPA molecules. Keywords: DNA replication, polymerase alpha, POLA1, CD437, lagging strand, strand uncoupling, RPA, ssDNA, replication catastrophe, ATR

Among the different natural sources of drinking water, aquifers are the most exposed to the environmental pressures posed by old landfills. Conventional monitoring methods to follow up the migration of leachate into groundwater and the generation of biogases in landfills are costly, time-consuming, and only provide a partial picture of these complex systems. Alternatively, we present the results of a non-invasive and cost-effective methodology applied to a non-engineered (i.e., no gas or liquid recovery systems) closed landfill in southern Ontario. The study combines, through a numerical approach, data from a direct current (DC) resistivity and an Induced Polarization (IP) survey, with measurements of methane concentrations taken over the landfill. We used a Dipole–Dipole array along four regional Lines of approximately 300 meters each, cutting crosswise and throughout the strike of the site. The interpretation of the inverted resistivity and IP data allowed us to recognize and describe some hydrogeological features that went unnoticed by using conventional monitoring techniques. We also applied a hybrid algorithm that incorporates fuzzy logic to neural networks (ANFIS) for causal variable forecasting of surface methane concentrations, using geoelectrical proxies of leachate accumulation as antecedent parameters (i.e. minimum resistivity and their corresponding IP values). The ANFIS provided a statistically significant inference of the main tendencies of methane concentrations along the surveyed Lines. Some coarse inferences appear to be locally associated with IP bright spots of anomalous metal ion content. Overall, a better inference seems to be hampered by the uncertainties involved in the generation of biogas and its upward flow through the soil cover. These results substantiate the feasibility of employing surface methane concentration data as a first diagnostic test to characterize the areal extent of the leachate plume underground.

International audience; DNA replication is an extremely complex process, involving thousands of replication forks progressing along chromosomes. These forks are requently slowed down or stopped by various obstacles, such as secondary DNA structures, chromatin-acting proteins or a lack of nucleotides. Thisslowing down, known as replicative stress, plays a central role in tumour development. Complex processes, which are not yet fully understood, are set up to respond to this stress. Certain nucleases, such as MRE11 and DNA2, degrade the neo-replicated DNA at the level of blocked forks, allowing the replication to restart. The interferon pathway is a defense mechanism against pathogens that detects the presence of foreign nucleic acids in the cytoplasm and activates the innate immune response. DNA fragments resulting from genomic DNA metabolism (repair, retrotransposition) can diffuse into the cytoplasm and activate this pathway. A pathological manifestation of this process is the Aicardi-Goutières syndrome, a rare disease characterized by chronic inflammation leading to neurodegenerative and developmental problems. In this encephalopathy, it has been suggested that DNA replication may generate cytosolic DNA fragments, but the mechanisms involved have not beencharacterized. SAMHD1 is frequently mutated in the Aicardi-Goutières syndrome as well as in some cancers, but its role in the etiology of these diseases was largely unknown. We show that cytosolic DNA accumulates in SAMHD1-deficient cells, particularly in the presence of replicative stress, activating the interferon response. SAMHD1 is important for DNA replication under normal conditions and for the processing of stopped forks, independent of its dNTPase activity. In addition, SAMHD1 stimulates the exonuclease activity of MRE11 in vitro. When SAMHD1 is absent, degradation of neosynthesized DNA is inhibited, which prevents activation of the replication checkpoint and leads to failure to restart the replication forks. Resection of the replication forks is performed by an alternative mechanism which releases DNA fragments into the cytosol, activating the interferon response. The results obtained show, for the first time, a direct link between the response to replication stress and the production of interferons. These results have important implications for our understanding of the Aicardi-Goutières syndrome and cancers related to SAMHD1. For example, we have shown that MRE11 and RECQ1 are responsible for the production of DNA fragments that trigger the inflammatory response in cells deficient for SAMHD1. We can therefore imagine that blocking the activity of these enzymes could decrease the production of DNA fragments and, ultimately, the activation of innate immunity in these cells. In addition, the interferon pathway plays an essential role in the therapeutic efficacy of irradiation and certain chemotherapeutic agents such as oxaliplatin. Modulating this response could therefore be of much wider interest in anti-tumour therapy; La réplication de l’ADN est un processus extrêmement complexe, impliquant des milliers de fourches de réplication progressant le long des chromosomes. Ces fourches sont fréquemment ralenties ou arrêtées par différents obstacles, tels que des structures secondaires de l’ADN, des protéines agissant sur la chromatine ou encore un manque de nucléotides. Ce ralentissement, qualifié de stress réplicatif, joue un rôle central dans le développement tumoral. Des processus complexes, qui ne sont pas encore totalement connus, sont mis en place pour répondre à ce stress. Certaines nucléases, comme MRE11 et DNA2, dégradent l’ADN néorépliqué au niveau des fourches bloquées, ce qui permet le redémarrage des réplisomes. La voie interféron est un mécanisme de défense contre les agents pathogènes qui détecte la présence d’acides nucléiques étrangers dans le cytoplasme et active la réponse immunitaire innée. Des fragments d’ADN issus du métabolisme de l’ADN génomique (réparation, rétrotransposition) peuvent diffuser dans le cytoplasme et activer cette voie. Une manifestation pathologique de ce processus est le syndrome d’Aicardi-Goutières, une maladie rare caractérisée par une inflammation chronique générant des problèmes neurodégénératifs et développementaux. Dans le cadre de cette encéphalopathie, il a été suggéré que la réplication de l’ADN pouvait générer des fragments d’ADN cytosoliques, mais les mécanismes impliqués n’avaient pas été caractérisés. SAMHD1 est fréquemment muté dans le syndrome d’Aicardi-Goutières ainsi que dans certains cancers, mais son rôle dans l’étiologie de ces maladies était jusqu’à présent largement inconnu. Nous montrons que de l’ADN cytosolique s’accumule dans les cellules déficientes pour SAMHD1, particulièrement en présence de stress réplicatif, activant la réponse interféron. Par ailleurs, SAMHD1 est important pour la réplication de l’ADN en conditions normales et pour le processing des fourches arrêtées, indépendamment de son activité dNTPase. De plus, SAMHD1 stimule l’activité exonucléase de MRE11 in vitro. Lorsque SAMHD1 est absent, la dégradation de l’ADN néosynthétisé est inhibée, ce qui empêche l’activation du checkpoint de réplication et entraine un défaut de redémarrage des fourches de réplication. De plus, la résection des fourches de réplication est réalisée par un mécanisme alternatif qui libère des fragments d’ADN dans le cytosol, activant la réponse interféron. Les résultats obtenus montrent, pour la première fois, un lien direct entre la réponse au stress réplicatif et la production d’interférons. Ces résultats ont des conséquences importantes dans notre compréhension du syndrome d’Aicardi Goutières et des cancers liés à SAMHD1. Par exemple, nous avons démontré que MRE11 et RECQ1 sont responsables de la production des fragments d’ADN qui déclenchent la réponse inflammatoire dans les cellules déficientes pour SAMHD1. Nous pouvons donc imaginer que bloquer l’activité de ces enzymes pourrait diminuer la production des fragments d’ADN et, in fine, l’activation de l’immunité innée dans ces cellules. Par ailleurs, la voie interférons joue un rôle essentiel dans l’efficacité thérapeutique de l’irradiation et de certains agents chimiothérapiques comme l’oxaliplatine. Moduler cette réponse pourrait donc avoir un intérêt beaucoup plus large en thérapie anti-tumorale

Summary As predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion. In most cells where cohesin loads without conferring cohesion, it does so by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen is neutralized loads and moves along chromatin despite failing to entrap DNAs. Thus, cohesin engages chromatin in non-topological, as well as topological, manners. Since hinge mutations, but not Smc-kleisin fusions, abolish entrapment, DNAs may enter cohesin rings through hinge opening. Mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin’s recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin’s hinge driven by cycles of ATP hydrolysis., Graphical Abstract, Highlights • Chromatid cohesion is mediated by co-entrapment of sister DNAs inside cohesin rings • Cohesin engages chromatin in non-topological as well as topological manners • Cohesin loads onto chromatin despite closure of any of the three ring interfaces. • Cohesin’s hinge domain is critical for non-topological and topological DNA association., Cohesin can selectively engage DNA without entrapping it, suggesting a new dimension to cohesin function.

A study of surface sediments from the Anllons riverbed (northwestern Spain) was conducted to assess the use of their magnetic properties as proxies of recent trace element contamination (i.e., As, Zn, Pb, Cr, Ni and Cu). Thermomagnetic curves, hysteresis parameters, specific magnetic susceptibility χ, frequency-dependent magnetic susceptibility κ FD%, magnetic remanence and saturation isothermal remanent magnetization ratios (i.e., S and SIRM/χ) reveal the presence of mostly fine-grained magnetite and maghemite in these samples. The Tomlinson Pollution Load Index (PLI), as well as the magnetic parameters χ, κ FD%, SIRM/χ and S-ratios, vary more or less in agreement to each other when they are sequentially arranged throughout a 30-km-long transect that goes from the town of Carballo to the river mouth in Ponteceso. From that profile, it is possible to identify an anomaly of χ that lies between two small peaks of the PLI and seems to define a contaminated area probably linked to a diffuse low-impact source of pollution. There is also a conspicuous peak of the PLI that is likely associated to a nearby gold mine. The variations of the κ FD%’s along the transect are the ones that best reflect the changes in the PLI values, regardless of the level of contamination. This result suggests that adsorption is the main mechanism of trace elements removal by Fe oxides in these samples. Direct signal analyses, used to decompose the isothermal remanent magnetization curves, reveal the presence of four magnetic fractions, with varying relative concentrations all over the profile, namely single-domain magnetite, close to the superparamagnetic threshold (SD/SP) and/or maghemite ( $$\log B_{1/2}$$ ca 1.2), single-domain magnetite ( $$\log B_{1/2}$$ ca 2), hematite ( $$\log B_{1/2}$$ ca 2.5) and goethite ( $$\log B_{1/2}$$ ca 3.5). Integrating all the available information, it was sketched out a likely sequence of events that included magnetic minerals neoformation in parent soils, their subsequent chemical transformations and the contamination episodes that affected the region.

A relationship between hydrocarbons and their magnetic signatures has previously been alluded to but this is the first study to combine extensive geochemical and magnetic data of hydrocarbon-associated samples. We report a detailed study that identifies a connection between magnetic mineralogy and oil biodegradation within oil-bearing sedimentary units from Colombia, Canada Indonesia and the UK. Geochemical data reveal that all the oil samples are derived from mature type-II kerogens deposited in oxygen-poor environments. Biodegradation is evident to some extent in all samples and leads to a decrease in oil quality through the bacterially mediated conversion of aliphatic hydrocarbons to polar constituents. The percentage of oil components and the biodegradation state of the samples were compared to the magnetic susceptibility and magnetic mineralogy. A distinct decrease in magnetic susceptibility is correlated to decreasing oil quality and the amount of extractable organic matter present. Further magnetic characterization revealed that the high quality oils are dominated by pseudo-single domain grains of magnetite and the lower quality oils by larger pseudo-single domain to multidomain grains of magnetite and hematite. Hence, with decreasing oil quality there is a progressive dominance of multidomain magnetite as well as the appearance of hematite. It is concluded that biodegradation is a dual process, firstly, aliphatic hydrocarbons are removed thereby reducing oil quality and secondly, magnetic signatures are both created and destroyed. This complex relationship may explain why controversy has plagued previous attempts to resolve the connection between magnetics and hydrocarbon deposits. These findings reinforce the importance of bacteria within petroleum systems as well as providing a platform for the use of magnetization as a possible exploration tool to identify subsurface reservoirs and a novel proxy of hydrocarbon migration.

Giovanni N Roviello,1 Caterina Vicidomini,1 Vincenzo Costanzo,1 Valentina Roviello2 1CNR Istituto di Biostrutture e Bioimmagini, Via Mezzocannone site and Headquarters, 2Centro Regionale di Competenza (CRdC) Tecnologie, Via Nuova Agnano, Napoli, Italy Abstract: In the present study, we report the interaction of an artificial oligolysine (referred to as AOL) realized in our laboratory with targets of biomedical importance. These included polyinosinic acid (poly rI) and its complex with polycytidylic acid (poly I:C), RNAs with well-known interferon-inducing ability, and double-stranded (ds) DNA. The ability of the peptide to bind both single-stranded poly rI and ds poly I:C RNAs emerged from our circular dichroism (CD) and ultraviolet (UV) studies. In addition, we found that AOL forms complexes with dsDNA, as shown by spectroscopic binding assays and UV thermal denaturation experiments. These findings are encouraging for the possible use of AOL in biomedicine for nucleic acid targeting and oligonucleotide condensation, with the latter being a key step preceding their clinical application. Moreover, we tested the ability of AOL to bind to proteins, using serum albumin as a model protein. We demonstrated the oligolysine–protein binding by CD experiments which suggested that AOL, positively charged under physiological conditions, binds to the protein regions rich in anionic residues. Finally, the morphology characterization of the solid oligolysine, performed by scanning electron microscopy, showed different crystal forms including cubic-shaped crystals confirming the high purity of AOL. Keywords: nucleic acid binding, polyinosinic acid, double-stranded nucleic acids, oligolysine, circular dichroism

Summary The chemical identity and integrity of the genome is challenged by the incorporation of ribonucleoside triphosphates (rNTPs) in place of deoxyribonucleoside triphosphates (dNTPs) during replication. Misincorporation is limited by the selectivity of DNA replicases. We show that accumulation of ribonucleoside monophosphates (rNMPs) in the genome causes replication stress and has toxic consequences, particularly in the absence of RNase H1 and RNase H2, which remove rNMPs. We demonstrate that postreplication repair (PRR) pathways—MMS2-dependent template switch and Pol ζ-dependent bypass—are crucial for tolerating the presence of rNMPs in the chromosomes; indeed, we show that Pol ζ efficiently replicates over 1–4 rNMPs. Moreover, cells lacking RNase H accumulate mono- and polyubiquitylated PCNA and have a constitutively activated PRR. Our findings describe a crucial function for RNase H1, RNase H2, template switch, and translesion DNA synthesis in overcoming rNTPs misincorporated during DNA replication, and may be relevant for the pathogenesis of Aicardi-Goutières syndrome., Graphical Abstract Highlights ► Unprocessed rNMPs in the chromosomes sensitize cells to replication stress ► Postreplication repair pathways are essential to tolerating rNMP-containing templates ► DNA polymerase ζ efficiently bypasses rNMPs in DNA templates ► Postreplication repair is chronically activated in RNase H1/H2 defective cells

In this work we try to better characterise the shallow magnetic signature of hydrocarbon microseepage in oil fields from eastern and western Venezuela. To get a better insight of the processes involved, we attempt to find out the main magnetic phases responsible for the observed oil-related shallow magnetic anomalies. In this way, a new and alternative numerical approach to decompose Isothermal Remagnetization (IRM) curves is introduced. The method is based on a Direct Signal Analysis (DSA) of the IRM curve in order to identify the number and type of magnetic components. Representative wells from western (La Victoria) and eastern (San Joaquin) Venezuelan fields are studied. The DSA approach, together with rock magnetic experimental results, indicates that in the well from western Venezuela the main magnetic mineralogy associated to hydrocarbon microseepage is magnetite. Conversely, in the well from eastern Venezuela, these MS anomalies are mainly caused by the presence of Fe-sulphides (i.e. greigite). These results support the hypothesis of two different authigenic processes. For the well at the western field, we propose that a net electron transfer from the organic matter, degraded by hydrocarbon gas leakage, should occur precipitating Fe(II) magnetic minerals (e.g. magnetite). On the other hand, in the well of the eastern field, high concentrations of H2S at shallow levels, might allow the formation of secondary Fe-sulphides.

We have studied near-surface magnetic contrasts in nine oil wells from an oil prospective area in eastern Venezuela (compressive deformation front of the northeastern Maturin Sub-Basin). Samples are drill cuttings taken at intervals of about 15 m within approximately the first 1200 m of most of these wells. Rock magnetic experiments, electron paramagnetic resonance (EPR), extractable organic matter (EOM), X-ray diffraction and scanning electron microscopy (SEM) are used to discriminate between two anomalies in magnetic susceptibility (MS), related to either a reducing environment caused by the underlying reservoir (Type A), or to primary lithological contrasts (Type B). Contour maps of MS, S-ratio and organic-matter-free radical concentrations (OMFRC), for the A-like depth levels, show a major central zone of anomalous high values. This area is probably associated with either maximum accumulation of hydrocarbons in the reservoir or with their southerly migration from the northern petroleum source towards the deformation front. We argue that such a result could be used, for future exploration and production ventures in the region, as a preliminary characterization of the reservoir. We also show in a Day plot, that saturation remanence — saturation magnetization ratio (Mrs/Ms) and coercivity of remanence — coercive force ratio (Hcr/Hc) stand as additional criteria to discriminate between these two types of magnetic contrasts. In fact, for the area of study, hysteresis ratios show three distinct trends corresponding to samples from A-like, B-like and non-anomalous depth levels.

Ataxia telangiectasia (A-T) is a human disease caused by ATM deficiency characterized among other symptoms by radiosensitivity, cancer, sterility, immunodeficiency and neurological defects. ATM controls several aspects of cell cycle and promotes repair of double strand breaks (DSBs). This probably accounts for most of A-T clinical manifestations. However, an impaired response to reactive oxygen species (ROS) might also contribute to A-T pathogenesis. Here, we show that ATM promotes an anti-oxidant response by regulating the pentose phosphate pathway (PPP). ATM activation induces glucose-6-phosphate dehydrogenase (G6PD) activity, the limiting enzyme of the PPP responsible for the production of NADPH, an essential anti-oxidant cofactor. ATM promotes Hsp27 phosphorylation and binding to G6PD, stimulating its activity. We also show that ATM-dependent PPP stimulation increases nucleotide production and that G6PD-deficient cells are impaired for DSB repair. These data suggest that ATM protects cells from ROS accumulation by stimulating NADPH production and promoting the synthesis of nucleotides required for the repair of DSBs.

Repetitive DNA constitutes a large part of most vertebrate genomes. However, it is unclear how DNA containing repetitive sequences is organized in the context of chromatin and nuclear structures, h...

Thermal stimulated depolarization currents (TSDC), natural remanent magnetization (NRM), scanning electron microscopy (SEM) and thin section capillarity analyses (CA) have been used here to study adsorption and confinement effects of water aggregates of porous rock samples in the vicinity of a lithological contact between two sedimentary sequences from Cretaceous northeastern Venezuela. Two different locations (I and II) were studied and the lithological contact was dielectrically characterized by the TSDC technique. The direct signal analysis method was applied to find the mean overlapping processes and the characteristic relaxation parameters. Drying and rehydration treatments performed on these samples indicate that the observed spectra could be associated with physisorbed moisture. For location II, five main relaxations were identified in the TSDC spectra for the temperature range between 77 and 240 K. Similarly to results previously reported for location I, they were associated with water dipoles, hydrogen-bonded to different sites in the material. For both localities, the dielectric relaxation peaks, located from 200 to 320 K, were related to water clusters confined in the pores. The pore dimension was determined from SEM microphotographs and CA. Each location presents different behavior. At location II a confinement effect is observed, above and below the contact, in the dipolar species responsible for the high-temperature relaxations. For location I this effect is observed only above the contact. Below the contact these relaxations are mainly controlled by adsorption effects as the pore sizes increase. A threshold between two pore size ranges was observed. In pores below 12 μm, confinement effects seem to predominate whereas for larger pores, adsorption is the main effect. Due to the change in the environment seen by the adsorbed water molecules, a variation in the mean reorientation energy is observed as the contact is approached, in the same region where NRM anomalies have been reported.

High-resolution aeromagnetic surveys over oil fields and magnetic susceptibility (MS) measurements in soils, sediments, and drill cuttings have been proposed as complementary or alternative means of exploration and assessment of hydrocarbon reservoirs. Some micromagnetic anomalies detected by these studies could be the likely result of the existence, at shallow depths, of authigenic magnetite and/or Fe-sulphides. It has been hypothesized that such magnetic minerals are the chemical byproducts of primary Fe-oxides exposed to a reducing environment induced by the underlying reservoir. Secondary magnetite could appear as spherical aggregates (framboids) of submicronic crystals identifiable by scanning electron microscopy (SEM). The formation of these framboids seems linked either to inorganic chemical processes associated with the presence of hydrocarbons or to oil biodegradation. In the last few years, we have examined the causal relationship between framboids of magnetic minerals and hydrocarbons via rock magnetic and electron paramagnetic resonance (EPR) analyses of drill cuttings. The samples used have been taken at different depths (within the first 1500 m) of producing and nonproducing wells from two oil fields in southwestern Venezuela. We have complemented MS results with S-ratio logs, SEM, X-ray energy dispersion (EDX), IRM acquisition curves, and high-temperature susceptibility measurements. More recently we have also studied soil samples from an oil prospective area in western Venezuela, where rock magnetic and EPR results were compared with ethane gas analyses and remote sensing data. Rock magnetic and EPR studies have been performed in 14 oil wells from La Victoria (3) and Guafita (11) fields (Figure 1). Since our research has focused at the near-surface expression of hydrocarbon migration, the samples analyzed (drill cuttings) were taken from different depth at intervals of about 15 m between 100 and 1500 m. All samples belong to a single group of molasses from strata that lie far above the producing levels …

Changes in hippocampal function seem critical for cognitive impairment in Alzheimer's disease (AD). Although there is eventual loss of synapses in both AD and animal models of AD, deficits in spatial memory and inhibition of long-term potentiation (LTP) precede morphological alterations in the models, suggesting earlier biochemical changes in the disease. In the studies reported here we demonstrate that amyloid beta-peptide (Abeta) treatment of cultured hippocampal neurons leads to the inactivation of protein kinase A (PKA) and persistence of its regulatory subunit PKAIIalpha. Consistent with this, CREB phosphorylation in response to glutamate is decreased, and the decrease is reversed by rolipram, a phosphodiesterase inhibitor that raises cAMP and leads to the dissociation of the PKA catalytic and regulatory subunits. It is likely that a similar mechanism underlies Alphabeta inhibition of LTP, because rolipram and forskolin, agents that enhance the cAMP-signaling pathway, can reverse this inhibition. This reversal is blocked by H89, an inhibitor of PKA. These observations suggest that Alphabeta acts directly on the pathways involved in the formation of late LTP and agents that enhance the cAMP/PKA/CREB-signaling pathway have potential for the treatment of AD.

Magnetic susceptibility (MS) and organic matter free radical concentration (OMFRC) determined by electron paramagnetic resonance, have been measured in soil samples (≈1.5 m depth) from an oil prospective area located at the southern flank of the Venezuelan Andean Range. S-ratios close to 1, as well as high temperature susceptibility analyses, reveal magnetite as the chief magnetic phase in most of these samples. Ethane concentrations, MS and OMFRC normalized data have been plotted against the relative position of 22 sampling sites sequentially arranged from north to south. Although there is not a linear correlation between MS and OMFRC data, these two profiles seem to vary in like fashion. A MS and OMFRC southern anomaly coincides with the zone of highest ethane concentration that overlies a “Cretaceous kitchen”. OMFRC highs could be linked to the degradation or alteration of organic matter, the possible result of hydrocarbon gas leakage, whose surface expression is the stressed fern observed by remote sensing studies previously performed in the area. Ethane anomalies are associated to this seepage that also produces changes in the magnetic mineralogies detected as MS positive anomalies.

The cytotoxicity of SN1-type alkylating agents such as N-methyl-N'-nitrosourea (MNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or the cancer chemotherapeutics temozolomide, dacarbazine and streptozotocin has been ascribed to the persistence of O(6)-methylguanine ((me)G) in genomic DNA. One hypothesis posits that (me)G toxicity is caused by futile attempts of the mismatch repair (MMR) system to process (me)G/C or (me)G/T mispairs arising during replication, while an alternative proposal suggests that the latter lesions activate DNA damage signaling, cell cycle arrest and apoptosis directly. Attempts to elucidate the molecular mechanism of (me)G-induced cell killing in vivo have been hampered by the fact that the above reagents induce several types of modifications in genomic DNA, which are processed by different repair pathways. In contrast, defined substrates studied in vitro did not undergo replication. We set out to re-examine this phenomenon in replication-competent Xenopus laevis egg extracts, using either phagemid substrates containing a single (me)G residue, or methylated sperm chromatin. Our findings provide further support for the futile cycling hypothesis.

Electron Paramagnetic Resonance (EPR) and Magnetic Susceptibility (MS) measurements were carried out in near-surface samples of 6 wells (producers and non-producers) from some Venezuelan oil fields. EPR measurements were performed to determine the organic matter free radical concentration (OMFRC) in the samples. MS anomalous levels, where the restrictive presence of microscopic framboidal magnetic minerals has been recognized, were found only at the producer wells. Moreover for these same wells, anomalies of OMFRC were observed at depths close to MS anomalous levels. These preliminary results seem to suggest a relationship between the presence of OMFRC and MS anomalies, possibly associated with the underlying reservoir.

Rock magnetic and dielectric studies have been conducted in sedimentary rocks close to a lithological contact (Chimana and Querecual formations) at the Pertigalete sedimentary sequence (Cretaceous northeastern Venezuela). Rock magnetic results suggest that the presence of secondary magnetic minerals has been conditioned by the transitional zone between Chimana and Querecual. In fact, this contact might have acted as a conduit for fluid circulation and a focus of chemical alteration on most of its adjacent strata. Some of the secondary magnetic minerals (e.g. magnetite) must be the outcome of the anoxic conditions that prevailed during times when the sedimentary basin reached its maximum temperatures when oil and gas were generated (Lower Miocene). Some others (e.g. hematite) are the likely byproduct of alteration dating from times when oxidizing conditions were dominant. The Thermally Stimulated Depolarization Current (TSDC) technique is used here to dielectrically characterize the same contact. The complex TSDC spectra obtained in the temperature range from 77 to 230 K for the samples studied, indicate a notable difference between the Chimana (CH) and Querecual (QC) samples. Drying and rehydration treatments performed on these samples show that the whole depolarization spectrum is mainly due to moisture trapped in the microporosity of the material. The broad spectra, separated by using Direct Signal Analysis, reveal the presence of four overlapping peaks. TSDC results indicate a progressive change in the mean re-orientation energy with the lithology for the four relaxations. This variation, due to the change in the environment seen by the water dipoles hydrogen-bonded to different sites in the material, is observed for the same lithologies where magnetic anomalies have been reported. Thus, rock magnetic analyses, combined with the TSDC technique, seem to serve as a sensitive and alternative means of characterizing lithologies and stratigraphic discontinuities in a sedimentary sequence.

Topoisomerase I (Top1) releases torsional stress during DNA replication and transcription and is inhibited by camptothecin and camptothecin-derived cancer chemotherapeutics. Top1 inhibitor cytotoxicity is frequently linked to double-strand break (DSB) formation as a result of Top1 being trapped on a nicked DNA intermediate in replicating cells. Here we use yeast, mammalian cell lines and Xenopus laevis egg extracts to show that Top1 poisons rapidly induce replication-fork slowing and reversal, which can be uncoupled from DSB formation at sublethal inhibitor doses. Poly(ADP-ribose) polymerase activity, but not single-stranded break repair in general, is required for effective fork reversal and limits DSB formation. These data identify fork reversal as a means to prevent chromosome breakage upon exogenous replication stress and implicate proteins involved in fork reversal or restart as factors modulating the cytotoxicity of replication stress-inducing chemotherapeutics. © 2012 Nature America, Inc. All rights reserved., M.L. and A.R.C. are supported by a Swiss National Science Foundation grant (PP00A-114922). A.R.C. was supported by a European Molecular Biology Organization (EMBO) short-term fellowship. V.C. and Y.H. are supported by Cancer Research UK, the Lister Institute of Preventive Medicine, a European Research Council (ERC-206281) startup grant and the EMBO Young Investigator Program (YIP).

In higher eukaryotes the dynamics of replisome components during fork collapse and restart are poorly understood. Here, we reconstituted replication fork collapse and restart by inducing single-strand DNA (ssDNA) lesions that create a double-strand break (DSB) in one of the replicated sister chromatids after fork passage. We found that, upon fork collapse, the active CDC45–MCM–GINS (CMG) helicase complex loses its GINS subunit. A functional replisome is restored by the reloading of GINS and Pol epsilon onto DNA in a RAD51- and MRE11- dependent manner, but independently of replication origin assembly and firing. PCNA mutant alleles defective in break-induced replication (BIR) are unable to support restoration of replisome integrity. These results reveal that in higher eukaryotes replisomes are partially dismantled following fork collapse and fully re-established by a recombination-mediated process.

This study reports paleomagnetic results from nine localities (22 sites) of the Uairen Formation, the basal and oldest unit of the Roraima sedimentary sequence (Roraima Group) in the Precambrian Guayana shield (southeastern Venezuela). Three distinct natural remanent magnetizations (NRM's), U1N, U1R, and U2, were isolated by alternating-field and thermal demagnetization. U1N and U1R NRM's have mean directions D = 342°, I = 6° (k = 14, α95 = 10°, N = 14 sites) and D = 155°, I = 10° (k = 8, α95 = 17°, N = 8 sites), respectively. Because of their high coercivities (HC) and high (>580 °C) laboratory unblocking temperatures (TUB), it is believed that these NRM's are carried mainly by fine-grained magnetite and (or) hematite. The U2 NRM has D = 338°, I = −7° (k = 5, α95 = 25°, N = 6 sites). U2 is a low-HC (20 mT) and low-TUB (

The Keystone Symposium on Genome Instability and DNA Repair took place between 1 and 6 March 2009, in Taos, New Mexico, and was organized by M. Jasin, S.J. Elledge & M.S. Neuberger. ![][1] See Glossary for abbreviations used in this article DSBs occur when both strands of a DNA double helix are damaged by either the normal metabolic processes of the cell, or environmental factors such as ultraviolet light or radiation. Cells have evolved mechanisms to repair DSBs, to ensure that large pieces of the genome are not lost; however, the aberrant repair of DSBs can lead to genome rearrangements that scramble the information encoded in the DNA. The 2009 Keystone Symposium on Genome Instability and DNA Repair featured mechanistic studies of DSB repair, host responses to DSBs and recent phenomenology in human genomic disorders. This report highlights some of the key findings presented at the meeting. Space limitations have prevented us from discussing every contribution, and we apologize to those whose work is not highlighted in this report. One way to understand the consequences of aberrant DSB repair is to identify and characterize genomic rearrangements. M. Stratton (Cambridge, UK) used second‐generation whole‐genome DNA‐sequencing technologies to characterize the most common types of DNA rearrangement in a range of cancer cell lines and primary tumours. In addition to finding a large number of point mutations in these samples, preliminary analyses of the data indicate that many rearrangements result from intrachromosomal events, and, less frequently, interchromosomal breakage and rejoining events. Importantly, many of these rearrangements had not been seen previously because they are too small to be detected by conventional karyotype analyses. The analysis of junction sequences by the Stratton group also revealed that classical repair processes such as HR and NHEJ, probably have a role in the formation of these rearrangements. However, … [1]: /embed/graphic-1.gif

Exogenous and endogenous insults continuously damage DNA. DNA damage must be detected in order to prevent loss of vital genetic information. Cells respond to DNA damage by activating checkpoint pathways that delay the progression through the cell cycle, promote DNA repair or induce cell death. A regulatory network of proteins has been identified that participate in DNA damage checkpoint pathways. Central to this network are ATM, ATR and the Mre11/Rad50/Nbs1 (MRN) complex. Detailed biochemical analysis of ATM, ATR and the MRN dependent DNA damage responses has taken advantage of several in vitro model systems to understand the detailed mechanisms underlying their function. Here we describe some recent findings obtained analysing these pathways using in vitro model systems. In particular we focus on the studies performed in the Xenopus laevis egg cell free extract, which recapitulates the DNA damage response in the context of the cell cycle.

Background and purpose: Ataxia-telangiectasia (A-T) is a rare autosomal recessive disorder caused by alterations of the A-T mutated (ATM) gene. Although A-T is a noncurable disease, we, previously, documented a clear improvement of cerebellar functions during a short-term betamethasone trial. The aim of this study was to define the underlying biochemical mechanism. Methods: In six A-T patients receiving a short-term steroid therapy, intracellular glutathione (GSH) levels were evaluated with a colorimetric assay. The lipid peroxidation level and reactive oxygen species (ROS) production were evaluated using commercial assays. All the parameters were compared with the improvement of cerebellar functions expressed as delta (Δ) of the Scale for the Assessment and Rating of Ataxia (SARA). Results: We observed an inverse correlation between Δ SARA and the severity of cerebellar atrophy and between the latter and basal GSH values. Four of the five patients with the highest Δ SARA also had the highest GSH values. Moreover, even though basal ROS values were comparable in patients and controls, in the only patient studied at different time-points of therapy, a remarkable reduction in ROS levels was documented. Conclusion: We suggest that antioxidative mechanisms play a role in favouring the improvement of cerebellar functions observed in A-T patients receiving a short-term betamethasone trial.

DNA double-strand breaks (DSBs) can be processed by the Mre11–Rad50–Nbs1 (MRN) complex, which is essential to promote ataxia telangiectasia-mutated (ATM) activation. However, the molecular mechanisms linking MRN activity to ATM are not fully understood. Here, using Xenopus laevis egg extract we show that MRN-dependent processing of DSBs leads to the accumulation of short single-stranded DNA oligonucleotides (ssDNA oligos). The MRN complex isolated from the extract containing DSBs is bound to ssDNA oligos and stimulates ATM activity. Elimination of ssDNA oligos results in rapid extinction of ATM activity. Significantly, ssDNA oligos can be isolated from human cells damaged with ionizing radiation and injection of small synthetic ssDNA oligos into undamaged cells also induces ATM activation. These results suggest that MRN-dependent generation of ssDNA oligos, which constitute a unique signal of ongoing DSB repair not encountered in normal DNA metabolism, stimulates ATM activity.

Tipin and its interacting partner Tim1 (Timeless) form a complex at replication forks that plays an important role in the DNA damage checkpoint response. Here we identify Xenopus laevis Tipin as a substrate for cyclin E/cyclin-dependent kinases 2 that is phosphorylated in interphase and undergoes further phosphorylation upon entry into mitosis. During unperturbed DNA replication, the Tipin/Tim1 complex is bound to chromatin, and we were able to detect interactions between Tipin and the MCM helicase. Depletion of Tipin from Xenopus extracts did not significantly impair normal replication but substantially blocked the ability of stalled replication forks to recover after removal of a block imposed by aphidicolin. Tipin-depleted extracts also showed defects in the activation of Chk1 in response to aphidicolin, probably because of a failure to load the checkpoint mediator protein Claspin onto chromatin.

Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia Rad3-related (ATR) and the Mre11/Rad50/Nbs1 complex ensure genome stability in response to DNA damage. However, their essential role in DNA metabolism remains unknown. Here we show that ATM and ATR prevent accumulation of DNA double-strand breaks (DSBs) during chromosomal replication. Replicating chromosomes accumulate DSBs in Xenopus laevis egg extracts depleted of ATM and ATR. Addition of ATM and ATR proteins to depleted extracts prevents DSB accumulation by promoting restart of collapsed replication forks that arise during DNA replication. We show that collapsed forks maintain MCM complex but lose Pol epsilon, and that Pol epsilon reloading requires ATM and ATR. Replication fork restart is abolished in Mre11 depleted extracts and is restored by supplementation with recombinant human Mre11/Rad50/Nbs1 complex. Using a novel fluorescence resonance energy transfer-based technique, we demonstrate that ATM and ATR induce Mre11/Rad50/Nbs1 complex redistribution to restarting forks. This study provides direct biochemical evidence that ATM and ATR prevent accumulation of chromosomal abnormalities by promoting Mre11/Rad50/Nbs1 dependent recovery of collapsed replication forks.

Cell-free systems derived from Xenopus eggs represent a powerful tool, intermediate between in vitro and in vivo model systems. Here, we describe protocols to prepare cell-free extracts recapitulating several aspects of the DNA damage response, including the DNA damage-dependent activation of ATM/ATR protein kinases and several DNA damage checkpoint signaling pathways that inhibit initiation of DNA replication. We provide protocols to prepare cell-free extracts, DNA templates, protein kinase substrates, and to perform checkpoint assays. In addition, we describe related methods that provide useful readouts of the DNA damage response.

Department of Genetics and Development; Columbia University; New York, NewYork USA*Correspondence to: Jean Gautier, Ph.D.; Department of Genetics and Development;Columbia University; HHSC 1602A; 701 West 168th Street; New York, New York10032 USA; Tel.: 212.305.9586; Fax: 212.923.2090; Email: jg130@columbia.eduReceived 02/06/03; Accepted 02/07/03Previously published online as a

In this work we present a review of our current research on the subject of near-surface hydrocarbon-induced magnetic contrasts. Samples are drill cuttings from number of Venezuelan and Colombian oil fields, and some soils from an oil prospective area. Results from rock magnetic, electronic paramagnetic resonance (EPR), extractable organic matter (EOM) and scanning electron microscopy (SEM) are discussed in light of new experiments. Two types of magnetic susceptibility (MS) anomalies have been recognized in oil well samples (i.e. A and B-like). A-like anomalies are probably associated to a reducing environment caused by the underlying reservoirs, whereas B-like anomalies might reflect lithological contrasts. Samples from A-like anomalous levels reveal the restrictive presence of authigenic spherical aggregates of submicronic Fe-rich magnetic crystals. Peaks of organic matter free radical concentration (OMFRC) and EOM are also observed at depth levels that lie close to those with A-like anomalies. Hysteresis ratios J rs/Js and H cr/Hc could be used as additional criteria to recognize A-like magnetic contrasts. In soil samples, MS and OMFRC anomalies coincide with peaks of ethane concentration and an area underneath where source rock has reached appropriate conditions of pressure and temperature to generate hydrocarbons (Cretaceous kitchen). Different types of crude oils and subsurface structures could have also a direct effect on the observed amplitude of A-like anomalies.

Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery., The research was funded by the grants from the Fondazione AIRC per la Ricerca sul Cancro to M.F.; grants from Danish Cancer Society, Novo Nordisk Foundation, Danish National Research Foundation (project CARD), Swedish Research Council, Grant Agency of the Czech Republic (GACR 19-21325S), and Lundbeck Foundation to J.B.; and grants from the European Research Council (ERC2014 AdG669898 TARLOOP), the Spanish Ministry of Economy and Competitiveness (BFU2016-75058-P), and European Union Regional Funds (FEDER) to A.A. Author F.G-B. was a recipient of a predoctoral training fellowship of the Spanish Ministry of Education, Culture and Sports. Work of V.C. has been supported by Associazione Italiana per la Ricerca sul Cancro, AIRC-IG Ref: 21824 and by AIRC-FIRC fellowship assigned to M.A.R.-O., Ref: 2531. M.K. has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 707600, and from the Fondazione Umberto Veronesi (Milano, Italy), Post-doctoral Fellowships 2019 and 2020.