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.

Learning from Sisyphus: time to rethink our current, ineffective strategy on neurodevelopmental environmental toxicants.

BACKGROUND: The overwhelming number of potentially toxic chemicals in consumer products and in our daily environment makes it unrealistic to carry out in-depth analyses of each product with the objective of banning and eliminating toxic chemicals from our environment. OBJECTIVES: To present the challenges that environmental toxicology and epidemiology are currently facing in the context of ubiquitous chemical pollution. DISCUSSION: We propose a realistic and pragmatic approach to this Herculean problem.

DNA-Platinum Thin Films for Use in Chemoradiation Therapy Studies.

Dry films of platinum chemotherapeutic drugs covalently bound to plasmid DNA (Pt-DNA) represent a useful experimental model to investigate direct effects of radiation on DNA in close proximity to platinum chemotherapeutic agents, a situation of considerable relevance to understand the mechanisms underlying concomitant chemoradiation therapy. In the present paper we determine the optimum conditions for preparation of Pt-DNA films for use in irradiation experiments. Incubation conditions for DNA platination reactions have a substantial effect on the structure of Pt-DNA in the films. The quantity of Pt bound to DNA as a function of incubation time and temperature is measured by inductively coupled plasma mass spectroscopy. Our experiments indicate that chemical instability and damage to DNA in Pt-DNA samples increase when DNA platination occurs at 37(°)C for 24 hours, the condition which has been extensively used for in vitro studies. Platination of DNA for the formation of Pt-DNA films is optimal at room temperature for reaction times less than 2 hours. By increasing the concentration of Pt compounds relative to DNA and thus accelerating the rate of their mutual binding, it is possible to prepare Pt-DNA samples containing known concentrations of Pt while reducing DNA degradation caused by more lengthy procedures.

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.

Mono- and tri-cationic porphyrin-monoclonal antibody conjugates: photodynamic activity and mechanism of action.

Two cationic porphyrins bearing an isothiocyanate group for conjugation to monocolonal antibodies have been synthesized. The two porphyrins conjugated efficiently to three monoclonal antibodies (anti-CD104, anti-CD146 and anti-CD326), which recognize antigens commonly over-expressed on a range of tumour cells. In vitro, all conjugates retained the phototoxicity of the porphyrin and the immunoreactivity of the antibody. Mechanistic studies showed that conjugates formed from the mono- and tri-cationic porphyrin and anti-CD104 antibody mediated apoptosis following irradiation with non-thermal red light of 630 ± 15 nm wavelength. In vivo antibody conjugates caused suppression of human LoVo tumour growth in immunodeficient NIH III mice, similar to the commercial photodynamic therapy (PDT) agent Photofrin, but at administered photosensitizer doses that were more than two orders of magnitude lower. Positron emission tomography (PET) following PDT showed a large, early increase in uptake of (18) fluorodeoxyglucose (FDG) by tumours treated with the anti-CD104 conjugates. This effect was not observed with Photofrin or with conjugates formed from the same photosensitizers conjugated to an irrelevant antibody.

On the role of low-energy electrons in the radiosensitization of DNA by gold nanoparticles.

Four different gold nanoparticle (GNP) preparations, including naked GNPs and GNPs coated either with thiolated undecane (S-C(11)H(23)), or with dithiolated diethylenetriaminepentaacetic (DTDTPA) or gadolinium (Gd) DTDTPA chelating agents, were synthesized. The average diameters, for each type of nanoparticle, are 5 nm, 10 and 13 nm, respectively. Dry films of plasmid DNA pGEM-3Zf(-), DNA with bound GNPs and DNA with coated GNPs were bombarded with 60 keV electrons. The yields of single and double strand breaks were measured as a function of exposure by electrophoresis. The binding of just one GNP without coating to DNA containing 3197 base pairs increases single and double strand breaks by a factor of 2.3 while for GNPs coated with S-C(11)H(23) this factor is reduced to 1.6. The GNPs coated with DTDTPA and DTDTPA:Gd in the same ratio with the DNA, produce essentially no increment in damage. These results could be explained by the attenuation by the coatings of the intensity of the low-energy photoelectrons emitted from the GNPs. Thus, coatings of GNPs may considerably attenuate the short-range low-energy electrons emitted from gold, leading to a considerable decrease of radiosensitization. According to our results, the highest radiosensitization should be obtained with GNPs having the shortest possible ligand, directed to the DNA of cancer cells.

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.

DNA interstrand cross-links induced by ionizing radiation: an unsung lesion.

The induction of DNA interstrand cross-links by ionizing radiation has been largely ignored in favour of studies on double-strand break formation and repair. At least part of the problem is technical; it is difficult to detect and quantify interstrand cross-links when the same agent forms both cross-links and single strand breaks because the detection of interstrand cross-links generally involves a denaturation step. Our group has studied the induction of interstrand cross-links following irradiation of DNA containing bromouracil at specific sites. We found that the formation of interstrand cross-links requires the presence of a few (3-5) mismatched bases, comprising the bromouracil. In the absence of mismatched bases, no radiation-induced cross-linking was observed; however, even in the absence of bromouracil, cross-linking still occurred, albeit at a lower efficiency. Our molecular modelling studies demonstrate that the mobility of the bases in the mismatched region is essential for the cross-linking process. Thus, our hypothesis is that ionizing radiation induces DNA interstrand cross-links in non-hybridized regions of DNA. Some obvious examples of such DNA regions are replication forks, transcription bubbles and the D-loop of telomeres. However, an abundance of studies have made it clear that there must be many single-stranded regions in the genome, such as hairpins and cruciforms. For example, alpha satellite DNA, in centromere regions of human chromosomes, forms hairpins. Thus, a variety of non-B DNA structures (hairpins, slipped DNA and tetrahelical structures) exist in the genome and should be susceptible to the formation of radiation-induced interstrand cross-links. Although interstrand cross-links have thus far been virtually ignored in radiation biology, it will be worthwhile to develop methods to detect their presence following exposure of cells to biologically relevant levels of ionizing radiation, since, on a per lesions basis, they are probably more toxic than double-strand breaks.

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.

Periconceptional folate deficiency leads to autism-like traits in Wistar rat offspring.

BACKGROUND: Folates in their role as key one carbon donors, are essential for two major pathways: the synthesis of DNA and RNA precursors and DNA methylation. A growing body of evidence from epidemiological studies indicates a possible association between nutritional and functional deficiency in folates and Autism Spectrum Disorders (ASD). However, there are no available behavioral animal studies on periconceptional one­carbon donor deficiency during gestation and the autistic phenotype. OBJECTIVE: The objective of this study was to determine if the periconceptional alteration of one­carbon metabolism induced with a folate deficient diet would affect the behaviour of rat offspring. METHODS: Female Wistar rats were divided in two groups: control (basal diet, in compliance with standards of regular laboratory diets), or exposed during one month before breeding until Gestational Day 15 to a modified diet with no added folic acid (0.2mg/kg of food), reduced choline (750mg/kg of food), and added 1% SST (a non-absorbable antibiotic used to inhibit folate synthesis by gut bacteria). We administered behavioral tests to offspring, i.e., open field (P20), social interactions (P25), marble burying (P30), elevated plus maze (P35), and prepulse inhibition of the acoustic startle reflex (sensorimotor gating) (P45). Blood homocysteine levels were used to confirm the deficit in one­carbon donors. RESULTS: Compared to controls, offspring with the periconceptional deficit in folate showed: (i) congenital body malformations; (ii) reduced social interactions, with a ~30% decrease in social sniffing behavior; (iii) reduced exploration of the open arm by 50% in the elevated plus maze test, indicating increased anxiety; (iv) a ~160% increased number of marbles buried, indicating repetitive behaviors; and (v) altered sensorimotor gating, with a global 50% decrease in startle inhibition. CONCLUSION: Maternal periconceptional deficit in folate provokes alterations in the behavior of offspring relevant to the autistic-like phenotype.

Improved assessment of sensorimotor gating in animal models relevant to ASD: A data modelling approach to quantify PrePulse Inhibition of the acoustic startle reflex.

BACKGROUND: The PrePulse Inhibition (PPI) of the acoustic startle reflex is a neurobehavioral test frequently used in neurodevelopmental studies. Most PPI studies have used rodent models of schizophrenia; however, the currently used data analysis method does not take into account the variability present in autistic preclinical models. NEW METHOD: We propose a new data modelling approach for PPI data obtained from animals exposed to valproic acid or endocrine disruptors, using mixed modeling; and a new calculation of inhibition of the acoustic startle, which takes into account the habituation phenomenon. RESULTS: Habituation, or possibly exhaustion, occurred in all groups. The classic method of calculation of inhibition analysed with ANOVA indicated no group or sex effect for the overall inhibition of startle. In contrast, when analysed using mixed models, group and sex effects were observed. In addition, using the new method of calculation, both statistical analyses showed a sex effect, with females having decreased global inhibition but no group effect. ANOVA generated more false positive results for PPI in relation to prepulse intensities. COMPARISON WITH EXISTING METHOD: The current classic method of analysis of PPI test is a calculation of inhibition based on average startle amplitude throughout the test session and a statistical ANOVA analysis. This method does not take into account habituation/exhaustion and within-subject and -group variability. CONCLUSIONS: The results of this study demonstrate that use of ANOVA analysis leads to misinterpretation of PPI data in autistic preclinical models and we propose a new data analysis adapted to these models.

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.

Enhanced DNA damage induced by secondary electron emission from a tantalum surface exposed to soft x rays.

Both thick and thin films of pGEMR-3Zf- plasmid DNA deposited on a tantalum foil were exposed to soft X rays (effective energy of 14.8 keV) for various times in air under a relative humidity of 45% (Gamma approximately 6, where Gamma is the number of water molecules per nucleotide) and 84% (Gamma approximately 21), respectively. For a thick film, the DNA damage was induced chiefly by X-ray photons. For a thin film of DNA, X-ray-induced secondary electrons emitted from the tantalum result in a substantial increase in DNA damages. Different forms of plasmid DNA were separated and quantified by agarose gel electrophoresis and laser scanning. The exposure curves for the formation of nicked circular (single-strand break, SSB), linear (double-strand break, DSB), and interduplex crosslink forms 1 and 2 were obtained for both thick and thin films of DNA. The secondary electron enhancement factor for SSBs, DSBs and crosslinks of the thin film of DNA were derived to be 3.8 +/- 0.5, 2.9 +/- 0.7 and 7 +/- 3 at Gamma approximately 6 and 6.0 +/- 0.8, 7 +/- 1 and 3.9 +/- 0.9 at Gamma approximately 21, respectively. This study provides a molecular basis for understanding the enhanced biological effects at interfaces during diagnostic X-ray examination and radiotherapy.

Effective cross sections for production of single-strand breaks in plasmid DNA by 0.1 to 4.7 eV electrons.

We determined effective cross sections for production of single-strand breaks (SSBs) in plasmid DNA [pGEM 3Zf(-)] by electrons of 10 eV and energies between 0.1 and 4.7 eV. After purification and lyophilization on a chemically clean tantalum foil, dry plasmid DNA samples were transferred into a high-vacuum chamber and bombarded by a monoenergetic electron beam. The amount of the circular relaxed DNA in the samples was separated from undamaged molecules and quantified using agarose gel electrophoresis. The effective cross sections were derived from the slope of the yield as a function of exposure and had values in the range of 10(-15)- 10(-14) cm2, giving an effective cross section of the order of 10(-18) cm2 per nucleotide. Their strong variation with incident electron energy and the resonant enhancement at 1 eV suggest that considerable damage is inflicted by very low-energy electrons to DNA, and it indicates the important role of pi* shape resonances in the bond-breaking process. Furthermore, the fact that the energy threshold for SSB production is practically zero implies that the sensitivity of DNA to electron impact is universal and is not limited to any particular energy range.

Photodynamic activity of substituted zinc trisulfophthalocyanines: role of plasma membrane damage.

We recently reported that variations in cellular phototoxicity among a series of alkynyl-substituted zinc trisulfophthalocyanines (ZnPcS3Cn) correlates with their hydrophobicity, with the most amphiphilic derivatives showing the highest cell uptake and phototoxicity. In this study we address the role of the plasma membrane in the photodynamic response as it relates to the overall hydrophobicity of the photosensitizer. The membrane tracker dye 1-[4(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), which is incorporated into plasma membranes by endocytosis, was used to establish plasma membrane uptake by EMT-6 cells of the ZnPcS3C, by colocalization, and TMA-DPH membrane uptake rates after photodynamic therapy were used to quantify membrane damage. TMA-DPH colocalization patterns show plasma membrane uptake of the photosensitizers after short 1 h incubation periods. TMA-DPH plasma membrane uptake rates after illumination of the photosensitizer-treated cells show a parabolic relationship with photosensitizer hydrophobicity that correlates well with the phototoxicity of the ZnPcS3C,. After a 1 h incubation period, overall phototoxicity correlates closely with the postillumination rate of TMA-DPH incorporation into the cell membrane, suggesting a major role of plasma membrane damage in the overall PDT effect. In contrast, after a 24 h incubation, phototoxicity shows a stronger but imperfect correlation with total cellular photosensitizer uptake rather than TMA-DPH membrane uptake, suggesting a partial shift in the cellular damage responsible for photosensitization from the plasma membrane to intracellular targets. We conclude that plasma membrane localization of the amphiphilic ZnPcS3C6-C9 is a major factor in their overall photodynamic activity.

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.

Turing Revisited: Decoding the microRNA Messages in Brain Extracellular Vesicles for Early Detection of Neurodevelopmental Disorders.

The prevention of neurodevelopmental disorders (NDD) of prenatal origin suffers from the lack of objective tools for early detection of susceptible individuals and the long time lag, usually in years, between the neurotoxic exposure and the diagnosis of mental dysfunction. Human data on the effects of alcohol, lead, and mercury and experimental data from animals on developmental neurotoxins and their long-term behavioral effects have achieved a critical mass, leading to the concept of the Developmental Origin of Health and Disease (DOHaD). However, there is currently no way to evaluate the degree of brain damage early after birth. We propose that extracellular vesicles (EVs) and particularly exosomes, released by brain cells into the fetal blood, may offer us a non-invasive means of assessing brain damage by neurotoxins. We are inspired by the strategy applied by Alan Turing (a cryptanalyst working for the British government), who created a first computer to decrypt German intelligence communications during World War II. Given the growing evidence that microRNAs (miRNAs), which are among the molecules carried by EVs, are involved in cell-cell communication, we propose that decrypting messages from EVs can allow us to detect damage thus offering an opportunity to cure, reverse, or prevent the development of NDD. This review summarizes recent findings on miRNAs associated with selected environmental toxicants known to be involved in the pathophysiology of NDD.