Recognition of the polycistronic nature of human genes is critical to understanding the genotype-phenotype relationship.

Technological advances promise unprecedented opportunities for whole exome sequencing and proteomic analyses of populations. Currently, data from genome and exome sequencing or proteomic studies are searched against reference genome annotations. This provides the foundation for research and clinical screening for genetic causes of pathologies. However, current genome annotations substantially underestimate the proteomic information encoded within a gene. Numerous studies have now demonstrated the expression and function of alternative (mainly small, sometimes overlapping) ORFs within mature gene transcripts. This has important consequences for the correlation of phenotypes and genotypes. Most alternative ORFs are not yet annotated because of a lack of evidence, and this absence from databases precludes their detection by standard proteomic methods, such as mass spectrometry. Here, we demonstrate how current approaches tend to overlook alternative ORFs, hindering the discovery of new genetic drivers and fundamental research. We discuss available tools and techniques to improve identification of proteins from alternative ORFs and finally suggest a novel annotation system to permit a more complete representation of the transcriptomic and proteomic information contained within a gene. Given the crucial challenge of distinguishing functional ORFs from random ones, the suggested pipeline emphasizes both experimental data and conservation signatures. The addition of alternative ORFs in databases will render identification less serendipitous and advance the pace of research and genomic knowledge. This review highlights the urgent medical and research need to incorporate alternative ORFs in current genome annotations and thus permit their inclusion in hypotheses and models, which relate phenotypes and genotypes.

Associations of Childhood and Perinatal Blood Metals with Children's Gut Microbiomes in a Canadian Gestation Cohort.

BACKGROUND: The gut microbiome is important in modulating health in childhood. Metal exposures affect multiple health outcomes, but their ability to modify bacterial communities in children is poorly understood. OBJECTIVES: We assessed the associations of childhood and perinatal blood metal levels with childhood gut microbiome diversity, structure, species, gene family-inferred species, and potential pathway alterations. METHODS: We assessed the gut microbiome using 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing in stools collected from 6- to 7-year-old children participating in the GESTation and Environment (GESTE) cohort study. We assessed blood metal concentrations [cadmium (Cd), manganese (Mn), mercury (Hg), lead (Pb), selenium (Se)] at two time points, namely, perinatal exposures at delivery (N=70) and childhood exposures at the 6- to 7-y follow-up (N=68). We used multiple covariate-adjusted statistical models to determine microbiome associations with continuous blood metal levels, including linear regression (Shannon and Pielou alpha diversity indexes), permutational multivariate analysis of variance (adonis; beta diversity distance matrices), and multivariable association model (MaAsLin2; phylum, family, species, gene family-inferred species, and pathways). RESULTS: Children's blood Mn and Se significantly associated with microbiome phylum [e.g., Verrucomicrobiota (coef=-0.305, q=0.031; coef=0.262, q=0.084, respectively)] and children's blood Mn significantly associated with family [e.g., Eggerthellaceae (coef=-0.228, q=0.052)]-level differences. Higher relative abundance of potential pathogens (e.g., Flavonifractor plautii), beneficial species (e.g., Bifidobacterium longum, Faecalibacterium prausnitzii), and both potentially pathogenic and beneficial species (e.g., Bacteriodes vulgatus, Eubacterium rectale) inferred from gene families were associated with higher childhood or perinatal blood Cd, Hg, and Pb (q<0.1). We found significant negative associations between childhood blood Pb and acetylene degradation pathway abundance (q<0.1). Finally, neither perinatal nor childhood metal concentrations were associated with children's gut microbial inter- and intrasubject diversity. DISCUSSION: Our findings suggest both long- and short-term associations between metal exposure and the childhood gut microbiome, with stronger associations observed with more recent exposure. Future epidemiologic analyses may elucidate whether the observed changes in the microbiome relate to children's health. https://doi.org/10.1289/EHP9674.

Modeling the interactions of the nucleotide excision repair UvrA(2) dimer with DNA.

The UvrA protein initiates the DNA damage recognition process by the bacterial nucleotide excision repair (NER) system. Recently, crystallographic structures of holo-UvrA(2) dimers from two different microorganisms have been released (Protein Data Bank entries 2r6f , 2vf7 , and 2vf8 ). However, the details of the DNA binding by UvrA(2) and other peculiarities involved in the damage recognition process remain unknown. We have undertaken a molecular modeling approach to appraise the possible modes of DNA-UvrA(2) interaction using molecular docking and short-scale guided molecular dynamics [continuum field, constrained, and/or unrestricted simulated annealing (SA)], taking into account the three-dimensional location of a series of mutation-identified UvrA residues implicated in DNA binding. The molecular docking was based on the assumptions that the UvrA(2) dimer is preformed prior to DNA binding and that no major protein conformational rearrangements, except moderate domain reorientations, are required for binding of undamaged DNA. As a first approximation, DNA was treated as a rigid ligand. From the electrostatic relief of the ventral surface of UvrA(2), we initially identified three, noncollinear DNA binding paths. Each of the three resulting nucleoprotein complexes (C1, C2, and C3) was analyzed separately, including calculation of binding energies, the number and type of interaction residues (including mutated ones), and the predominant mode of translational and rotational motion of specific protein domains after SA to ensure improved DNA binding. The UvrA(2) dimer can accommodate DNA in all three orientations, albeit with different binding strengths. One of the UvrA(2)-DNA complexes (C1) fulfilled most of the requirements (high interaction energy, proximity of DNA to mutated residues, etc.) expected for a natural, high-affinity DNA binding site. This nucleoprotein presents a structural organization that is designed to clamp and bend double-stranded DNA. We examined the binding site in more detail by docking DNAs of significantly different (AT- vs CG-enriched) sequences and by submitting the complexes to DNA-unrestricted SA. It was found that in a manner independent of the DNA sequence and applied MD protocols, UvrA(2) favors binding of a bent and unwound undamaged DNA, with a kink positioned in the proximity of the Zn3 hairpins, anticollinearly aligned at the bottom of the ventral protein surface. It is further hypothesized that the Zn3 modules play an essential role in the damage recognition process and that the apparent existence of a family of DNA binding sites might be biologically relevant. Our data should prove to be useful in rational (structure-based) mutation studies.

Reaching for the other side: generating sequence-dependent interstrand cross-links with 5-bromodeoxyuridine and gamma-rays.

Interstrand cross-links impede critical cellular processes such as transcription and replication and are thus considered to be one of the most toxic types of DNA damage. Although several studies now point to the existence of gamma-radiation-induced cross-links in cellular DNA, little is known about the characteristics required for their creation. Recently, we reported the formation of interstrand cross-links that were specific for mismatched nucleotides within 5-bromo-2'-deoxyuridine-substituted DNA. Given the structural specificity for interstrand cross-link formation, it is likely that open or mismatched regions of DNA in cells may be particularly favorable for cross-link production. Herein, we investigated the effect of the local DNA sequence on the formation of interstrand cross-links, using 5-bromo-2'-deoxyuridine to generate radicals in a mismatched region of DNA. We investigated a total of 12 variations of bases in the mismatched region. The oligonucleotides were irradiated with gamma-rays, and interstrand cross-link formation was analyzed by denaturing gel electrophoresis. We found that the efficiency of cross-link formation was highly dependent on the nature of mismatched bases and, on the basis of electrophoretic mobility, observed several distinctive cross-link structures with specific DNA sequences. This study provides new insights into the reactivity of mismatched DNA and the mechanisms leading to interstrand cross-link formation. The potential application of 5-bromo-2'-deoxyuridine-induced interstrand cross-links to the field of DNA repair is discussed.

Gamma-radiation induced interstrand cross-links in PNA:DNA heteroduplexes.

Peptide nucleic acids (PNAs) efficiently hybridize with DNA and are promoted as versatile gene-targeting analytical tools and pharmaceuticals. However, PNAs have never been exploited as radiopharmaceuticals, and radiation-induced physicochemical modifications of PNA:DNA heteroduplexes have not been studied. Drug- and radiation-induced creation of covalent cross-links in DNA obstruct crucial cell survival processes such as transcription and replication and are thus considered genotoxic events with a high impact in anticancer therapies. Here we report that gamma-irradiation of complementary PNA:DNA heteroduplexes, wherein the PNA contains l-lysine, free amino, or N-methylmorpholinium N- and C-capping groups, results in the formation of irreversible interstrand cross-links (ICL). The number of detected ICL corresponds to the number of available amino functional groups on the PNA. The effect of DNA sequence on the formation of ICL was studied by modifying the terminal nucleotides of the DNA oligonucleotide to create deletions and overhangs. The involvement of abasic sites (ABS) on the DNA strand in the cross-linking reaction was confirmed by independent experiments with synthetic ABS-containing oligonucleotides. Molecular modeling and molecular dynamics (MD) simulations were applied to elucidate the conformation of the N- and C-capping groups of the PNA oligomer and their interactions with the proximal terminus of the DNA. Good agreement between experimental and modeling results was achieved. Modeling indicated that the presence of positively charged capping groups on the PNA increases the conformational flexibility of the PNA:DNA terminal base pairs and often leads to their melting. This disordered orientation of the duplex ends provides conditions for multiple encounters of the short (amino) and bulky (Lys) side chains with nucleobases and the DNA backbone up to the third base pair along the duplex stem. Dangling duplex ends offer favorable conditions for increased accessibility of the radiation-induced free radicals to terminal nucleotides and their damage. It is suggested that the ICL are produced by initial formation of Schiff base adducts between the PNA amino functions and the opposed DNA oxidation-damaged bases or abasic 2'-deoxyribose-derived aldehydic groups. The subsequent reduction by solvated electrons (e(-)(aq)) or other radiation-produced reducing species results in irreversible covalent interstrand cross-links. The simultaneous involvement of oxidizing, (*)OH, and reducing, e(-)(aq), radicals presents a case in which multiple ionization events along a gamma-particle path lead to DNA injuries that also encompass ICL as part of the multiply damaged sites (MDS). The obtained results may find applications in the development of a new generation of gene-targeted radiosensitizers based on PNA vectors.

Radiosensitization of DNA by gold nanoparticles irradiated with high-energy electrons.

Zheng, Y., Hunting, D. J., Ayotte, P. and Sanche, L. Radiosensitization of DNA by Gold Nanoparticles Irradiated with High-Energy Electrons. Radiat. Res. 168, 19-27 (2008). Thin films of pGEM-3Zf(-) plasmid DNA were bombarded by 60 keV electrons with and without gold nanoparticles. DNA single- and double-strand breaks (SSBs and DSBs) were measured by agarose gel electrophoresis. From transmission electron micrographs, the gold nanoparticles were found to be closely linked to DNA scaffolds, probably as a result of electrostatic binding. The probabilities for formation of SSBs and DSBs from exposure of 1:1 and 2:1 gold nanoparticle:plasmid mixtures to fast electrons increase by a factor of about 2.5 compared to neat DNA samples. For monolayer DNA adsorbed on a thick gold substrate, the damage increases by an order of magnitude. The results suggest that the enhancement of radiosensitivity is due to the production of additional low-energy secondary electrons caused by the increased absorption of ionizing radiation energy by the metal, in the form of gold nanoparticles or of a thick gold substrate. Since short-range low-energy secondary electrons are produced in large amounts by any type of ionizing radiation, and since on average only one gold nanoparticle per DNA molecule is needed to increase damage considerably, targeting the DNA of cancer cells with gold nanoparticles may offer a novel approach that is generally applicable to radiotherapy treatments.

Conformational rearrangement of 1,2-d(GG) intrastrand cis-diammineplatinum crosslinked DNA is driven by counter-ion penetration within the minor groove of the modified site.

The major structural aberrations of DNA induced by a cis-diammineplatinum (II) 1,2-d(GG) intrastrand cross-link (CPT) have been known for decades. To gain deeper insights into the structural dynamics of the sequence-dependent DNA distortions adjacent to the CPT adduct, we employed molecular modeling and molecular dynamics (MD) simulations. The structural dynamics of native (N-DNA) and cisPt 1,2-d(GG) crosslinked (CPT-DNA) in the form of symmetric 36 nt d(G2T15G*G*T15G2)●C2A15CCA15C2) oligonucleotide duplexes is compared. The selected sequence context enabled tracking of the origin of the DNA axis curvature at the YpR flexible points (N-DNA), the enhancement of axis bending, and further distortions due to steric/electrostatic perturbations arising from the CPT-crosslink. In addition to the known structural distortions of CPT-DNA: helix bend towards the major groove; local helix unwinding; high roll angle between cross-linked guanine bases; and adoption of A-form DNA on the 5'-side of the CPT-crosslink (TpG junction); our results show the existence of a singular irreversible and reproducible conformational rearrangement, not previously observed, resulting in two stable CPT-DNA1 and CPT-DNA2 conformers. The CPT-DNA2 conformation presents an enhanced DNA axis bend and a wider and shallower minor grove with increased solvent accessibility within the modified site. It is concluded that the polymorphous (unstable) DNA environment near the cisPt 1,2-d(GG) unit in synergy with specific dynamic events, such as prolonged minor groove retention of particular Na+ ions and water redistribution within the d(TG*G*T) site, together with the formation of extra and more stable H-bonds between Pt(NH3)2 amines and neighboring nucleotides, are cooperatively responsible for the initiation of the conformational rearrangement leading to the CPT-DNA2 conformer, which, surprisingly, closely resembles the HMGB1-bound CPT-DNA structure. Graphical abstract Superimposed averaged structures of normal (N-DNA, green) and cisplatin intrastrand cross-linked (CPT-DNA, orange). Global DNA axes: N-DNA (blue beads); CPT-DNA (red beads); PT (yellow sphere); crosslinked dGs viewed from the minor groove (bold).

Deep transcriptome annotation enables the discovery and functional characterization of cryptic small proteins.

Recent functional, proteomic and ribosome profiling studies in eukaryotes have concurrently demonstrated the translation of alternative open-reading frames (altORFs) in addition to annotated protein coding sequences (CDSs). We show that a large number of small proteins could in fact be coded by these altORFs. The putative alternative proteins translated from altORFs have orthologs in many species and contain functional domains. Evolutionary analyses indicate that altORFs often show more extreme conservation patterns than their CDSs. Thousands of alternative proteins are detected in proteomic datasets by reanalysis using a database containing predicted alternative proteins. This is illustrated with specific examples, including altMiD51, a 70 amino acid mitochondrial fission-promoting protein encoded in MiD51/Mief1/SMCR7L, a gene encoding an annotated protein promoting mitochondrial fission. Our results suggest that many genes are multicoding genes and code for a large protein and one or several small proteins.

Electron transfer in DNA duplexes containing 2-methyl-1,4-naphthoquinone.

2-methyl-1,4-naphthoquinone (menadione, MQ) was linked to synthetic oligonucleotides and exposed to near-UV light to generate base radical cations in DNA. This model system of electron transfer induced alkali-labile breaks at GG doublets, similar to anthraquinone and metallointercalators systems. In sharp contrast to other systems, the photolysis of MQ-DNA duplexes gave interstrand cross-links and alkali-labile breaks at bases on the complementary strand opposite the MQ moiety. For sequences with an internal MQ, the formation of cross-links with A and C opposite the MQ moiety was 2- to 3-fold greater than that with G and T. The yield of cross-links was more than 10-fold greater than that of breaks opposite MQ, which in turn was more than 2-fold greater than breaks at GG doublets. The yield of damage at GG doublets greatly increased for a sequence with a terminal MQ. The distribution of base damage was measured by enzymatic digestion and HPLC analysis (dAdo > dThd > dGuo > dCyd). The formation of novel products in MQ-DNA duplexes was attributed to the ability of excited MQ to generate the radical cations of all four DNA bases; thus, this photochemical reaction provides an ideal model system to study the effects of ionizing radiation and one-electron oxidants.

Maternal gut and fetal brain connection: Increased anxiety and reduced social interactions in Wistar rat offspring following peri-conceptional antibiotic exposure.

BACKGROUND: A growing body of evidence indicates that gut microbiota characteristics may be closely related to mental dysfunctions. However, no studies have investigated fetal brain development in relation to the maternal gut microbiota, despite the extensive use of antibiotics in obstetric practice. OBJECTIVE: To determine how periconceptional exposure to SuccinylSulfaThiazole (SST), a non-absorbable antibiotic, can affect behavior in rat offspring. This antibiotic drug has previously been shown to substantially perturb the gut microbiota in rats following a 28-day exposure. METHODS: Female Wistar rats were divided in two groups: control, or exposed during one month before breeding until gestational day 15 to a diet containing 1% SST. We administered behavioral tests to offspring, i.e., open field (post-natal day 20), social interactions (P25), marble burying (P30), elevated plus maze (P35), and prepulse inhibition of the acoustic startle reflex (sensory gating) (P45). RESULTS: Both male and female offspring exposed peri-conceptionally to SST showed reduced social interactions, with a decrease of about half in time spent in social interactions compared to controls, reduced exploration of the open arm by 20% in the elevated plus maze test indicating increased anxiety and altered sensorimotor gating, with a 1.5-2-fold decrease in startle inhibition. CONCLUSION: Maternal periconceptional exposure to SST provokes alterations in offspring behavior in the absence of maternal infection. Because we administered SST, a non-absorbable antibiotic, only to the dam, we conclude that these neurobehavioral alterations in the offspring are related to maternal gut microbiota alterations.

Correlation between energy deposition and molecular damage from Auger electrons: A case study of ultra-low energy (5-18 eV) electron interactions with DNA.

PURPOSE: The present study introduces a new method to establish a direct correlation between biologically related physical parameters (i.e., stopping and damaging cross sections, respectively) for an Auger-electron emitting radionuclide decaying within a target molecule (e.g., DNA), so as to evaluate the efficacy of the radionuclide at the molecular level. These parameters can be applied to the dosimetry of Auger electrons and the quantification of their biological effects, which are the main criteria to assess the therapeutic efficacy of Auger-electron emitting radionuclides. METHODS: Absorbed dose and stopping cross section for the Auger electrons of 5-18 eV emitted by(125)I within DNA were determined by developing a nanodosimetric model. The molecular damages induced by these Auger electrons were investigated by measuring damaging cross section, including that for the formation of DNA single- and double-strand breaks. Nanoscale films of pure plasmid DNA were prepared via the freeze-drying technique and subsequently irradiated with low-energy electrons at various fluences. The damaging cross sections were determined by employing a molecular survival model to the measured exposure-response curves for induction of DNA strand breaks. RESULTS: For a single decay of(125)I within DNA, the Auger electrons of 5-18 eV deposit the energies of 12.1 and 9.1 eV within a 4.2-nm(3) volume of a hydrated or dry DNA, which results in the absorbed doses of 270 and 210 kGy, respectively. DNA bases have a major contribution to the deposited energies. Ten-electronvolt and high linear energy transfer 100-eV electrons have a similar cross section for the formation of DNA double-strand break, while 100-eV electrons are twice as efficient as 10 eV in the induction of single-strand break. CONCLUSIONS: Ultra-low-energy electrons (<18 eV) substantially contribute to the absorbed dose and to the molecular damage from Auger-electron emitting radionuclides; hence, they should be considered in the dosimetry calculation of such radionuclides. Moreover, absorbed dose is not an appropriate physical parameter for nanodosimetry. Instead, stopping cross section, which describes the probability of energy deposition in a target molecule can be an appropriate nanodosimetric parameter. The stopping cross section is correlated with a damaging cross section (e.g., cross section for the double-strand break formation) to quantify the number of each specific lesion in a target molecule for each nuclear decay of a single Auger-electron emitting radionuclide.

A single subexcitation-energy electron can induce a double-strand break in DNA modified by platinum chemotherapeutic drugs.

The sensitization of malignant cells to ionizing radiation is the clinical rationale for the use of platinum-drug-based concurrent chemoradiotherapy (CCRT) for cancer treatment; however, the specific mechanisms of radiosensitization and their respective contributions still remain unknown. Biological mechanisms such as inhibition of DNA repair may contribute to the efficacy of CCRT; nevertheless, there is a dearth of information on the possible contribution of nanoscopic mechanisms to the generation of lethal DNA lesions, such as double-strand breaks (DSB). The present study demonstrates that the abundant near zero-eV (0.5 eV) electrons, created by ionizing radiation during radiotherapy, induce DSB in supercoiled plasmid DNA modified by platinum-containing anticancer drugs (Pt drugs), but not in unmodified DNA. They do so more efficiently than other types of radiation, including soft X-rays and 10 eV electrons. The formation of DSB by 0.5 eV electrons is found to be a single-hit process. These findings reveal insights into the radiosensitization mechanism of Pt drugs that can have implications for the development of optimal clinical protocols for platinum-based CCRT and the deployment of in situ sources of subexcitation-energy electrons (e.g., Auger electron-emitting radionuclides) to efficiently enhance DSB formation in DNA modified by Pt drugs in malignant cells.

Loss of cellular transformation efficiency induced by DNA irradiation with low-energy (10 eV) electrons.

Low energy electrons (LEEs) of energies less than 20 eV are generated in large quantities by ionizing radiation in biological matter. While LEEs are known to induce single (SSBs) and double strand breaks (DSBs) in DNA, their ability to inactivate cells by inducing nonreparable lethal damage has not yet been demonstrated. Here we observe the effect of LEEs on the functionality of DNA, by measuring the efficiency of transforming Escherichia coli with a [pGEM-3Zf (-)] plasmid irradiated with 10 eV electrons. Highly ordered DNA films were prepared on pyrolitic graphite by molecular self-assembly using 1,3-diaminopropane ions (Dap(2+)). The uniformity of these films permits the inactivation of approximately 50% of the plasmids compared to <10% using previous methods, which is sufficient for the subsequent determination of their functionality. Upon LEE irradiation, the fraction of functional plasmids decreased exponentially with increasing electron fluence, while LEE-induced isolated base damage, frank DSB, and non DSB-cluster damage increased linearly with fluence. While DSBs can be toxic, their levels were too low to explain the loss of plasmid functionality observed upon LEE irradiation. Similarly, non-DSB cluster damage, revealed by transforming cluster damage into DSBs by digestion with repair enzymes, also occurred relatively infrequently. The exact nature of the lethal damage remains unknown, but it is probably a form of compact cluster damage in which the lesions are too close to be revealed by purified repair enzymes. In addition, this damage is either not repaired or is misrepaired by E. coli, since it results in plasmid inactivation, when they contain an average of three lesions. Comparison with previous results from a similar experiment performed with γ-irradiated plasmids indicates that the type of clustered DNA lesions, created directly on cellular DNA by LEEs, may be more difficult to repair than those produced by other species from radiolysis.

Cisplatin enhances the formation of DNA single- and double-strand breaks by hydrated electrons and hydroxyl radicals.

The synergistic interaction of cisplatin with ionizing radiation is the clinical rationale for the treatment of several cancers including head and neck, cervical and lung cancer. The underlying molecular mechanism of the synergy has not yet been identified, although both DNA damage and repair processes are likely involved. Here, we investigate the indirect effect of γ rays on strand break formation in a supercoiled plasmid DNA (pGEM-3Zf-) covalently modified by cisplatin. The yields of single- and double-strand breaks were determined by irradiation of DNA and cisplatin/DNA samples with (60)Co γ rays under four different scavenging conditions to examine the involvement of hydrated electrons and hydroxyl radicals in inducing the DNA damage. At 5 mM tris in an N2 atmosphere, the presence of an average of two cisplatins per plasmid increased the yields of single- and double-strand breaks by factors of 1.9 and 2.2, respectively, relative to the irradiated unmodified DNA samples. Given that each plasmid of 3,200 base pairs contained an average of two cisplatins, this represents an increase in radiosensitivity of 3,200-fold on a per base pair basis. When hydrated electrons were scavenged by saturating the samples with N2O, these enhancement factors decreased to 1.5 and 1.2, respectively, for single- and double-strand breaks. When hydroxyl radicals were scavenged using 200 mM tris, the respective enhancement factors were 1.2 and 1.6 for single- and double-strand breaks, respectively. Furthermore, no enhancement in DNA damage by cisplatin was observed after scavenging both hydroxyl radicals and hydrated electrons. These findings show that hydrated electrons can induce both single- and double-strand breaks in the platinated DNA, but not in unmodified DNA. In addition, cisplatin modification is clearly an extremely efficient means of increasing the formation of both single- and double-strand breaks by the hydrated electrons and hydroxyl radicals created by ionizing radiation.

Absolute cross section for low-energy-electron damage to condensed macromolecules: a case study of DNA.

Cross sections (CSs) for the interaction of low-energy electrons (LEE) with condensed macromolecules are essential parameters for accurate modeling of radiation-induced molecular decomposition and chemical synthesis. Electron irradiation of dry nanometer-scale macromolecular solid films has often been employed to measure CSs and other quantitative parameters for LEE interactions. Since such films have thicknesses comparable with electron thermalization distances, energy deposition varies throughout the film. Moreover, charge accumulation occurring inside the films shields a proportion of the macromolecules from electron irradiation. Such effects complicate the quantitative comparison of the CSs obtained in films of different thicknesses and limit the applicability of such measurements. Here, we develop a simple mathematical model, termed the molecular survival model, that employs a CS for a particular damage process together with an attenuation length related to the total CS, to investigate how a measured CS might be expected to vary with experimental conditions. As a case study, we measure the absolute CS for the formation of DNA strand breaks (SBs) by electron irradiation at 10 and 100 eV of lyophilized plasmid DNA films with thicknesses between 10 and 30 nm. The measurements are shown to depend strongly on the thickness and charging condition of the nanometer-scale films. Such behaviors are in accord with the model and support its validity. Via this analysis, the CS obtained for SB damage is nearly independent of film thickness and charging effects. In principle, this model can be adapted to provide absolute CSs for electron-induced damage or reactions occurring in other molecular solids across a wider range of experimental conditions.

Preferential tumour accumulation of gold nanoparticles, visualised by Magnetic Resonance Imaging: radiosensitisation studies in vivo and in vitro.

PURPOSE: To investigate the radiosensitisation of gold nanoparticles (GNP) with an average diameter of 5 nm coated with the gadolinium chelating agent dithiolated diethylenetriaminepentaacetic gadolinium (Au@DTDTPA:Gd) in vitro and in mice bearing tumours (MC7-L1). MATERIALS AND METHODS: In vivo, the gadolinium chelate coating allows one to perform real-time Magnetic Resonance Imaging (MRI) pharmacokinetic analysis during intravenous infusion. Experiments were performed following treatment with 10 Gy of 150 kVp X-rays. In vitro experiments were also performed with clonogenic assays to generate dose response curves for the same cells. RESULTS: We observed a preferential accumulation of Au@DTDTPA:Gd in tumours; a substantial toxicity for tumour cells in vitro, but no obvious toxicity for mice; and the absence of a synergistic effect with Au@DTDTPA:Gd and radiation in all experiments. CONCLUSIONS: The additional absorption of radiation and the subsequent increase in secondary electrons, attributable to the presence of gold in Au@DTDTPA:Gd, does not lead to radiosensitisation. However, this chelating agent exhibits a chemotherapeutic action which warrants further investigation. When compared to positive results obtained by others on radiosensitisation by GNP, the present study suggests that the chemotherapeutic and radiosensitising properties of GNP may depend strongly on the nature of the coating.

Probing the interactions of the solvated electron with DNA by molecular dynamics simulations: II. bromodeoxyuridine-thymidine mismatched DNA.

The interaction of solvated electrons (e(-)(aq)) with DNA results in various types of DNA lesions. The in vitro and in vivo sensitisation of DNA to (e(-)(aq))-induced damage is achieved by incorporation of the electron-affinity radiosensitiser bromodeoxyuridine (BUdR) in place of thymidine. However, in DNA duplexes containing single-stranded regions (bulged BUdR-DNA), the type of lesion is different and the efficiency of damage is enhanced. In particular, DNA interstrand crosslinks (ICL) form at high efficiency in bulged DNA but are not detectable in completely duplex DNA. Knowledge about the processes and interactions leading to these differences is obscure. Previously, we addressed the problem by applying molecular modelling and molecular dynamics (MD) simulations to a system of normal (BUdR.A)-DNA and a hydrated electron, where the excess electron was modelled as a localised e(-)(H2O6) anionic cluster. The goal of the present study was to apply the same MD simulation to a wobble DNA-e(-)(aq) system, containing a pyrimidine-pyrimidine mismatched base pair, BUdR.T. The results show an overall dynamic pattern similar to that of the e(-)(aq) motion around normal DNA. However, the number of configuration states when e(-)(aq)) was particularly close to DNA is different. Moreover, in the (BUdR.T)-wobble DNA system, the electron frequently approaches the brominated strand, including BUdR, which was not observed with the normal (BUdR.A)-DNA. The structure and exchange of water at the sites of e(-)(aq) immobilisation near DNA were also characterised. The structural dynamics of the wobble DNA is prone to more extensive perturbations, including frequent formation of cross-strand (cs) interatomic contacts. The structural deviations correlated with e(-)(aq) approaching DNA from the major groove side, with sodium ions trapped deep in the minor groove. Altogether, the obtained results confirm and/or throw light on dynamic-structure determinants possibly responsible for the enhanced radiation damage of wobble DNA.

Probing the interactions of the solvated electron with DNA by molecular dynamics simulations: bromodeoxyuridine substituted DNA.

Solvated electrons ((e-)(aq)) are produced during water radiolysis and can interact with biological substrates, including DNA. To augment DNA damage, radiosensitizers such as bromo-deoxyuridine (BUdR), often referred to as an "electron affinic radiosensitizer", are incorporated in place of isosteric thymidine. However, little is known about the primary interactions of (e-)(aq) with DNA. In the present study we addressed this problem by applying molecular modeling and molecular dynamics (MD) simulations to a system of normal (BUdR.A)-DNA and a hydrated electron, where the excess electron was modeled as a localized (e-)(H2O)6 anionic cluster. Our goals were to evaluate the suitability of the MD simulations for this application; to characterize the motion of (e-)(aq) around DNA (e.g., diffusion coefficients); to identify and describe configurational states of close (e-)(aq) localization to DNA; and to evaluate the structural dynamics of DNA in the presence of (e-)(aq). The results indicate that (e-)(aq) has distinct space-preferences for forming close contacts with DNA and is more likely to interact directly with nucleotides other than BUdR. Several classes of DNA - (e-)(aq) contact sites, all within the major groove, were distinguished depending on the structure of the intermediate water layer H-bonding pattern (or its absence, i.e., a direct H-bonding of (e-)(aq) with DNA bases). Large-scale structural perturbations were identified during and after the (e-)(aq) approached the DNA from the major groove side, coupled with deeper penetration of sodium counterions in the minor groove.

Role of secondary low-energy electrons in the concomitant chemoradiation therapy of cancer.

Solid films of DNA with and without the chemotherapeutic agent cisplatin bonded to guanine were bombarded with electrons of 1, 10, 100, and 60,000 eV causing single and double strand breaks. In the presence of cisplatin this damage was increased by factors varying from 1.3 to 4.4 owing to an increase in bond dissociation triggered by the formation of transient anions. This mechanism may lie at the basis of the efficiency of concomitant cisplatin-radiation therapy.

5-Bromodeoxyuridine radiosensitization: conformation-dependent DNA damage.

DNA structure has recently emerged as one of the key factors governing the ability of 5-bromodeoxyuridine (BrdU) to radiosensitize DNA. Here, we report the dependence of the specific damage induced by BrdU for different DNA conformations. Strand breaks are specific for B-form DNA, whereas A-DNA only undergoes formation of piperidine-sensitive DNA lesions. Interstrand cross-links are only found in semi-complementary B-DNA. DNA conformation was altered by gradually rehydrating lyophilized DNA samples, which induces an A- to B-form transition. These results were also validated by irradiating DNA in solution, in the presence or absence of 80% ethanol to induce an A- or B-form, respectively. Alkali-labile DNA lesions were revealed using hot piperidine to transform both base and sugar lesions into strand breaks. We also analyzed the location of damage as a function of DNA structure: piperidine-sensitive lesions were observed exclusively at the site of BrdU substitution, whereas strand breaks were able to migrate along the DNA strand, with a clear preference for the adenine 5' of the BrdU. Thus, not only the hybridization state but also the DNA conformation affect the degree of sensitization by BrdU by influencing the amount and type of damage produced. Although clinical trials using BrdU as a radiosensitizer have been disappointing up to this point, these new findings point to several key features of BrdU radiosensitization that may alter future radiotherapeutic studies.