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The increased use of engineered nanomaterials (ENM) such as SiO2 and TiO2 in industrial products, especially in food, raises concerns with regard to their effect on human health. In particular, ENM-induced genotoxicity is crucial to investigate, since DNA damage can cause induction or promotion of carcinogenesis. However, current in vitro and in vivo nanogenotoxicological data are highly contradictory, which impedes interpretation and extrapolation. Hence, robust, reliable, and ideally scalable in vitro methods for nanogenotoxicity assessment are of great interest. This work aimed at evaluating the suitability of flow cytometry-based micronuclei scoring for reliable nanogenotoxicological assessment in human intestinal cells. Therefore, we have evaluated the genotoxicity of differently sized SiO2 and TiO2 from different sources (food-relevant, commercially available, and laboratory-synthesized) using the well-established alkaline single cell gel electrophoresis (Comet assay) and the micronucleus (MN) assay employing a flow cytometric readout. Our study demonstrates that physiologically relevant doses of several types of SiO2 and TiO2 did not cause genotoxicity, as assessed by the Comet assay, and the MN flow cytometry assay under the particular experimental conditions described. To improve data reliability, we identified ENM-induced interferences with flow cytometric scoring employing a set of interference controls, which is generally applicable for any nanomaterial and any cell line. In conclusion, flow cytometry-based MN scoring appears to be a promising methodology in nanogenotoxicity testing since data acquisition and analysis are significantly faster, highly scalable in terms of throughput, and less operator-dependent compared to the traditional microscopic evaluation. In particular, ENM-induced false-positive or false-negative results, which have not been addressed sufficiently in the literature, can be detected easily, thus enhancing data reliability.

International audience; Ionizing radiation induces genomic instability in living organisms, and several studies reported an ageing-dependent radiosensitivity. Chemical compounds, such as scavengers, radioprotectors, and modifiers, contribute to reducing the radiation-associated toxicity. These compounds are often antioxidants, and therefore, in order to be effective, they must be present before or during exposure to radiation. However, not all antioxidants provide radioprotection. In this study, we investigated the effects of procaine and of a procaine-based product Gerovital H3 (GH3) on the formation of endogenous and X-ray-induced DNA strand breaks in peripheral blood mononuclear cells (PBMCs) isolated from young and elderly individuals. Interestingly, GH3 showed the strongest radioprotective effects in PBMCs from young subjects, while procaine reduced the endogenous amount of DNA strand breaks more pronounced in aged individuals. Both procaine and GH3 inhibited lipid peroxidation, but procaine was more effective in inhibiting mitochondria free radicals' generation, while GH3 showed a higher antioxidant action on macrophage-induced low-density lipoprotein oxidation. Our findings provide new insights into the mechanisms underlying the distinct effects of procaine and GH3 on DNA damage.

Sulfur mustard (SM) is a toxicant and chemical warfare agent with strong vesicant properties. The mechanisms behind SM-induced toxicity are not fully understood and no antidote or effective therapy against SM exists. Both, the risk of SM release in asymmetric conflicts or terrorist attacks and the usage of SM-derived nitrogen mustards as cancer chemotherapeutics, render the mechanisms of mustard-induced toxicity a highly relevant research subject. Herein, we review a central role of the abundant cellular molecule nicotinamide adenine dinucleotide (NAD+) in molecular mechanisms underlying SM toxicity. We also discuss the potential beneficial effects of NAD+ precursors in counteracting SM-induced damage.

A redox steady state is important in maintaining vital cellular functions and is therefore homeostatically controlled by a number of antioxidative agents, the most important of which are enzymes. Oxidative Stress (OS) is associated with (or/and caused by) excessive production of damaging reactive oxygen and/or nitrogen species (ROS, RNS), which play a role in many pathologies. Because OS is a risk factor for many diseases, much effort (and money) is devoted to early diagnosis and treatment of OS. The desired benefit of the “identify (OS) and treat (by low molecular weight antioxidants, LMWA)” approach is to enable selective treatment of patients under OS. The present work aims at gaining understanding of the benefit of the antioxidants based on interrelationship between the concentration of different OS biomarkers and LMWA. Both the concentrations of a variety of biomarkers and of LMWA were previously determined and some analyses have been published by the MARK-AGE team. For the sake of simplicity, we assume that the concentration of an OS biomarker is a linear function of the concentration of a LMWA (if the association is due to causal relationship). A negative slope of this dependence (and sign of the correlation coefficient) can be intuitively expected for an antioxidant, a positive slope indicates that the LMWA is pro-oxidative, whereas extrapolation of the OS biomarker to [LMWA] = 0 is an approximation of the concentration of the OS biomarker in the absence of the LMWA. Using this strategy, we studied the effects of 12 LMWA (including tocopherols, carotenoids and ascorbic acid) on the OS status, as observed with 8 biomarkers of oxidative damage (including malondialdehyde, protein carbonyls, 3-nitrotyrosine). The results of this communication show that in a cross-sectional study the LMWA contribute little to the redox state and that different “antioxidants” are very different, so that single LMWA treatment of OS is not scientifically justified assuming our simple model. In view of the difficulty of quantitating the OS and the very different effects of various LMWA, the use of the “identify and treat” approach is questionable.

Increased engineered nanomaterial (ENM) production and incorporation in consumer and biomedical products has raised concerns about the potential adverse effects. The DNA damaging capacity is of particular importance since damaged genetic material can lead to carcinogenesis. Consequently, reliable and robust in vitro studies assessing ENM genotoxicity are of great value. We utilized two complementary assays based on different measurement principles: (1) comet assay and (2) FADU (fluorimetric detection of alkaline DNA unwinding) assay. Assessing cell viability ruled out false-positive results due to DNA fragmentation during cell death. Potential structure–activity relationships of 10 ENMs were investigated: three silica nanoparticles (SiO2-NP) with varying degrees of porosity, titanium dioxide (TiO2-NP), polystyrene (PS-NP), zinc oxide (ZnO-NP), gold (Au-NP), graphene oxide (GO) and two multi-walled carbon nanotubes (MWNT). SiO2-NPs, TiO2-NP and GO were neither cytotoxic nor genotoxic to Jurkat E6-I cells. Quantitative interference corrections derived from GO results can make the FADU assay a promising screening tool for a variety of ENMs. MWNT merely induced cytotoxicity, while dose- and time-dependent cytotoxicity of PS-NP was accompanied by DNA fragmentation. Hence, PS-NP served to benchmark threshold levels of cytotoxicity at which DNA fragmentation was expected. Considering all controls revealed the true genotoxicity for Au-NP and ZnO-NP at early time Points. published

Density-driven dissolution of carbon dioxide in water is a well-known and much described mechanism in geological sequestration of this greenhouse gas. It is remarkable that such enhanced dissolutio...

The level of understanding of coal combustion compared to the requirements of predictive engineering is still insufficient due to the complexity of the processes and breadth of scales involved. In particular, in modern oxy-coal firing, where the fuel is burned in an oxygen/carbon-dioxide atmosphere to reduce the emission of greenhouse gases and pollutants, both experimental investigations and detailed numerical simulations are sparse. To understand and model the phenomena involved, the community follows a stepwise approach from generic to close-to-application combustion systems by steadily increasing the size, complexity and thermal power of the investigated burners as well as the complexity of the fuel. Here, an investigation of the flue gas of an intermediate range oxy-fuel burner is presented. To simplify the fuel in a first step, the combustion of methane in air and two oxygen/carbon dioxide atmospheres (oxy-fuel) is investigated. The concentrations of multiple species as well as the path-integrated temperatures are measured using three independent spectrometer systems based on tunable diode laser absorption spectroscopy (TDLAS). The rate of burnout and fuel-slip in the flue gas is studied through measurements of the concentration of CH4, whereas a quasi-simultaneously applied OH-measurement system allows for the detection of remaining combustion zones in the flue gas. An acetylene (C2H2) measurement as a chemical precursor provides insights to the formation of soot. The O2-concentration is measured for an investigation of the oxygen-excess, and, in combination with measurements of the CO, CO2 and H2O concentration at various positions allows to detect the main components and pollutants of the oxy-fuel flue gas. An Allan-Werle variance is utilized to study the time scales occurring in the combustion process. A quasi-simultaneous measurement of the path-integrated H2O and CO2 temperature allows for investigating the progression of mixing of the different flows within the combustion chamber. This measurement reveals, that a complete mixing of all flows is only achieved close to the outlet.

Background:It remains unclear whether combination antiretroviral therapy (ART) regimens differ in their ability to fully suppress human immunodeficiency virus (HIV) replication. Here, we report the results of two cross-sectional studies that compared levels of cell-associated (CA) HIV markers between individuals receiving suppressive ART containing either a non-nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI).Methods:CA HIV unspliced RNA and total HIV DNA were quantified in two cohorts (n = 100, n = 124) of individuals treated with triple ART regimens consisting of two nucleoside reverse transcriptase inhibitors (NRTIs) plus either an NNRTI or a PI. To compare CA HIV RNA and DNA levels between the regimens, we built multivariable models adjusting for age, gender, current and nadir CD4+ count, plasma viral load zenith, duration of virological suppression, NRTI backbone composition, low-level plasma HIV RNA detectability, and electronically measured adherence to ART.Results:In both cohorts, levels of CA HIV RNA and DNA strongly correlated (rho = 0.70 and rho = 0.54) and both markers were lower in NNRTI-treated than in PI-treated individuals. In the multivariable analysis, CA RNA in both cohorts remained significantly reduced in NNRTI-treated individuals (padj = 0.02 in both cohorts), with a similar but weaker association between the ART regimen and total HIV DNA (padj = 0.048 and padj = 0.10). No differences in CA HIV RNA or DNA levels were observed between individual NNRTIs or individual PIs, but CA HIV RNA was lower in individuals treated with either nevirapine or efavirenz, compared to PI-treated individuals.Conclusions:All current classes of antiretroviral drugs only prevent infection of new cells but do not inhibit HIV RNA transcription in long-lived reservoir cells. Therefore, these differences in CA HIV RNA and DNA levels by treatment regimen suggest that NNRTIs are more potent in suppressing HIV residual replication than PIs, which may result in a smaller viral reservoir size.Funding:This work was supported by ZonMw (09120011910035) and FP7 Health (305522).

The analysis of copper (Cu) and zinc (Zn) along with their major serum carriers, albumin (Alb) and ceruloplasmin (Cp), could provide information on the capacity of humans to maintain homeostasis of metals (metallostasis). However, their relationship with aging, sex, body mass index, as well as with nutritional and inflammatory markers was never investigated in a large-scale study. Here, we report results from the European large-scale cross-sectional study MARK-AGE in which Cu, Zn, Alb, Cp, as well as nutritional and inflammatory parameters were determined in 2424 age-stratified participants (35–75 years), including the general population (RASIG), nonagenarian offspring (GO), a well-studied genetic model of longevity, and spouses of GO (SGO). In RASIG, Cu to Zn ratio and Cp to Alb ratio were higher in women than in men. Both ratios increased with aging because Cu and Cp increased and Alb and Zn decreased. Cu, Zn, Alb, and Cp were found associated with several inflammatory as well as nutritional biomarkers. GO showed higher Zn levels and higher Zn to Alb ratio compared to RASIG, but we did not observe significant differences with SGO, likely as a consequence of the low sample size of SGO and the shared environment. Our results show that aging, sex, body mass index, and GO status are characterized by different levels of Cu, Zn, and their serum carrier proteins. These data and their relationship with inflammatory biomarkers support the concept that loss of metallostasis is a characteristic of inflammaging.

There are conflicting data about localization of poly(ADP-ribose)polymerase-1 and its product poly(ADP-ribose) in mitochondria. To finally clarify the discussion, we investigated with biochemical and cell biological methods the potential presence of poly(ADP-ribose) polymerase-1 in these organelles. Our data show that endogenous and overexpressed poly(ADP-ribose)polymerase 1 is only localized to the nucleus with a clear exclusion of cytosolic compartments. In addition, highly purified mitochondria devoid of nuclear contaminations do not contain poly(ADP-ribose)polymerase-1. Although no poly(ADP-ribose)polymerase-1 enzyme is detectable in mitochondria, a shorter variant of its product poly(ADP-ribose) is present, associated specifically with a small subset of mitochondrial proteins as revealed by immunoprecipitation and protein fingerprint analysis. These proteins are located at key-points of the Krebs-cycle, are chaperones involved in mitochondrial functionality and quality-control, and are RNA-binding proteins important for transcript stability, respectively. Of note, despite the fact that especially poly(ADP-ribose)polymerase-1 is its own major target for modification, we could not detect this enzyme by mass spectrometry in these organelles. These data suggests a new way of targeted nuclear-mitochondrial signaling, mediated by nuclear poly(ADP-ribosyl)ation dependent on poly(ADP-ribose)polymerase-1. published

Wound healing is a complex process, and disturbance of even a single mechanism can result in chronic ulcers developing after exposure to the alkylating agent sulfur mustard (SM). A possible contributor may be SM-induced chronic senescent mesenchymal stem cells (MSCs), unable to fulfil their regenerative role, by persisting over long time periods and creating a proinflammatory microenvironment. Here we show that senescence induction in human bone marrow derived MSCs was time- and concentration-dependent, and chronic senescence could be verified 3 weeks after exposure to between 10 and 40 µM SM. Morphological changes, reduced clonogenic and migration potential, longer scratch closure times, differences in senescence, motility and DNA damage response associated genes as well as increased levels of proinflammatory cytokines were revealed. Selective removal of these cells by senolytic drugs, in which ABT-263 showed initial potential in vitro, opens the possibility for an innovative treatment strategy for chronic wounds, but also tumors and age-related diseases. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-020-02946-5.

The regular use of medication may interfere with micronutrient metabolism on several levels, such as absorption, turnover rate, and tissue distribution, and this might be amplified during aging. This study evaluates the impact of self-reported medication intake on plasma micronutrients in the MARK-AGE Project, a cross-sectional observational study in 2217 subjects (age- and sex-stratified) aged 35&ndash, 75 years from six European countries that were grouped according to age. Polypharmacy as possible determinant of micronutrient concentrations was assessed using multiple linear regression models adjusted for age-group, dietary fruit, vegetables, and juice intake, and other confounders. Younger participants reported taking fewer drugs than older participants. Inverse associations between medication intake and lutein (&minus, 3.31% difference per increase in medication group), &beta, carotene (&minus, 11.44%), &alpha, 8.50%) and positive associations with retinol (+2.26%), &alpha, tocopherol/cholesterol (+2.89%) and &gamma, tocopherol/cholesterol (+1.36%) occurred in multiple adjusted regression models. Combined usage of a higher number of medical drugs was associated with poorer status of carotenoids on the one hand and higher plasma concentrations of retinol, &alpha, and &gamma, tocopherol on the other hand. Our results raise concerns regarding the safety of drug combinations via the significant and surprisingly multifaceted disturbance of the concentrations of relevant micronutrients.

Background and AimWhen exceeding the homeostatic range, manganese (Mn) might cause neurotoxicity, characteristic of the pathophysiology of several neurological diseases. Although the underlying mechanism of its neurotoxicity remains unclear, Mn-induced oxidative stress contributes to disease etiology. DNA damage caused by oxidative stress may further trigger dysregulation of DNA-damage-induced poly(ADP-ribosyl)ation (PARylation), which is of central importance especially for neuronal homeostasis. Accordingly, this study was designed to assess in the genetically traceable in vivo model Caenorhabditis elegans the role of PARylation as well as the consequences of loss of pme-1 or pme-2 (orthologues of PARP1 and PARP2) in Mn-induced toxicity.MethodsA specific and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify PARylation in worms. Next to monitoring the PAR level, pme-1 and pme-2 gene expression as well as Mn-induced oxidative stress was studied in wildtype worms and the pme deletion mutants.Results and ConclusionWhile Mn failed to induce PARylation in wildtype worms, toxic doses of Mn led to PAR-induction in pme-1-deficient worms, due to an increased gene expression of pme-2 in the pme-1 deletion mutants. However, this effect could not be observed at sub-toxic Mn doses as well as upon longer incubation times. Regarding Mn-induced oxidative stress, the deletion mutants did not show hypersensitivity. Taken together, this study characterizes worms to model PAR inhibition and addresses the consequences for Mn-induced oxidative stress in genetically manipulated worms. published

Poly(ADP-ribosyl)ation regulates numerous cellular processes like genome maintenance and cell death, thus providing protective functions but also contributing to several pathological conditions. Poly(ADP-ribose) (PAR) molecules exhibit a remarkable heterogeneity in chain lengths and branching frequencies, but the biological significance of this is basically unknown. To unravel structure-specific functions of PAR, we used PARP1 mutants producing PAR of different qualities, i.e. short and hypobranched (PARP1\G972R), short and moderately hyperbranched (PARP1\Y986S), or strongly hyperbranched PAR (PARP1\Y986H). By reconstituting HeLa PARP1 knockout cells, we demonstrate that PARP1\G972R negatively affects cellular endpoints, such as viability, cell cycle progression and genotoxic stress resistance. In contrast, PARP1\Y986S elicits only mild effects, suggesting that PAR branching compensates for short polymer length. Interestingly, PARP1\Y986H exhibits moderate beneficial effects on cell physiology. Furthermore, different PARP1 mutants have distinct effects on molecular processes, such as gene expression and protein localization dynamics of PARP1 itself, and of its downstream factor XRCC1. Finally, the biological relevance of PAR branching is emphasized by the fact that branching frequencies vary considerably during different phases of the DNA damage-induced PARylation reaction and between different mouse tissues. Taken together, this study reveals that PAR branching and chain length essentially affect cellular functions, which further supports the notion of a ‘PAR code’.

Despite its worldwide ban, the warfare agent sulfur mustard (SM) still represents a realistic threat, due to potential release in terroristic attacks and asymmetric conflicts. Therefore, the rigorous and quantitative detection of SM exposure is crucial for diagnosis, health risk assessment, and surveillance of international law. Alkylation adducts of nucleic acids can serve as valuable toxicologically relevant 'biomarkers of SM exposure'. Here, we developed a robust and versatile bioanalytical platform based on isotope dilution UPLC-MS/MS to quantify major SM-induced DNA and RNA adducts, as well as adducts induced by the monofunctional mustard 2-chloroethyl ethyl sulfide. We synthesized 15N/13C-labeled standards, which allowed absolute quantitation with full chemical specificity and subfemtomole sensitivities. DNA and RNA mono-alkylation adducts and crosslinks were carefully analyzed in a dose- and time-dependent manner in various matrices, including human cancer and primary cells, derived of the main SM-target tissues. Nucleic acid adducts were detected up to 6 days post-exposure, indicating long persistence, which highlights their toxicological relevance and proves their suitability as forensic and medical biomarkers. Finally, we investigated ex vivo-treated rat skin biopsies and human blood samples, which set the basis for the implementation into the method portfolio of Organization for the Prohibition of Chemical Weapons-designated laboratories to analyze authentic samples from SM-exposed victims. published

The bi-functional chemical warfare agent sulfur mustard (SM), whose release in asymmetric conflicts or terrorist attacks represents a realistic threat, induces several kinds of biomolecular adducts, including highly toxic DNA adducts. Isotope dilution liquid chromatographic tandem mass spectrometry (ID-LC-MS/MS) is considered the gold standard for highly accurate, precise, specific and sensitive quantification of DNA adducts in general. Recently, a number of LC-MS/MS approaches have been established to analyze SM-induced protein and DNA adducts in cell culture and rodent animal models. As DNA adducts are mechanism-based biomarkers for SM exposure, results from such studies provide a deeper understanding of the etiology of SM-induced pathologies, especially of long-term effects such as cancer formation. As a result, medical treatment of SM-exposed individuals might be improved. Yet, despite the progress that has been made during the last years, there is still a need for advanced methods of ID-LC-MS/MS for the detection and quantitation of SM adducts.

Psychological stress has been associated with DNA damage, thus increasing the risk of numerous diseases including cancer. Here, we investigate the effect of acute and chronic stress on poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage and DNA repair initiator. In order to mimic the chronic release of epinephrine, human peripheral blood mononuclear cells (PBMCs) were treated repeatedly with the sympathomimetic drug isoproterenol. We found significant induction of DNA strand breaks that remained unrepaired 24 h after ex vivo incubation. Isoproterenol-induced DNA strand breaks could be partially prevented by pre-treatment with the β-adrenergic receptor antagonist propranolol. Furthermore, the level of PARP-1 protein and PARP activity decreased and the levels of the PARP substrate nicotinamide adenine dinucleotide (NAD+) and of adenosine triphosphate (ATP), necessary to replenish NAD+ pools, were lowered by isoproterenol treatment. In conclusion our data provide novel insights into the mechanisms of isoproterenol-induced genotoxicity linking β-adrenergic stimulation and PARP-1.

We present an experimental study on the effects of an oxy-fuel atmosphere with 30 %Vol. O2/70 %Vol. CO2 content on a laminar, non-premixed methane flame based on measured spatial profiles of gas temperature as well as H2O, CH4, OH and CO concentrations, with the aim of providing thermochemical validation data for numerical simulations in the oxy-fuel context. The in situ measurements were performed using temporally multiplexed laser spectrometer based on direct laser absorption spectroscopy in combination with two-line thermometry. The system is validated on a well-characterized air-blown flame at the same burner and found to be in good agreement with reference data. The different behaviors of the oxy-fuel compared to the conventional air-blown flame with respect to the flame structure, species concentrations and temperature distribution are discussed and explained by the altered thermo-physical properties of the oxy-fuel atmosphere and the reactions involved. The altered stoichiometry of the oxy-fuel flame leads to increased CO and H2O concentrations, whereas the increased heat transfer and heat capacity caused a reduction in peak flame temperature.

Poly(ADP-ribosyl)ation (PARylation) is a complex and reversible posttranslational modification catalyzed by poly(ADP-ribose)polymerases (PARPs), which orchestrates protein function and subcellular localization. The function of PARP1 in genotoxic stress response upon induction of oxidative DNA lesions and strand breaks is firmly established, but its role in the response to chemical-induced, bulky DNA adducts is understood incompletely. To address the role of PARP1 in the response to bulky DNA adducts, we treated human cancer cells with benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE), which represents the active metabolite of the environmental carcinogen benzo[a]pyrene [B(a)P], in nanomolar to low micromolar concentrations. Using a highly sensitive LC-MS/MS method, we revealed that BPDE induces cellular PAR formation in a time- and dose-dependent manner. Consistently, PARP1 activity significantly contributed to BPDE-induced genotoxic stress response. On one hand, PARP1 ablation rescued BPDE-induced NAD+ depletion and protected cells from BPDE-induced short-term toxicity. On the other hand, strong sensitization effects of PARP inhibition and PARP1 ablation were observed in long-term clonogenic survival assays. Furthermore, PARP1 ablation significantly affected BPDE-induced S- and G2-phase transitions. Together, these results point towards unresolved BPDE-DNA lesions triggering replicative stress. In line with this, BPDE exposure resulted in enhanced formation and persistence of DNA double-strand breaks in PARP1-deficient cells as evaluated by microscopic co-localization studies of 53BP1 and γH2A.X foci. Consistently, an HPRT mutation assay revealed that PARP inhibition potentiated the mutagenicity of BPDE. In conclusion, this study demonstrates a profound role of PARylation in BPDE-induced genotoxic stress response with significant functional consequences and potential relevance with regard to B[a]P-induced cancer risks. Electronic supplementary material The online version of this article (10.1007/s00204-017-2115-6) contains supplementary material, which is available to authorized users.

The recent clinical availability of the PARP inhibitor olaparib (Lynparza) opens the door for potential therapeutic repurposing for non-oncological indications. Considering (a) the preclinical efficacy data with PARP inhibitors in non-oncological diseases and (b) the risk-benefit ratio of treating patients with a compound that inhibits an enzyme that has physiological roles in the regulation of DNA repair, we have selected indications, where (a) the severity of the disease is high, (b) the available therapeutic options are limited, and (c) the duration of PARP inhibitor administration could be short, to provide first-line options for therapeutic repurposing. These indications are as follows: acute ischaemic stroke; traumatic brain injury; septic shock; acute pancreatitis; and severe asthma and severe acute lung injury. In addition, chronic, devastating diseases, where alternative therapeutic options cannot halt disease development (e.g. Parkinson's disease, progressive multiple sclerosis or severe fibrotic diseases), should also be considered. We present a preclinical and clinical action plan for the repurposing of PARP inhibitors. This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Using two tetraphenylbenzene isomers differing only by the anchoring points to the gold electrodes, we investigate the influence of quantum interference on the single molecule charge transport. The distinct anchor points are realized by selective halogen-mediated binding to the electrodes by formation of surface-stabilized isomers after iodine cleavage. Both isomers are essentially chemically identical and only weakly perturbed by the electrodes avoiding largely parasitic effects, which allows us to focus solely on the relation between quantum interference and the intrinsic molecular properties. The conductance of the two isomers differs by over 1 order of magnitude and is attributed to constructive and destructive interference. Our ab initio based transport calculations compare very well with the accompanying scanning tunneling microscope break junction measurements of the conductance. The findings are rationalized using a two level model, which shows that the interorbital coupling plays the decisive role for the interference effects.

Oxidative stress and antioxidants play a role in age-related diseases and in the aging process. We here present data on protein carbonyls, 3-nitrotyrosine, malondialdehyde, and cellular and plasma antioxidants (glutathione, cysteine, ascorbic acid, uric acid,α-tocopherol, and lycopene) and their relation with age in the European multicenter study MARK-AGE. To avoid confounding, only data from countries which recruited subjects from all three study groups (five of eight centers) and only participants aged ≥55 years were selected resulting in data from 1559 participants. These included subjects from (1) the general population, (2) members from long-living families, and (3) their spouses. In addition, 683 middle-aged reference participants (35–54 years) served as a control. After adjustment for age, BMI, smoking status, gender, and country, there were differences in protein carbonyls, malondialdehyde, 3-nitrotyrosine,α-tocopherol, cysteine, and glutathione between the 3 study groups. Protein carbonyls and 3-nitrotyrosine as well as cysteine, uric acid, and lycopene were identified as independent biomarkers with the highest correlation with age. Interestingly, from all antioxidants measured, only lycopene was lower in all aged groups and from the oxidative stress biomarkers, only 3-nitrotyrosine was increased in the descendants from long-living families compared to the middle-aged control group. We conclude that both lifestyle and genetics may be important contributors to redox biomarkers in an aging population.

In order to appraise the residence time calculation for a fluid element within a complex combustion system, which features oxy-fuel combustion, large-eddy simulations (LES) are carried out under cold flow conditions in a complex swirled flow generic laboratory-scaled combustor. The quantities, like residence time distribution, mean residence time, cumulative distribution function, variance and skewness, are used to characterize the configuration under investigation. To accurately account for the influence of the flow and to capture the tracer concentration evolution, LES are first assessed by comparison with statistical moments from experiments and by various indices of quality and error analysis. They show that the investigated configuration features a non-ideal reactor based on the flow and tracer transport. The calculated residence time distribution is analyzed and compared with experimental findings providing an estimated mean residence time of about τ=1.9 s. The derived residence time and functions are afterwards used to make predictions of tracer concentration at the reactor outlet. It turns out that such an appraised LES methodology is able to capture the residence time distribution in an accurate manner which allows its further extension to reacting conditions.

Recently, Weber et al. published a thorough investigation of the age-dependency of oxidative stress (OS) determined by the steady state concentrations of different compounds - oxidation products and antioxidants - that are in common use as biomarkers of OS in 2207 healthy individuals of the cross-sectional MARK-AGE Project. The correlations among biomarkers were significant but weak. These findings may indicate different manifestations of OS and must further be evaluated. Here, we report a refined analysis of OS based on the above-mentioned original data. We show that malondialdehyde (MDA) appears to be sensitive to both gender and age. It is significantly lower and shows a greater age-dependence in women than in men. The age-dependency of MDA in women arises in a stepwise fashion. The age-dependent slope of the steady state concentration is maximal at the age between 50 and 55 years, indicating that it may be attributed to the change of metabolism in the post-menopause. Interestingly, total glutathione (GSH) decreased with age simultaneously with the increase in MDA.Different biomarkers yield different gender- and age-dependencies. Unlike the concentration of MDA, the concentrations of the other two oxidation products, i.e. protein carbonyls and 3-nitrotyrosine were similar in men and women and appeared to be independent of age in the healthy study population. The analyzed antioxidants exhibited different gender- and age-dependencies. In conclusion, it appears that all the biomarkers assessed here reflect different types of OS and that MDA and GSH reflect the same type of OS. Keywords: Oxidative stress, Age-dependency, Gender-associated differences, Biomarkers

Due to multiple domains and in part intrinsically disordered regions, structural analyses of p53 remain a challenging task, particularly in complex with DNA and other macromolecules. Here, we applied a novel attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic approach to investigate changes in secondary structure of full-length p53 induced by non-covalent interactions with DNA and poly(ADP-ribose) (PAR). To validate our approach, we confirmed a positive regulatory function of p53’s C-terminal domain (CTD) with regard to sequence-specific DNA binding and verified that the CTD mediates p53–PAR interaction. Further, we demonstrate that DNA and PAR interactions result in distinct structural changes of p53, indicating specific binding mechanisms via different domains. A time-dependent analysis of the interplay of DNA and PAR binding to p53 revealed that PAR represents p53’s preferred binding partner, which efficiently controls p53–DNA interaction. Moreover, we provide infrared spectroscopic data on PAR pointing to the absence of regular secondary structural elements. Finally, temperature-induced melting experiments via CD spectroscopy show that DNA binding stabilizes the structure of p53, while PAR binding can shift the irreversible formation of insoluble p53 aggregates to higher temperatures. In conclusion, this study provides detailed insights into the dynamic interplay of p53 binding to DNA and PAR at a formerly inaccessible molecular level. published

DNA replication stress is a major source of genomic instability and is closely linked to tumor formation and progression. Poly(ADP-ribose)polymerases1/2 (PARP1/2) enzymes are activated in response to replication stress resulting in poly(ADP-ribose) (PAR) synthesis. PARylation plays an important role in the remodelling and repair of impaired replication forks, providing a rationale for targeting highly replicative cancer cells with PARP1/2 inhibitors. The human oncoprotein DEK is a unique, non-histone chromatin architectural protein whose deregulated expression is associated with the development of a wide variety of human cancers. Recently, we showed that DEK is a high-affinity target of PARylation and that it promotes the progression of impaired replication forks. Here, we investigated a potential functional link between PAR and DEK in the context of replication stress. Under conditions of mild replication stress induced either by topoisomerase1 inhibition with camptothecin or nucleotide depletion by hydroxyurea, we found that the effect of acute PARP1/2 inhibition on replication fork progression is dependent on DEK expression. Reducing DEK protein levels also overcomes the restart impairment of stalled forks provoked by blocking PARylation. Non-covalent DEK-PAR interaction via the central PAR-binding domain of DEK is crucial for counteracting PARP1/2 inhibition as shown for the formation of RPA positive foci in hydroxyurea treated cells. Finally, we show by iPOND and super resolved microscopy that DEK is not directly associated with the replisome since it binds to DNA at the stage of chromatin formation. Our report sheds new light on the still enigmatic molecular functions of DEK and suggests that DEK expression levels may influence the sensitivity of cancer cells to PARP1/2 inhibitors.

DNA replication stress is a major source of genomic instability and is closely linked to tumor formation and progression. Poly(ADP-ribose)polymerases1/2 (PARP1/2) enzymes are activated in response to replication stress resulting in poly(ADP-ribose) (PAR) synthesis. PARylation plays an important role in the remodelling and repair of impaired replication forks, providing a rationale for targeting highly replicative cancer cells with PARP1/2 inhibitors. The human oncoprotein DEK is a unique, non-histone chromatin architectural protein whose deregulated expression is associated with the development of a wide variety of human cancers. Recently, we showed that DEK is a high-affinity target of PARylation and that it promotes the progression of impaired replication forks. Here, we investigated a potential functional link between PAR and DEK in the context of replication stress. Under conditions of mild replication stress induced either by topoisomerase1 inhibition with camptothecin or nucleotide depletion by hydroxyurea, we found that the effect of acute PARP1/2 inhibition on replication fork progression is dependent on DEK expression. Reducing DEK protein levels also overcomes the restart impairment of stalled forks provoked by blocking PARylation. Non-covalent DEK-PAR interaction via the central PAR-binding domain of DEK is crucial for counteracting PARP1/2 inhibition as shown for the formation of RPA positive foci in hydroxyurea treated cells. Finally, we show by iPOND and super resolved microscopy that DEK is not directly associated with the replisome since it binds to DNA at the stage of chromatin formation. Our report sheds new light on the still enigmatic molecular functions of DEK and suggests that DEK expression levels may influence the sensitivity of cancer cells to PARP1/2 inhibitors. published

Doxorubicin, bleomycin, vinblastine sulfate, and dacarbazine (ABVD) is associated with severe toxicity in older patients, particularly from bleomycin-induced lung toxicity (BLT). Therefore, using bleomycin has been questioned in older Hodgkin lymphoma (HL) patients, especially in early-stage HL. We therefore analyzed feasibility, toxicity, and efficacy of ABVD or AVD in 287 older early-stage favorable HL patients. We included patients ≥60 years of age in the German Hodgkin Study Group HD10 and HD13 trials randomized to either 2 cycles of ABVD (2×ABVD; n = 137) or AVD (2×AVD; n = 82), each followed by involved-field radiotherapy (IF-RT), with patients randomized to 4×ABVD+IF-RT (n = 68). Patients' median age was 65 years (range, 60-75) with comparable patient and disease characteristics. Grade III-IV adverse event rates were similar in patients receiving 2×AVD and 2×ABVD (40% and 39%, respectively), but considerably higher in patients receiving 4×ABVD (65%). Similarly, BLT was rare in patients receiving 2×ABVD/AVD, but occurred in 7/69 (10%) of patients randomized to 4×ABVD, with 3 lethal events. In conclusion, no effects of bleomycin on toxicity rates were detectable in older patients receiving 2 cycles of chemotherapy. However, we found a high risk of severe toxicity of bleomycin in older HL patients receiving more than 2 cycles of ABVD. These trials are registered at www.clinicaltrials.gov and www.isrctn.com as #NCT00265018 (HD10) and #ISRCTN63474366 (HD13).

Halogen-free organophosphorus flame retardants are considered as replacements for the phased-out class of polybrominated diphenyl ethers (PBDEs). However, toxicological information on new flame retardants is still limited. Based on their excellent flame retardation potential, we have selected three novel 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) derivatives and assessed their toxicological profile using a battery of in vitro test systems in order to provide toxicological information before their large-scale production and use. PBDE-99, applied as a reference compound, exhibited distinct neuro-selective cytotoxicity at concentrations ≥10 µM. 6-(2-((6-oxido-6H-dibenzo[c,e][1,2]oxaphosphinin-6-yl)amino)ethoxy)-6H-dibenzo[c,e][1,2]oxaphosphinine 6-oxide (ETA-DOPO) and 6,6′-(ethane-1,2-diylbis(oxy))bis(6H-dibenzo[c,e][1,2]oxaphosphinine-6-oxide) (EG-DOPO) displayed adverse effects at concentrations >10 µM in test systems reflecting the properties of human central and peripheral nervous system neurons, as well as in a set of non-neuronal cell types. DOPO and its derivative 6,6′-(ethane-1,2-diylbis(azanediyl))bis(6H-dibenzo[c,e][1,2]oxaphosphinine-6-oxide) (EDA-DOPO) were neither neurotoxic, nor did they exhibit an influence on neural crest cell migration, or on the integrity of human skin equivalents. The two compounds furthermore displayed no inflammatory activation potential, nor did they affect algae growth or daphnia viability at concentrations ≤400 µM. Based on the superior flame retardation properties, biophysical features suited for use in polyurethane foams, and low cytotoxicity of EDA-DOPO, our results suggest that it is a candidate for the replacement of currently applied flame retardants. Electronic supplementary material The online version of this article (doi:10.1007/s00204-016-1680-4) contains supplementary material, which is available to authorized users.

About 30% of all Hodgkin lymphoma (HL) patients are ≥60 years old. As lenalidomide has promising single agent activity in multiple relapsed HL, we replaced bleomycin in ABVD with lenalidomide in this phase-I trial. Patients aged ≥60 years with early-unfavourable- or advanced-stage HL (Eastern Cooperative Oncology Group performance status ≤2, Cumulative Illness Rating Scale for Geriatrics score 0-7) received 4-8 cycles of AVD (doxorubicin, vinblastine, dacarbazine) and lenalidomide in escalation with overdose control. Dose-limiting toxicities (DLTs) included thromboembolism ≥grade 2, severe haematological toxicity, neutropenic fever and prolonged therapy delay. Twenty-five patients with a median age of 68 years were included, 68% had advanced-stage HL. A pre-defined stopping criterion for dose escalation after DLT evaluation of 20/24 patients suggested a recommended phase II dose (RPTD) of 20 mg. DLTs occurred in 10/24 evaluable patients, all treated with ≥20 mg, however, median relative dose intensity was 97% (interquartile range 49-104%). Grade 3 or higher toxicities occurred in all 22 patients at ≥20 mg lenalidomide but no treatment-related deaths occurred. Overall response rate was 80% for all patients (20/25) and 86% (19/22) at ≥20 mg lenalidomide. Three-year estimates for progression-free survival and OS were 69·7% (95% CI: 50·3-89·1%) and 83·8% (95%-CI: 69·3-98·4%), respectively. In conclusion, AVD with lenalidomide 20 mg is feasible and highly effective in older HL patients.

Objective Acute stress induces redistribution of circulating leucocytes in humans. Whereas effects on lymphocytes as adaptive immune cells are well-understood, the mechanisms underlying stress effects on granulocytes and monocytes as innate immune blood cells are still elusive. We investigated whether the stress hormone norepinephrine (NE) and α-adrenergic receptors (α-ADRs) may play a mediating role.Methods In a stress study, we cross-sectionally tested in 44 healthy men for associations between stress-induced NE increases and simultaneous granulocyte and monocyte cell count increases, as measured immediately before and several times after the Trier Social Stress Test. In a subsequent infusion study, 21 healthy men participated in three different experimental trials with sequential infusions of 1 and 15-min duration with varying substances [saline as placebo, the non-specific α-ADR blocker phentolamine (2.5mg/min), and NE (5μg/min)]: trial1=saline+saline, trial2=saline+NE, trial3=phentolamine+NE. Granulocyte and monocyte cell numbers were assessed before, immediately after, 10min, and 30min after infusion procedures.Results In the stress study, higher NE related to higher neutrophil stress changes (β=.31,p=.045, R2change=.09), but not epinephrine stress changes. In the infusion study, saline+NE induced significant increases in neutrophil (F(3/60)=43.50,p2=.69) and monocyte (F(3/60)=18.56,p2=.48) numbers compared to saline+saline. With phentolamine+NE, neutrophil (F(3/60)=14.41,p2=.42) and monocyte counts (F(2.23/44.6)=4.32,p=.016,η2=.18) remained increased compared to saline+saline but were lower compared to saline+NE (neutrophils: F(3/60)=19.55,p2=.494, monocytes: F(3/60)=2.54,p=.065,η2=.11) indicating partial mediation by α-ADRs. Trials did not differ in eosinophil and basophil count reactivity.Conclusions Our findings suggest that NE-induced immediate increases in neutrophil and monocyte numbers resemble psychosocial stress effects and can be reduced by blockade of α-ADRs. published

Metallothionein (MT) family are cysteine-rich proteins that regulate zinc (Zn) homeostasis and protect against oxidative damage. Studies in transgenic mice have shown that MT favorably influence longevity, although their role in human aging is not completely understood. Within the European multicenter study MARK-AGE, we analyzed MT induction after Zn treatment in peripheral blood mononuclear cells (PBMCs) and its relation with redox biomarkers in 2,936 age-stratified subjects (35–75 years) including the general population (RASIG), centenarian offspring (GO), and their spouses (SGO). We found that the lymphocyte capability to induce MT in response to Zn is not affected by aging. However, GO participants showed lower Zn-induced MT and increased basal expression of MT1A, MT1X, and ZnT-1 genes than RASIG subjects. Moreover, Zn-induced MT levels were found to be inversely related with oxidative stress markers (plasma protein carbonyls, 3-nitrotyrosine, and malondialdehyde) in the whole population, but not in GO subjects. In conclusion, our results support the hypothesis that the response to Zn is attenuated in PBMCs of centenarian offspring compared to the general population as a consequence of a tighter control of Zn homeostasis which is likely to provide them constant protection against stress stimuli over the whole lifespan. published

The post-translational modification poly(ADP-ribosyl)ation (PARylation) plays key roles in genome maintenance and transcription. Both non-covalent poly(ADP-ribose) binding and covalent PARylation control protein functions, however, it is unknown how the two modes of modification crosstalk mechanistically. Employing the tumor suppressor p53 as a model substrate, this study provides detailed insights into the interplay between non-covalent and covalent PARylation and unravels its functional significance in the regulation of p53. We reveal that the multifunctional C-terminal domain (CTD) of p53 acts as the central hub in the PARylation-dependent regulation of p53. Specifically, p53 bound to auto-PARylated PARP1 via highly specific non–covalent PAR-CTD interaction, which conveyed target specificity for its covalent PARylation by PARP1. Strikingly, fusing the p53-CTD to a protein that is normally not PARylated, renders this a target for covalent PARylation as well. Functional studies revealed that the p53–PAR interaction had substantial implications on molecular and cellular levels. Thus, PAR significantly influenced the complex p53–DNA binding properties and controlled p53 functions, with major implications on the p53-dependent interactome, transcription, and replication-associated recombination. Remarkably, this mechanism potentially also applies to other PARylation targets, since a bioinformatics analysis revealed that CTD-like regions are highly enriched in the PARylated proteome. published

Due to their interesting physicochemical properties, gold nanoparticles (Au-NPs) are the focus of increasing attention in the field of biomedicine and are under consideration for use in drug delivery and bioimaging, or as radiosensitizers and nano-based vaccines. Thorough evaluation of the genotoxic potential of Au-NPs is required, since damage to the genome can remain undetected in standard hazard assessments. Available genotoxicity data is either limited or contradictory. Here, we examined the influence of three surface modified 3–4 nm Au-NPs on human A549 cells, according to the reactive oxygen species (ROS) paradigm. After 24 h of Au-NP treatment, nanoparticles were taken up by cells as agglomerates; however, no influence on cell viability or inflammation was detected. No increase in ROS production was observed by H2- DCF assay; however, intracellular glutathione levels reduced over time, indicating oxidative stress. All three types of Au- NPs induced DNA damage, as detected by alkaline comet assay. The strongest genotoxic effect was observed for positively charged Au-NP I. Further analysis of Au-NP I by neutral comet assay, fluorimetric detection of alkaline DNA unwinding assay, and γH2AX staining, revealed that the induced DNA lesions were predominantly at alkali-labile sites. As highly controlled repair mechanisms have evolved to remove a wide range of DNA lesions with great efficiency, it is important to focus on both acute cyto- and genotoxicity, alongside post-treatment effects and DNA repair. We demonstrate that Au-NPinduced DNA damage is largely repaired over time, indicating that the observed damage is of transient nature. published

Background and Purpose Few neuropharmacological model systems use human neurons. Moreover, available test systems rarely reflect functional roles of co-cultured glial cells. There is no human in vitro counterpart of the widely used 1-methyl-4-phenyl-tetrahydropyridine (MPTP) mouse model of Parkinson's disease

Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project 'MARK-AGE'. The results provide evidence for an age-related decline of TET1, TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly. published

For improving the design of combustors, the knowledge of residence-time distributions (RTD) is important as they influence exhaust gas compositions. Measuring RTDs in combustors is challenging, due to high temperatures, chemical reactions, the presence of particles or corrosive species as well as high turbulence levels. This paper presents a technique for the in situ measurement of RTDs in combustors. Based on tunable diode laser absorption spectroscopy (TDLAS), the temporal evolution of the concentration of tracers is tracked simultaneously at the combustion chamber inlet and outlet. Using either air or mixtures of oxygen and carbon dioxide (oxy-fuel atmosphere) as oxidants, the method is applied to reacting and non-reacting operating conditions in a 20-kWth methane combustor. For reacting conditions, hydrogen chloride is used as a tracer, whereas for non-reacting conditions methane was chosen. Depending on the tracer, for a repetition rate of approximately 2 kHz detection limits of 16–40 ppmV are achieved. For deriving RTDs, low-pass filtering is compared to reactor network modeling. Different RTDs observed for varying operating conditions are discussed.

Poly(ADP-ribosyl)ation (PARylation), i.e., the formation of the nucleic acid-like biopolymer poly(ADP-ribose) (PAR), is an essential posttranslational modification carried out by poly(ADP-ribose) polymerases (PARPs). While PAR levels are low under physiological conditions, they can transiently increase more than 100-fold upon induction of genotoxic stress. The accurate quantitation of cellular PAR with high sensitivity is of critical importance to understand the role of PARylation in cellular physiology and pathophysiology and to determine the pharmacodynamic efficiencies of clinically relevant PARP inhibitors, which represent a novel class of promising chemotherapeutics. Previously, we have developed a bioanalytical platform based on isotope dilution mass spectrometry (LC-MS/MS) to quantify cellular PAR with unequivocal chemical specificity in absolute terms with femtomol sensitivity (Martello et al. ACS Chem Biol 8(7):1567-1575, 2013). This method enables the analysis of steady-state levels, as well as stress-induced levels of PAR in various biological systems including cell lines, mouse tissues, and primary human lymphocytes. It has a wide range of potential applications in basic research, as well as in drug development (Martello et al. ACS Chem Biol 8(7):1567-1575, 2013; Mangerich et al. Toxicol Lett 244:56-71, 2016). Here, we present an improved and adjusted version of the original protocol by Martello/Mangerich et al., which uses UPLC-MS/MS instrumentation.

NAD+ is an essential cofactor for enzymes catalyzing redox-reactions as well as an electron carrier in energy metabolism. Aside from this, NAD+ consuming enzymes like poly(ADP-ribose) polymerases and sirtuins are important regulators involved in chromatin-restructuring processes during repair and epigenetics/transcriptional adaption. In order to replenish cellular NAD+ levels after cleavage, synthesis starts from precursors such as nicotinamide, nicotinamide riboside or nicotinic acid to match the need for this essential molecule. In the present study, we investigated the impact of supplementation with nicotinic acid on resting and proliferating human mononuclear blood cells with a focus on DNA damage and repair processes.We observed that nicotinic acid supplementation increased NAD+ levels as well as DNA repair efficiency and enhanced genomic stability evaluated by micronucleus test after x-ray treatment. Interestingly, resting cells displayed lower basal levels of DNA breaks compared to proliferating cells, but break-induction rates were identical. Despite similar levels of p53 protein upregulation after irradiation, higher NAD+ concentrations led to reduced acetylation of this protein, suggesting enhanced SIRT1 activity. Our data reveal that even in normal primary human cells cellular NAD+ levels may be limiting under conditions of genotoxic stress and that boosting the NAD+ system with nicotinic acid can improve genomic stability. published

Poly(ADP-ribose) (PAR) is a complex and reversible post-translational modification that controls protein function and localization through covalent modification of, or noncovalent binding to target proteins. Previously, we and others characterized the noncovalent, high-affinity binding of the key nucleotide excision repair (NER) protein XPA to PAR. In the present study, we address the functional relevance of this interaction. First, we confirm that pharmacological inhibition of cellular poly(ADP-ribosyl)ation (PARylation) impairs NER efficacy. Second, we demonstrate that the XPA–PAR interaction is mediated by specific basic amino acids within a highly conserved PAR-binding motif, which overlaps the DNA damage-binding protein 2 (DDB2) and transcription factor II H (TFIIH) interaction domains of XPA. Third, biochemical studies reveal a mutual regulation of PARP1 and XPA functions showing that, on the one hand, the XPA–PAR interaction lowers the DNA binding affinity of XPA, whereas, on the other hand, XPA itself strongly stimulates PARP1 enzymatic activity. Fourth, microirradiation experiments in U2OS cells demonstrate that PARP inhibition alters the recruitment properties of XPA-green fluorescent protein to sites of laser-induced DNA damage. In conclusion, our results reveal that XPA and PARP1 regulate each other in a reciprocal and PAR-dependent manner, potentially acting as a fine-tuning mechanism for the spatio-temporal regulation of the two factors during NER.

Background: Previous research reveals an association between traumatic stress and an increased risk for numerous diseases, including cancer. At the molecular level, stress may increase carcinogenesis via increased DNA damage and impaired DNA repair mechanisms. We assessed DNA breakage in peripheral blood mononuclear cells from individuals with post-traumatic stress disorder (PTSD) and measured the cellular capacity to repair single-strand breaks after exposure to ionizing X-radiation. We also investigated the effect of psychotherapy on both DNA breakage and DNA repair. Methods: In a first study we investigated DNA breakage and repair in 34 individuals with PTSD and 31 controls. Controls were subdivided into 11 trauma-exposed subjects and 20 individuals without trauma exposure. In a second study, we analysed the effect of psychotherapy (Narrative Exposure Therapy) on DNA breakage and repair. Thirty-eight individuals with PTSD were randomly assigned to either a treatment or a waitlist control condition. Follow-up was performed 4 months and 1 year after therapy. Results: In study 1 we found higher levels of basal DNA breakage in individuals with PTSD and trauma-exposed subjects than in controls, indicating that traumatic stress is associated with DNA breakage. However, single-strand break repair was unimpaired in individuals with PTSD. In study 2, we found that psychotherapy reversed not only PTSD symptoms, but also DNA strand break accumulation. Conclusion: Our results show - for the first time in vivo - an association between traumatic stress and DNA breakage; they also demonstrate changes at the molecular level, i.e., the integrity of DNA, after psychotherapeutic interventions., publishedVersion

The effect of surface plasmons on the conductance of single-molecule junctions is studied using a squeezable break junction setup. We show that the conductance of 2,7-diaminofluorene single-molecule junctions can be enhanced upon laser irradiation. Our experimental approach enables us to show that this enhancement is due to the plasmon-induced oscillating field within the nanoscale metal gap of the junctions. The effective plasmon field enhancement within the gap is calculated to be ∼1000. The experimental procedure presented in this work, which enables one to explore the coupling between plasmons and molecular excitations via transport measurements, could potentially become a valuable tool in the field of plexcitonics.

Accumulation of DNA damage deriving from exogenous and endogenous sources has significant consequences for cellular survival, and is implicated in aging, cancer, and neurological diseases. Different DNA repair pathways have evolved in order to maintain genomic stability. Genetic and environmental factors are likely to influence DNA repair capacity. In order to gain more insight into the genetic and environmental contribution to the molecular basis of DNA repair, we have performed a human twin study, where we focused on the consequences of some of the most abundant types of DNA damage (single-strand breaks), and some of the most hazardous lesions (DNA double-strand breaks). DNA damage signaling response (Gamma-H2AX signaling), relative amount of endogenous damage, and DNA-strand break repair capacities were studied in peripheral blood mononuclear cells from 198 twins (94 monozygotic and 104 dizygotic). We did not detect genetic effects on the DNA-strand break variables in our study.

Poly(ADP-ribosyl)ation is an essential post-translational modification with the biopolymer poly(ADP-ribose) (PAR). The reaction is catalyzed by poly(ADP-ribose) polymerases (PARPs) and plays key roles in cellular physiology and stress response. PARP inhibitors are currently being tested in clinical cancer treatment, in combination therapy, or as monotherapeutic agents by inducing synthetic lethality. We have developed an accurate and sensitive bioanalytical platform based on isotope dilution mass spectrometry in order to quantify steady-state and stress-induced PAR levels in cells and tissues and to characterize pharmacological properties of PARP inhibitors. In contrast to existing PAR-detection techniques, the LC-MS/MS method uses authentic isotope-labeled standards, which provide unequivocal chemical specificity to quantify cellular PAR in absolute terms with femtomol sensitivity. Using this platform we analyzed steady-state levels as well as stress-induced dynamics of poly(ADP-ribosyl)ation in a series of biological systems including cancer cell lines, mouse tissues, and primary human lymphocytes. Our results demonstrate a rapid and transient stress-induced increase in PAR levels by >100-fold in a dose- and time-dependent manner with significant differences between cell types and individual human lymphocyte donors. Furthermore, ex vivo pharmacodynamic studies in human lymphocytes provide new insight into pharmacological properties of clinically relevant PARP inhibitors. Finally, we adapted the LC-MS/MS method to quantify poly(ADP-ribosyl)ation in solid tissues and identified tissue-dependent associations between PARP1 expression and PAR levels in a series of different mouse organs. In conclusion, this study demonstrates that mass spectrometric quantification of cellular poly(ADP-ribosyl)ation has a wide range of applications in basic research as well as in drug development.

Metabolism is essential to understand human health. To characterize human metabolism, a high-resolution read-out of the metabolic status under various physiological conditions, either in health or disease, is needed. Metabolomics offers an unprecedented approach for generating system-specific biochemical definitions of a human phenotype through the capture of a variety of metabolites in a single measurement. The emergence of large cohorts in clinical studies increases the demand of technologies able to analyze a large number of measurements, in an automated fashion, in the most robust way. NMR is an established metabolomics tool for obtaining metabolic phenotypes. Here, we describe the analysis of NMR-based urinary profiles for metabolic studies, challenged to a large human study (3007 samples). This method includes the acquisition of nuclear Overhauser effect spectroscopy one-dimensional and J-resolved two-dimensional (J-Res-2D) 1H NMR spectra obtained on a 600 MHz spectrometer, equipped with a 120 μL flow probe, coupled to a flow-injection analysis system, in full automation under the control of a sampler manager. Samples were acquired at a throughput of ∼20 (or 40 when J-Res-2D is included) min/sample. The associated technical analysis error over the full series of analysis is 12%, which demonstrates the robustness of the method. With the aim to describe an overall metabolomics workflow, the quantification of 36 metabolites, mainly related to central carbon metabolism and gut microbial host cometabolism, was obtained, as well as multivariate data analysis of the full spectral profiles. The metabolic read-outs generated using our analytical workflow can therefore be considered for further pathway modeling and/or biological interpretation.

Blood micronutrient status may change with age. We analyzed plasma carotenoids, α-/γ-tocopherol, and retinol and their associations with age, demographic characteristics, and dietary habits (assessed by a short food frequency questionnaire) in a cross-sectional study of 2118 women and men (age-stratified from 35 to 74 years) of the general population from six European countries. Higher age was associated with lower lycopene and α-/β-carotene and higher β-cryptoxanthin, lutein, zeaxanthin, α-/γ-tocopherol, and retinol levels. Significant correlations with age were observed for lycopene (r = -0.248), α-tocopherol (r = 0.208), α-carotene (r = -0.112), and β-cryptoxanthin (r = 0.125; all p < 0.001). Age was inversely associated with lycopene (-6.5% per five-year age increase) and this association remained in the multiple regression model with the significant predictors (covariables) being country, season, cholesterol, gender, smoking status, body mass index (BMI (kg/m²)), and dietary habits. The positive association of α-tocopherol with age remained when all covariates including cholesterol and use of vitamin supplements were included (1.7% vs. 2.4% per five-year age increase). The association of higher β-cryptoxanthin with higher age was no longer statistically significant after adjustment for fruit consumption, whereas the inverse association of α-carotene with age remained in the fully adjusted multivariable model (-4.8% vs. -3.8% per five-year age increase). We conclude from our study that age is an independent predictor of plasma lycopene, α-tocopherol, and α-carotene. published