Abstract  OBJECTIVE: To establish a method for determination 1-bromopropane (1-BP) in workplace air by gas chromatography-flame ionization detector (GC-FID).

METHOD: 1-bromopropane in workplace air was collected with activated charcoal tube, then desorbed by carbon disulfide and determined by GC-FID. 1-bromopropane was quantitatively measured using retention time and peak area.

RESULTS: Linear regression formula was Y = 3353.4x-10064 in a range of 2.50 ∼ 500.00 µg/ml with regression coefficient R = 0.9998. Detection limit was 0.25 µg/ml and the lowest detection concentration of 1-brmopropane in air was 0.14 mg/m(3) (at air volume 1.8L). The mean recoveries of 1-BP were between 96.8% and 102.6%, and relative standard deviation of inter and intra-assay was less than 10%. The average desorption efficiencies were between 93.2% and 104.4%. The samples in activated charcoal tube could be stably stored for 5 days at room temperature.

CONCLUSION: The method could be feasible to determine 1-bromopropane in workplace air.

Abstract  The purposes of the present study are (1) to develop a sensitive analytical method to measure 1-bromopropane (1-BP) in urine, (2) to examine if 1-BP or bromide ion (Br) in urine is a useful biomarker of exposure to 1-BP, and (3) to identify the lowest 1-BP exposure concentration the method thus established can biomonitor. A factory survey was carried out on Friday, and 33 workers (all men) in cleaning and painting workshops participated; each worker was equipped with a diffusive sampler (carbon cloth KF-1500 as an adsorbent) to monitor 1-BP vapour for an 8-h shift, and offered a urine sample at the end of the shift for measurement of 1-BP and Br in urine. In addition, 10 non-exposed men offered urine samples as controls. The performance of the carbon cloth diffusive sampler was examined to confirm that the sampler is suitable for monitoring time-weighted average 1-BP vapour exposure. A head-space GC technique was employed for analysis of 1-BP in urine, whereas Br in urine was analysed by ECD-GC after derivatization to methyl bromide. The workers were exposed to vapours of seven other solvents (i.e. toluene, xylenes, ethylbenzene, acetone, etc.) in addition to 1-BP vapour; the 1-BP vapour concentration was 1.4 ppm as GM and 28 ppm as the maximum. Multiple regression analysis however showed that 1-BP was the only variable that influenced urinary 1-BP significantly. There was a close correlation between 1-BP in urine and 1-BP in air; the correlation coefficient (r) was >0.9 with a narrow variation range, and the regression line passed very close to the origin so that 2 ppm 1-BP exposure can be readily biomonitored. The correlation of Br in urine with 1-BP in air was also significant, but the r (about 0.7) was smaller than that for 1-BP, and the background Br level was also substantial (about 8 mg l-1). Thus, it was concluded that 1-BP in end-of-shift urine is a reliable biomarker of occupational exposure to 1-BP vapour, and that Br in urine is less reliable.

Abstract  Ethylene oxide (EO) and propylene oxide (PO) are direct acting mutagens with high Swain-Scott s-values, which indicate that they react preferentially with ring nitrogens in the DNA. We have previously described that in the X-linked recessive lethal (RL) assay in Drosophila postmeiotic male germ cells EO is, per unit exposure dose, 5-10 times more mutagenic than PO. Furthermore, at the higher dose range of EO tested, 62.5-1000 ppm, up to 20-fold enhanced mutation rates were measured in the absence of maternal nucleotide excision repair (NER) compared to repair proficient conditions. The lower dose range of EO tested, 2-7.8 ppm, still produced a small increased mutation rate but without a significant elevated effect when the NER system is being suppressed. The lowest dose of PO tested, 15.6 ppm, produced only in NER- condition an increased mutation rate. The aim of the present study was to compare the mutagenic effect of EO and PO in the RL assay under XPG proficient and deficient conditions with the formation of N-7-(2-hydroxyethyl)guanine (7-HEG) and N-7-(2-hydroxypropyl)guanine (7-HPG), respectively, the major DNA adducts formed. The formation of 7-HEG and 7-HPG was investigated in Drosophila males exposed to EO and PO as a measure of internal dose for exposures ranging from 2 to 1000 or 2000 ppm, respectively, for 24h. Analysis of 7-HEG and 7-HPG, using a highly sensitive 32P-postlabelling assay, showed a linear increase of adduct levels over the entire dose range. The non-linear dose-response relationship for mutations could therefore not be explained by a reduced inhalation or increased detoxification at higher exposure levels. In analogy with the four times higher reactivity of EO the level of N-7-guanine alkylation per ppm was for EO 3.5-fold higher than that for PO. Per unit N-7-guanine alkylation EO was found to be slightly more mutagenic than PO, whereas PO was the more potent clastogenic agent. While this research has not identified the DNA lesions that cause the increase in repair deficient flies, it supports the hypothesis that efficient error-free repair of some N-alkylation products can explain why these agents tend to be weakly genotoxic or even inactive in repair-competent (premeiotic) germ cells of the mouse and the Drosophila fly.

Abstract  Two mononuclear Cu(I) complexes based on 2-(2-pyridyl)benzimidazolyl derivative ligand containing electron-transporting 1,3,4-oxadiazole group (L), [Cu(L)(PPh(3))2](BF(4)) and [Cu(L)(DPEphos)](BF(4)), where L = 1-(4-(5-(4-tert-butylphenyl)-1,3,4-oxadiazol-2-yl)benzyl)-2-(pyridin-2-yl)benzimidazole and DPEphos = bis[2-(diphenylphosphino)phenyl]ether, have been successfully synthesized and characterized. The X-ray crystal structure analyses of the ligand L and the complex [Cu(L)(PPh(3))2](BF(4)) were described. The photophysical properties of the complexes were examined by using UV-vis, photoluminescence spectroscopic analysis. The doped light-emitting devices using the Cu(I) complexes as dopants were fabricated. With no electron transporting layers employed in the devices, yellow electroluminescence from Cu(I) complexes were observed. The devices based on the complex [Cu(L)(DPEphos)](BF4) possess better performance as compared with the devices fabricated by the complex [Cu(L)(PPh(3))2](BF(4)). The devices with the structure of ITO/MoO(3) (2 nm)/NPB (40 nm)/CBP:[Cu(L)(DPEphos)](BF(4)) (8 wt%, 30 nm)/BCP (30 nm)/LiF (1 nm)/Al (150 nm) exhibit a maximum efficiency of 3.04 cd/A and a maximum brightness of 4,758 cd/m(2).

Abstract  The in vitro metabolism of 1-propyl halides (chloride, bromide, and iodide) by hepatic microsomes from phenobarbital-induced rats was examined. The following metabolites were detected: propene, 1,2-epoxypropane, 1,2-propanediol, propionic acid, and undefined species bound to protein (for propyl chloride). The addition of exogenous glutathione to the incubation mixture led to the production of S-(1'-propyl)glutathione and S-(2'-hydroxy-1'-propyl)glutathione. The ratio of the metabolites resulting from C1-C2 functionalization [propene, 1,2-propanediol, and S-(2'-hydroxy-1'-propyl)glutathione] to that resulting from C1 functionalization (propionic acid) increased as the halide progressed down the halide order chloride bromide, and iodide. Mechanisms which rationalize the distribution of propyl halide metabolites as a function of the halide are discussed. The preferred mechanism interprets that the results obtained are a consequence of the partitioning of the initial metabolic transformation between alpha-hydroxylation and halogen oxygenation pathways.

Abstract  Many states are considering, and some states have actively pursued, banning the use of perchloroethylene (PERC) in dry cleaning establishments. Proposed legislation has led many dry cleaners to consider the use of products that contain greater than 90% n-propyl bromide (n-PB; also called 1-bromopropane or 1-BP). Very little information is known about toxicity and exposure to n-PB. Some n-PB-containing products are marketed as nonhazardous and "green" or "organic." This has resulted in some users perceiving the solvent as nontoxic and has resulted in at least one significant poisoning incident in New Jersey. In addition, many dry cleaning operators may not realize that the machine components and settings must be changed when converting from PERC to n-PB containing products. Not performing these modifications may result in overheating and significant leaks in the dry cleaning equipment. A preliminary investigation was conducted of the potential exposures to n-PB and isopropyl bromide (iso-PB; also called 2-bromopropane or 2-BP) among dry cleaners in New Jersey who have converted their machines from PERC to these new solvent products. Personal breathing zone and area samples were collected using the National Institute for Occupational Safety and Health Sampling and Analytical Method 1025, with a slight modification to gas chromatography conditions to facilitate better separation of n-PB from iso-PB. During the preliminary investigation, exposures to n-PB among some workers in two of three shops were measured that were greater than the American Conference of Governmental Industrial Hygienists (ACGIH) threshold limit value (TLV) for n-PB. The highest exposure measured among a dry cleaning machine operator was 54 parts per million (ppm) as an 8-hr time-weighted average, which is more than 5 times the ACGIH TLV of 10 ppm. The preliminary investigation also found that the work tasks most likely to result in the highest short-term exposures included the introduction of solvent to the machine, maintenance of the machine, unloading and handling of recently cleaned clothes, and interrupting the wash cycle of the machine. In addition, this assessment suggested that leaks may have contributed to exposure and may have resulted from normal machine wear over time, ineffective maintenance, and from the incompatibility of n-PB with gasket materials.

Abstract  nfxB encodes a negative regulator of the mexCD-oprJ genes for drug efflux in the opportunistic pathogen Pseudomonas aeruginosa. Inactivating mutations in this transcriptional regulator constitute one of the main mechanisms of resistance to ciprofloxacin (Cip(r)). In this work, we evaluated the use of nfxB/Cip(r) as a new test system to study mutation spectra in P. aeruginosa. The analysis of 240 mutations in nfxB occurring spontaneously in the wild-type and mutator backgrounds or induced by mutagens showed that nfxB/Cip(r) offers several advantages compared with other mutation detection systems. Identification of nfxB mutations was easy since the entire open reading frame and its promoter region were sequenced from the chromosome using a single primer. Mutations detected in nfxB included all transitions and transversions, 1-bp deletions and insertions, >1-bp deletions and duplications. The broad mutation spectrum observed in nfxB relies on the selection of loss-of-function changes, as we confirmed by generating a structural model of the NfxB repressor and evaluating the significance of each detected mutation. The mutation spectra characterized in the mutS, mutT, mutY and mutM mutator backgrounds or induced by the mutagenic agents 2-aminopurine, cisplatin and hydrogen peroxide were in agreement with their predicted mutational specificities. Additionally, this system allowed the analysis of sequence context effects since point mutations occurred at 85 different sites distributed over the entire nfxB. Significant hotspots and preferred sequence contexts were observed for spontaneous and mutagen-induced mutation spectra. Finally, we demonstrated the utility of a luminescence-based reporter for identification of nfxB mutants previous to sequencing analysis. Thus, the nfxB/Cip(r) system in combination with the luminescent reporter may be a valuable tool for studying mutational processes in Pseudomonas spp. wherein the genes encoding the NfxB repressor and the associated efflux pump are conserved.

Abstract  Radiation-induced bystander effect (RIBE) has been proposed to have tight relationship with the irradiation-caused secondary cancers beyond the irradiation-treated area after radiotherapy. Our previous studies demonstrated a protective effect of low concentration carbon monoxide (CO) on the genotoxicity of RIBE after α-particle irradiation. In the present work, a significant inhibitory effect of low-dose exogenous CO, generated by tricarbonyldichlororuthenium (II) dimer [CO-releasing molecule (CORM-2)], on both RIBE-induced proliferation and chromosome aberration was observed. Further studies on the mechanism revealed that the transforming growth factor β1/nitric oxide (NO) signaling pathway, which mediated RIBE signaling transduction, could be modulated by CO involved in the protective effects. Considering the potential of exogenous CO in clinical applications and its protective effect on RIBE, the present work aims to provide a foundation for potential application of CO in radiotherapy.

Abstract  In the Upper Valley of Río Negro and Río Neuquén in Argentina, agriculture represents the second most important economic activity. Azinphos-methyl has been found in water from this region throughout the year at a maximum concentration of 22.48 μg L(-1) during the application period. Toxicological studies on local non-target species have been performed mostly on vertebrates, while mollusks, which could be more sensitive, have not been studied so far. This work aims to characterize cholinesterase (ChE) and carboxilesterase (CE) activities of Chilina gibbosa, a freshwater gastropod native to southern Argentina and Chile. These enzymes, together with neurotoxicity signals, are evaluated herein after as sensitive biomarkers of exposure to azinphos-methyl at environmentally relevant concentrations. Effects of azinphos-methyl on antioxidant defenses: glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) are also studied in order to complete a set of biomarkers with different sensitivity and specificity, to propose C. gibbosa as a sentinel species. The highest specific activity was obtained with acetylthiocholine as substrate, followed by propionylthiocholine (83% in comparison to acetylthiocholine) and butyrylthiocholine (19%).The lowest Km and the highest efficiency for ChE were obtained with acetylthiocholine. Regarding CEs activities, a higher efficiency was obtained with p-nitrophenyl butyrate than with p-nitrophenyl acetate. Eserine produced significant inhibition of ChE activity (81% with 0.001 mM and 98% with 1mM) while iso-OMPA did not produce any significant effect on ChE. Our results show that C. gibbosa ChE is very sensitive to azinphos-methyl (CI50 0.02 μg L(-1)) while CEs are inhibited at higher concentrations (CI50 1,000 μg L(-1)). CEs have been reported to be more sensitive to OPs than ChEs in most of the aquatic invertebrates protecting the organisms from neurotoxic effects. In contrast, C. gibbosa, has ChE which are much more sensitive to azinphos-methyl than CEs and shows marked signs of neurotoxicity. Regarding antioxidant defenses, GSH levels were significantly increased by 0.02 and 20 μg L(-1) azinphos-methyl (80 and 103%, respectively), CAT activity was increased 85% only at 0.02 μg L(-1) and SOD and GST did not show any significant response. Since ChE activity, neurotoxicity signs, GSH and CAT are sensitive biomarkers of acute exposure to azinphos-methyl at environmental concentrations C. gibbosa could be included as sentinel species in monitoring programs of pesticide hazard in regions of Argentina and Chile.

Abstract  While scientific knowledge of the potential health significance of chemical exposures has grown, experimental methods for predicting the carcinogenicity of environmental agents have not been substantially updated in the last two decades. Current methodologies focus first on identifying genotoxicants under the premise that agents capable of directly damaging DNA are most likely to be carcinogenic to humans. Emphasis on the distinction between genotoxic and non-genotoxic carcinogens is also motivated by assumed implications for the dose-response curve; it is purported that genotoxicants would lack a threshold in the low dose region, in contrast to non-genotoxic agents. However, for the vast majority of carcinogens, little if any empirical data exist to clarify the nature of the cancer dose-response relationship at low doses in the exposed human population. Recent advances in scientific understanding of cancer biology-and increased appreciation of the multiple impacts of carcinogens on this disease process-support the view that environmental chemicals can act through multiple toxicity pathways, modes and/or mechanisms of action to induce cancer and other adverse health outcomes. Moreover, the relationship between dose and a particular outcome in an individual could take multiple forms depending on genetic background, target tissue, internal dose and other factors besides mechanisms or modes of action: inter-individual variability and susceptibility in response are, in turn, key determinants of the population dose-response curve. New bioanalytical approaches (e.g., transcriptomics, proteomics, and metabolomics) applied in human, animal and in vitro studies could better characterize a wider array of hazard traits and improve the ability to predict the potential carcinogenicity of chemicals. Published by Elsevier B.V

Abstract  Locked nucleic acid (LNA) is a modified RNA nucleotide that can be incorporated at specific positions to generate probes with the desired length, melting temperature (TM), and specificity. Here, we describe a method of multiplex genotyping based on dramatic shifts in the TM of a single dual-labeled LNA probe. Using this method, two varieties of the hairtail fish Trichiurus lepturus can be distinguished from each other, as well as from Trichiurus japonicus, based on a 1- to 2-bp difference in a fragment of mitochondrial cytochrome oxidase subunit 1. The shift in TM was 15 °C for a 1-bp mismatch and 27 °C for a 2-bp mismatch, indicating remarkable specificity. We anticipate that the method will be widely useful in applications such as species identification that require accurate, multiplex, and efficient detection of DNA polymorphisms.

Abstract  BACKGROUND: Ralstonia solanacearum is a vascular soil-borne plant pathogen with an unusually broad host range. This economically destructive and globally distributed bacterium has thousands of distinct lineages within a heterogeneous and taxonomically disputed species complex. Some lineages include highly host-adapted strains (ecotypes), such as the banana Moko disease-causing strains, the cold-tolerant potato brown rot strains (also known as R3bv2) and the recently emerged Not Pathogenic to Banana (NPB) strains.

RESULTS: These distinct ecotypes offer a robust model to study host adaptation and the emergence of ecotypes because the polyphyletic Moko strains include lineages that are phylogenetically close to the monophyletic brown rot and NPB strains. Draft genomes of eight new strains belonging to these three model ecotypes were produced to complement the eleven publicly available R. solanacearum genomes. Using a suite of bioinformatics methods, we searched for genetic and evolutionary features that distinguish ecotypes and propose specific hypotheses concerning mechanisms of host adaptation in the R. solanacearum species complex. Genome-wide, few differences were identified, but gene loss events, non-synonymous polymorphisms, and horizontal gene transfer were identified among type III effectors and were associated with host range differences.

CONCLUSIONS: This extensive comparative genomics analysis uncovered relatively few divergent features among closely related strains with contrasting biological characteristics; however, several virulence factors were associated with the emergence of Moko, NPB and brown rot and could explain host adaptation.

Abstract  KEY MESSAGE: Altered starch quality with full knockout of GBSS gene function in potato was achieved using CRISPR-Cas9 technology, through transient transfection and regeneration from isolated protoplasts. Site-directed mutagenesis (SDM) has shown great progress in introducing precisely targeted mutations. Engineered CRISPR-Cas9 has received increased focus compared to other SDM techniques, since the method is easily adapted to different targets. Here, we demonstrate that transient application of CRISPR-Cas9-mediated genome editing in protoplasts of tetraploid potato (Solanum tuberosum) yielded mutations in all four alleles in a single transfection, in up to 2 % of regenerated lines. Three different regions of the gene encoding granule-bound starch synthase (GBSS) were targeted under different experimental setups, resulting in mutations in at least one allele in 2-12 % of regenerated shoots, with multiple alleles mutated in up to 67 % of confirmed mutated lines. Most mutations resulted in small indels of 1-10 bp, but also vector DNA inserts of 34-236 bp were found in 10 % of analysed lines. No mutations were found in an allele diverging one bp from a used guide sequence, verifying similar results found in other plants that high homology between guide sequence and target region near the protospacer adjacent motif (PAM) site is essential. To meet the challenge of screening large numbers of lines, a PCR-based high-resolution fragment analysis method (HRFA) was used, enabling identification of multiple mutated alleles with a resolution limit of 1 bp. Full knockout of GBSS enzyme activity was confirmed in four-allele mutated lines by phenotypic studies of starch. One remaining wild-type (WT) allele was shown sufficient to maintain enough GBSS enzyme activity to produce significant amounts of amylose.

Abstract  Microspores can be induced to develop homozygous doubled haploid plants in a single generation. In the present experiments androgenic microspores of wheat have been genetically transformed and developed into mature homozygous transgenic plants. Two different transformation techniques were investigated, one employing electroporation and the other co-cultivation with Agrobacterium tumefaciens. Different tissue culture and transfection conditions were tested on nine different wheat cultivars using four different constructs. A total of 19 fertile transformants in five genotypes from four market classes of common wheat were recovered by the two procedures. PCR followed by DNA sequencing of the products, Southern blot analyses and bio/histo-chemical and histological assays of the recombinant enzymes confirmed the presence of the transgenes in the T0 transformants and their stable inheritance in homozygous T1∶2 doubled haploid progenies. Several decisive factors determining the transformation and regeneration efficiency with the two procedures were determined: (i) pretreatment of immature spikes with CuSO4 solution (500 mg/L) at 4°C for 10 days; (ii) electroporation of plasmid DNA in enlarged microspores by a single pulse of ∼375 V; (iii) induction of microspores after transfection at 28°C in NPB-99 medium and regeneration at 26°C in MMS5 medium; (iv) co-cultivation with Agrobacterium AGL-1 cells for transfer of plasmid T-DNA into microspores at day 0 for <24 hours; and (v) elimination of AGL-1 cells after co-cultivation with timentin (200-400 mg/L).

Abstract  We present a study of five children from three unrelated Irish Traveller families presenting with primary ciliary dyskinesia (PCD). As previously characterized disorders in the Irish Traveller population are caused by common homozygous mutations, we hypothesised that all three PCD families shared the same recessive mutation. However, exome sequencing showed that there was no pathogenic homozygous mutation common to all families. This finding was supported by histology, which showed that each family has a different type of ciliary defect; transposition defect (family A), nude epithelium (family B) and absence of inner and outer dynein arms (family C). Therefore, each family was analysed independently using homozygosity mapping and exome sequencing. The affected siblings in family A share a novel 1 bp duplication in RSPH4A (NM_001161664.1:c.166dup; p.Arg56Profs*11), a radial-spoke head protein involved in ciliary movement. In family B, we identified three candidate genes (CCNO, KCNN3 and CDKN1C), with a 5-bp duplication in CCNO (NM_021147.3:c.258_262dup; p.Gln88Argfs*8) being the most likely cause of ciliary aplasia. This is the first study to implicate CCNO, a DNA repair gene reported to be involved in multiciliogenesis, in PCD. In family C, we identified a ∼3.5-kb deletion in DYX1C1, a neuronal migration gene previously associated with PCD. This is the first report of a disorder in the relatively small Irish Traveller population to be caused by >1 disease gene. Our study identified at least three different PCD genes in the Irish Traveller population, highlighting that one cannot always assume genetic homogeneity, even in small consanguineous populations.

Abstract  Mutation and selection are both thought to impact significantly the nucleotide composition of bacterial genomes. Earlier studies have compared closely related strains to obtain mutation patterns based on the hypothesis that these bacterial strains had diverged so recently that selection will not have had enough time to play its role. In this study, we used a SOLiD autosequencer that was based on a dual-base encoding scheme to sequence the genome of Staphylococcus aureus with a mapping coverage of over 5,000×. By directly counting the variation obtained from these ultradeep sequencing reads, we found that A → G was the predominant single-base substitution and 1 bp deletions were the major small indel. These patterns are completely different from those obtained by comparison of closely related S. aureus strains, where C → T accounted for a larger proportion of mutations and deletions were shown to occur at an almost equal frequency to insertion. These findings suggest that the genomic differences between closely related bacterial strains have already undergone selection and are therefore not representative of spontaneous mutation.

Abstract  INTRODUCTION: Experimental studies indicate that some chemicals with UV blocking properties (known as UV filters) can act as endocrine disruptors. UV filters are used in sunscreens and other cosmetic- and personal care products, as well as in other consumer products such as food packaging, clothing and furniture textiles to protect the products against UV radiation. Here we present the urinary excretion of suspected endocrine active UV filters in Danish children and adolescents recruited from the general population.

METHODS: The content of benzophenone (BP), benzophenone-1 (BP-1), benzophenone-2 (BP-2), benzophenone-3 (BP-3), 5-chloro-2- hydroxybenzophenone (BP-7), 4-hydroxybenzophenone (4-HBP), 4-methyl-benzophenone (4-MBP), 3-(4- methylbenzylidene)-camphor (4-MBC) and 3-benzylidene camphor (3-BC) were monitored in 24h urine and two consecutive first morning samples from 129 healthy Danish children and adolescents (6-21 yrs). All 387 samples were collected during the autumn (Nov. 2007) and were analyzed by a new on-line TurboFlow-LC-MS/MS method developed for simultaneous biomonitoring of these nine UV filters in urine.

RESULTS: BP-3 and BP-1 were detected in more than 80% of the 24h samples and were significantly correlated (R(2)=0.815). BP, 4-HBP and BP-2 were found in 43, 15 and 5% of the samples, respectively. The median (range) concentrations of the UV-filters in 24-h urine were as follows: BP-3, 0.92 (LOD-115); BP-1, 0.54 (LOD-44.6); BP,<LOD (LOD-48.5); 4-HBP,<LOD (LOD-10.5); and BP-2,<LOD (LOD-8.43) ng/mL. In general BP-1 and BP-3 were higher in girls compared to boys and were also higher in the group of adolescent girls (16-21 yrs) compared to the younger age groups of girls. None of the other UV filters; BP-7, 4-MBP, 4-MBC or 3-BC were detected in urine. A highly significant correlation between first morning and 24h urine levels of BP-3, BP-1 and 4-HBP from the same day was observed.

CONCLUSION: Our project on UV filters analyzed by a new robust and sensitive LC-MS/MS method in Danish children and adolescents showed that almost all individuals were exposed to UV filters. Sun protection products have been claimed to be a major source of exposure to sunscreens. However since all children in the present study were exposed during autumn where sunscreens are not needed in Denmark our study also indicates that other sources and routes of exposure might be of relevance.

Abstract  The fruit of Phyllanthus emblica Linn. (PE) has been widely consumed as a functional food and folk medicine in Southeast Asia due to its remarkable nutritional and pharmacological effects. Previous research showed PE delays mitotic progress and increases genomic instability (GIN) in human colorectal cancer cells. This study aimed to investigate the similar effects of PE by the biomarkers related to spindle assembly checkpoint (SAC), mitotic aberrations and GIN in human NCM460 normal colon epithelial cells. Cells were treated with PE and harvested differently according to the biomarkers observed. Frequencies of micronuclei (MN), nucleoplasmic bridge (NPB) and nuclear bud (NB) in cytokinesis-block micronucleus assay were used as indicators of GIN. Mitotic aberrations were assessed by the biomarkers of chromosome misalignment, multipolar division, chromosome lagging and chromatin bridge. SAC activity was determined by anaphase-to- metaphase ratio (AMR) and the expression of core SAC gene budding uninhibited by benzimidazoles related 1 (BubR1). Compared with the control, PE-treated cells showed (1) decreased incidences of MN, NPB and NB (p < 0.01); (2) decreased frequencies of all mitotic aberration biomarkers (p < 0.01); and (3) decreased AMR (p < 0.01) and increased BubR1 expression (p < 0.001). The results revealed PE has the potential to protect human normal colon epithelial cells from mitotic and genomic damages partially by enhancing the function of SAC.

Abstract  The nucleolus is the site of ribosome biogenesis and forms around the actively transcribed ribosomal RNA (rRNA) genes. However, the nucleolus is also implicated in cell cycle regulation, tumour suppression and chromosome segregation and nucleolar disfunction is linked to a wide range of human diseases. Interestingly, the nucleolus is also required for genome reprogramming and the establishment of heterochromatin in the mammalian embryo. Mammalian oocytes contain a subnuclear structure that is believed to be the precursor of the functional nucleolus, the Nucleolar Precursor Body (NPB). But the NPB is also required for the organisation of the zygotic heterochromatin and the establishment of pluripotency. We found that disruption of the mouse Upstream Binding Factor (UBF (UBTF)) gene caused disassembly of somatic nucleoli and the accumulation of the key rRNA gene transcription factors into dense subnuclear foci resembling NPBs. Here we show that UBF deletion causes the rRNA genes to collapse onto their centromere-proximal chromosomal sites spatially distinct from NPB-like structures, and that these structures contain rRNA gene transcription factors but not all nucleolar proteins. We further find that embryonic NPBs and their surrounding heterochromatin are both disrupted in UBF-null mouse embryos. These embryos also display subnuclear foci containing the rRNA gene transcription factors and arrest development before completing the forth cleavage division. The data suggest that the rRNA gene transcription factors have an intrinsic ability to interact and form a discrete nuclear compartment even in the absence of any rRNA gene activity and that the formation or maintenance of the zygotic NPB and surrounding heterochromatin requires UBF.

Abstract  Mitochondrial DNA depletion syndromes (MDS) are often serious autosomal recessively inherited disorders characterized by tissue-specific mtDNA copy number reduction. Many genes, including MPV17, are associated with the hepatocerebral form of MDS. MPV17 encodes for a mitochondrial inner membrane protein with a poorly characterized function. Several MPV17 mutations have been reported in association with a heterogeneous group of early-onset manifestations, including liver disease and neurological problems. Mpv17-deficient mice present renal and hearing defects. We describe here a MPV17 truncation mutation in dogs. We found a 1-bp insertion in exon 4 of the MPV17 gene, resulting in a frameshift and early truncation of the encoded protein. The mutation halves MPV17 expression in the lymphocytes of the homozygous dogs and the truncated protein is not translated in transfected cells. The insertion mutation is recurrent and exists in many unrelated breeds, although is highly enriched in the Boxer breed. Unexpectedly, despite the truncation of MPV17, we could not find any common phenotypes in the genetically affected dogs. The lack of observable phenotype could be due to a late onset, mild symptoms or potential tissue-specific compensatory mechanisms. This study suggests species-specific differences in the manifestation of the MPV17 defects and establishes a novel large animal model to further study MPV17 function and role in mitochondrial biology.

Abstract  Some studies based on ambulatory blood pressure (BP) monitoring (ABPM) have reported a reduction in sleep-time relative BP decline towards a more non-dipping pattern in the elderly, but rarely have past studies included a proper comparison with younger subjects, and no previous report has evaluated the potential role of hypertension treatment time on nighttime BP regulation in the elderly. Accordingly, we evaluated the influence of age and time-of-day of hypertension treatment on the circadian BP pattern assessed by 48-h ABPM. This cross-sectional study involved 6147 hypertensive patients (3108 men/3039 women), 54.0 ± 13.7 (mean ± SD) yrs of age, with 2137 (978 men/1159 women) being ≥60 yrs of age. At the time of study, 1809 patients were newly diagnosed and untreated, and 4338 were treated with hypertension medications. Among the later, 2641 ingested all their prescribed BP-lowering medications upon awakening, whereas 1697 ingested the full daily dose of ≥1 hypertension medications at bedtime. Diagnosis of hypertension in untreated patients was based on ABPM criteria, specifically an awake systolic (SBP)/diastolic (DBP) BP mean ≥135/85 mm Hg and/or an asleep SBP/DBP mean ≥120/70 mm Hg. Collectively, older in comparison with younger patients were more likely to have diagnoses of microalbuminuria, chronic kidney disease, obstructive sleep apnea, metabolic syndrome, anemia, and/or obesity. In addition, the group of older vs. younger patients had higher glucose, creatinine, uric acid, triglycerides, and fibrinogen, but lower cholesterol, hemoglobin, and estimated glomerular filtration rate. In older compared with younger patients, ambulatory SBP was significantly higher and DBP significantly lower (p < .001), mainly during the hours of nighttime sleep and initial hours after morning awakening. The prevalence of non-dipping was significantly higher in older than younger patients (63.1% vs. 41.1%; p < .001). The largest difference between the age groups was in the prevalence of a riser BP pattern, i.e., asleep SBP mean greater than awake SBP mean (19.9% vs. 4.9% in older vs. younger patients, respectively; p < .001). The sleep-time relative SBP decline was mainly unchanged until ~40 yrs of age, and then significantly and progressively decreasing with increasing age at a rate of .28%/yr (p < .001), reaching a minimum value of 4.38% ± .47% for patients ≥75 yrs of age. Treated compared with untreated patients showed lower awake and asleep SBP means, although the predictable changes of SBP and DBP with age were equivalent in both groups. As a consequence, there were no significant differences between untreated and treated patients in the changes of the sleep-time relative SBP and DBP declines with age. Additionally, the asleep SBP and DBP means were significantly lower and the sleep-time relative SBP and DBP declines significantly higher at all ages in patients ingesting ≥1 BP-lowering medications at bedtime as compared with those ingesting all medications upon awakening. Our findings document a significantly elevated prevalence of a blunted nighttime BP decline with increasing age ≥40 yrs. The prevalence of a riser BP pattern, associated with highest cardiovascular risk among all possible BP patterns, was 4 times more prevalent in patients ≥60 yrs of age than those <60 yr of age. Most important, there was an attenuated prevalence of a blunted nighttime BP decline at all ages when ≥1 hypertension medications were ingested at bedtime as compared with when all of them were ingested upon awakening. These findings indicate that older age should be included among the conditions for which ABPM is recommended for proper cardiovascular risk assessment.

Abstract  MYB transcription factors exist in a large copy number and control various plant phenotypes. We cloned R2R3 MYB-type transcription factors that determine the coloration of basal sheaths in barley and wheat coleoptiles. These genes are highly homologous to maize C1 and rice OsC1, regulators for anthocyanin biosynthesis, but they control seed pigmentation in maize and rice. On the basis of high homology, barley and wheat counterparts are designated HvC1 and TaC1, respectively. HvC1 gene is located on the short arm of chromosome 7H, and TaC1 genes are located on the short arms of chromosomes 7A, 7B, and 7D (TaC1-A1, B1, and D1, respectively). HvC1 is a strong candidate for Ant1 because of (1) complete co-segregation of anthocyanin pigmentation phenotype of the basal sheath with the HvC1 genotype in genetic mapping, and (2) complete deletion of the HvCl gene in two anthocyanin-decreased allelic mutants (ant1.1 and ant1.2) that were induced by irradiation. In contrast, colorless coleoptile wheat lines had lesions in all three genomes consisting of a single-nucleotide substitution or a 1-bp deletion of TaC1-A1, a 1.7-kb insertion of TaC1-B1, and a 2.0-kb insertion of TaC1-D1. At least one normal TaC1 gene appears to be sufficient to produce anthocyanin pigments in wheat coleoptiles. Previous crossing experiments localized Rc (red coleoptile) genes to homoeologous group 7 chromosomes and deduced Rc genotypes of several wheat lines. Their TaC1 gene sequence variation coincided with deduced Rc genotypes; therefore, the present molecular genetic study demonstrates that TaC1 is a strong candidate for Rc in wheat.

Abstract  We identified the minimal locus of 163-kb plasmid pSV1 of Streptomyces violaceoruber for the replication in S. lividans. This locus comprised a repA gene and an upstream 407-bp sequence containing two inverted repeats (IR-III and IR-IV) within an iteron, an AT-rich region and a 300-bp noncoding sequence (NCS). RepA protein bound specifically to a 94-bp sequence covering the intact IR-III and IR-IV to form multimers of DNA/protein complexes, but was unable to bind specifically to the NCS and the promoter of repA gene. Interestingly, this 'bound' region also leaves eight 1-bp 'unbound' spacers at 7-11-9-11-9-11-9-11-8-bp intervals. RepA protein-protein interaction could form dimers or trimers in vitro. These results suggest that a higher-order complex between pSV1 RepA protein and the long inverted repeats may be formed during the initiation of plasmid replication.